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Rett syndrome genetics {#Sec1}
======================
It is now 50 years since Andreas Rett reported his observations on 22 girls with similar clinical features, subsequently known as Rett syndrome (RTT). Classic RTT is defined by a regression phase and subsequent stabilization of diagnostic criteria, which include partial or complete loss of spoken language, dyspraxic gait and stereotypic hand movements such as 'hand mouthing' \[[@CR1]\]. With very few familial cases available, it took a further 33 years before mutations affecting a protein called methyl-CpG-binding protein 2 (MeCP2) were shown to be the almost exclusive cause of this neurological disorder. The *MECP2* gene is located on the X chromosome, and RTT is classified as an X-linked dominant disorder. Thus, as expected, males are more severely affected and rarely survive infancy. Rett syndrome therefore overwhelmingly affects females, who, owing to X chromosome inactivation, have a mixture of cells that express either the wild-type or mutant version of MeCP2. This cellular mosaicism defines RTT and highlights the importance of MeCP2 for proper neuronal function. In the light of new insights into the function of MeCP2 and the novel gene therapy technologies currently being developed, here we discuss recent progress in understanding the molecular pathogenesis of this complex disease and the search for a therapy.
Model systems for studying Rett syndrome {#Sec2}
========================================
Mouse models are a vital tool for studying RTT as MeCP2 deficiency closely mimics the clinical features of the human disorder, including motor defects and breathing arrhythmia. Indeed, a recent study \[[@CR2]\] of animals expressing three *Mecp2* mutations with differing average clinical severity showed a matching severity spectrum in the mice (Fig. [1](#Fig1){ref-type="fig"}) \[[@CR3]\]. These findings reflect the high conservation of the MeCP2 amino acid sequence between human and mouse (95% identical) and the parallel dynamics of MeCP2 expression during brain development. Thus, despite differences in the brain structure and developmental timing between human and mouse, these striking similarities suggest that the molecular consequences of MeCP2 mutation are similar between the two species. Early indications that RTT results exclusively from absence of MeCP2 in the brain have recently been reinforced by a mouse model that has normal levels of central nervous system MeCP2, but lacks this protein in the rest of the body, and shows none of the major phenotypes associated with RTT-like mice \[[@CR4]\].Fig. 1Analysis of point mutations responsible for Rett syndrome (*RTT*) in human and mouse. **a** The primary protein structure of methyl-CpG-binding protein 2 (MeCP2), which is a chromosomal protein that binds to methylated DNA, highlights two key functional domains---a methyl-CpG-binding domain (MBD) and a NCoR/SMRT co-repressor interaction domain (NID). Shown as *red vertical lines* below the schematic are the positions of all RTT-causing missense mutations (RettBASE; <http://mecp2.chw.edu.au/>). The positions of three particular RTT-causing missense mutations---R133C, T158M and R306C, reflecting the spectrum of clinical severity---are indicated above the schematic (modified from \[[@CR6]\]). **b** The approximate clinical severity of patients possessing the specific missense mutations T158M (*red*), R306C (*blue*) or R133C (*green*), based on independent studies using a variety of clinical severity score systems, for example \[[@CR3]\]. **c** Scores of phenotypic severity of mouse models containing the T158M (*red*), R306C (*blue*) and R133C (*green*) missense mutations, in comparison with those of wild-type mice (*dark gray solid line*) and *Mecp2*-null mice (*pale gray broken line*). The *asterisks* indicate where no animals of that genotype survived beyond the indicated time-point. The data are adapted from Brown et al. \[[@CR2]\] and are reproduced with permission of Oxford University Press
Advances in cellular technologies have allowed further development of human tissue-culture models of RTT \[[@CR5], [@CR6]\]. The ability to study a homogeneous population of human neurons eliminates the complexity of the brain, allows more precise genetic manipulation and simplifies the interpretation of findings \[[@CR5]\]. It also streamlines screening of potential therapeutics and viral delivery vectors in a human neuronal setting. In addition to mice, a *Mecp2*-null rat model has been produced (Sage Labs), and there has been progress towards generating non-human primate models of RTT \[[@CR7]\] that should be beneficial for testing once further phenotypic characterization is available.
MeCP2---a genome-wide transcriptional repressor {#Sec3}
===============================================
Progress in understanding the molecular aetiology of RTT has been facilitated by combining data from clinical genetics and mouse models with cellular and biochemical investigations. Importantly, nearly all RTT missense mutations cluster in two discrete domains of MeCP2: the methyl-CpG-binding domain (MBD) and the NCoR/SMRT co-repressor interaction domain (NID) (Fig. [1](#Fig1){ref-type="fig"}) \[[@CR6]\]. With a crystal structure of the MBD bound to methylated DNA available, we can understand the loss of DNA binding caused by RTT mutations in the MBD (reviewed in \[[@CR6]\]). Likewise, most amino acids in the NID---which interact with NCoR/SMRT co-repressor complexes---when mutated cause RTT syndrome, and all NID mutations tested so far prevent the interaction with this large multi-component complex. The NCoR/SMRT complex includes the transcriptional-repression-associated histone deactylase HDAC3, thus supporting the hypothesis that MeCP2 recruits this complex to methylated sites within the genome to downregulate transcription (reviewed in \[[@CR6]\]).
The notion that MeCP2 recruits gene silencing machinery is well supported by a variety of experimental data, but it has taken time to link this model convincingly to the effects of MeCP2 loss on transcription patterns in the brain. Recent studies, however, have uncovered striking three-way proportionalities between DNA methylation levels, MeCP2 binding-site occupancy and transcriptional inhibition \[[@CR8], [@CR9]\]. This progress has depended on new information regarding the DNA binding-site specificity of MeCP2 showing that the di-nucleotide methyl-CA (mCA), as well as the canonical methyl-CG (mCG), both constitute target sites. In addition, improved mapping of MeCP2 binding in vivo using chromatin immunoprecipitation has increasingly taken account of the high frequency of mCG and mCA (\~1 per 100 bp) in the neuronal genome, which rendered conventional peak-finding approaches problematic. Finally, well-controlled and highly replicated RNA-sequencing analysis is required for sensitive transcriptional profiling. What has emerged from these precision studies is that MeCP2 exerts a restraining effect on transcription. This effect is global, gene-body-dependent and subtle, but correlates well with DNA methylation density, in particular affecting a large proportion of brain-specific genes that are unusually long \[[@CR8], [@CR9]\] (reviewed in \[[@CR6]\]). Thus, in contrast to early expectations that MeCP2 would alter the expression of a few discrete target genes, new results portray it as a global transcriptional repressor that modulates gene expression programmes in a DNA-methylation-dependent manner. It remains unclear, however, how loss of this genome-wide modulation of gene expression patterns contributes to the observed RTT pathologies.
Reversibility and the search for a cure {#Sec4}
=======================================
A major contribution of mouse models has been the proof-of-principle demonstration that RTT is a reversible (i.e. curable) disorder. Restoration of the gene in *Mecp2*-deficient animals with advanced Rett-like signs led to dramatic restoration of normal breathing, mobility and other features (reviewed in \[[@CR6]\]). These results have given great impetus to the search for a cure. One approach is to seek small molecules that reverse the downstream consequences of MeCP2 deficiency, whereas an alternative is to rectify the genetic cause of the disorder through gene therapy, protein replacement or gene correction \[[@CR1]\].
As a global regulator that tunes gene expression, and perhaps reduces transcriptional noise, it is feasible that a loss of MeCP2 function causes a transcriptional imbalance leading to a 'sub-optimal' brain. This underlying mechanistic heterogeneity has implications for therapeutic approaches for RTT. With many genes mildly misregulated, it might be difficult to identify a single pathway that is susceptible to small-molecule regulation that could cure RTT. More positively, it might be possible to identify drugs to target certain biochemical pathways that are crucial for brain function, and thereby ameliorate a number of aspects of RTT. Pharmacological readjustment of key pathways could be of therapeutic value, even if many other processes remain un-corrected \[[@CR1]\]. In support of this argument, a screen for genetic suppressors of the mouse Rett-like phenotype detected multiple hits (cited in \[[@CR6]\]), indicating that inhibition of specific pathways can indeed be beneficial. These results indicate that it is premature to rule out small-molecule-based therapeutic approaches at this stage.
Two laboratories have shown that delivery of the *Mecp2* gene using an adeno-associated virus (AAV) vector is efficient enough to rescue some of the phenotypes of *Mecp2*-null male and heterozygous female mice (reviewed in \[[@CR1]\]). Initial concerns about inducing MeCP2 overexpression syndrome, especially in female mice that express a wild-type copy of the gene in approximately half of their cells, have so far been ameliorated by the use of endogenous promoters and systemic injection routes. There is a long way to go, but initial results suggest that gene therapy could be of clinical utility. The most desirable therapeutic scenario of all would be to correct the point mutation itself using CRISPR--Cas9 or related techniques. In vivo gene disruption of the *Mecp2* allele in wild-type mice using AAV vectors to deliver Cas9 and single guide RNA (sgRNA) has been successful, achieving a knockout of MeCP2 in approximately 70% of cells in the hippocampus \[[@CR10]\]. This and other findings raise the possibility that in vivo correction of *MECP2* mutations will one day be feasible. To achieve targeting specificity, the RTT mutation itself (or single-nucleotide polymorphisms between the two alleles) could be used to distinguish mutant and wild-type copies of *MECP2*, and thus encourage targeting of only the mutant *MECP2* allele. Technical problems remain---most notably the restricted payload of AAV vectors requires the simultaneous use of multiple viruses for delivery of the many, large gene-editing components, which would reduce the efficiency of such an approach. Despite these current limitations, gene editing remains a promising therapeutic option.
Concluding remarks {#Sec5}
==================
Pre-clinical research strongly suggests that RTT could be one of the first curable neurological disorders. The application of in-depth, genome-wide approaches and increasingly sophisticated model systems are painting a clearer picture of the functional role of MeCP2 in neurons, and this new information promises to guide future therapeutic strategies. At the same time, multiple therapies are currently being developed---for example gene therapy, reactivation of the inactive X chromosome and modulation of neurotransmitter signalling pathways. As to which of these diverse approaches could be of therapeutic value, only time will tell.
AAV
: Adeno-associated virus
Cas9
: CRISPR-associated protein 9
CRISPR
: Clustered regularly interspaced short palindromic repeat
mCA
: Methyl-CA
mCG
: Methyl-CG
MDB
: Methylated-DNA binding domain
MeCP2
: Methyl-CpG-binding protein 2
NID
: NCoR/SMRT co-repressor interaction domain
RTT
: Rett syndrome
sgRNA
: Short guide RNA
SNP
: Single-nucleotide polymorphism
We thank Jim Selfridge and Jacky Guy for useful comments on the manuscript. Funding was provided by the Wellcome Trust (\[099841\], \[091580\], \[107930\] and \[092076\]) and by the Rett Syndrome Research Trust. We apologise for omitting many deserved citations owing to space constraints.
Authors' contributions {#FPar1}
======================
Both authors read and approved the final manuscript.
Competing interests {#FPar2}
===================
The authors declare that they have no competing interests.
| {
"pile_set_name": "PubMed Central"
} |
1. Introduction {#sec1-nutrients-09-01369}
===============
Recently, a lot of studies have focused on pro-health and curative properties of natural products, including bee products, and bee pollen is among these valuable apitherapeutics. It is a product of plant origin, collected and partly processed by bees. It is produced from pollen grains enriched with nectar, honey and honeybee salivary glands secretion. Bee pollen is composed of more than 250 various substances. The type and role of separate compounds in the total mass is diverse, and depend on plant species, the climate zone and season when it was collected \[[@B1-nutrients-09-01369],[@B2-nutrients-09-01369],[@B3-nutrients-09-01369]\].
Bee pollen is characterized by high nutritious value and different biological activities. Nutritious properties of bee pollen result from the presence of such substances as proteins, amino acids, carbohydrates, lipids (including ω-3 and ω-6 acids), vitamins and bio-elements. Therapeutic and protective effects are related to the content of functional compounds such as polyphenols \[[@B4-nutrients-09-01369],[@B5-nutrients-09-01369],[@B6-nutrients-09-01369],[@B7-nutrients-09-01369]\].
Polyphenols are the main ingredients that determine antioxidant activity of bee pollen \[[@B8-nutrients-09-01369],[@B9-nutrients-09-01369]\]. Their content may vary significantly depending on the origin of the material \[[@B10-nutrients-09-01369],[@B11-nutrients-09-01369],[@B12-nutrients-09-01369]\]. The profile of polyphenolic compounds in bee pollen may serve as an important indicator of the product quality due to its specificity as well as qualitative and quantitative stability \[[@B13-nutrients-09-01369],[@B14-nutrients-09-01369]\]. Polyphenols in bee pollen are mainly phenolic acids and flavonoids, and their biological activity is conditioned by their specific chemical structure. The presence of a benzene ring in the particle structure is a common characteristic of the compounds. High antioxidant capacity of polyphenols is closely related to the presence of conjugated double bonds and the number and location of hydroxyl groups in an aromatic ring \[[@B15-nutrients-09-01369],[@B16-nutrients-09-01369]\]. Since polyphenols can neutralize free radicals, they have the following activities: anti-inflammatory, antiallergenic, antiviral, anti-clotting, anticancer, immunostimulating, hepatoprotective, and they also inhibit specific enzymes. Furthermore, polyphenols have a protective function in cardio-vascular diseases caused by oxidative stress \[[@B17-nutrients-09-01369],[@B18-nutrients-09-01369],[@B19-nutrients-09-01369]\].
Cardio-vascular diseases, including cardiac artery disease, are a frequent cause of death in the majority of highly developed countries. Atherosclerosis is a specific form of a chronic inflammatory process in the aorta and medium-sized arteries. Pathogenesis of atherosclerosis is caused by various factors. The development of atherosclerosis is modulated by disturbances of lipid metabolism, oxidative stress, fibrinolysis and coagulation in the vascular wall as well as disturbed homeostasis of the renin-angiotensin-aldosterone system, which impairs the function of endothelial cells \[[@B20-nutrients-09-01369],[@B21-nutrients-09-01369]\].
Taking into consideration various biological activities of bee pollen ingredients, the aim of the current study was to determine the effect of polyphenol-rich EEP on atherosclerosis induced by a high-fat diet in ApoE-knockout mice. The latter constitute an internationally recognized animal model for studying atherosclerosis and hypercholesterolemia.
2. Materials and Methods {#sec2-nutrients-09-01369}
========================
2.1. Reagents and Drugs {#sec2dot1-nutrients-09-01369}
-----------------------
Xylene, hematoxylin, eosin aqueous solution (1%), anhydrous ethyl alcohol (99.8%), acetone, phosphate buffered saline (PBS), formalin were purchased from POCh (Gliwice, Poland). DPX mounting medium for histology (mixture of Distyrene, a Plasticizer, and Xylene) was purchased from Fluka (Dresden, Germany). Tiopental was purchased from Sandoz (Warsaw, Poland), total cholesterol Test (Cat. No. OSR 6116) was purchased from Beckman Coulter (Praha, Czech Republic), ELISA Kit for mouse oxidized low density lipoprotein (Cat. No. E90527Mu) was purchased from Uscn Life Science Inc. (Wuhan, China), ADMA ELISA Kit for the determination of ADMA (Cat. No. K 3001) was purchased from Immundiagnostik AG (Bensheim, Germany), ELISA Kit for mouse angiotensin I converting enzyme (ACE) (Cat. No. E90004Mu) was purchased from Uscn Life Science Inc. (Wuhan, China), Angiotension II (Human, Rat, Mouse, Porcine, Caniane) EIA Kit (Cat. No. EK-002-12) was purchased from Phoenix Pharmaceutical Inc. (Karlsruhe, Germany).
2.2. Preparation of Ethanol Extract of Bee Pollen (EEP) {#sec2dot2-nutrients-09-01369}
-------------------------------------------------------
The material for the tests were ground pollen loads obtained from ecologically clean harvest areas in the south of Poland. Samples of polyfloral bee pollen were collected from an apiary at Kamianna (GPS N 49°31′527, E 20°56′116), Poland, in the Beskidy Mountains. From May to July, bee pollen samples were collected by beekeepers with the use of pollen traps mounted on selected beehives. Mainly, they were pollens of common dandelion *Taraxacum officinale*, rapeseed *Brassica napus*, European raspberry *Rubus ideaeus*, acacia *Robinia pseudoacacia*, buckwheat *Fagopyrum esculentum*, linden *Tilia mordata*, clover *Trifolium repens*. Ethanol extract was prepared according to a slightly modified method of Almaraz-Abarca et al. \[[@B8-nutrients-09-01369]\]. The ethanol extract of bee pollen was prepared by weighing 20 g of ground bee pollen, with accuracy of 0.01 g. Then, the bee pollen sample was extracted 5 times with 50% (*v*/*v*) ethanol aqueous solution, in 200 mL portions, and shaken each time for 60 min at room temperature, in order to macerate the sample. After each extraction, the sample was filtered under reduced pressure with the use of a water pump. The filtrate was collected, and substrate was extracted again with another portion of ethanol aqueous solution. The obtained filtrate was centrifuged at 10,000 rpm for 10 min, and then it was evaporated under reduced pressure in a rotary vacuum evaporator (UNIPAN-PRO 350P). The evaporated extract was dried in a laboratory incubator at 38 °C to obtain solid mass. Studies of EEP focused on the content of polyphenols and flavonoids, and their antioxidant effect \[[@B22-nutrients-09-01369]\].
2.3. Animals and Treatments {#sec2dot3-nutrients-09-01369}
---------------------------
The study was conducted on 56 females of C~57~BL~6~ ApoE-knockout mice, aged 4 weeks, 25 ± 5 g of body mass. During the experiment, the animals were kept in standard breeding conditions, i.e., in groups of 10 animals in polypropylene cages, in rooms of constant temperature (23 ± 2 °C), and constant air humidity (50--70%), keeping the daytime rhythm in the inflow of light (a 12-h cycle). The high-fat diet (HFD) comprised High Fat Rodent Diet supplemented with 21% lard and 0.15% cholesterol (Special Diets Services, Witham, Essex, UK). The standard diet (SD) was standard feed (Labofeed B) containing 17% protein, 3.5% fat and 38% carbohydrates (Animal Feed Manufacturer "Morawski" in Kcynia). EEP was added to the feed in a dose of 0.1 and 1 g/kg BM, respectively. Two dosing levels of EEP used in the research were calculated based on the daily ingestion of polyphenols in the diet of the inhabitants of Central Europe \[[@B23-nutrients-09-01369]\].
Experimental animals were divided into 6 groups according to the following scheme: 5 study groups: HFD, HFD-0.1, HFD-1, SD-0.1, SD-1 (10 animals in each group), and a control group SD (6 animals). Characteristics of the groups: HFD: high-fat diet, HFD-0.1: high-fat diet supplemented with EEP (0.1 g/kg BM), HFD-1: high-fat diet supplemented with EEP (1 g/kg BM), SD-0.1: standard diet supplemented with EEP (0.1 g/kg BM), SD-1: standard diet supplemented with EEP (1 g/kg BM), and SD: standard diet.
The project with the use of animal models was approved by the Local Ethics Committee on Animal Experimentation of the Medical University of Silesia in Katowice.
2.4. Collecting Biological Material for Tests {#sec2dot4-nutrients-09-01369}
---------------------------------------------
The material for tests was collected in the fasted state, in the 5th, 10th, 12th, 14th, and 16th week of the experiment, after general anesthesia with a drug called Tiopental in a dose of 20 mg/kg BM, administered by injection.
Blood for analyses was collected by puncturing the tip of the heart with a cannula. Blood was collected to chemically pure test tubes in order to obtain serum. Blood after clotting and clot retraction was centrifuged, and serum was frozen at −80 °C, and stored for further tests.
The collection of material for histopathological tests was as follows: before the material was collected, perfusion was performed by decompression surgery in the 1/3 of descending abdominal aorta. Perfusion was performed with phosphate buffered saline (PBS) until the light pink translucent liquid flew out in the decompressing orifice. Then, perfusion with 10% PBS-buffered formaldehyde was performed for 2 min to obtain preliminary fixing of the heart and trunks of major blood vessels. Next, both, the heart and the brachiocephalic trunk were fixed in 10% PBS-buffered formaldehyde, and kept for further tests.
2.5. Histopathological Tests {#sec2dot5-nutrients-09-01369}
----------------------------
Histopathological evaluation of the heart and the brachiocephalic trunk was conducted in the 16th week of the study. The collected material was fixed in a 10% formalin solution for minimum 24 h. After fixing, the tissues were treated with the aqueous solutions of ethyl alcohol. The material was subsequently rinsed with acetone and xylene, respectively. Then, tissues were placed in a xylene and paraffin mixture at a 1:1 ratio and then in liquid paraffin. After solidification, the paraffin was cut into 3--5 micron thick bands. Paraffin bands were treated with xylene, an ethyl alcohol/xylene mixture at a 1:1 ratio and aqueous solutions of ethyl alcohol, and then rinsed in distilled water. The material prepared in this way was stained with the standard hematoxylin-eosin (HE) method, which included staining with alkaline solution of hematoxylin, rinsing in distilled water, staining with acid solution of eosin, and rinsing in distilled water. Later, preparations were treated with aqueous solutions of ethyl alcohol, ethanol/xylene mixture at 1:1 ratio and rinsed in xylene in order to finally dehydrate and radiate the tissue. Slides were covered with a cover glass with DPX-medium.
Histopathological preparations were evaluated in 40×, 100×, and 200× microscopy objectives magnification using Olympus BX60 microscope equipped with XC50 digital camera and Olympus cellSens Standard software (Olympus Corp., Tokyo, Japan).
2.6. Biochemical Tests {#sec2dot6-nutrients-09-01369}
----------------------
We tested serum obtained in the scheduled study periods, i.e., in the 5th, 10th, 12th, 14th, and 16th week of the experiment for the study groups HFD, HFD-0.1, HFD-1, SD-0.1, SD-1, and in the 1st, 10th, and 16th week for the control group (SD). The levels of ox-LDL, ADMA, ACE, ANG II were determined with Asys HiTech UVM 340 microplate reader and Micro Win 4.35.
### 2.6.1. Determination of Total Cholesterol (TC) {#sec2dot6dot1-nutrients-09-01369}
Total cholesterol serum level was determined with an enzymatic method. A reagent set from Beckman Coulter (Cat. No. OSR 6116) was used for the determination, which was conducted according to the manufacturer's instruction for Beckman Coulter AU 680 analyzer. Total cholesterol level was given in mg/dL.
### 2.6.2. Determination of Oxidized Low Density Lipoprotein (Ox-LDL) Concentration {#sec2dot6dot2-nutrients-09-01369}
The concentration of ox-LDL was determined with a sandwich ELISA using Mouse Oxidized Low Density Lipoprotein ELISA Kit. The analysis was conducted according to the manufacturer's instruction.
The microtiter plate was pre-coated with an antibody specific to ox-LDL. Samples diluted 10 times or standards were then added to the appropriate microtiter plate wells, and incubated for 2 h (37 °C). Then, the liquid was removed and a biotin-conjugated polyclonal antibody specific for ox-LDL was added. The plate was incubated for 1 h at 37 °C. Then the plate was washed, and avidin conjugated to horseradish peroxidase (HRP) was added to each microplate well, and the plate was incubated for 1 h (37 °C). Next, a 3,3′,5,5′-tetramethyl-benzidine (TMB) substrate solution was added to each well and incubated for 30 min (37 °C). Only those wells that contained ox-LDL, biotin-conjugated antibody and enzyme-conjugated avidin exhibited a change in color. The enzyme-substrate reaction was terminated by the addition of a sulfuric acid solution, and the color change was measured spectrophotometrically at a wavelength of 450 nm. The concentration of ox-LDL (ng/mL) in the samples was then determined by comparing the optical density (O.D.) of the samples to the standard curve.
### 2.6.3. Determination of Asymmetric Dimethylarginine (ADMA) Concentration {#sec2dot6dot3-nutrients-09-01369}
The concentration of ADMA was determined with ADMA ELISA Kit for the determination of ADMA. The assay was conducted according to the manufacture's instruction.
This assay is based on the method of competitive enzyme-linked immunoassays. The sample preparation includes the addition of a derivatization-reagent for ADMA coupling. Afterwards, the treated samples and the polyclonal ADMA-antiserum are incubated in wells of microplate coated with ADMA-derivative (tracer) for 18 h at 4--8 °C. During the incubation period, the target ADMA in the sample competes with the tracer immobilized on the wall of the microtiter wells for the binding of the polyclonal antibodies. The ADMA in the sample displaces the antibodies out of the binding to the tracer. Therefore the concentration of the tracer-bound antibody is inversely proportional to the ADMA concentration in the sample. During the second incubation step (for 1 h at 18--26 °C), a peroxidase-conjugated antibody is added to each microtiter well to detect the anti-ADMA antibodies. After washing away the unbound components, 3,3′,5,5′-tetramethyl-benzidine (TMB) is added as a substrate for peroxidase and then incubated for 10 min at 18--26 °C. Finally, the enzymatic reaction is terminated by acidic stop solution. The color changes from blue to yellow and the absorbance is measured in the photometer at 450 nm. The intensity of the yellow color is inversely proportional to the ADMA concentration in the sample; this means high ADMA concentration in the sample reduces the concentration of tracer-bound antibodies and lowers the photometric signal. The concentration of ADMA (μmol/L) in the samples is then determined by comparing the O.D. of the samples to the standard curve.
### 2.6.4. Determination of Angiotensin-Converting Enzyme (ACE) Concentration {#sec2dot6dot4-nutrients-09-01369}
The concentration of ACE was determined with a sandwich ELISA using mouse angiotensin I converting enzyme (ACE) ELISA Kit. The assay was conducted according to the manufacturer's instruction.
The microtiter plate in this kit was pre-coated with an antibody specific to ACE. Samples diluted 10 times or standards were added to the appropriate microtiter plate wells and incubated for 2 h (37 °C). Then the liquid was removed and a biotin-conjugated polyclonal antibody specific for ACE was added. The plate was incubated for 1 h at 37 °C. Then the plate was washed, and avidin conjugated to horseradish peroxidase (HRP) was added to each microplate well, and incubated for 1 h (37 °C). Then a 3,3′,5,5′-tetramethyl-benzidine (TMB) substrate solution was added to each well and incubated for 15 min (37 °C). Only those wells that contained ACE, biotin-conjugated antibody and enzyme-conjugated avidin would exhibit a change in color. The enzyme-substrate reaction was terminated by the addition of a sulphuric acid solution and the color change was measured spectrophotometrically at a wavelength of 450 nm. The concentration of ACE (ng/mL) in the samples was then determined by comparing the O.D. of the samples to the standard curve.
### 2.6.5. Determination of Angiotensin II (ANG II) Concentration {#sec2dot6dot5-nutrients-09-01369}
The concentration of ANG II was determined with Angiotension II (Human, Rat, Mouse, Porcine, Caniane) EIA Kit. The assay was carried out according to the manufacturer's instruction.
The microtiter plate in this kit was pre-coated with an antibody specific to ANG II. Samples diluted 10 times or standards were then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for ANG II and incubated for 2 h (20--23 °C). Then streptavidin conjugated to Horseradish Peroxidase (SA-HRP) was added to each microplate well and incubated 1 for hour (20--23 °C). Next a 3,3′,5,5′-tetramethyl-benzidine (TMB) substrate solution was added to each well and incubated for 1 h (20--23 °C). Only those wells that contained ANG II, biotin-conjugated antibody and enzyme-conjugated streptavidin would exhibit a colour change. The enzyme-substrate reaction was terminated by the addition of a hydrochloric acid solution, and the colour change was measured spectrophotometrically at a wavelength of 450 nm. The concentration of ANG II (ng/mL) in the samples was then determined by comparing the O.D. of the samples to the standard curve.
2.7. Statistical Analysis {#sec2dot7-nutrients-09-01369}
-------------------------
The obtained results in particular groups were given as the mean ± standard deviation (±SD), and checked for normal distribution and homogeneity of these groups. The Shapiro-Wilk test was performed for normality of the obtained results. Homogeneity of variance was evaluated with the Levene's test with two variables. The comparison of homogenous groups and the effect of parameters (mouse model, diet type, supplement dose) on group differentiation were analyzed with ANOVA and the Least Significant Differences (LSD) test.
The differences were considered to be statistically significant when a significance level was less than 0.05 (*p* ≤ 0.05). The calculations were performed with Statistica 10.0 (Polish version), and Microsoft Excel.
3. Results and Discussion {#sec3-nutrients-09-01369}
=========================
3.1. Histopathological Tests of Arteries {#sec3dot1-nutrients-09-01369}
----------------------------------------
In our study, we evaluated the anti-atherogenic effect of polyphenol-rich extract of bee pollen on the development of atherosclerosis induced by a high-fat diet in ApoE-knockout mice. We showed that supplementation of a high-fat diet with EEP in a dose of 1 g/kg BM protected heart arteries from development of atherosclerotic plaque. Histopathological presentation did not reveal any proliferative changes which would be evidence of atherosclerosis development ([Figure 1](#nutrients-09-01369-f001){ref-type="fig"}C). Supplementing a high-fat diet with EEP in a dose of 0.1 g/kg BM significantly limited the growth of atherosclerotic plaque ([Figure 1](#nutrients-09-01369-f001){ref-type="fig"}B), while severe atherosclerotic changes occurred in mice on a high-fat diet without supplementation. Near the aortic arch, atherosclerotic plaque almost completely filled the vascular lumen ([Figure 1](#nutrients-09-01369-f001){ref-type="fig"}A). In mice on a standard diet, the control group SD ([Figure 1](#nutrients-09-01369-f001){ref-type="fig"}F), and in the mice on a standard diet supplemented with EEP in a dose of 0.1 g/kg BM ([Figure 1](#nutrients-09-01369-f001){ref-type="fig"}D), and 1 g/kg BM ([Figure 1](#nutrients-09-01369-f001){ref-type="fig"}E) atherosclerotic characteristics were not observed.
The mechanism of inhibitory capacity of EEP, as far as atherosclerosis is concerned, is difficult to explain. Our study has led to a conclusion that the mechanism is related to a significant decrease in total cholesterol, oxidatively modified pro-atherogenic ox-LDL molecules, and the decrease of ADMA and ANG II level. To the best of our knowledge, the current study is the first report on the subject.
EEP used in our study was characterized by a high content of polyphenols (27 mg GAE/g) and flavonoids (20 mg QE/g), which corresponded to a strong antioxidant effect resulting from reduction of DPPH (EC~50~ = 57.5 μmol/g), free radicals, and ABTS^•+^ (TEAC = 0.692 mmol/g). Rutin was a dominant flavonoid, which is a glycoside of quercetin. Other flavonoids present in the extract are: mireycetin \> quercetin \> isorhamnetin \> kaempferol. The main phenolic acids which are present in EEP are: gallic acid \> trans-cinnamic acid \> 4-hydroxycinnaminic acid \> felluric acid \> 4-trans-p-coumaric acid \> caffeic acid. Detailed characteristics of particular phenolic acid and flavonoid content in EEP used in research were presented in a previously published paper \[[@B22-nutrients-09-01369]\]. These compounds are characterized by a strong antioxidant effect reducing oxidative stress, and strongly inhibiting lipid peroxidation, which probably was crucial for preventing the formation of atherosclerotic plaque in ApoE-knockout mice.
In vitro studies showed that quercetin and catechin inhibit platelet aggregation, limit pro-clotting activity and block phosphoinositide cascade \[[@B24-nutrients-09-01369]\]. Flavonoids interact with platelet receptors. Quercetin decreases reactivity of platelets by blocking activation that depends on GPVI receptors \[[@B25-nutrients-09-01369]\]. Quercetin and catechin inhibit oxidative stress in platelets, and simultaneously limit activation of a fibrogen receptor, and increase NO synthesis \[[@B26-nutrients-09-01369]\]. In studies conducted on mice with apolipoprotein E deficiency, it was shown that polyphenol mixture, e.g., catechin, caffeic acid and resveratrol, decreases progression of atherosclerotic changes in the aortic arch. According to authors, the inhibition of endothelin 1 synthesis is one of the mechanisms in which polyphenols affect the balance between vasoconstrictive and vasodilating factors \[[@B27-nutrients-09-01369]\]. Hayek et al. \[[@B28-nutrients-09-01369]\] obtained significant reduction of atherosclerotic plaque on the surface of the aortic intima-media in ApoE-knockout mice after administration of quercetin.
The anti-atherosclerotic effect of propolis, an important apitherapeutic, was described in the literature. The reduction of early and advanced atherosclerotic changes due to a polyphenol fraction from green, brown, and red propolis was confirmed in in vitro studies conducted on genetically modified mice without LDL receptor (LDLr-/- mice). Polyphenols from red propolis had the strongest effect, and they reduced atherosclerotic changes in the aortic sinus. This was related to improving the lipid profile, reducing the level of pro-inflammatory cytokines, monocyte chemotactic protein-1 (MCP1) and interleukin 6 (IL6), chemokine, and angiogenic factors \[[@B29-nutrients-09-01369]\]. Limitation of atherosclerotic progression in the aortic sinus was observed in the case of a polyphenol fraction from Chilean propolis. Authors claim that this can result from the synergic effect of a polyphenol complex present in propolis that involves a significant reduction of the expression of a pro-angiogenic vascular endothelial growth factor A (VEGF-A) \[[@B30-nutrients-09-01369]\].
3.2. Effect of EEP on Total Cholesterol (TC) {#sec3dot2-nutrients-09-01369}
--------------------------------------------
Improper diet can impair lipid management and contribute to the development of cardio-vascular diseases. Disorders of lipid metabolism, manifested by hypercholesterolemia, are recognized and important factors which increase the risk of atherosclerosis \[[@B31-nutrients-09-01369]\].
In the current study, we have evaluated the effect of polyphenol-rich extract of bee pollen (EEP) on total cholesterol level in ApoE-knockout mice on high-fat and standard diets. Total cholesterol level in ApoE-knockout mice is presented in [Figure 2](#nutrients-09-01369-f002){ref-type="fig"}.
After five weeks, a high-fat diet in genetically modified animals resulted in an increase of TC to 779 mg/dL ([Figure 2](#nutrients-09-01369-f002){ref-type="fig"}A), i.e., by 101% compared with the average level (388 mg/dL, [Figure 2](#nutrients-09-01369-f002){ref-type="fig"}B) in the controls. The use of a high-fat diet for 16 weeks further increased the level of TC to 1303 mg/dL ([Figure 2](#nutrients-09-01369-f002){ref-type="fig"}A), i.e., by 67% compared with values recorded after five weeks. In this group, the average TC level was the highest and the difference in comparison with all remaining experimental groups was statistically significant (*p* \< 0.05, [Figure 2](#nutrients-09-01369-f002){ref-type="fig"}B).
Supplementing a high-fat diet with EEP in a dose of 0.1 g/kg BM decreased TC level, which reached the value of 856 mg/dL ([Figure 2](#nutrients-09-01369-f002){ref-type="fig"}A) in the 16th week, and was 34% lower when compared with an unsupplemented high-fat diet. Supplementation of a high-fat diet with EEP in a dose of 1 g/kg BM stabilized TC level as early as in the 5th week. This parameter level in all experimental periods was similar to the control group level, and the difference was not statistically significant ([Figure 2](#nutrients-09-01369-f002){ref-type="fig"}B). In the 16th week, TC level was 398 md/dL ([Figure 2](#nutrients-09-01369-f002){ref-type="fig"}A), i.e., 69% lower than in an unsupplemented high-fat diet.
Supplementation of a standard diet with EEP in a dose of 0.1 g/kg did not significantly change TC level (*p* \> 0.05), but a dose of 1 g/kg MB resulted in a high decrease in TC level (*p* \< 0.05) when compared with the control group ([Figure 2](#nutrients-09-01369-f002){ref-type="fig"}A,B).
A significant decrease of TC level due to polyphenol fraction in bee pollen extract (EEP), which was recorded in our study, can be an effect of various mechanisms. It can be a sign of decreased biosynthesis of cholesterol in the liver. It can be a result of increased excretion of cholesterol with bile, or a result of the activation of peroxisome proliferator-activated receptors (PPAR), which regulate adipocyte maturation and lipid storage; therefore, they are regulators of hepatic metabolism of lipids.
It can be concluded from published data that polyphenols from natural products affect lipid metabolism as well as having cardio- and angio-protective effect. During in vitro studies, quercetin activates PPAR-γ receptors by increasing their expression. It also increases the expression of ATP-binding cassette transporter (ABCA1), which plays a key role in reverse cholesterol transport. Quercetin-induced modulation of ABCA1 expression reduces cholesterol build-up in macrophages, and increases cholesterol outflow from macrophages, and therefore reduces formation of foam cells, lowering the risk of atherosclerosis \[[@B32-nutrients-09-01369]\]. The lipid level in LDLr-knockout mice is normalized by a polyphenol fraction from different types of propolis. The polyphenol fraction from red propolis had the strongest protective effect. This fraction caused the most significant reduction of triglyceride level, and TC level as well as an increase in HDL. The anti-atherogenic effect was weaker in the case of green and brown propolis. This may be related to different content of main polyphenol components, in particular, types of propolis. The following were mostly present in red propolis: 3-hydroxy-8,9-dimethoxypterocarpan, medicarpin, daidzein, whereas artepellin C, pinocembrin, kampferol in green propolis, and pinocembrin, caffeic acid phenyl ester, quercetin, galangin in brown propolis \[[@B29-nutrients-09-01369]\].
The current study shows that EEP in a dose of 1 g/kg BM significantly decreases TC levels in ApoE-knockout mice on both, high-fat and standard diets. Consequently, it reduces hypercholesterolemia, which is a risk factor for atherosclerosis.
3.3. Effect of EEP on Oxidized Low Density Lipoprotein (Ox-LDL) {#sec3dot3-nutrients-09-01369}
---------------------------------------------------------------
Oxidized LDL molecules are one of the main factors responsible for atherosclerosis development. They are formed due to free radical activity during the process of chemical modification, namely, oxidation of low density lipoproteins (LDL). Ox-LDL are highly atherogenic molecules, since they have pro-inflammatory activity and they cause focal inflammation of arterial vascular endothelium \[[@B33-nutrients-09-01369]\].
In our study, we have investigated the effect of polyphenol-rich EEP on ox-LDL levels in ApoE-knockout mice on high-fat and standard diets. Ox-LDL level in ApoE-knockout mice is presented in [Figure 3](#nutrients-09-01369-f003){ref-type="fig"}.
The highest levels of ox-LDL in all experimental periods were recorded in mice on a high-fat diet, whereas the lowest ones were noted when a standard diet was supplemented with EEP at a dose of 1 g/kg BM ([Figure 3](#nutrients-09-01369-f003){ref-type="fig"}A,B). Since the fifth week, a high-fat diet resulted in a significant increase of ox-LDL level, which was 867 ng/mL in the 16th week ([Figure 3](#nutrients-09-01369-f003){ref-type="fig"}A) and was 91% higher than the average level (453 ng/mL, [Figure 3](#nutrients-09-01369-f003){ref-type="fig"}B) in the controls. Supplementing a high-fat diet with EEP as early as in the fifth week resulted in lowering ox-LDL levels, which diminished to 526 ng/mL ([Figure 3](#nutrients-09-01369-f003){ref-type="fig"}A), i.e., by 40% in the case of a dose of 0.1 g/kg BM, and to 355 ng/mL ([Figure 3](#nutrients-09-01369-f003){ref-type="fig"}A), i.e., by 59% when the dose was 1 g/kg BM compared with an unsupplemented high-fat diet. In Apo-E-knockout mice, supplementation of a high-fat diet with EEP regardless of the dose, causes statistically significant reduction of average ox-LDL level to a level similar to the one of the controls ([Figure 3](#nutrients-09-01369-f003){ref-type="fig"}B).
The literature indicates that polyphenols are inhibitors of oxidative modification of LDL. They play an important role in prevention of many degenerative diseases, including cardio-vascular diseases. Polyphenols inhibit progression of atherosclerosis by reducing oxidative stress, and inflammatory biomarkers of atherosclerosis, as well as by improving lipid profile and insulin sensitivity, and by making endothelial function more efficient \[[@B17-nutrients-09-01369],[@B34-nutrients-09-01369],[@B35-nutrients-09-01369],[@B36-nutrients-09-01369]\].
Loke et al. \[[@B37-nutrients-09-01369]\], in their studies on ApoE-knockout mice showed that quercetin reduced hydrogen peroxide and leukotrien B4 in vessels, and P-selectin serum level as well as increased the activity of endothelial nitric oxide synthase (eNOS). Theaflavin (catechin dimer) and epicatechin have a similar but weaker effect. In vitro studies of olive oil polyphenols have shown that they reduce intracellular levels of reactive oxygen species (ROS) and the expression of nuclear factor-κB (NFκB) transcription. Reduction of NFκB expression has been related to lowering metalloproteinase 9 level \[[@B36-nutrients-09-01369]\]. Oxidative stress in ApoE-knockout mice is also reduced by cocoa polyphenols. This effect has resulted from reducing expression of vascular cell adhesion molecule-1 (VCAM1) and intercellular adhesion molecule-1 (ICAM1) \[[@B38-nutrients-09-01369]\]. Olive oil supplemented with epigallocatechin gallate (EGCG) significantly improves vascular endothelium function in patients with early atherosclerotic dysfunction of endothelium due to the levels of ICAM, monocytes and lymphocytes \[[@B39-nutrients-09-01369]\]. Polyphenols inhibit LDL oxidation, reduce thrombocyte aggregation, inhibit the activity of enzymes that mediate the immune cells response to LDL, and reduce TC levels \[[@B40-nutrients-09-01369]\].
The presented effect of the polyphenol fraction of bee pollen extract on ox-LDL level in ApoE-knockout mice has been reported for the first time. Any information on the effect of polyphenols from bee pollen on ox-LDL level has not been found in the literature. Our study can lead to the conclusion that a polyphenol fraction from bee pollen significantly reduces ox-LDL level, and it results, most probably, from high antioxidant capacity of EEP. Reduction of proatherogenic ox-LDL level and consequently, limitation of the development of atherosclerotic changes are the signs that ethanol extract of bee pollen is highly efficient at reducing oxidative stress. Therefore, it can be useful as a potential anti-atherogenic substance.
3.4. Effect of EEP on Asymmetric Dimethylarginine (ADMA) {#sec3dot4-nutrients-09-01369}
--------------------------------------------------------
ADMA is an endogenous inhibitor of endothelial nitric oxide synthase (eNOS). ADMA participates in one of the mechanisms limiting bioavailability of nitric oxide (NO), which is an endogenous strongly anti-atherogenic substance \[[@B41-nutrients-09-01369],[@B42-nutrients-09-01369]\]. Impairment of endothelial functions, which is observed in various disorders, results from high levels of ADMA. This allows us to single out ADMA as an early biochemical marker of endothelial dysfunction in the prophylaxis of cardio-vascular diseases and insulin resistance \[[@B42-nutrients-09-01369],[@B43-nutrients-09-01369],[@B44-nutrients-09-01369]\].
We have studied the effect of bee pollen extract on ADMA level in ApoE-knockout mice on both, high-fat and standard diets. ADMA level is presented in [Figure 4](#nutrients-09-01369-f004){ref-type="fig"}.
After five weeks, a standard diet led to an increases in ADMA level to 0.653 μmol/L ([Figure 4](#nutrients-09-01369-f004){ref-type="fig"}A), i.e., by 26%, and up to 0.704 μmol/L ([Figure 4](#nutrients-09-01369-f004){ref-type="fig"}A), i.e., by 36% after 16 weeks when compared with an average level (0.519 μmol/L, [Figure 4](#nutrients-09-01369-f004){ref-type="fig"}B) in the controls.
Sixteen-week supplementation of a high-fat diet with EEP in a dose of 0.1 g/kg BM did not significantly change this parameter level. When a high-fat diet was supplemented with EEP in a dose of 1 g/kg BM, the lowest ADMA level was recorded in the 14th week (0.431 μmol/L, [Figure 4](#nutrients-09-01369-f004){ref-type="fig"}A), while in the 16th week ADMA level was 0.648 μmol/L ([Figure 4](#nutrients-09-01369-f004){ref-type="fig"}A), 8% lower than in the unsupplemented group. The lowest levels of this parameter were obtained for a standard diet supplemented with bee pollen extract in a dose of 1 g/kg BM ([Figure 4](#nutrients-09-01369-f004){ref-type="fig"}A,B). A high-fat diet in ApoE-knockout mice caused a statistically significant increase of ADMA level. Supplementing a high-fat diet with EEP in a dose of 1 g/kg BM in ApoE-knockout mice resulted in a statistically significant decrease of average ADMA levels ([Figure 4](#nutrients-09-01369-f004){ref-type="fig"}B).
According to the literature, polyphenols added to diet lower ADMA. Li Volti's et al. \[[@B45-nutrients-09-01369]\] in vitro studies showed that supplementation with silibinin---flavonoid belonging to the group of flavonolignans---lowered ADMA serum level in mice, limited endothelial dysfunction, and reduced insulin resistance. A significant decrease of ADMA level, ox-LDL level and C-reactive protein (CRP) level, as a result of polyphenol-rich olive oil consumption, was observed in studies carried out in a group of young women with slight hypertension or hypertension stage 1. A significant decrease of hypertension and improving endothelial function was recorded \[[@B46-nutrients-09-01369]\].
There are no published reports on the effect of bee pollen extract on ADMA level. Our results show that a polyphenol fraction from bee pollen reduces ADMA level, and consequently increases NO bioavailability, by limiting endothelial dysfunction and development of atherosclerotic changes. It is very important for prevention of cardio-vascular diseases, because an increase of ADMA level is an early risk factor of vascular endothelium dysfunction, and limiting its excessive synthesis can be used in atherosclerotic prevention.
3.5. Effect of EEP on Angiotensin-Converting Enzyme (ACE) and Angiotensin II (ANG II) {#sec3dot5-nutrients-09-01369}
-------------------------------------------------------------------------------------
The renin-angiotensin-aldosterone system (RAA) is a key mechanism of physiologic regulation of blood pressure and electrolyte balance. Individual constituents of the system can participate in pathogenesis of hypertension and organ changes related to hypertension such as cardiac and vascular remodeling, atherosclerosis, myocardial fibrosis, and renal fibrosis \[[@B47-nutrients-09-01369]\].
ACE belongs to zinc metalloproteinases. It is an enzyme that participates in blood pressure regulation through converting inactive angiotensin I into biologically active angiotensin II, with a vassopresor-like activity \[[@B48-nutrients-09-01369]\].
ANG II has a harmful effect on vascular endothelium and cardiac muscle. It causes endothelial dysfunction, increases oxidative stress, and contributes to atherosclerotic plaque rupture. ANG II also accelerates apoptosis of cardiac muscle cells, and increases tissue-specific insulin resistance \[[@B49-nutrients-09-01369]\]. By contributing to ox-LDL formation, ANG II can indirectly lead to dysfunctions of endothelial function related to clotting and fibrinolysis \[[@B50-nutrients-09-01369]\]. ANG II can also cause a change in endothelial function from anti-adhesive to pro-adhesive one, thus affecting the expression of endothelial adhesion molecules (ICAM, VCAM) \[[@B51-nutrients-09-01369]\].
In our study, we have investigated the effect of EEP on ACE and ANG II level in ApoE-knockout mice on high-fat and standard diets. ACE level is presented in [Figure 5](#nutrients-09-01369-f005){ref-type="fig"}.
The highest levels of ACE in particular experimental periods were recorded in animals on a high-fat diet, whereas the lowest ones were observed for a standard diet supplemented with bee pollen extract in a dose of 1 g/kg BM ([Figure 5](#nutrients-09-01369-f005){ref-type="fig"}A). After five weeks, a high-fat diet increased ACE level, which was 161 ng/mL ([Figure 5](#nutrients-09-01369-f005){ref-type="fig"}A) in the 16th week, i.e., 30% higher than the average level (124 ng/mL, [Figure 5](#nutrients-09-01369-f005){ref-type="fig"}B) in the control group. Supplementing a high-fat diet with EEP resulted in lowering this parameter level to 152 ng/mL after 16 weeks, i.e., by 6% for 0.1 g/kg BM, and to 124 ng/mL ([Figure 5](#nutrients-09-01369-f005){ref-type="fig"}A) i.e., by 23% for a dose of 1 g/kg BM when compared with the unsupplemented group.
The highest level of ANG II was recorded in mice on a high-fat diet. In the fifth and sixth week, ANG II level was 2.16 ng/mL ([Figure 6](#nutrients-09-01369-f006){ref-type="fig"}A), i.e., 95% higher than the average level (1.11 ng/mL, [Figure 6](#nutrients-09-01369-f006){ref-type="fig"}B) in the control group. Supplementing a high-fat diet with EEP decreased this parameter as early as in the fifth week, and after 16 weeks ANG II level was reduced to 1.31 ng/mL ([Figure 6](#nutrients-09-01369-f006){ref-type="fig"}A), i.e., by 39% for a dose of 0.1 g/kg BM, and to 1.09 ng/mL ([Figure 6](#nutrients-09-01369-f006){ref-type="fig"}A), i.e., by 50% for a dose of 1 g/kg BM compared with the unsupplemented group. The lowest ANG II level was recorded in mice on a standard diet supplemented with EEP in a dose of 1 g/kg BM ([Figure 6](#nutrients-09-01369-f006){ref-type="fig"}A). Supplementation of a high-fat diet with bee pollen extract, regardless of the dose, caused a statistically significant decrease of ANG II level ([Figure 6](#nutrients-09-01369-f006){ref-type="fig"}B).
The published data reveals that polyphenols are hypotensive, which results from their antioxidant capacity. This feature leads to lowering ROS concentration and to weakening ACE activity \[[@B19-nutrients-09-01369],[@B51-nutrients-09-01369],[@B52-nutrients-09-01369]\]. Xu et al. \[[@B53-nutrients-09-01369]\], both in studies on rats and in vitro studies showed that genistein had reduced expression and activity of ACE in endothelium and serum. The estrogen receptor activating a pathologic path of ERK1/2 participated in this activity. Genistein stimulates the synthesis of nitrogen oxide in endothelial cells through a mechanism dependent on cyclic 3′5′-adenosine monophosphate. Taxifolin inhibits ACE activity in the rat aorta \[[@B52-nutrients-09-01369]\], while quercetin lowers blood pressure in spontaneously hypertensive rats (SHR), and eliminates all pathological changes in their blood vessels \[[@B54-nutrients-09-01369]\]. The antihypertensive effect of polyphenols in SHRs was related to activation of eNOS, and inhibition of metalloproteinase-2 \[[@B55-nutrients-09-01369]\]. Lowering blood pressure by polyphenols can also result from modelling the renin-angiotensin-aldosterone system through reducing oxidative stress \[[@B56-nutrients-09-01369]\]. Polyphenols from various tea types inhibit ACE activity in in vivo studies \[[@B57-nutrients-09-01369]\]. Furthermore, also bee pollen extract has inhibited ACE activity in in vitro studies, which results from its high antioxidant potential \[[@B58-nutrients-09-01369]\].
Based on our study, it can be concluded that supplementing a high-fat diet and a standard diet with EEP decreases angiotensin II level in ApoE-knockout mice. This is related to weakening the activity of ACE. The available literature does not offer any data on the effect of bee pollen on ANG II level; this issue has been presented in our study for the first time. Our study may lead to a conclusion that a polyphenol fraction from bee pollen, due to its high antioxidant capacity, affects the modulation of the renin-angiotensin-aldosterone system; therefore, it improves endothelial function, and consequently it can inhibit the development of atherosclerotic changes.
In spite of the fact that many studies have been conducted, the mechanism of the anti-atherosclerotic effect has not been yet determined. According to the published data, it can be assumed that the main protective effect from the development of atherosclerosis consists improvement of endothelial function, oxidative reduction, LDL modification, decrease of total cholesterol, inhibiting the synthesis of pro-inflammatory cytokines such as TNF-α, IL-6, IL-8, and inhibiting the adhesion of ICAM-1, VCAM-1 molecules as well as the stimulation of eNOS synthase, and intensification of NO synthase \[[@B59-nutrients-09-01369]\].
4. Conclusions {#sec4-nutrients-09-01369}
==============
Supplementing a high-fat diet with polyphenol-rich EEP modulates lipid profile in ApoE-knockout mice by lowering total cholesterol level. EEP is protective for cardiac arteries since it significantly limits the development of atherosclerotic changes (dose of 0.1 g/kg BM) or prevents their development (dose of 1 g/kg BM). This results from lowering the level of strongly pro-atherosclerotic ox-LDL. Due to high antioxidant activity, EEP efficiently reduces oxidative stress, and is hence an inhibitor of LDL oxidation. The protective effect of EEP results from lowering ADMA level, which increases NO bioavailability, limits endothelial dysfunction, and prevents the development of atherosclerotic changes. An increase in ADMA level is an early risk factor of endothelium dysfunction, and limiting its excessive synthesis is crucial and can be used in prophylaxis of atherosclerosis as well as other cardio-vascular diseases. EEP prevents and inhibits the development of atherosclerotic changes in ApoE-knockout mice through inhibiting the activity of ACE and lowering ANG II level. Consequently, it affects modulation of the renin-angiotensin-aldosterone system, and can limit endothelial dysfunction in this way. Further studies should aim at comprehensive explanation of both, cellular and genetic mechanisms of a cardioprotective effect of polyphenols, including their stability, absorption, distribution, and biotransformation.
To the best of our knowledge, we have been the first to prove that in experimental conditions, EEP efficiently reduces oxidative stress through limiting atherosclerotic changes or eliminating their development. Hence, EEP can be useful as a potential anti-atherogenic agent.
This work was supported by the Medical University of Silesia in Katowice (Grant No. KNW-1-033/N/7/O).
Anna Rzepecka-Stojko conceived the study, designed and performed the experiments, analyzed the data and wrote the manuscript. Jerzy Stojko designed and performed the experiments and critically revised the manuscript. Krzysztof Jasik performed the experiment and analyzed the results histopathological tests of arteries. Ewa Buszman critically revised the manuscript. All authors read and approved the final manuscript.
The authors declare no conflict of interest. The founding sponsor had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, nor in the decision to publish the results.
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{#nutrients-09-01369-f006}
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1. Introduction
===============
Because nurses care for their patients around the clock in hospitals, they see themselves as primarily responsible for their patient's well-being and the main role they play in the health care team is to serve as a key guardian of patient safety. However, injuries in health care today are all too frequent. Safety is defined as the freedom from accidental injury [@BIB1]. Many injuries occur as the result of errors, defined as the failure of a planned action to be completed as intended, or the use of a wrong plan to achieve an aim [@BIB1]. An adverse event is an injury resulting from a medical intervention [@BIB1], such as scarring following an infiltrated intravenous line containing chemotherapy.
While errors and adverse events represent a difficult problem, increasing data suggest that information technology may be a powerful tool for improving safety[@BIB2]. Most of the work in this area takes either a multidisciplinary or physician-oriented perspective, but many of these tools are particularly helpful to nursing and many injuries may be prevented by nurses. In this paper, we discuss the international epidemiology of iatrogenic injury, illustrate these with case examples, and suggest tools and interventions for improving safety, viewed in particular through a nursing lens.
1.1. The epidemiology of latrogenic injury
------------------------------------------
In the US, the Institute of Medicine (IOM) reported, "To Err is Human: Building a Safer Health System," galvanized the public and the health care industry's interest in this area (2000). It stated that safety was a major problem in the U.S, resulting in large numbers of injuries and deaths. Furthermore, it argued that health care organizations must develop a "culture of safety" so that the workforce and processes focus on improving the reliability and safety of patient care.
A key study regarding the risk of hospitalization, which the IOM used to estimate numbers of injuries and deaths, was The Harvard Medical Practice Study (MPS), which evaluated the frequency of iatrogenic injury in patients discharged from hospitals in New York, in 1984 [@BIB3], [@BIB4]. The primary outcome of this study was the "adverse event," defined as injuries caused by medical mismanagement resulting in disability at discharge or prolonged length of stay. Adverse events occurred in 3.7% of hospitalizations, of which 28% were judged to be due to negligence. Another US study, using the same methodology in a random sample of patients from Colorado and Utah, found adverse events in 2.9% of admissions as compared to 3.7% in the New York study [@BIB5], suggesting that these rates are probably reasonably representative for the U.S. Data are now available from a number of large studies around the world regarding the frequency of adverse events, and essentially all of these demonstrate that adverse events are significant problems [@BIB6], [@BIB7], [@BIB8], [@BIB9], [@BIB10]. Most data so far come from the developed world. For example, the Quality in Australian Healthcare Study (QAHCS) identified an adverse event rate of 16.6% [@BIB6]. A subsequent study, comparing the adverse event rate in the QAHCS to that from the Medical Practice study, found that most of the differences in incidence were related to methodological differences between the two studies, although there was also a higher adverse event rate in Australia [@BIB7]. Vincent found that in United Kingdom, 10.7% of patients experienced one or more adverse events [@BIB8]. In New Zealand, an adverse event rate of 12.9% was identified in one study [@BIB9]. Numerous studies are currently underway in other countries, including Canada and Japan.
In the developing world, relatively limited data are available, though some studies are now being done, and the issue is often highly charged. For example, in Brazil, a study of 212 patients showed that among 46 patients, 80 errors had occurred relating to pressure ulcers, mechanical ventilation, IV catheters and medications [@BIB10]. Korea has no detailed data about iatrogenic accidents, such as adverse drug events, hospital infection, even though some physicians and nurses realize the importance of a systematic approach about these issues. In part for cultural reasons, iatrogenic injuries represent an especially sensitive and political issue in Korea. While the medical malpractice case rate has increased recently, and there is increasing public concern, there are still no organized systems to monitor adverse event rates. The few websites addressing iatrogenic accidents mostly focused on legal issues, such as reimbursement and insurance claims. These data make it clear that iatrogenic injury and adverse events are major international issues
Moreover, many adverse events can be detected, ameliorated or prevented by nurses. For example, adverse drug events are the leading cause of injury in most studies and nurses are in an excellent position to play an active role in this area, since they administer most drugs and interact with patients and families more frequently than other providers. Pressure ulcer prevention is primarily a nursing issue. Surgical adverse events and nosocomial infections are also important. These can be both identified and, in many instances, prevented by nurses. In a study by Needleman, who looked at staffing ratios and level of outcomes for patients in a large multistate study, there was consistent evidence of an association between higher levels of staffing by registered nurses and lower rates of adverse outcomes, such as urinary tract infections, upper gastrointestinal bleeding and hospital-acquired pneumonia [@BIB11].
In addition to the harm that they cause to patients, iatrogenic injuries are costly to health care systems. The Medical Practice Study estimated that the total cost for injuries that occurred during 1984 in New York was \$878 million in 1989, including medical care costs of \$161 million [@BIB3], [@BIB4]. The QAHCS estimated that adverse events accounted for 8% of hospital bed days and cost the Australian health care system \$4.7 billion (Australian dollars) per year [@BIB6]. A British study suggested that adverse events were responsible for an average of 8.5 additional bed days, and resulted in a direct cost of £290,000 to the trust concerned, and the overall estimate was that preventable events cost the National Health Service approximately 1 billion pounds annually [@BIB8].
Taken together, these data suggest that adverse events are highly important international problems with major financial consequences, and that many of the adverse events could be prevented, identified and treated by nurses.
1.2. Nursing and safety
-----------------------
In staff nurses' workflow, they consistently double-check orders, confirm questions about medications with pharmacists and physicians, and report their concerns about patient safety. Surprisingly, in a recent study conducted by Clarion Health Systems, a nurse had only an average of 20--50 min per patient, of direct patient contact, over a 12-h period [@BIB12]. The rest of the time was spent primarily managing and coordinating the communication of patient information to other departments, physicians, and members of the health care team. However, time spent in managing communication and information is not sufficient to ensure a safe health care environment. Other approaches, including greatly expanded use of information and communication technology, are needed to help nurses prevent errors. The use of a Clinical Information System (CIS) can allow the staff to be more efficient and to provide more time with patients [@BIB13].
A CIS is not the only tool that can help nursing improve safety. The International Council of Nurses (ICN) Code of Ethics was established in 1953. It has been revised many times. It is available on their website, and one of the elements includes technology and safety. "The nurse, in providing care, ensures that use of technology and scientific advances are compatible with the safety, dignity and rights of people." This statement links the use of technology, safety and ethical conduct such that the nurse is aware of the patient's safety when using technology and of the ethical implementation of new technologies [@BIB14]. The ICN also has a position statement on safety adopted in 2002 [@BIB15]. ICN believes nurses and national nurses associations have a responsibility to:•Inform patients and families of potential risks.•Report adverse events to the appropriate authorities promptly.•Take an active role in assessing the safety and quality of care.•Improve communication with patients and other healthcare professionals.•Lobby for adequate staffing levels.•Support measures that improve patient safety.•Promote rigorous infection control programmes.•Lobby for standardized treatment policies and protocols that minimise errors.•Liase with the professional bodies representing pharmacists, physicians and others to improve packaging and labelling of medications.•Collaborate with national reporting systems to record, analyse and learn from adverse events.•Develop mechanisms, for example through accreditation, to recognise the characteristics of healthcare providers that offer a benchmark for excellence in patient safety.•These responsibilities are available to all nurses when needing support in situations where safety is an issue.
The health care system quality components and implementation varies significantly nationwide in Brazil. The initiatives to achieve patient safety improvement are recent. One important advance was the establishment by law in 26 January 1999 of the ANVISA (National Health Surveillance Agency), with the mission "To protect and promote the population's health, ensuring the sanitary safety of products and services and taking part in developing access to it". The agency is an independently administered, financially autonomous regulatory agency within the structure of Federal Public Administration and it is linked to the Ministry of Health. In addition to the regulatory mission, the ANVISA has created a website for health care providers and consumers to report adverse events. This includes information ranging from problems with generic medications to news updates about severe acute respiratory syndrome (SARS). However, it is important to emphasise that Brazilian nursing is developing a more proactive leadership role in this field, as they essentially perform and control the majority of direct patient care tasks [@BIB16].
1.3. Health care team
---------------------
The IOM has suggested that safety is a systems property, and that achieving safety requires a team effort. Nurses clearly represent a key part of the health care team, especially in the hospital. Ideally, both nurses and pharmacists should be included in patient rounds. In terms of hospital safety, the health care team must look at patient care from admission to discharge, and beyond. Safety teams should be created that include not only physician and nurses, but also physical therapists, pharmacists, IT staff, environmentalists, radiologists, laboratory personnel and administrators [@BIB17].
Including nurses in decision-making teams can change the culture of safety and how errors are perceived by nursing staff. Larson reported on a two-week pilot study where staff could anonymously call a consultant to report errors and present ideas for improving safety [@BIB17]. During the two-week period, consultants received 400 reports (15 per day). The staff did not have to go to their supervisors and they did not have to identify themselves. This type of reporting can greatly enhance identification of problematic areas within a health care system. The data could not have been gathered if the staff thought there might be repercussion from reporting errors. In the US, the IOM has suggested that Congress enact laws to protect confidentiality in volunteer reporting systems, and such legislation has been introduced.
2. Information technology (IT) and nursing: what tools will make a difference?
==============================================================================
2.1. Effective design of a hospital unit
----------------------------------------
While not considered to be related to information technology, the basic structural design of a hospital unit and workspace can clearly have a significant impact on safety [@BIB17]. For example, the layout of a nursing unit can enhance communications, and improve visualisation of patients. Hand-held or mobile devices (laptops on carts) can enhance decision support. A team approach is best in designing hospital units or community clinics to prevent errors. For example, design teams were created for the new facility at St. Joseph's Community Hospital of West Bend, WI, and followed guiding principles to develop the new facility. The design was patient-centred, created a healing environment, was efficient, safe, technologically advanced and staff-friendly [@BIB17].
While designing a new structure is attractive, such opportunities are infrequent; more often, renovating existing designs and ensuring that optimum use of technology, such as infra-red scanning for medication and patient location, ensure safety and prevent errors. Standardisation of nursing units in terms of layout, placement of equipment and consistency in location of supplies are relatively inexpensive and cost-efficient.
2.2. Safety education
---------------------
Throughout their curriculum, student nurses are taught about safety measures. Safety is highlighted in all clinical courses. Students can fail a clinical course by not applying appropriate safety measures. However, learning to use IT to ensure optimum patient safety is just as important. Immediate reporting of errors and adverse events is essential. In teaching about particular areas, such as medication administration, nursing faculty must include problems that can occur and how to manage them. Students need to know that errors are sometimes made by others, e.g. pharmacists and physicians. Students need to know about where errors are most likely to occur and why it is important to always be vigilant. Electronically reporting of errors and adverse events is increasingly used, and is associated with higher reporting rates. These tools make it possible to obtain coded data about reports.
Today, nursing students have the opportunity to avoid many errors because computer education is now integrated into school curricula. Nursing informatics empowers nurses to be influential partners in the work environment. Computer systems are now a part of nurses' daily routines and the use of the Internet is an important source of health care information, which represents an increasingly important knowledge resource in health care. While some nursing schools already have labs to simulate patient care and separate computer labs to teach applications with nursing scenarios, there should be computers present in the patient care labs to simulate real life. A simulated computerized patient record should be part of the skills lab when educating nurses so that they enter the work force with these skills in place. The University of Kansas School of Nursing has started a technology-based approach to education and has combined forces with the Cerner Corporation to develop such a skills lab [@BIB18].
Nursing classifications should be integrated into nursing schools at the basic level of education. These classifications have been developed to describe nursing care and help enhance the nursing process. Their use in computer-based systems should be requested from nursing but they are not usually taught in basic level education. Two examples of these classifications are (1) perioperative nursing data set created by the American Operating Room Nurses (<http://www.aorn.org/research/pnds.htm>) and (2) the Home Health Care Classification, created by Dr. Virginia Saba (<http://www.sabacare.com>). At the international level, the International Classification for Nursing Practice (ICNP) is used. The International Council of Nurses (ICN) advocates the ICNP, which is a terminology for nursing practice that facilitates cross-mapping of local terms and existing vocabularies and classifications [@BIB19], [@BIB21]. The use of nursing vocabularies can be an important tool in tracking nursing care.
The Netherlands carries out a yearly national prevalence survey in different health care institutions, to determine the prevalence and severity of pressure ulcers. Feedback from the surveys is provided to individual institutions which may increase the consciousness of pressure ulcer problems among health care workers. This feedback may result in better prevention strategies and therefore in a decrease in prevalence [@BIB22].
2.3. Safety standards
---------------------
Errors can be made either because standards are lacking, or because approved standards are in place, but are not followed. Even if appropriate standards are in place, a mechanism is necessary to enforce their use. The following case studies demonstrate a lapse in safety standards:
A 13-year-old boy was treated for leukaemia in The Netherlands, in a special paediatric cancer unit of a large university hospital. In addition to his chemotherapy, he was receiving pain medication, and suffered from side effects, such as fatigue, anorexia and vomiting. There was also concern that he was depressed. On day 2 of admission, he reported feeling uncomfortable, had a stomachache, and was listless. On day 3, he was encouraged to be more active, and to take a bath. Although, an established nursing care standard required that any depressed or sedated child be monitored during bathing, the boy wanted privacy and the nurse was busy with other children, so the nurse made an exception and allowed him to bathe unsupervised. The boy was found in the bathtub apneic, and could not be resuscitated.
A 66-yr-old man from Portugal had a complicated stroke with complications and was on a medical ward for 9 days. On the afternoon of the third day, his nurse noted that he was constipated and began nursing interventions to facilitate bowel elimination. The nurse referred the situation to the patient's physician, who prescribed daily laxatives. However, 2 days after treatment, the patient developed diarrhoea. Another nurse called the physician, who ordered an anti-diarrheal, without discontinuing the laxatives. The patient then was receiving both medications, when in fact the diagnosis was fecal impaction, which did not resolve until the patient developed severe discomfort and was manually disimpacted.
### 2.3.1. IT tools
In the case studies presented, errors could have been prevented by using a well-designed interdisciplinary application and alert system. A system of alerts would help health professionals be aware of data that influence decisions. Having an alert, such as a pop-up message stating the boy was on medications that impaired his cognitive ability and that the standard was for supervised bathing, would remind the nurse to be present during the child's bathing. In the second case, there were several errors: the patient should have been started on a bowel regimen after the stroke (this should be standard); the nurse may have needed an electronic prompt to assess the patient's bowel situation; and the system should have alerted the nurse and physician that the patient was simultaneously receiving both a laxative and a medication to treat diarrhoea. The case studies presented indicate ways by which information systems can promote patient safety by all health care providers.
2.4. Knowledge gaps
-------------------
A 76-year-old man in the United States had bleeding oesophageal varices, and his doctor ordered an intravenous pitressin drip. The pharmacy misinterpreted the drug as pitocin, and delivered the wrong medication. The nurse injected the pitocin and the patient subsequently suffered a severe stroke.
A 7-year-old patient in Brazil, with renal disease and hypertension, presented to a clinic in hypertensive crisis. The physician wanted to give the patient captopril, but had no paediatric dosing tools available. They estimated a dose based on the adult dosage, but this represented a five-fold overdose and the child suffered a cardiorespiratory arrest.
### 2.4.1. IT tools
Medication administration standards are part of every nursing curriculum. Still, mistakes occur in today's systems [@BIB23]. High noise levels, interruptions, difficult-to-read equipment displays, illegible dosage labels, and bottles that have similar shapes, colors and sizes can all contribute to medication errors [@BIB24]. Both of these case studies illustrate errors in the medication system that could have been prevented with IT tools.
In the first example, if bar-coding had been implemented, the nurse would have been notified immediately that it was the wrong drug. The use of this technology for administering medication is available and its benefits are being recognized [@BIB25]. Bar-coding is also very useful as a means to identify patients uniquely within a hospital, and can prevent "wrong patient" errors as well as wrong drug errors [@BIB26].
In the second case, the physician needed information about the appropriate dose of a medication, but it was not readily available. Such issues are distressingly frequent: in a study by Leape et al. [@BIB27], knowledge gaps were the most frequent systems cause of serious medication errors. Dosing information is readily available today, either from a desktop or a handheld device. In addition, tools that facilitate dose calculations are available, and computers are much more reliable than humans in making correct calculations [@BIB2]. Dosing errors are a particularly important problem in paediatrics, and appropriate dose forms are often unavailable [@BIB28].
Increasingly, many hospitals are using computerized provider order entry (CPOE) [@BIB29]. This tool eliminates the problem of deciphering orders, and allows provision of decision support to providers. As an order is typed into the computer, it is checked for problems and, when complete, sent directly to the pharmacy for verification. This system does away with many of the steps once used to fill medication orders. It is estimated that every time an order is transcribed, a 15% chance of error is introduced [@BIB29]. The Johns Hopkins Hospital in Baltimore, Maryland, US, instituted new guidelines for prescribing orders within their institution in 2003 [@BIB30]. If an order is written with prohibited abbreviations, the staff is instructed to ask the prescriber to discontinue that order and rewrite it without the prohibited abbreviations. [Table 1](#TBL1){ref-type="table"} is a test that was developed so that employees can learn the new changes. [Table 2](#TBL2){ref-type="table"} explains why certain abbreviations are incorrect and should not be used.Table 1Which is the correct way of writing an order?110 unitsOr10 u2×3 daysOr×3d3.5Or0.546Or6.0512 μgOr12 mcg6NoOrØ7MSO~4~ or MSOrMorphineTable 2Correct answers and rationale110 unitsThe letter "u" for "units" can be mistaken for a "0"2×3 days"×3d" is ambiguous; could mean "times 3 days" or "times 3 doses"30.5With lack of leading zero could be read as five46With trailing zero could be read as 60512 mcgGreek symbol μ could be mistaken for "m" for milligram6NoØ could be mistaken for another number, particularly 4, 6 or 97MorphineConfusion between morphine sulphate and magnesium sulphate; no need for word "sulphate" with morphine
Many low-technology, common-sense approaches formally studied improve medication safety. For example, writing orders in plain English and not using shorthand abbreviations or arcane Latin letters can make medication orders clear and simple [@BIB31]. Another example is having a pharmacist make rounds with the team in the intensive care unit. This has been shown to improve medication safety [@BIB32].
2.5. Knowledge gaps
-------------------
A nurse measures a patient's blood glucose (BG) at the bedside and documents the BG of 400 on the chart that requires the nurses name, the date and time of the measurement, the BG level, and the identification number of the machine. This is done to maintain quality assurance. The physician must then be notified of the high level of the BG, by using a text pager or making a phone call and paging the physician. The nurse also documents the BG on the medication administration record (MAR). Then, the bedside flowsheet is updated. After the nurse delivers the appropriate dose of insulin and documents the BG in the numerous required locations, new interventions for this patient are considered. They have tried many interventions, which were unsuccessful, and now the nurse needs guidance for different approaches.
### 2.5.1. IT Tools
These multiple charting requirements can lead to missed charting and errors; it also creates job stress. A clinical information system with integrated CPOE is the best solution for maintaining accurate and up-to-date charting, while minimising errors. At Brigham and Women's Hospital in Boston, an 84% decrease in serious medication errors was reported by using a CIS with CPOE [@BIB32]. One benefit of having a CIS is that decision support is built in, and offers the health care worker suggestions for appropriate patient interventions. A CIS can link to bibliographic databases, such as PubMed. Another benefit is the ability to use standardized languages, such as nursing diagnoses, outcomes and interventions [@BIB19], [@BIB20], [@BIB21]. The nurse can refer to a list of nursing interventions appropriate for a diabetic patient, providing links between interventions, outcomes and nursing diagnoses. The nurse can then develop a pathway that can be accessed by other care providers. CIS vendors, in the past, have included nursing diagnoses, but have not linked them to complete standardized vocabularies and are only now seeking nursing input [@BIB12].
3. Views regarding the future
=============================
3.1. Hospital setting
---------------------
Nursing shortages are a problem around the world. There are several issues that contribute to this shortage, and they lead to job stress and a higher rate of errors than would occur with adequate staffing. Nurses in hospital jobs have patients with high acuity, and fewer staff to share the burden. A recent study on a model for predicting burnout in Korean nurses showed that Korean nurses reported higher levels of burnout than nurses in western countries, such as Germany, Canada, the United Kingdom and the US [@BIB33]. These issues can have serious safety consequences. A recent study at University of Pennsylvania Hospital regarding job satisfaction among nursing staff indicated that job dissatisfaction among nurses with high patient-to-staff ratios was associated with higher patient mortality [@BIB34]. Another study by Aiken [@BIB35] on nurse satisfaction in five countries shows that nurses leave the profession when they perceive that system inefficiencies compromise the quality of care they are able to give.
Nurses are looking towards IT to streamline work and reduce unnecessary and redundant activities, which may in turn allow them to spend more time with patients and have higher job satisfaction. Specific areas affected by IT are charting, care standards and medication administration. Nurses need to know that they are a significant part of the health care system. Nurse administrators and leaders are seeking ways to support their professional staff, by listening to ideas for role improvement and keeping an open line of communication with all nurses. Nurse managers need to discuss safety issues that occur on the nursing unit with their staff, as well as with the multidisciplinary team, to assess ways that errors can be reduced. Discussing journal articles or forming a "Nursing Journal Club" can be an effective way for sharing and promoting evidence-based practice. Asking interested nurses to be a representative on a department committee and giving them time to do so can make staff members feel appreciated and part of the process.
Equipment, such as intravenous pumps and bedside monitoring, will be directly connected to network systems throughout the hospital, not just in ICUs. Data can be captured at the source without transcriptions errors and automatically entered into the patient's electronic chart.
CIS can help streamline the change of shift report and create an outline for the nurse's daily activities based on each patient assignment. This same connection to patient information provides data to health care providers so that information need not be entered more than once. This can be very helpful when patients transfer to other institutions, such as a nursing home or rehabilitation center.
3.2. Outpatient setting
-----------------------
The CIS can facilitate provision of patient discharge instructions; this will benefit the home health nurse who must closely monitor the patient. All information can be updated prior to discharge. A recent study showed that medication use improved in 50% of the home care patients whose medications were reviewed by a pharmacist, versus 38% of control patients. Concurrently, there were no increases in nurse home visits to intervene because of medication errors [@BIB36].
Because patients are being discharged earlier from hospitals, home monitoring becomes very important. Home health nurses are using laptop computers to transfer data and communicate with physicians while still in the patient's home.
4. Conclusions
==============
Data now demonstrate clearly that patient safety is an international problem. Many adverse events--such as adverse drug events, pressure ulcers and nosocomial infections--can be prevented or detected by nurses. Increasingly, information and communication technology is playing an important role in improving safety. Nursing needs to be closely involved with the development and application of this technology worldwide. It has the potential both to free nurses to return to more direct patient interaction, and to dramatically improve the safety of health care.
| {
"pile_set_name": "PubMed Central"
} |
Background {#Sec1}
==========
Psychosis, principally including schizophrenia, is a severe pathological condition that commonly develops in one's twenties and often leads to a chronic course. Individuals with schizophrenia may experience years of disability, which also imposes a considerable burden on their family caregivers \[[@CR1]\]. The burden of care is defined by the disorder's impact and consequences on caregivers and has objective and subjective components. The subjective components relate to the perception of situations related to care, which can cause a psychological burden \[[@CR2]--[@CR4]\]. Relatives caring for patients with early stages of psychosis tend to suffer from distress, comparable to those caring for chronic patients \[[@CR5]--[@CR7]\]. Their distress can be attributed, directly or indirectly via emotional over-involvement, to anxiety for the patient \[[@CR8], [@CR9]\]. It is important to improve tendencies towards anxiety of family caregivers of young patients with psychotic disorders.
Family psychoeducation has been established as an evidence-based practice that primarily targets avoiding the relapse and rehospitalisation of patients with schizophrenia \[[@CR10]\]. This intervention also consistently improves caregivers' knowledge and self-efficacy, but whether the intervention has beneficial effects on their psychological wellbeing, care burden, or expressed emotion is as yet unclear \[[@CR11]\]. In terms of emotional stress, involving anxiety, depression, or anger, a few studies have reported that psychoeducational interventions improved caregivers' negative emotions, compared to control conditions \[[@CR12], [@CR13]\]. The trial conducted by Hazel et al. \[[@CR8]\] showed that multiple-family group treatment reduced the integrated distress of caregivers, which was operationalised as the standardised and averaged outcomes of depression, anxiety, anger, and perceived stress. However, other randomised controlled trails have failed to demonstrate a significant difference between multi-family groups and control groups \[[@CR14]--[@CR17]\]. The primary outcome of many other studies was not the effect of family psychoeducation on caregivers' emotional stress \[[@CR18]--[@CR20]\]. In addition to the inconsistency, few trials have focused on caregivers of young people with schizophrenia, who are more likely to be in earlier stages of psychosis and share similar problems relevant to the younger generation (e.g. work, marriage). A homogenous group intervention might be more effective to address the concern caregivers have about young patients with the mental disorder.
The Japanese Network of Psychoeducation and Family Support Program (JNPF) has developed a new approach, named SM-FPE \[[@CR21]\]. This model focuses on the strength of caregivers, which is defined as the power a family has for dealing with difficulties when caring for people with severe mental disorders. In the intervention, the affirmation of caregivers' coping behaviours is incorporated into problem-solving techniques; this design allows caregivers to reframe their viewpoints on problems, and helps them realise their inner strengths as well as alleviate their tendencies towards emotional stress. A quasi-randomised controlled trial found that SM-FPE lowered the relapse rate of schizophrenia \[[@CR22]\]. However, there has been little evidence regarding the positive effects of SM-FPE on the mental health of people who are engaged in informal care of an individual with a mental disorder \[[@CR23]--[@CR25]\]. This study thus investigated whether SM-FPE plus TAU is more effective than TAU alone for reducing anxiety and other burdens in family caregivers of young patients with psychotic disorders.
Methods {#Sec2}
=======
Participants {#Sec3}
------------
Subjects in this study were patients with psychotic disorders and their primary caregivers. Inclusion criteria for patients were to be aged between 15 and 39 years, to currently receive outpatient treatment, and to meet the diagnostic criteria of the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revision (DSM-IV-TR) for schizophrenia, brief psychotic disorder, schizophreniform disorder, schizoaffective disorder, or delusional disorder \[[@CR26]\]. The diagnosis of the patients was confirmed by the study's investigators according to the DSM-IV-TR. We excluded patients who were diagnosed with mental retardation or cluster B personality disorders by psychiatrists in charge.
Inclusion criteria for caregivers were to be aged between 20 and 74 years, to be classified as having one of the following relationships with a patient: parent, spouse, sibling, or to have been living with the patient for more than 3 months, and to play a primary role in the care of the patient. We excluded caregivers who had been judged not to be suitable for participating in this study for any reason by a psychiatrist in charge of the patients.
Study design {#Sec4}
------------
This was a parallel-group study stratified by recent-onset/chronic psychosis and implementation site in Japan. The clinical stages of psychosis were distinguished as either ≤59 or ≥ 60 months after the onset of psychotic symptoms \[[@CR27]\]. The study settings were four mental hospitals that covered the western side of the medical regions of a prefecture in the middle of Japan.
Randomisation of families to either a 'SM-FPE plus TAU' group or a 'TAU alone' group, stratified by time since the onset of psychotic symptoms and hospital, was performed by an independent statistician, according to a 1:1 allocation sequence \[[@CR28]\]. Allocation was concealed from investigators enrolling and assessing participants, in that the statistician generated the sequence at his office and informed the investigators of the results only after enrolled participants had completed baseline assessments.
Study setting {#Sec5}
-------------
The programme was carried out at four mental hospitals: Yagoto Hospital, Minami-chita Hospital, Kusunoki Mental Hospital, and Toyota-nishi Hospital in Aichi Prefecture. Yagoto and Kusunoki Mental Hospital are in Nagoya, which is the third largest city in Japan. Toyota-nishi Hospital is in Toyota, which is a typical medium-size city in the country. Minami-chita Hospital covers the rural medical region of the Chita Peninsula.
Interventions {#Sec6}
-------------
SM-FPE was composed of an educational session (45 min), a break (15 min), and a group session (60 min), which took place every 2 weeks over a course of 8 weeks. The group-format programme was conducted by a multidisciplinary team of three to six members chosen from psychiatrists, clinical psychologists, nurses, occupational therapists, and psychiatric social workers. Individual groups included three to five primary caregivers, but not their ill relatives. The educational sessions were provided to a group of caregivers as interactive lectures adapted to the problems that young patients with psychosis often face. The content of the lectures covered diagnosis, prognosis, aetiology, symptoms, drug treatment, communication skills, and social resources. The information was based on the normalization approach, which regards psychotic symptoms as lying on a continuum of psychotic-like experiences that healthy individuals can experience \[[@CR29]\]. A problem-solving approach was applied in the group sessions, but SM-FPE further focused on the strength of caregivers and did not include the patients.
Patients in both the intervention and the control groups received TAU. Their attending psychiatrists provided outpatient treatment, which primarily consisted of pharmacotherapy and supportive psychotherapy on a bi-weekly or 4-weekly basis. Case management, occupational therapy, or day-care programs were available depending on individual patient needs. If caregivers had been members of self-help groups before the allocation, they continued to attend their group sessions. Additionally, if caregivers used psychiatric services and/or psychotropic drugs before the allocation, they continued to use them.
Training and fidelity assessment {#Sec7}
--------------------------------
All staff members completed a 2-day workshop that was approved by the JNPF. The first, third, and fourth authors are SM-FPE instructors certified by the same organisation, but they were assigned the same roles as all other staff in the programme. In order to confirm the fidelity of the programme implementation, all group sessions were recorded, and, after the completion of all sessions, 20% of them were randomly selected and assessed on a fidelity scale with eight items related to structured group work and eight items related to staff roles. A certified instructor evaluated the former items with two options (yes/no) and the later items with three grades (all play/some play/none play). This instrument was developed by the JNPF, but its validity and reliability have not been confirmed.
Assessment measures {#Sec8}
-------------------
All outcome measures were assessed at baseline, post-intervention (10 weeks), and 1 month after the end of the intervention (14 weeks). The primary outcome was the trait anxiety of family caregivers at 14 weeks. The reason for the selection of the primary outcome was to investigate whether the strength-based intervention could modify the predisposition to anxiety behind state and psychosis-specific anxiety through changing how to perceive stressful situations. A previous study \[[@CR30]\] suggested that a preliminary intervention had the potential to modify traits related to anxiety. Since a brief family intervention did not show long-term effects \[[@CR15]\], the trait reduction was considered important because it could represent a persistent effect on caregivers' responses to anxiety-provoking situations. The secondary outcomes for family caregivers were state anxiety, psychological distress, care burden, expressed emotion, and stigma. In addition to the caregivers' outcomes, the patient's overall level of functioning was evaluated as a secondary outcome. We also assessed the kinds of antipsychotic drugs prescribed to patients and the total antipsychotic dose converted to chlorpromazine equivalent \[[@CR31]\].
### State-trait anxiety inventory (STAI) {#Sec9}
The STAI is a 40-item self-report questionnaire that measures trait and state anxiety. Trait anxiety (T-anxiety) is the extent to which an individual is predisposed to become anxious, whereas state anxiety (S-anxiety) is the severity of the anxiety experienced by an individual at a given time. Half of the items are reversed as measures of positive trait and state, which represents the absence of anxiety. Both types of anxiety are separately assessed and each total score ranges from 0 to 60. T-anxiety items include, 'I worry too much over something that really doesn't matter' and 'I am content'. S-anxiety items include, 'I am presently worrying over possible misfortunes' and 'I feel content' \[[@CR32]\]. The reliability and validity of the Japanese version of this questionnaire have been confirmed \[[@CR33]\].
### K6 {#Sec10}
This is a short (six-item) self-report screening tool that was originally developed to detect depressive and anxiety disorders in the general population \[[@CR34]\]. The total score ranges from 0 to 24 and a cut-off of 9 was adopted in a validation study in Japan. K6 items include, 'How often do you feel so depressed that nothing could cheer you up?' and 'How often do you feel nervous?' \[[@CR35]\]. In addition to its use in screening, the K6 has been used as a measure of severity of psychological distress due to depression and anxiety \[[@CR36]\].
### Japanese version of the Zarit burden interview Short version (J-ZBI_8) {#Sec11}
The Zarit Burden Interview is a 22-item self-report scale to assess the burden of care in the impaired elderly \[[@CR37]\]. The burden is associated with caregiver's depression and applicable to assess caregivers of patients with psychosis \[[@CR38], [@CR39]\]. The reliability and validity of the Japanese version have been confirmed \[[@CR40]\]. Arai et al. developed the eight-item short version, with a total score ranging from 0 to 32. The J-ZBI_8 comprises two factors: 'personal strain' and 'role strain'. The former includes items such as, 'Do you feel embarrassed over your relative's behaviour?'; the latter includes items such as, 'Do you feel your social life has suffered because you are caring for your relative?' \[[@CR41], [@CR42]\].
### Family attitude scale (FAS) {#Sec12}
The FAS is a 30-item self-report scale to assess expressed emotion (EE). This emotion can be characterised as the caregivers' feelings of anger and anxiety regarding their state of hostility towards, criticism of, or over-involvement with patients with schizophrenia \[[@CR9]\]. Each item score is summed up to provide a total score that ranges from 0 to 120. FAS items include, 'He deliberately causes me problems' and 'I find myself saying nasty or sarcastic things to him' \[[@CR43]\]. A higher total score is significantly correlated with higher levels of criticism (r = 0.44) and hostility (r = 0.41) in the Camberwell Family Interview \[[@CR44]\]. The Japanese version of the FAS has been validated \[[@CR45]\].
### Link's stigma scale (LSS) {#Sec13}
This self-report instrument with 12 questions is designed to measure the devaluation and discrimination that patients, family members, and general citizens perceive regarding mental illnesses. Each question score is summed up to provide a total score that ranges from 0 to 36. LSS items include, 'Most people think less of a person who has been in a mental hospital' and 'Most people in my community would threat a former mental patient just as they would treat anyone' \[[@CR46]\]. The reliability and validity of the Japanese version have been confirmed \[[@CR47]\].
### Global assessment of functioning (GAF) {#Sec14}
This scale is used to report patients' overall functioning. The GAF is scored from 0 to 100 with respect to psychological, social, and occupational functioning \[[@CR26]\]. High scores on the GAF correspond to better functioning. Psychiatrists in charge evaluated the patients' level of functioning and, to ensure blinding, participants were asked not to inform the doctors of assignment results.
Sample size {#Sec15}
-----------
We referred to a previous single-arm study that explored the effects of a psychoeducational intervention on T-anxiety of 46 relatives of patients with schizophrenia \[[@CR30]\]. The study results indicated that each group needed a sample size of 35 participants, assuming a 10% dropout rate, to detect a reduction of 6 points (SD = 7.2) in the total T-anxiety score at 14 weeks with a two-tailed significance level of 5% and a power of 90%.
Statistical analysis {#Sec16}
--------------------
All analyses were conducted in accordance with the intent-to-treat (ITT) model. If there were no missing values, analysis of covariance (ANCOVA) was used to examine group effects after adjusting for the baseline scores. If missing values were observed, we used linear mixed models. Subsequently, pre-specified subgroup analyses were conducted to investigate the differences between recent-onset and chronic groups.
In post-hoc analyses, structural equation modelling (SEM) was used to examine group effects on the overall state of caregivers' mental health. An exploratory factor analysis of S-anxiety, K6, J-ZBI_8, FAS, and LSS at baseline was carried out, using the principal factor method, with eigenvalues \> 1 being used as the criterion to select the number of factors. Variables with low communality were dropped to determine the components of the poor mental health state. Means and SDs of all variables used in the SEM were calculated and were correlated with each other. In the SEM, we created a path model of the conceptual framework of the caregivers' mental health state according to the flow of this randomised controlled trial (RCT). The fit of the model to the data was computed in terms of a chi-squared (CMIN), comparative fit index (CFI) and the root mean square error of approximation (RMSEA). According to conventional criteria, a good fit would be indicated by CMIN/*df* \< 2, CFI \> 0.97, and RMSEA \< 0.05, and an acceptable fit by CMIN/*df* \< 3, CFI \> 0.95, and RMSEA \< 0.08 \[[@CR48]\]. Since we were specifically interested in young people with schizophrenia in earlier stages of psychosis, a multiple-group analysis was performed to explore the differences in the intervention effects between recent-onset and chronic psychoses. Beginning with a non-constrained model, we compared a more constrained model with a less constrained model. The null hypothesis was defined as the model with less constrains being correct. If the χ^2^-values of the two models did not differ at a statistically significant level, we assumed that the model with more constrains was correct. A significant difference between the two stages of schizophrenia was indicated when the *z*-value of a paired comparison was the critical ratio of \> 1.96.
A two-tailed *p-*value of \< 0.05 was set to test the null hypotheses. All statistical analyses were calculated using PASW Statistics version 20 and Amos version 20 for Windows (IBM Software Japan, Tokyo, Japan). The statistician who performed the analyses was blinded to the allocation in the study.
Results {#Sec17}
=======
Enrolment and baseline characteristics of the participants {#Sec18}
----------------------------------------------------------
The trial started in July 2012 and ended in January 2016. Fig. [1](#Fig1){ref-type="fig"} shows the study flow; we screened 284 family caregivers, 238 of whom met the eligibility criteria. Out of 74 participants, 37 were randomly assigned to receive SM-FPE plus TAU and 37 to receive TAU alone. The number of participants at each facility was 24 (Yagoto Hospital), 21 (Kusunoki Mental Hospital), 12 (Toyota-nishi Hospital), and 17 (Minami-chita Hospital). A protocol deviation occurred for one participant in the intervention group. We noticed after randomisation that her daughter still remained hospitalised. We estimated however that this violation had only a very small influence on the outcomes, because she was discharged on the day of the first session. Table [1](#Tab1){ref-type="table"} shows sociodemographic and clinical characteristics at baseline. Clinical characteristics were similar between the assigned arms. Most caregivers were female (82.2%), and predominantly mothers (96.7%). Almost all patients were diagnosed with schizophrenia (97.3%), about one third in the recent-onset condition (35.6%). Although the ratio of female to male was 1:1.9 and that of compulsory to post-compulsory education was 1:2.8, most patients were unemployed (76.7%), unmarried (95.9%), and dependent on the caregivers for their lives (90.4%). Fig. 1Participant flow diagram Table 1Sociodemographic and clinical characteristics of the participantsCharacteristics (caregivers)SM-FPE + TAUTAU aloneAll participants(*n* = 36)(*n* = 37)(*n* = 73)Age, mean (SD), years58.9(6.4)58.2(9.4)58.4(8.0)Sex, n (%) Female30(83.3)30(81.1)60(82.2) Male6(16.7)7(18.9)13(17.8)Relationship, n (%) Mother30(83.3)29(78.4)58(80.8) Father6(16.7)6(16.2)12(16.4) Spouse01(2.7)1(1.4) Sibling01(2.7)1(1.4)Education, n (%) \< High school01(2.7)1(1.4) High school22(61.1)16(43.2)38(52.1) Two-year college10(27.8)12(32.4)22(30.1) University4(11.1)8(21.6)12(16.4)Occupation, n (%) Employed, full-time12(33.3)15(40.5)27(37.0) Employed, part-time8(22.2)9(24.3)17(23.3) Homemaker12(33.3)8(21.6)20(27.4) Retirement4(11.1)5(13.5)9(12.3)Marital status, n (%) Unmarried1(2.8)1(2.7)2(2.7) Married29(80.6)28(75.7)57(78.1) Divorced3(8.3)6(16.2)9(12.3) Widowed3(8.3)2(5.4)5(6.8) Psychotropic use/psychiatrist's visit, n (%)4(11.1)7(18.9)11(15.1) Self-help group, n (%)6(16.7)6(16.2)12(16.4) K6 ≥ 9, n (%)13(36.1)14(37.8)27(37.0) Age, mean (SD), years29.7(5.6)30.5(5.7)30.1(5.6)Sex, n (%) Female11(30.6)14(37.8)25(34.2) Male25(69.4)23(62.2)48(65.8)Education, n (%) \< High school8(22.2)11(29.7)19(26.0) High school12(33.3)12(32.4)24(32.9) Two-year college6(16.7)6(16.2)12(16.5) University10(27.8)8(21.6)18(24.7)Occupation, n (%) Unemployed27(75.0)29(78.4)56(76.7) Employed, full-time2(5.6)2(5.4)4(5.5) Employed, part-time4(11.1)4(10.8)8(11.0) Homemaker1(2.8)1(2.7)2(2.7) Sheltered work2(5.6)1(2.7)3(4.1)Marital status, n (%) Unmarried34(94.4)36(97.3)70(95.9) Married1(2.8)1(2.7)2(2.7) Divorced1(2.8)01(1.4)Living status, n (%) Living alone03(8.1)3(4.1) Living with a participating caregiver33(91.7)33(89.2)66(90.4) Living with someone not engaging in care2(5.6)1(2.7)3(4.1) Group home1(2.8)01(1.4)Diagnosis, n (%) Schizophrenia35(97.2)36(97.3)71(97.3) Brief psychotic disorder1(2.8)01(1.4) Schizoaffective disorder01(2.7)1(1.4)Duration of the disorders Mean (SD), months86.6(67.1)101(69.5)93.9(68.2) \< 59 months13(36.1)13(35.1)26(35.6) ≥ 60 months23(63.9)24(64.9)47(64.4) Number of hospitalisations, mean (SD)1.7(1.2)1.8(1.7)1.8(1.5)
Attrition and study integrity {#Sec19}
-----------------------------
### Attrition {#Sec20}
One mother withdrew her consent to participate in this trial just after the randomisation. Thus, data from 73 participants were available for ITT analyses. All 36 participants in the SM-FPE plus TAU group completed the intervention, and they attended a mean number of 4.8 of five sessions (SD = 0.49). No data were missing for the participants who were included in the analyses. In the TAU alone group, one participant started a new psychotropic/psychiatric therapy and two participants entered new self-help groups during the study period.
### Fidelity assessment {#Sec21}
Ten randomly selected sessions satisfied 100% of eight items for the structured group work and 81% of eight items for the roles of staff (all play/some play). Both percentages demonstrate that the implementation adequately adhered to the standard model of the JNPF.
### GAF assessment {#Sec22}
The κ-values for agreement between the allocation states and those speculated by psychiatrists in charge were 0.23 (95% CI: 0.01--0.46) at 10 weeks and 0.18 (95% CI: − 0.04--0.41) at 14 weeks. Both values suggest that the blinding level of the assessments was acceptable.
### Patient hospitalisation {#Sec23}
The numbers of patients who were admitted during the study period were one and three in the intervention and control group, respectively.
### Primary and other outcomes {#Sec24}
ANCOVA was conducted on T-anxiety of family caregivers (Table [2](#Tab2){ref-type="table"}). No statistically significant differences were detected between the two groups at week 10 (*p* = 0.19) and week 14 (*p* = 0.24), after adjusting for group differences in baseline scores. ANCOVA was also used to analyse secondary outcomes (Table [2](#Tab2){ref-type="table"}). The analyses did not detect statistically significant differences between groups in S-anxiety, K6, J-ZBI_8, FAS, LSS, GAF, or the kinds and amounts of antipsychotics at the two assessment points, after adjusting for group differences in respective baseline scores. Table 2Adjusted results for the outcomes of the participants, mean (SE), ANCOVABaseline10 Weeks14 WeeksMeasureSM-FPE + TAUTAU aloneSM-FPE + TAUTAU alone*Fp*SM-FPE + TAUTAU alone*Fp*Caregivers Trait anxiety46.6(1.7)49.4(1.9)42.9(1.5)46.0(1.7)1.730.1941.6(1.6)44.2(1.6)1.680.20 State anxiety48.5(1.8)49.0(1.5)41.0(1.8)46.4(1.7)1.840.1841.7(1.7)45.2(1.8)2.060.16 K66.8(0.8)7.2(0.9)4.9(0.7)5.1(0.7)0.110.744.3(0.6)5.7(0.7)0.580.45 J-ZBI_812.3(1.3)10.9(1.2)9.3(1.1)10.9(1.2)\< 0.010.949.1(1.1)9.1(1.1)0.010.95 FAS46.8(4.2)46.9(3.5)36.6(3.6)45.4(3.7)0.750.3937.8(3.9)42.2(3.4)0.790.38 LSS35.7(6.0)35.7(0.8)34.0(0.9)35.9(1.0)0.570.4534.4(1.1)35.2(1.0)0.500.48Patients GAF46.6(2.4)48.5(1.9)47.5(2.3)50.1(2.2)0.600.4447.1(2.5)50.6(2.4)0.770.38 Kinds of APs1.8(0.2)1.9(0.2)1.8(0.2)1.8(0.2)0.140.711.8(0.2)1.8(0.2)0.110.75 Amount of APs, mg810(112)780(96)806(116)760(95)0.070.80795(116)744(94)0.080.77
In the post-hoc analyses, the principle factor method found a single factor with an eigenvalue \> 1; the scree plot also indicated the presence of a single factor. We excluded LSS from the measurement model of the overall state of caregivers' mental health, because the communality of LSS (0.039) was clearly lower than that of S-anxiety, K6, J-ZBI_8, or FAS (0.649, 0.716, 0.602, or 0.481, respectively). Variables were more significantly correlated with each other except for assignment in chronic psychosis than in recent-onset psychosis (Table [3](#Tab3){ref-type="table"}). The path model provided acceptable fit to the data: CMIN/*df* = 1.671, CFI = 0.962, and RMSEA = 0.097. However, when the results of the recent-onset and chronic groups were compared in the original model, the chi-square difference between the non-constrained and measurement models reached a statistically significant level of 0.05 (*p* = 0.025). This suggested that both groups could be regarded as different in the above model, which provided acceptable fit to the data: CMIN/df = 1.496, CFI = 0.945, and RMSEA = 0.084. Fig. [2](#Fig2){ref-type="fig"} illustrates that the intervention effects were not significant at 10 and 14 weeks in the recent-onset stage (*p* = 0.429 and 0.445, respectively), while in the chronic stage, they were significant at the end of the programme (*p* = 0.012) but not at a 1-month follow-up (*p* = 0.361). Between the two stages, the paired comparisons of both intervention effects at 10 and 14 weeks did not reach the level of statistical significance (*z* = 0.46 and 0.20, respectively). Table 3Correlations among variables used in structural equation modelling123456789101112131. Assignment (1, SM-FPE + TAU, 2 TAU alone)--−.21.00−.21.08.13.10.00.15.01.17−.11.042. State anxiety, base line.15--.45\*.60\*\*.56\*\*.69\*\*\*.57\*\*.71\*\*\*.57\*\*.66\*\*\*.57\*\*.68\*\*\*.55\*\*3. K6, base line.08.58\*\*--.61\*\*.64\*\*\*.28.38.52\*\*.50\*.52\*\*.43\*.51\*\*.62\*\*4. J-ZBI, base line−.01.61\*\*\*.61\*\*\*--.58\*\*.45\*.47\*.72\*\*\*.49\*.44\*.35.83\*\*\*.57\*\*5. FAS, base line−.05.50\*\*\*.48\*\*.74\*\*\*--.25.30.51\*\*.89\*\*\*.26.41\*.67\*\*\*.88\*\*\*6. State anxiety, week 10.35\*.55\*\*\*.41\*\*.40\*\*.45\*\*--.78\*\*\*.77\*\*\*.45\*.84\*\*\*.74\*\*\*.57\*\*.367. K6, week 10−.02.54\*\*\*.71\*\*\*.54\*\*\*.60\*\*\*.52\*\*\*--.78\*\*\*.48\*.76\*\*\*.86\*\*\*.59\*\*.378. J-ZBI_8, week 10.18.54\*\*\*.55\*\*\*.76\*\*\*.60\*\*\*.58\*\*\*.69\*\*\*--.69\*\*\*.77\*\*\*.77\*\*\*.83\*\*\*.61\*\*9. FAS, week 10.22.51\*\*\*.45\*\*.63\*\*\*.81\*\*\*.63\*\*\*.73\*\*\*.79\*\*\*--.39\*.60\*\*.65\*\*\*.87\*\*10. State anxiety, week 14.29\*.57\*\*\*.48\*\*.38\*\*.44\*\*.79\*\*\*.57\*\*\*.55\*\*\*.61\*\*\*--.81\*\*.52\*\*.40\*11. K6, week 14.21.47\*\*.61\*\*\*.34\*.39\*\*.62\*\*\*.68\*\*\*.52\*\*\*.54\*\*\*.68\*\*\*--.56\*\*.49\*12. J-ZBI_8, week 14.06.48\*\*.49\*\*\*.78\*\*\*.64\*\*\*.50\*\*\*.61\*\*\*.89\*\*\*.74\*\*\*.54\*\*\*.50\*\*\*--.71\*\*\*13. FAS, week 14.14.34\*.38\*\*.54\*\*\*.78\*\*\*.55\*\*\*.65\*\*\*.70\*\*\*.89\*\*\*.60\*\*\*.50\*\*\*.76\*\*\*--Mean1.552.08.212.446.747.46.210.842.349.46.89.743.61.546.96.311.147.041.74.39.740.340.24.08.838.0SD0.59.35.38.325.211.34.37.122.810.64.56.323.50.59.84.87.022.310.03.67.022.69.43.36.621.0Upper figure reflects recent-onset psychosis (*n* = 26), lower figure reflects chronic psychosis (*n* = 47): \**p* \< .05; \*\**p* \< .01; \*\*\**p* \< .001 Fig. 2Path model of the stratification of recent-onset psychosis (right) and chronic psychosis (left) according to the study flow
Discussion {#Sec25}
==========
We failed to demonstrate effects of SM-FPE on T-anxiety and other individual outcomes of family caregivers of young adults with schizophrenia. Our analysis indicates that our failure to prove the usefulness of the family intervention can be partly attributed to a lack of effectiveness in the integrated outcome of the caregivers of the recent-onset patients, in which the factor loadings of anxiety and depression were higher than those of care burden and expressed emotion.
A main reason for these negative results may be the use of general measures to assess negative emotions. Although our primary interest was the caregivers' predisposition to anxiety, a psychosis-specific questionnaire might be more sensitive to evaluate their state anxiety (e.g. the Involvement Evaluation Questionnaire) \[[@CR49]\]. Another reason may be floor effects in the assessment measures. In the case of the STAI, scores of 20.5% for T-anxiety and 30.1% for S-anxiety at baseline were less than 30% of each standard score, corresponding to the category of low anxiety \[[@CR50]\]. Other possible reasons are the natural course of anxiety and social desirability biases, which can be seen in the decrease of 5.2 points in T-anxiety and that of 7.3 points in S-anxiety in the TAU alone group, from baseline to 14 weeks, although the patients' condition had undergone very little change. These types of reductions could enhance floor effects on the outcomes. Despite these factors that might undermine the evaluation of the usefulness of the intervention, this pragmatic study did not exclude caregivers with subthreshold anxieties, because such selection does not usually occur in a clinical setting. Furthermore, the SM-FPE has been considered incapable of improving caregivers' emotional distress \[[@CR25]\]. The multiple-group analysis suggests that the standard intervention needs to be improved to alleviate the anxiety and depression of caregivers of young people with recent-onset psychosis more effectively.
The internal validity of this RCT was supported by low attrition and good adherence. According to a review of psychoeducational studies for families of people with schizophrenia, 17% of studies presented recruitment, retention, and engagement problems, and 38% offered no explicit data on these matters \[[@CR11]\]. In our study, we observed almost no attrition from the randomisation to the post-intervention follow-up. Furthermore, almost all participants adhered to the programme. This implies that they considered it meaningful to continue with it, although we did not assess perceived usefulness of the programme through qualitative interviews with the participants. In the lecture component, caregivers could accept psychotic symptoms more easily by using the normalisation. The approach explained that people could hear hallucinations in normal situations, such as the voices that stranded climbers experienced in winter mountains \[[@CR29]\]. In the group format component, the discussed problems were categorised into four themes: 'how to deal with the symptoms and treatment of the illness', 'concerns about patients' present and future lives', 'how to face communication with the patients', and 'how to support patients' social engagement'. In terms of external validity, the trial was carried out in four psychiatric hospitals, which are located in the metropolis, a provincial city, and a rural area in central Japan. The multi-site implementation thus increased the representativeness of the study population and demonstrated that a multi-family group intervention could be integrated into routine practices and delivered by staff in mental hospitals across Japan.
The present study has a number of limitations. First, the characteristics of participants might differ from those of non-participants. According to the protocol approved by the Institutional Review Board and Ethics Committee, we had to guarantee the right that screened people could cease participation without reason. The rate of refusal in the screening (about 58%) might reduce the external validity of this study. Second, participants and researchers were aware of the allocation arm due to the nature of the RCT for psychological interventions. Lack of blinding might influence outcome assessments \[[@CR51]\]. We had an independent data analyst to reduce detection bias, and patient outcome assessors were blinded to the allocation. Third, the participants were followed up for only 4 weeks from the end of the intervention. In chronic psychosis, the direct effects of SM-FPE on the overall state of caregivers' mental health decreased during this short period. This suggests that booster sessions are needed to maintain the improvements. Fourth, the positive effects were revealed only by the post-hoc analyses that integrated different concepts in caregiving. However, the reason for the use of the SEM model was that it was considered to be a close representation of the clinical responses of the caregivers observed in the intervention. In further research, an a priori defined conceptual framework is needed to evaluate caregivers' mental health in family interventions more robustly.
Conclusions {#Sec26}
===========
The implementation of the SM-FPE did not modify caregivers' T-anxiety or improve other individual outcomes. When their burdens were considered overall, there was evidence of an intervention benefit on the overall mental health state of the caregivers of the chronic patients. However, such intervention effects were not observed in the integrated outcome of those of the recent-onset patients. The intervention programme requires stronger evidence that it supports families before wider dissemination.
ANCOVA
: Analysis of covariance
AP
: Antipsychotic
CB
: Care Burden
CFI
: Comparative fit index
CMIN
: Chi-squared index
DSM-IV-TR
: Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revision
EE
: Expressed emotion
FAS
: Family Attitude Scale
GAF
: Global Assessment of Functioning
ITT
: Intent-to-treat
JNPF
: Japanese Network of Psychoeducation and Family Support Program
J-ZBI_8
: Japanese version of the Zarit Burden Interview Short Version
LSS
: Link's Stigma Scale
PMHS
: Poor Mental Health State
RCT
: Randomised controlled trial
RMSEA
: Root mean square error of approximation
SA
: State anxiety
S-anxiety
: State anxiety
SEM
: Structural equation modelling
SM-FPE
: Standard model of family psychoeducation
STAI
: State-Trait Anxiety Inventory
T-anxiety
: Trait anxiety
TAU
: Treatment as usual
**Publisher's Note**
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
We thank the responsible therapists (Maho Komori, PSW, at Yagoto Hospital; Hirotomo Kataoka, PSW, at Minami-chita Hospital; Hikari Katsuragawa, PSW, at Kusunoki Mental Hospital; Yukihito Igami, RN, at Toyota-nishi Hospital), administrative staff (Shinichi Yoshida, MD, PhD, at Yagoto Hospital; Sachiko Maki, MD, at Minami-chita Hospital; Jun Ito, MD, at Kusunoki Mental Hospital; Shigehiro Tsuboi, MD, at Toyota-nishi Hospital), and an independent data analyst (Toshinori Kitamura, MD, PhD at his own Institute of Mental Health Tokyo). We also thank Tatsuya Isomura at the Clinical Study Support for conducting the central allocation.
Availability of data and material {#FPar1}
=================================
The data sets analysed during the present study are not publicity available due to the restriction put by the Institutional Review Board and Ethics Committee of Nagoya City University Graduate School of Medical Science but are available from the corresponding author on reasonable request.
NS conceived the design of the study, managed the implementation of recruitment, intervention, and data collection, and drafted the manuscript. NW and TA were responsible for the trial supervision. KF and HS were major contributors in creating the programme and training the participating staff. NW, KF, and TA revised the draft, and all authors approved the final manuscript.
This study was supported by a Grant-in-Aid for Scientific Research \[No. 15 K17300\] from the Japanese Ministry of Education, Science, and Technology. The funding body did not play any role in the design of the study; the collection, analysis, or interpretation of the data; or the writing of the manuscript.
This study was approved by the Institutional Review Board and Ethics Committee of Nagoya City University Graduate School of Medical Science (no. 708) and conducted in accordance with the principles laid down in the Declaration of Helsinki. After an in-depth explanation of the study's purpose and methods, written consent was obtained from all eligible patients and caregivers.
Not applicable.
NS has received a lecture fee from Dainippon-Sumitomo. NW has received research funds from the Japanese Ministry of Health Labor and Welfare, the Japanese Ministry of Education, Science, and Technology, from the National Center of Neurology and Psychiatry, as well as an Intramural Research Grant for Neurological and Psychiatric Disorders. He has also received royalties from Sogensha and Akatsuki. KF has received research funds from the Japanese Ministry of Education, Science, and Technology. She has also received lecture fees from MSD. HS declares no conflicts of interest. TA has received lectures fees and/or research funds from Daiichi-Sankyo, Dainippon-Sumitomo, Eizai, Hisamitsu, Lilly, MSD, Meiji-seika Pharma, Mochida, Pfizer, Novartis, Shionogi, Takeda, Tanabe, Terumo, and Yoshitomi. He has received royalties from Igaku-Shoin, Kagakuhyoron-sha, and Seiwa shoten. The Japanese Ministry of Education, Science, and Technology and the Japanese Ministry of Health, Labor, and Welfare, Nagoya City University, the Japan Agency for Medical Research and Development, the Foundation for Promotion of Cancer Research, and the Yuumi Memorial Foundation for Home Health Care have funded his research projects.
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INTRODUCTION {#sec1-1}
============
Oral squamous cell carcinoma (OSCC) is the sixth most common tumor in the world.\[[@ref1]\] There are several strategies for OSCC treatments involving chemotherapy, surgery, radiation, or a combination of these methods. However, the adequate understanding of cell biology of oral oncogenesis has not been explored, and the development of drug resistance to cancer chemotherapy has been the most critical problem.\[[@ref2]\] Therefore, finding the new type of agents to treat OSCC and elucidating their potential mechanisms have great scientific and practical values.\[[@ref3]\]
To find potential anticancer agents from natural products and their derivatives is one of the most convenient and valuable methods.\[[@ref4]\] In traditional Asian medicine, *Cordyceps militaris* has attracted a great attention. Some constituents obtained from *C. militaris*, such as nucleosides and polysaccharides, were reported to have antitumor,\[[@ref5][@ref6]\] immunomodulatory,\[[@ref7][@ref8]\] and anti-inflammatory\[[@ref9][@ref10]\] activities. In our original research, *C. militaris* fraction (CMF) has been demonstrated to possess antiproliferative property in human chronic myeloid leukemia K562 cells.\[[@ref11]\]
In the last two decades, many studies have proposed that diverse phytochemicals and various botanical formulations have potential anticancer effects via inducing apoptosis.\[[@ref12]\] Thus, activating the process of cell death has been proved to be a valuable method in cancer therapy.\[[@ref13]\] The intrinsic or mitochondrial apoptotic pathway is controlled by the proteins of Bcl-2 family which regulate the permeability of mitochondrial membrane.\[[@ref14]\] The released cytochrome c could recruit Apaf-1 and activate caspase-9 and caspase-3, resulting in apoptosis. Additionally, induction of cell cycle arrest is another way to control tumor. The G2/M cell cycle procedure is positively regulated by the members of cyclin-dependent kinase (CDK) family.\[[@ref15]\] In particular, the phosphorylation of Tyr15 of cdc2 suppresses the activity of cdc2/cyclin B1 kinase complex, while the dephosphorylation of Tyr15 of cdc2 by cdc25 phosphatases decides cell entry into mitosis.\[[@ref16]\] The G2 phase is also can be regulated by the CDK inhibitor (CKI), which can induce cell cycle arrest in G2 phase, thereby inhibiting cell proliferation.\[[@ref17]\] Cell cycle checkpoint kinase 2 (CHK2), a serine/threonine protein kinase, contributes to phosphorylate a number of proteins involved in cell cycle arrest, apoptosis, and DNA repair.\[[@ref18]\]
Additionally, the mitogen-activated protein kinase (MAPK) family has been identified to play pivotal roles in a variety of cell functions, including cell cycle and apoptosis, and different MAPK members have different functions.\[[@ref15]\] Studies have shown that c-Jun N-terminal kinases (JNK) is sensitive to stress signals, which mediate cellular steps in the apoptosis of some cell types.\[[@ref19][@ref20]\] As a target of JNK pathway, the specifically phosphorylated c-Jun plays a central role in diverse functions of AP-1 complex.\[[@ref21]\]
In the present study, we investigated the antiproliferative effect of CMF and to explore its mechanism in oral squamous carcinoma KB cells. Furthermore, we first demonstrated that the inhibition of proliferation of KB cells by CMF was involved with the induction of apoptosis and G2/M phase arrest via JNK activation.
MATERIALS AND METHODS {#sec1-2}
=====================
Fraction preparation and reagents {#sec2-1}
---------------------------------
Cultured *C. militaris* was purchased from Honghao Biological Company of Jiangmen (Guangdong, China). CMF was isolated, identified, and purified as per our previous report.\[[@ref11]\] CMF stock solution was prepared into 1000 μg/ml concentration in 1640 complete medium and stored at 4°C. The antibodies for p-c-Jun, c-Jun, p-ERK, ERK, p-p38, p38, p-JNK, JNK, GAPDH, PARP, p-p53, cyclin B1, cdc2, cdc25c, caspase-3, and caspase-9 were obtained from Cell Signaling Technology, Inc. (Boston, MA, USA). Antibodies for Bax, Bcl-2, and cytochrome c were purchased from Abcam Ltd. (Cambridge, UK). SB203580, SP600125, and 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma-Aldrich, Inc. (Saint Louis, MO, USA). Roswell Park Memorial Institute (RPMI)-1640 medium and fetal bovine serum (FBS) were purchased from Gibco/Life Technologies Inc. (Carlsbad, CA, USA).
Cell culture and treatment {#sec2-2}
--------------------------
The human oral squamous carcinoma cancer (KB) cells were purchased from the American Type Culture Collection (Manassas, VA, USA), cells were cultured in RPMI-1640 supplemented with 10% FBS, and incubated at 37°C with 5% CO~2~ and 95% humidity.
3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay for cell viability {#sec2-3}
-------------------------------------------------------------------------------------
The KB cells were seeded in 96-well plates 3.5 × 10^3^ cells per well and then treated with 0.625, 1.25, 2.5, 5, 10, and 20 μg/ml of CMF for 24, 48, or 72 h. After the treatment, 20 μl of MTT working solution was added to each well for another 4 h. The culture supernatant was removed from the well and 200 μl of dimethyl sulfoxide was added to resuspend the dark formazan crystals, and the absorbance was measured at 570 nm.
Quantification of apoptotic cells by flow cytometry {#sec2-4}
---------------------------------------------------
Apoptotic/necrotic cells were quantitatively detected with Annexin V-FITC/propidium iodide (PI) apoptosis detection kit (Nanjing KeyGen Biotech Co. Ltd., Nanjing, China).\[[@ref3]\] Briefly, KB cells were treated with CMF at concentrations of 2.5, 5, 10, and 20 μg/ml for 48 h. Then, cells were suspended in binding buffer and stained with Annexin V-FITC and PI in the dark at room temperature. Cell fluorescence was detected by a flow cytometer after 10 min. When SP600125 was used, KB cells were incubated with the desired concentration of the JNK inhibitor for 1 h before addition of CMF.
4',6-diamidino-2-phenylindole staining assay {#sec2-5}
--------------------------------------------
Cells were plated at a density of 5 × 10^4^ cells/ml and exposed to different concentrations of CMF for 48 h. Treated cells for the indicated time period were collected, washed three times, and fixed with 4% paraformaldehyde for 10 min at room temperature. Fixed cells were washed and stained with 4′,6-diamidino-2-phenylindole (DAPI) solution for 15 min in the dark. The morphological changes were then observed via a fluorescence microscope (Carl Zeiss, Germany). Ten fields were randomly selected to observe and images were taken.
Cell cycle analysis {#sec2-6}
-------------------
Cells were treated with CMF at concentrations of 2.5, 5, 10, and 20 μg/ml for 24 h and the whole cells were harvested, pelleted at 2000 rpm, and fixed with 70% alcohol on ice. Fixed cells were resuspended and washed again with PBS and incubated with RNase A (100 mg/ml) for 30 min, and then PI Flow Cytometry Kit was added for another 30 min at room temperature.\[[@ref22]\] The cell cycle distribution was then detected using a flow cytometer. The percentage of cells on each phase was determined using MultiCycle AV for Windows Version 295 (Beckman Coulter, Brea, CA, USA).
Western blot analysis {#sec2-7}
---------------------
A total of 2.0 × 10^5^ cells were cultured in 6-well plates. After treatment with indicated concentrations of CMF for 8 or 24 h, the adherent and nonadherent KB cells were harvested by 200 μl of RIPA lysis buffer supplemented with protease inhibitors (1 mM Na~3~VO~4~ and 1 mM phenylmethanesulfonyl fluoride) for 30 min in ice to incubate. The protein content was measured by bicinchoninic acid (BCA) protein assay kit (Beyotime, Nanjing, China). For Western blot analysis, the equal amount of protein was loaded onto SDS-PAGE Running Buffer and transferred to polyvinylidene difluoride membrane (Millipore Bedford, MA, USA) by electroblotting, and the membranes were treated with the desired primary antibodies overnight, incubated with the diluted enzyme-linked secondary antibody for 2 h, and detected with an ECL detection kit.\[[@ref23]\] GAPDH was used as an internal control.
Fluorescence staining for confocal imaging {#sec2-8}
------------------------------------------
KB cells were treated with different concentrations of CMF for 24 h, and then the immunofluorescence staining was performed as described previously.\[[@ref24][@ref25]\] Cells were fixed using 2% paraformaldehyde at 4°C for 10 min, followed by permeabilized 0.1% Triton X-100 in TBS and blocking with 1% bovine serum albumin for 2 h. The cells were incubated with the primary antibody against p-c-Jun (1:800) overnight at 4°C and with Alexa Fluor^®^ 488 secondary antibody (Invitrogen, Grand Island, NY, USA) for 2 h. Samples were mounted and analyzed by the use of Confocal Microscope Detection System, Leica TCS SP5 (LeicaBiosystems Nussloch GmbH, Heidelberg, Germany).
Statistical analysis {#sec2-9}
--------------------
Data were expressed as means ± standard deviation of more than three separate assays. Statistical Package for the Social Sciences version 20.0 software (SPSS, Chicago, IL, USA) and GraphPad Prism Version 6.0 (GraphPad Software Inc., San Diego, CA, USA) were used for statistical analysis. Differences between treatment groups were calculated using Student\'s *t*-test, with the following symbols of significance levels: \*P \< 0.05 and \*\*P \< 0.01.
RESULTS {#sec1-3}
=======
*Cordyceps militaris* fraction inhibits proliferation of KB cells {#sec2-10}
-----------------------------------------------------------------
The viability of KB cells was determined by MTT assay. IC~50~ values of KB cell line were around 18.1 μg/ml for 24 h treatment, 4.32 μg/ml for 48 h treatment, and 3.94 μg/ml for 72 h treatment \[[Figure 1](#F2){ref-type="fig"}\].
{#F2}
*Cordyceps militaris* fraction induces apoptosis through the intrinsic apoptotic pathway {#sec2-11}
----------------------------------------------------------------------------------------
To examine whether CMF inhibits KB cell survival through the induction of apoptosis, the morphological changes of KB cells were preliminarily investigated. As indicated by arrows in [Figure 2a](#F3){ref-type="fig"}, apoptotic shrinkage and chromatin condensation occurred in the cells treated with CMF, whereas these features of apoptotic cells were not observed in control cells. Apoptotic cell death was further detected by flow cytometry. As shown in [Figure 2b](#F3){ref-type="fig"}, CMF induced the proportions of late apoptosis cells, which is consistent with the results by DAPI staining.
{#F3}
Substantial evidence supports the idea that Bcl-2 family members regulate the release of mitochondrial proteins.\[[@ref26]\] Cytochrome c is a key component for the activation of the caspase cascade.\[[@ref27]\] To further unravel the mechanism of apoptotic signaling in CMF-treated KB cells, the intrinsic signaling pathway of apoptosis was investigated. As shown in [Figure 2c](#F3){ref-type="fig"}, the level of cytochrome c was increased and the pro-apoptosis protein Bax as the last gateway of cytochrome c release was subsequently increased. In contrast, CMF suppressed Bcl-2 expression. Furthermore, CMF significantly increased the expression of cleaved caspase-9, caspase-3, and PARP \[[Figure 2d](#F3){ref-type="fig"}\]. These results suggested that an increased ratio of Bax/Bcl-2 can induce cytochrome c release, thereby increasing apoptosis.
*Cordyceps militaris* fraction induces G2/M arrest in KB cells and inhibits the expression of key mitotic proteins {#sec2-12}
------------------------------------------------------------------------------------------------------------------
To test if CMF induce cell cycle arrest, we determined the cell cycle distribution. The results demonstrated that an increase in G2 phase from 5.09% to 42.0% and a decrease in G1 phase from 58.8% to 38.5% in comparison with the control, after the cells were treated with CMF (20 μg/ml) for 24 h \[Figure [3a](#F4){ref-type="fig"} and [b](#F4){ref-type="fig"}\].
{#F4}
Next, the effects of CMF on the expression of cell cycle-related proteins were determined. As shown in Figure [3c](#F4){ref-type="fig"} and [d](#F4){ref-type="fig"}, CMF concentration dependently increases the expression of p21 and CHK2, while the expression of phosphorylated retinoblastoma protein (p-Rb) was downregulated. Report has shown that p53 was involved in response to DNA damage by upregulated transcription of p21 gene.\[[@ref28]\] However, CMF had no effect on the expression of p53. It suggested that CMF upregulated the expression of p21 through the p53-independent pathway.
The important role of CDKs and cyclins in regulate cell cycle progression was observed.\[[@ref15]\] The cyclin B1/cdc2 complex was also proposed to control the G2/M phase, which is regulated by cdc25c. And then, the effects of CMF modulating the expression of cell cycle regulatory molecules were determined. The result showed that the levels of cyclin B1, cdc2, and cdc25c were significantly decreased by CMF \[[Figure 3d](#F4){ref-type="fig"}\]. The data were consistent with those of cell cycle analysis by flow cytometry. These results suggested that CMF was proposed to inhibit the proliferation of KB cells through the cell cycle arrest at G2/M phase.
The activation of JNK and c-Jun in KB cells was proposed to be induced by CMF {#sec2-13}
-----------------------------------------------------------------------------
The effect of CMF on activation of MAPKs and c-Jun was examined to identify the molecular target of CMF in KB cells. Western blot experiment demonstrated that phosphorylation of JNK, p38, and c-Jun was significantly increased by CMF, whereas phosphorylation level of ERK was not changed \[Figure [4a](#F5){ref-type="fig"} and [b](#F5){ref-type="fig"}\].
{#F5}
We then determined whether these phosphorylation events can be suppressed by specific kinase inhibitors, KB cells were incubated with CMF in the presence of SP600125 (JNK inhibitor) or SB203580 (p38 inhibitor). The pretreatment with SP600125 markedly decreased CMF-induced p-JNK and p-c-Jun induction, while treated with SB203580 did not change the expression of p-p38 and p-c-Jun \[Figure [4c](#F5){ref-type="fig"}--[e](#F5){ref-type="fig"}\].
The experiment results indicated that JNK pathway plays an important role in CMF-induced apoptosis. It is also proposed that JNK, not p38 and ERK, was induced by CMF for the expression of c-Jun.
*Cordyceps militaris* fraction increases the nuclear localization of c-Jun {#sec2-14}
--------------------------------------------------------------------------
The immunofluorescence microscopic analysis was performed to further corroborate the notion that KB cells are defective in c-Jun upregulation in response to DNA damage and cell cycle arrest. As shown in [Figure 5](#F6){ref-type="fig"}, p-c-Jun mostly localized in nuclei in a dose-dependent manner after KB cells was treated with CMF. It demonstrated that JNK/c-Jun pathway could be responding to CMF, which then results in a series of related reactions.
{#F6}
*Cordyceps militaris* fraction induces apoptosis and cell cycle arrest in a manner dependent on JNK/c-Jun signaling pathway {#sec2-15}
---------------------------------------------------------------------------------------------------------------------------
To unravel the possible roles of JNK pathway in CMF-induced apoptosis and cell cycle arrest, KB cells were incubated with CMF and JNK inhibitor. As shown in Figure [6a](#F7){ref-type="fig"} and [b](#F7){ref-type="fig"}, SP600125 significantly inhibited CMF-induced apoptosis and G2/M phase cell cycle arrest. In addition, SP600125 not only inhibited the CMF-induced cleavage of PARP, but also reversed the upregulation of p21 expression and downregulation of mitotic cyclin/kinase/phosphatase-related protein levels \[Figure [6c](#F7){ref-type="fig"} and [d](#F7){ref-type="fig"}\]. Taken together, the results show that JNK signaling pathway is directly related to the apoptosis and cell cycle arrest caused by CMF in KB cells.
{#F7}
DISCUSSION {#sec1-4}
==========
Natural products have drawn more and more attention due to their growth inhibition of various types of cancers through complex signaling pathways.\[[@ref29]\] The results proposed that CMF could efficiently inhibit the proliferation of KB cells by targeting JNK-mediated signaling pathways. It also changed a broad spectrum of signaling effectors, including c-Jun, Bcl-2, Bax, and p21, thus regulate cell cycle and cell survival.
We are aware that a successful cancer therapy depends on the complex network of cell signaling pathways, and activation of the MAPK cascade precedes apoptosis and cell cycle arrest.\[[@ref30]\] Some investigations have shown that JNK might decrease cell proliferation by activating its downstream effector c-Jun.\[[@ref31]\] At the present experiment, the results showed that the levels of phosphorylated JNK, p38, and c-Jun were significantly increased by CMF. In another experiment, CMF-induced c-Jun expression was abrogated by the pharmacological JNK inhibitor SP600125. However, no obvious effect on c-Jun expression was observed while using p38 inhibitor SB203580 in CMF-treated KB cells. Therefore, we analyzed that p38 MAP kinase signaling was not mediated by growth inhibition in KB cells. Additionally, CMF stimulated the activation of JNK, leading to increased nuclear localization of c-Jun, supporting a role of JNK, not p38 and ERK, in controlling the expression of c-Jun in KB cells.
Besides c-Jun nuclear localization, JNK-induced apoptosis can also be regulated through JNK cytoplasmic substrates, such as proapoptotic proteins Bax, Puma, and Bim activity,\[[@ref25]\] or can be regulated via Bcl-2 family proteins Bcl-2, Bcl-x L, and Mcl-1 activity.\[[@ref32]\] In this work, the pro-apoptosis effects of CMF were also shown in KB cells. We observed that CMF significantly increased the ratio of Bcl-2/Bax and induced the release of cytochrome c, thus resulting in a significant proteolytic cleavage of caspase-9 and PARP. In addition, JNK inhibitor significantly abrogated the cleavage of caspase-9 and PARP caused by CMF and consequently reduced the level of apoptosis \[Figure [6a](#F7){ref-type="fig"} and [c](#F7){ref-type="fig"}\]. Altogether, these data suggested that activation of the intrinsic apoptotic pathway might be a mechanism underlying CMF-induced antiproliferative effect in KB cells.
Many types of stimuli have been shown to inhibit cell cycle progression from one phase to another. During G2 phase, the main regulator is the cdc2/cyclin B1 complex. The decreased formation of cdc2/cyclin B1 complex inhibits cell cycle progression from G2 phase to M phase, and this complex can be kept inactive via phosphorylation at Thr14 and Tyr15 of cdc2,\[[@ref33]\] or inhibited by p21. Additionally, CHK2 as a key operator in eliciting DNA repair, cell cycle arrest, or apoptosis in response to DNA damage\[[@ref15][@ref18]\] and both p-Rb and p53 are key cell cycle regulatory proteins in mediating certain functions of p21. In the present study, CMF resulted in G2/M phase arrest in a dose-dependent manner. CMF decreased the expression of cyclin B1, cdc2, cdc25c, and p-Rb, whereas it induced an increase in the protein levels of p21 and CHK2. However, CMF had no effect on the expression of p53. It is well known that the JNK pathway has antiproliferative effects and regulates the cell cycle.\[[@ref15][@ref34]\] The treatment with CMF plus JNK inhibitor led to an increase in cdc2/cyclin B1 and a decrease in the expression of p21, and consequently reduced the levels of cell cycle arrest compared with CMF treatment alone \[[Figure 6d](#F7){ref-type="fig"}\]. These results suggested that CMF enhanced p21 expression, which induced G2/M phase arrest by a p53-independent pathway.
Interestingly, previous reports had shown that c-Jun transcription factor could control a wide range of molecular targets, such as Bim,\[[@ref30]\] Bcl-2,\[[@ref35]\] caspase-3,\[[@ref36]\] and CDK inhibitors, which could regulate the cellular processes, including proliferation, differentiation, migration, survival, or death. Thus, we propose that CMF activated JNK signaling pathway, promoted the nuclear localization of c-Jun, and then regulated the key apoptosis-related proteins and cell cycle-related proteins, eventually inhibiting KB cell proliferation.
CONCLUSIONS {#sec1-5}
===========
The present paper reported the potent anticancer activities of CMF fraction. The mechanism of action might result from CMF-induced apoptosis and G2/M cell cycle arrest through JNK pathway. This preliminary elucidating of JNK and c-Jun also provide new insights for further development of this agent. Therefore, CMF may possess great potential as a candidate for therapy of OSCC.
Financial support and sponsorship {#sec2-16}
---------------------------------
Nil.
Conflicts of interest {#sec2-17}
---------------------
There are no conflicts of interest.
| {
"pile_set_name": "PubMed Central"
} |
Background
==========
The functionally distinct repertoire of secreted cytokines of type 1 and type 2 CD4^+^ T helper cells (Th1 and Th2 cells) has been shown to play an important role in the pathogenesis of human allergy and inflammatory diseases \[[@B1]\]. Effector Th1 cells secrete predominantly interferon-γ (IFN-γ) and interleukin-2 (IL-2) and regulate cell-mediated immunity against intracellular pathogens, whereas differentiated Th2 cells produce IL-4 and IL-5 and promote antibody-mediated humoral immune responses \[[@B2], [@B3]\]. In several immunological disorders the balance between the type 1 and type 2 cells is disturbed and favors either a predominant Th1 response (autoimmune diseases) or enhanced Th2 response (allergic inflammation and atopic disorders) \[[@B1]\]. Cytokines IL-12 and IL-4 play a major role in selectively regulating the development and differentiation of CD4^+^ T cell subsets to IFN-γ and IL-2 producing Th1 or IL-4 and IL-5 secreting Th2, respectively. This differentiation process can be mimicked *in vitro.*
DNA microarray techniques can be used to study gene expression on a large scale \[[@B4], [@B5]\]. The expression of a large number of genes can be monitored simultaneously and the expression profiles in different samples compared. Recent approaches in immunology exploiting this technology include the expression monitoring of changes caused by cytokines \[[@B6], [@B7]\], viruses \[[@B8], [@B9]\], inflammation \[[@B10], [@B11]\] or cancers of hematopoietic origin \[[@B12], [@B13], [@B14]\]. The profiles of mRNA expression in human type 1 and type 2 Th cells are still largely unknown. An earlier report describing differentiating Th cells using an oligonucleotide array analysis led to the production of a large volume of data \[[@B15]\], but because of the experimental design the gene expression profile of Th2 cells remained obscure. We chose to study the expression of polarized T helper cells and describe the comparative analysis of effector Th1/Th2 cells. As a preliminary screen, a restricted number (250) of inflammation-related genes was included for validation. In a parallel analysis a quantitative reverse transcription polymerase chain reaction (RT-PCR) method (TaqMan) was used to evaluate the results. We show that an oligonucleotide microarray complemented by quantitative RT-PCR is an excellent method for gene expression profiling.
Results
=======
Probe selection and oligonucleotide array design
------------------------------------------------
The Roche PA-1 oligonucleotide array contained 66,176 features that correspond to 517 inflammation-related genes and was fabricated by Affymetrix. Among these, 250 genes were of human origin whereas the others represented mouse and rat genes. Each gene is monitored by 64 pairs of features that consist of 25-mer oligonucleotides, one perfectly complementary to the gene sequence and the other identical except for a single mismatch located in the center of the oligonucleotide.
Specificity, sensitivity and linearity of the detection methods used
--------------------------------------------------------------------
The Specificity and sensitivity of the current type of oligonucleotide array used are based on several criteria. First, the design of the antisense oligonucleotides was targeted toward a well annotated full-length cDNA sequence (without introns) for each gene. Second, the number of both perfectly and mismatching oligonucleotides (features) for each sequence was 64. When directly converted, the 32 specific oligonucleotides (25-mers) overlap 800 bp of sequence data. Third, for this preliminary screen the amount of mRNA used for *in vitro* transcription reaction was rather high, 1 μg. In principle this amount should represent even the low abundant messages needed for difference detection \[[@B16], [@B17], [@B18]\]. Lastly, the hybridization mixture was used only once and discarded. However, from our earlier studies \[[@B18]\] it was known that for abundant messages (such as IFN-γ) or very rare transcripts (IL-4) the linearity of detection in this type of hybridization-based method is largely compromised (10^2^ to 10^3^). We therefore have chosen a real-time RT-PCR method that facilitates quantitation of the transcripts over at least five orders of magnitude \[[@B19]\]. To confirm the linearity of the detection for each gene dilution curves were created (see Materials and methods). In addition, on the basis of our earlier work \[[@B20]\], we knew that the differences in the expression for several of the key genes in effector type 1 and type 2 cells would be very small. To minimize individual variation the oligonucleotide array was first hybridized twice with a type 1 or type 2 sample that had been previously validated with known marker genes. A second aliquot of the same mRNA was then independently prepared and hybridized to a new oligonucleotide array manufactured within the same batch. After this, the analyzed data was confirmed in samples from other individuals with a real-time RT-PCR method.
Expression profiles of Th1 and Th2 cells
----------------------------------------
The expression of a total of 250 human inflammation-related genes was screened with an oligonucleotide array by performing two independent hybridizations with a Th1 or Th2 cell sample. The phenotype of these cells had been determined using ELISA as type 1 or type 2 (secretion of high IFN-γ, low IL-4, or high IL-4, low IFN-γ, respectively, data not shown). The differentially expressed human RNA transcripts of Th1 and Th2 cells are shown in Figures [1](#F1){ref-type="fig"} and [2](#F2){ref-type="fig"}. The level of expression is shown as the average fluorescence intensity value for each gene in each sample. Collectively, 34 transcripts were recorded as differentially expressed in these cells (14 in type 1 and 20 in type 2 cells). The transcript of a proinflammatory cytokine INF-γ is abundant in type 1 T cells and very low in type 2 cells (Figure [1a](#F1){ref-type="fig"}). IFN-γ mRNA had routinely been measured with real-time RT-PCR from our cell cultures and the actual difference between the type 1 and type 2 cells was in the order of 10^3^- to 10^5^-fold. An oligonucleotide array, however, indicated less than 10^2^-fold difference in type 1 and type 2 cells (78-fold, calculated from average fluorescence intensity values for IFN-γ: Th1 1567.8, SD 129.1 and Th2 \< 20, SD 3.3). In clear contrast to IFN-γ are the transcripts of the IL-4/IL-13 cluster known to promote humoral type 2 responses. Despite their all (IL-4, IL-5 and IL-13) being detected as differentially expressed in T-cell samples by an oligonucleotide array (Figure [2b](#F2){ref-type="fig"}), reliable detection of both IL-4 and IL-5 failed in a customized RT-PCR setup, probably because of their low level of expression.
{#F1}
{#F2}
The other transcripts expressed at a higher level by Th1 than Th2 cells are presented in Figure [1a,b](#F1){ref-type="fig"}. In addition to IFN-γ, these include IL-8, tumor necrosis factor-α (TNF-α) and granzyme B (fluorescence intensity \> 400) as well as granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage inhibitory protein-1α (MIP-1α), MIP-1β, RANTES, IL-7 receptor (IL-7R), IL-12Rβ2, SLAM, CCR1, CCR2, CCR5, IκB, TIMP-1, c-Jun, IRF-1, caspase-1 and clusterin (fluorescence intensity \< 400). Transcripts of MIF, IL-4R and STAT4 gave the highest relative fluorescence intensity values (\> 300) in Th2 samples and their relative expression is shown as a separate graph (Figure [2a](#F2){ref-type="fig"}). The fluorescence values for all other genes that were expressed at a higher level in Th2 cells compared to Th1 cells were lower (\< 300; Figure [2b](#F2){ref-type="fig"}). In Th2 cells, preferentially expressed genes include in addition to IL-4, IL-5 and IL-13, the transcripts for IFN-αβR, IFN-γRβ, fibroblast growth factor receptor (FGFR), CCR4, Smad2, BAX, CAS and caspase-6.
Gene expression according to real-time RT-PCR
---------------------------------------------
Hybridizations of oligonucleotide arrays were used to screen a panel of genes in T cells that are differentially expressed by polarized Th1 or Th2 cells (Figures [1](#F1){ref-type="fig"},[2](#F2){ref-type="fig"}). To validate the observations, the same Th1 and Th2 samples were quantitated together with samples of cultured Th1/Th2 cells originating from other individuals by using a real-time RT-PCR (TaqMan) method. We designed probe and primer sets for the quantitative detection of mRNAs for 14 genes (Table [1](#T1){ref-type="table"}; caspase-1, CCR1, CCR2, CCR4, CCR5, clusterin, FGFR, GM-CSF, IκB, IL-4R, RANTES, STAT4, TIMP-1 and TNF-α). In addition, we had earlier confirmed a differential expression of SLAM, IFN-γRβ and IL-12Rβ2 transcripts in these cells \[[@B20]\]. All TaqMan RT-PCR measurements were carried out for each gene in duplicate. After three individual measurements the fold change in mRNA expression in Th1 cells compared to Th2 cells was calculated (Table [2](#T2){ref-type="table"}). The overall transcriptional preferences recorded with a real-time RT-PCR method correspond well with the differences recorded by the oligonucleotide microarrays. The measurements from Th1 or Th2 cultures of other individuals further confirm this. When the calculated fold differences by the different methods in Th1 and Th2 samples are compared they are in most cases reminiscent but not equal. Because of a broader dynamic range of the real-time RT-PCR detection, we consider TaqMan results to be more reliable in this respect. In the case of the chemokine receptors CCR1, CCR2 and CCR3, where the expression level in Th2 cells is low, the sensitivity of the method becomes limiting and leads to higher values of standard deviation. The reason for the significantly higher fold difference values recorded by the oligonucleotide arrays for TIMP-1 and IL-4 is currently unknown. Most notably, even when the fold difference for some of the target genes (CCR5, IκB, STAT4) is low (\< twofold) according to an oligonucleotide array analysis, the transcriptional preference in all Th1/Th2 samples is similar and the statistical significance high (*p* \< 0.01).
######
TaqMan primers and probes used in this study
Gene Accession 1\) 5\' - (FAM)-probe-(TAMRA) - 3\'
----------- ----------- -------------------------------------
Caspase-1 M87507 1\) AA GACGTGTGCGGCTTGACTTGTCC
2\) AATACTGTCAAATTCTTCATTGCAGATAAT
3\) AAGTCGGCAGAGATTTATCCAATAA
CCR1 L10918 1\) TGGCCATCGTCCACGCCG
2\) TCCTGCTGACGATTGACAGGTA
3\) GTGCCCGCAAGGCAAAC
CCR2 U03882 1\) AGTGCTTCGCAGATGTCCTTGATGCTC
2\) GCGTTTAATCACATTCGAGTGTTT
3\) CCACTGGCAAATTAGGGAACAA
CCR4 X85740 1\) TTACTCTGCTGACACCCCCAGCTCATC
2\) CAATACTGTGGGCTCCTCCAA
3\) ATCCATGGTGGACTGCGTGTA
CCR5 U54994 1\) TGCAAAAGGCTGAAGAGCATGACTGACAT
2\) GCTGGTCATCCTCATCCTGATAA
3\) ATGGCCAGGTTGAGCAGGTA
Clusterin X14728 1\) TGCCAGCCGGGACACCCAG
2\) CTTCGCCTTGCGTGAGGT
3\) GAGCAGCTGAACGAGCAGTTT
FGFR X51803 1\) CCGTAGCTCCATATTGGACATCCCCAG
2\) AGATAACACCAAACCAAACCGTATG
3\) GCATGCAATTTCTTTTCCATCTT
GM-CSF M11222 1\) CCCCTTTGACTGCTGGGAGCCAG
2\) CCTGAAGGACTTTCTGCTTGTCA
3\) CTCATCTGGCCGGTCTCACT
IκB M69043 1\) TTCTAGTGTCAGCTGGCCCAGCTGC
2\) TCTCTGGCAGCATCTGAAGGT
3\) CCCAAGCACCCGGATACAG
IL-4R X52425 1\) AATGCCACTGCCCAGGCTGTCC
2\) GTGGCAGGTAAGGGCTGAGTAGA
3\) CACCTCGCAGTCCGCAGA
RANTES M21121 1\) TTGGCACACACTTGGCGGTTCTTC
2\) TCCCGAACCCATTTCTTCTCT
3\) CCCAGCAGTCGTCTTTGTCA
STAT4 L78440 1\) AGTCTCGCAGGATGTCAGCGAATGG
2\) GCTGAGAGCTGTAGTGTTTACCGA
3\) AATAAAGGCCGGTTGTCTGCT
TIMP-1 X03124 1\) CAATTCCGACCTCGTCATCAGGGC
2\) CACCCACAGACGGCCTTCT
3\) TCTGGTGTCCCCACGAACTT
TNF-α M10988 1\) CATCTTCTCGAACCCCGAGTGACAAGC
2\) TGGCCCAGGCAGTCAGA
3\) GGTTTGCTACAACATGGGCTACA
Abbreviations: FAM, 6-carboxyfluorescein; TAMRA, 6-carboxytetramethyl-rhodamine.
######
Fold differences in gene expression in human Th1/Th2 cells
Oligonucleotide Real-time RT-PCR
----------- ----------- ----------------- ------------------ ------- ------ ------- ------ ------ --------
Th1 \>Th2 GM-CSF 4.1 3.6 6.73 0.80 8.86 0.83 4.4 ^\*\*^
TNF-α 12.3 6.2 2.91 0.28 4.40 0.92 2.8 ^\*\*^
RANTES 3.1 4.0 1.23 0.72 3.88 0.71 6.3 ^\*\*^
CCR1 5.1 122.0 7.18 0.85 12.73 1.95 46.7 ^\*\*^
CCR2 8.9 30.7 7.57 0.55 10.95 2.10 10.4 ^\*\*^
CCR5 1.9 109.0 10.64 0.46 15.08 1.92 21.7 ^\*\*^
IκB 1.7 1.4 1.76 0.65 2.92 0.55 2.2 ^\*\*^
TIMP-1 6.6 1.2 3.39 0.48 3.48 0.80 1.1 n.s.
Caspase-1 2.9 1.9 8.08 0.72 8.87 0.75 1.7 ^\*\*^
Clusterin 2.4 1.4 3.50 0.60 4.02 0.12 1.4 ^\*\*^
Th2 \>Th1 IL-4R 7.0 2.3 7.90 0.48 6.31 1.01 3.0 n.s.
FGFR 3.8 1.9 10.10 0.53 9.35 0.18 1.7 ^\*\*^
CCR4 3.1 3.2 7.37 0.47 5.23 0.18 4.3 ^\*\*^
STAT4 1.8 1.7 7.07 0.43 6.06 0.55 2.0 ^\*\*^
^†^For calculations, see Materials and methods. ^‡^Same sample measured with both methods. ^§^Data from Th1/Th2 culture samples representing three individuals were used. According to analysis of variance: n.s., not significant (*p* \> 0.05); ^\*^significant (0.01 \< *p* \< 0.05); ^\*\*^highly significant (*p* \< 0.01).
Activation-induced changes in polarized T helper cells
------------------------------------------------------
Because cell activation is known to affect the transcription of genes in T cells \[[@B21]\], we next asked whether the cell activation also changes the level of transcription of genes in polarized T helper cells. Real-time RT-PCR measurements of gene expression were carried out with a set of 14 genes (Table [1](#T1){ref-type="table"}) on three separate T-cell cultures. Polarized (day 14 cells) CD4^+^ Th1 or Th2 cells were triggered with antibodies for CD3 and CD28 for 2, 6 or 24 hours and gene expression was quantitated (Figure [3](#F3){ref-type="fig"}). During the time course of activation a general trend of downregulation of transcription was observed in the genes studied. The expression of a housekeeping gene, however, was unaffected (data not shown). The downregulation occurred within 24 hours in both type 1 and type 2 cells. Even the transcripts for GM-CSF, TNF-α and IκB, which were rapidly upregulated in the early activation period were downregulated soon after. Downregulation of expression was also seen for CCR1, CCR2, CCR5, caspase-1 and clusterin. Both T-cell subsets seem to exhibit very similar patterns of changes in the mRNA levels during activation of these genes, with the following exceptions. First, the early induction of GM-CSF and TNF-α expression is more pronounced in Th1 cells than in Th2 cells. Second, the downregulation of transcripts at 24 hours of activation for CCR1, CCR2 and CCR5 in Th2 cells is more effective compared to Th1 cells. The mRNA levels for RANTES, CCR4, FGFR and STAT4 in Th1 or Th2 cells did not respond to an anti-CD3/CD28 mediated activation signal or the observed changes were low (\< two- to threefold). In conclusion, genes that responded promptly to the activation signal were GM-CSF, TNF-α, IκB, CCR1, CCR2, CCR5, caspase-1 and clusterin. Most importantly, activation-induced changes in expression of these genes did not affect the preferential expression pattern in Th1 and Th2 cells.
{#F3}
Discussion
==========
We have used high-density oligonucleotide arrays to profile gene expression of cultured polarized human Th1 and Th2 cells. Many research groups, including ours, have intensively searched for genes that would be differentially regulated in human type 1 or type 2 cells and would potentially explain their distinct functions in immunological disorders \[[@B22]\]. Until recently, only a few genes, such as those for cytokines, have been defined as differentially expressed in human CD4^+^ T helper subsets \[[@B1], [@B2]\]. Classically, the definition of a type 1 or type 2 Th cell was based on measurements of a limited number of cytokines secreted by these cells after activation (by phorbol myristoyl acetate and calcium ionophore). Current developments in RNA technologies enable the basal level of gene expression to be studied in these cells. The research has been inspired by the discoveries of differentially expressed genes, first in mouse cells and then often followed by confirmatory discoveries in human T helper cells. These discoveries in human cell systems have included the differential expression of the receptor chains IL-12Rβ2 and IFN-γ Rβ \[[@B23], [@B24]\], chemokine receptors CCR2, CCR4, CCR5 \[[@B25], [@B26]\] and SLAM \[[@B20]\]. Several attempts have been made to delineate a specific Th1/Th2 transcription factor, which would determine the development of different T-cell phenotypes \[[@B27]\]. The role of such transcription factors as c-Maf \[[@B28]\] and GATA-3 \[[@B29]\] in Th2-cell development and T-bet \[[@B30]\] in Th1 development is currently being extensively studied mainly in mouse models. In order to broaden the view on gene expression in human type 1 and type 2 cells we designed a microarray to screen for 250 inflammation-related human genes. With careful experimental considerations we are able to report subtle differences in the expression of 34 genes in polarized human CD4^+^ T helper cells that characterize their phenotypes and participate in their function.
The immune response is mediated by the coordinated expression of growth factors and chemokines. It is characterized by the presence of several secreted mediator molecules (ligands) in variable doses surrounding the cell simultaneously. Therefore, both the type and the quantity of the ligands surrounding the cell, as well as the receptors on the cell surface, dictate the early events in a signaling cascade. Microarray profiles of human type 1 and type 2 cells reveal a large number of molecules known to potentiate inflammation responses. In addition to the proinflammatory molecules IFN-γ and TNF-α, the transcripts for GM-CSF, IL-8, MIP-1α, MIP-1β and RANTES were also preferentially expressed in Th1 cells as compared to Th2 cells. GM-CSF is often produced together with the proinflammatory molecules TNF-α and IFN-γ and its production by activated human T cells has been reported \[[@B1], [@B31]\]. The expression profile of the type 2 cells has somewhat fewer inflammatory mediators than Th1 cells. In addition to interleukines IL-4, IL-5 and IL-13, only MIF mRNA was found to be expressed at a higher level in Th2 cells than in Th1 cells. The simultaneous expression of the classical Th2 cytokines IL-4, IL-5 and IL-13 is not surprising however, as it was recently discovered that they are all transcribed from a single gene cluster as a result of coordinated regulation \[[@B32], [@B33]\]. Increased expression of MIF has been observed in activated mouse Th2 cell clones \[[@B34]\].
Chemokines IL-8, MIP-1α, MIP-1β and RANTES are known to recruit neutrophils, eosinophils, macrophages and other lymphocytes to the site of inflammation \[[@B35]\]. MIP-1α, MIP-1β and RANTES have previously been shown to be direct chemoattractants of Th1 cells \[[@B36], [@B37]\]. These chemokines are promiscuously used by several receptors, but all three are ligands for CCR5, which has been shown to be preferably expressed in type 1 cells \[[@B25], [@B38], [@B39]\]. Therefore, it is of interest to note that the CC-chemokines and their receptors are coexpressed in Th1 cells. The coexpression is not only Th1 cell specific, but the similar expression pattern applies to Th2 cells that express CCR4 (this study, and \[[@B15], [@B25], [@B26]\]) and its ligand TARC \[[@B15]\] to which Th2 cells are also responsive \[[@B39]\]. Because the gene expression profiles in this study reflect pure cell cultures of effector cell populations, it seems plausible that these chemotactic signals are actively used within the type 1 or type 2 cells.
The comparison of the expression profiles of polarized T helper cells also reveals several differences in the expression of signaling receptor chains. Differences in expression of IL-12Rβ2 \[[@B23]\], IFN-γ Rβ \[[@B24]\] and SLAM \[[@B20]\] in human type 1 and type 2 cells have been previously described. T cells differed also in their expression for IL-7R, IFN-αβR and FGFR. Signaling through all three receptors is poorly characterized. Both IL-7 and type 1 interferons increase the survival of activated T cells \[[@B40], [@B41]\]. Interestingly, it was recently shown that a specific effector dendritic cell (DC2) that induces primarily type 2 differentiation in T cells is the natural source of type 1 interferon in human blood \[[@B42]\]. Our results indicate that at least the receptor chain IFN-αβR of the IFN type 1 interferons is preferentially expressed in Th2 cells and in lesser amounts in Th1 cells.
At present it is not known why the basal level of transcription of STAT4 appears to be higher in Th2 cells compared to Th1 cells. This is surprising because under the culture conditions used for Th2 cells, the IL-12 is neutralized in the medium with an antibody, whereas in the Th1 cells the STAT4 is strongly stimulated. Also, there is no literature available as to whether the STAT4 expression levels are affected by signals from the type 1 interferon receptors. Functional receptors for FGF in some human T cells have been reported by Zhao *et al.* \[[@B43]\]. However, our finding that FGFR is preferentially expressed in Th2 cells conflicts with a report by Rogge *et al.* \[[@B15]\], in which a 6.9-fold higher expression in Th1 cells was found. It is possible that the expression of FGFR does vary in the course of T helper cell development, depending on the state of differentiation and cell activation, because the observations in these papers are from different phases of T-cell differentiation.
The oligonucleotide array used here measured the transcripts for relatively few transcription factors and, for example, c-Maf, GATA-3 and T-bet were not included. However, the differential expression of these transcription factors during human T helper cell differentiation has been confirmed in these cells (E. Ylikoski and R. Lund, unpublished observations). In the Th2 effector cell profile the preferential expression of Smad2 is still worth mentioning. The details of transforming growth factor-β (TGF-β) receptor signaling in T cell development, for which the Smads form the signaling cascade to the nucleus, are still poorly characterized. It has been suggested that TGF-β modulates growth and development of T-cell precursors during the early differentiation phase \[[@B44]\]. In addition, more recent papers show that the inhibition of type 2 T-cell differentiation by TGF-β actually occurs by inhibiting the expression of GATA-3 in mouse \[[@B45], [@B46]\] and that TGF-β regulates the Th2-related airway hyperreactivity in an animal model of asthma \[[@B47]\].
The differential expression of several cell death- and apoptosis-related genes in Th1 and Th2 cells is also evident from the profiles created. Previous studies have indicated that T cells exploit different pathways for cell death \[[@B37], [@B38]\]. Mouse Th1 cells were recently shown to be susceptible to activation-induced cell death (AICD) by Fas ligand, whereas Th2 cells were shown to be resistant to it \[[@B48], [@B49]\]. This resistance was suggested to be characteristic of asthma \[[@B48], [@B50]\]. A distinct panel of cell death-related genes preferentially expressed in Th1 cells includes those for granzyme B, caspase-1 and clusterin, whereas Th2 cells preferably expressed Bax, CAS and caspase-6. The differential expression of clusterin that was also sustained during T-cell activation is also interesting in the light of recent data suggesting that clusterin can act as a secreted chaperone in mammalian cells \[[@B51]\] and protects cells from the cytotoxicity of TNF-α \[[@B52]\]. The preferential expression of TNF-α and clusterin in Th1 cells, together with the differentially expressed IκB and c-Jun, suggests that in addition to Fas/Fas ligand-mediated death, signaling through the TNF receptor family could also serve as an additional pathway to control cell survival and death in these cells. Systematic studies on cell death in Th1 and Th2 cells are needed to clarify this.
To identify differentially expressed genes for the human T helper subsets that would not be affected by cell activation status, the effect of activation through CD3 and CD28 in human Th1 or Th2 cells was studied. During activation, most of the genes studied responded by downregulating the transcription to a basal or even lower level of transcription. This was also true for the transcripts of GM-CSF, TNF-α and IκB, which had been rapidly induced and subsequently downregulated back to the basal level. Most importantly, the transitional preference favoring Th1 cells, which have a higher level of expression of GM-CSF, TNF-α, IκB, CCR1, CCR2, CCR5, caspase-1 and clusterin compared to Th2 cells, did not change during activation, a feature potentially important with clinical samples of mixed cell phenotypes.
In conclusion, microarray technology in combination with quantitative RT-PCR proved to be powerful in profiling the expression of inflammation-related genes in human CD4^+^ Th1 and Th2 cell populations. The expression patterns of type 1 and type 2 cells described here potentially reflect also the transcriptional patterns common in other type 1 and type 2 cells of the immune response \[[@B53]\]. What we have shown here is that differences in the basal levels of gene transcription can be extremely small and yet can indicate essential functional outcomes of that particular cell type. We believe that the results presented here will provide valuable new insights into gene array discovery. In addition, they also provide a challenge to bioinformaticians to develop accurate algorithms for expression profiling.
Materials and methods
=====================
Human CD4^+^ T cells
--------------------
Human CD4^+^ T helper cells were isolated from neonatal cord blood and cultured in polarizing conditions for 14 days as previously described. Briefly, the stimulation of the cells was carried out by plating 1.0 × 10^6^ T cells per ml and 0.5 × 10^6^ per ml of irradiated (6400 rad) CD32-B7 transfected mouse L fibroblasts \[[@B54]\] and 100 ng/ml PHA (Murex Diagnostics, France) on 24-well flat-bottomed plates (Lindbro, ICN Biomedicals). Cells were grown in Yssel\'s medium supplemented with 1% of human AB serum (Gemini Bioproducts) and 100 U/ml human recombinant IL-2 (Cellular Products). Cell differentiation was primed with either 2.5 ng/ml human recombinant IL-12 (R & D Systems) or with 10 ng/ml human recombinant IL-4 (R & D Systems,) for Th1 or Th2 cells, respectively. Culture media for Th2 cells contained 10 μg/ml human recombinant α-IL-12 (R & D Systems). The cells were fed every other day and split at day 3-4 after stimulation. At day 7, cells were restimulated and cultured as described above for another 7 days. When cells were harvested for RNA isolation, an additional immunomagnetic purification step of CD4^+^ cells was performed (DYNABEADS M-450 CD4, Dynal A.S). The protocol for activating the cells through CD3/CD28 for 2, 6 and 24 h has been previously described \[[@B55]\]. Monoclonal antibodies for CD3 and CD28 were from Immunotech and goat F(ab\')~2~ antibody from BioSource International. The phenotype of the cells was defined by measuring secreted IFN-γ and IL-4, corresponding to type 1 and type 2 cells, respectively, by ELISA (Genzyme).
RNA sample preparation and hybridization
----------------------------------------
Poly(A)^+^ mRNA (1 μg) was isolated by two rounds of Oligotex purification (Qiagen). cDNA synthesis of mRNA, *in vitro* transcription and the production of a biotin-labeled RNA probe for oligonucleotide arrays were performed as previously described \[[@B8]\]. The hybridization mixture contained 12.5 μg of the fragmented sample cRNA together with defined amounts of control *Escherichia coli* and bacteriophage P1 gene cRNAs, produced for spiking purposes in an identical manner as the sample, and a biotin labeled control oligo (5\'-biotin-GTCAAGATCGTACCGTTCAG-3\') from Genset. Subsequent hybridization and washing steps were done as described by Wodicka *et al.* \[[@B17]\]. Chips were washed with GeneChip WashB program on a fluidics station and scanned with the GeneChip scan program (Affymetrix).
Oligonucleotide microarray data analysis
----------------------------------------
The principles of quantitative analysis of hybridization intensities have previously been described \[[@B16], [@B17], [@B56]\]. Data analysis was carried out under low or high stringency using the GeneChip software program. Low-stringency parameter values were: difference threshold (DT) = 20; ratio threshold (RT) = 1.5; change threshold (CT) = 15, and percent change threshold (PCT) = 30. High-stringency parameter values were: DT = 50; RT = 1.5; CT = 50 and PCT = 30. Comparative data were obtained by analyzing results from IL-4-treated RNA samples (Th2 cells) compared to those from IL-12-treated RNA samples (Th1 cells) as baseline. All intensity values below 20 were assigned a threshold value 20. The data reported here is from the more rigorous high stringency analysis (cutoff range \> 1.5-fold). The GenBank accession numbers of the sequences used for the initial microarray design and reported here are as presented in Table [1](#T1){ref-type="table"} and as follows: BAX (U19599); c-Jun (J04111); granzyme B (M28879); IFN-αβR (X77722); IFN-γ (X13274); IFN-γRβ (U05875); IL-4 (M13982); IL-5 (X04688); IL-8 (M17017); IL-13 (L06801); IL-7R (M29696); IL-12Rβ2 (U64198); IRF-1 (X14454); MIF (M25639); MIP-1α (M23452); MIP-1β (J04130); SLAM (U33017) and Smad2 (U59911).
Real-time quantitative RT-PCR
-----------------------------
The principles of the real-time RT-PCR detection with hydrolysis probes (TaqMan) have been previously described \[[@B19], [@B57]\]. TaqMan probes and primers for the quantitative detection of target mRNAs were designed (Table [1](#T1){ref-type="table"}) by using Primer Express computer software (Applied Biosystems). The real-time measurements were carried out with the ABI PRISM 7700 SDS instrument (Applied Biosystems) as described in Hamalainen *et al.* \[[@B20]\]. Samples were analyzed in duplicate in three independent runs. Statistical significance was determined by ANOVA model. The C~T~ value is defined as the cycle number in which the detected fluorescence exceeds the threshold value \[[@B19], [@B57]\]. In all experiments the threshold value used to determine C~T~ during analysis was kept constant. For each probe and primer pair the linearity of detection was confirmed to have a correlation coefficient of at least 0.98 over the detection area by measuring a fourfold dilution curve with cDNA isolated from T helper cells. Fold difference in expression in Th1 and Th2 cells was therefore calculated assuming 100% efficient PCR where each C~T~ was normalized to GAPDH:
\(1\) Fold difference =
2 ^\|\|C~T~1(target)\ -\ C~T~1(GAPDH)\|\ -\ \|\ C~T~2(target)\ -\ C~T~2(GAPDH)\|\ \|^
\(2\) Fold difference =
2^\|\|deltaC~T~1\|\ -\ \|deltaC~T~2\|\|^
Where C~T~1(target) and C~T~2(target) represent the C~T~ values for the target gene of Th1 and Th2 samples, respectively. C~T~1(GAPDH) and C~T~2(GAPDH) represent the C~T~ values for the GAPDH gene of the Th1 and Th2 samples, respectively.
Acknowledgements
================
We thank Tuija Kyrola and Paula Suominen for technical assistance and Nina Johansson for critical review of the manuscript. This study was supported by grants from the Academy of Finland, National Technology Agency (TEKES) and Turku University Hospital Fund.
| {
"pile_set_name": "PubMed Central"
} |
Introduction
============
Fatigue is a common symptom in stroke \[[@B1],[@B2]\]. It can be considered to be 'a feeling of early exhaustion, weariness and aversion to effort' \[[@B3]\], or a 'lack of energy with an increased need to rest' \[[@B4]\]. The extent of fatigue has been shown to increase with stroke severity\[[@B5]\]. It can have a considerable impact upon lifestyle and has, for example, been shown to be an independent predictor for the need to move into an institutional setting post-stroke \[[@B6]\]. It has also been shown to have association with depression, and sleeping problems \[[@B7]\].
Given the importance of post-stroke fatigue, several fatigue scales have been used to ascertain the extent of fatigue experienced. Examples include the Fatigue Assessment Scale \[[@B8]\]; the Multidimensional Fatigue Inventory (MFI-20) \[[@B9]\] the Fatigue Severity Scale \[[@B10]\]; and the Brief Fatigue Inventory \[[@B11]\]. A recent review of some of these scales suggested varying levels of reliability and validity, with no one scale showing satisfactory results across all psychometric quality indicators \[[@B12]\]. Consequently it has been argued that a more exact definition of fatigue is needed, and then more valid scales or other technical instruments to quantify fatigue \[[@B13]\]. One such scale, the Neurological Fatigue Index (NFI-MS), was developed from theory and the experiences of those with multiple sclerosis \[[@B14],[@B15]\].
This current paper sets out to examine if the thematic structure relating to fatigue which emerged from that MS study is also consistent with those who have experienced a stroke, and to test the reliability and validity of the NFI-MS in stroke (any valid subscales would then also be known as NFI-Stroke).
Methods
=======
The study had approval from the local research ethics committee (Sefton EC115.03 and 05/Q1501/24). All subjects received written information on the study and gave written informed consent prior to participation.
Sample and materials
--------------------
### Qualitative construct validation
Semi-structured interviews with stroke patients were used to identify the features which defined the concept of fatigue. Subjects with radiologically confirmed stroke were recruited, non-purposively, to undergo a semi-structured interview, as they attended the out patient clinic in the Department of Medicine for the Elderly at University Hospitals Aintree, Liverpool and the Neurology Rehabilitation Unit, Walton Centre for Neurology and Neurosurgery, Liverpool, UK. Patients were excluded if they either had marked impairment of communication or they had another neurological condition. The same interviewer (SD), blinded to the results of the NFI-MS, was used throughout and the face-to-face interviews audio-taped and later transcribed. The 'framework approach' \[[@B16]\] was used for the qualitative analysis of the interview transcripts. This part of the study mirrored the qualitative work which had already been undertaken in forty patients with multiple sclerosis and which formed the basis of the NFI-MS; the method is described in detail elsewhere \[[@B14]\].
The post-stroke qualitative analysis was examined for thematic equivalence against the MS data. It was decided *a priori* that if post-stroke fatigue was found to be qualitatively identical to MS fatigue, then the existing NFI-MS item set would be used, otherwise further interviews would be undertaken, and new items would be generated to represent any stroke-specific features of fatigue.
### NFI-MS
The NFI-MS consists of 23 items in four subscales of Physical (8 items), Cognitive (4 items), Relief by diurnal sleep or rest (6 items) and Abnormal nocturnal sleep and sleepiness (5 items). A 10-item Summary Scale derived from physical and cognitive items is also available. Wording of the scales is both simple and concise; the use of the word 'fatigue' was deliberately avoided because of its associated semantic ambiguities. All items are worded in such a way as to be scored in the same direction. Each item has a four point, Likert response option \[[@B17]\] with headings of 'strongly disagree', 'disagree', 'agree' and 'strongly agree', which progress in the natural reading direction (*i.e.* left to right), and are scored 0, 1, 2, 3. There is a single sentence instruction at the start of the scale asking respondents to consider their experience over the previous four weeks.
### Data collection
A pack containing the NFI-MS, other measures and questions on demographics and basic disease information, was mailed to a random cross-section of stroke patients identified from the Aintree Stroke Register held at the University Hospital Aintree, Liverpool, UK. All patients had one or more radiologically confirmed stroke(s) in the previous 50 months. The type of stroke (ischaemic or intracerebral haemorrhage) was known from the stroke register but the Oxfordshire Community Stroke Project subtype \[[@B18]\] was not available. There were 4,276 patients in the registry with the clinical and demographic details having been obtained prospectively during admission to hospital.
The Fatigue Severity Scale (FSS) \[[@B10]\], a nine item scale with a seven-point response option, was co-administered as a comparator measure. The FSS is the most frequently used scale in stroke fatigue \[[@B19]\]. In addition, a 10 cm, modified, vertical, visual analogue scale (VAS) with anchors of 'lively and alert' and 'absolutely no energy to do anything at all' was co-administered; similar vertical visual analogue scales have been widely used \[[@B20],[@B21]\].
Estimation of disability was made by administration of the Stroke Impact Scale-16 (SIS) \[[@B22]\]. This is a short form version of the Stroke Impact Scale v2.0 \[[@B21]\]. with items based on mobility and activities of daily living, each having a 5-point Likert response; minimum possible score was zero (no meaningful disability) and the maximum was 64. Hemianopia and visual neglect, which might interfere with completion the response option, were assessed by copy of a clock face.
In total, 999 people received the pack. Retesting was performed at 2 to 4 weeks on the first 80 respondents to the main mailout; estimates of the level of fatigue would be correlated between initial and retest time points accepting a Spearman's rho of ≥0.7. Invariance of mean person estimates at each time point would be confirmed by paired *t*-test. For analysis of the Rasch fit criteria, only the initial time point data and not the retest data, were included. Data were transcribed to a computer database (transcription error based on checking a random 10% sample was \<0.1%, missing data accounted for 3.8% of total).
If the response to the mailout was less than 50%, then non-response bias would be assessed by *t*-test or chi square comparison for: age at onset of most recent stroke, sex, previous stroke, previous transient ischaemic attack (TIA) and stroke type. This would be performed to exclude any gross bias in the responders, but it must be stressed that population representativeness is *not* a requirement for Rasch analysis, but rather a wide range of person 'ability' (in this case levels of fatigue) is needed \[[@B23]\].
### Quantitative psychometric analysis
#### Measurement and the rasch model
The internal construct validity of the NFI-MS in stroke was examined by fit of data to the Rasch measurement model \[[@B24]\]. Full details of the process of Rasch analysis are given elsewhere \[[@B25],[@B26]\]. Briefly, the process is concerned with whether or not the data meets the model expectations, and provides an assessment of the suitability of the response scale, the fit of individual items, differential item functioning, and the dimensionality and targeting of the scale as a whole.
In summary, fit of data to the Rasch model was deemed acceptable if the following criteria were fulfilled:
1\) ordered item category thresholds;
2\) both total chi-square probability and individual item chi-square probability values non-significant (5% alpha with Bonferroni correction for the number of items);
3\) individual item fit residual, by convention, within ±2.5;
4\) mean and SD of both item fit residual and person fit residual approaching 0 and 1 respectively;
5\) person-item separation index (PSI) (reliability) greater than 0.70 for group use and 0.85 for individual use;
6\) ANOVA probability for differential item functioning (DIF) non-significant (5% alpha with Bonferroni correction) for the following factors: sex, age and whether had help (as a scribe) completing the scale, as well as time point (*i.e.* initial and retest). This is undertaken with a two way ANOVA with class interval (grouped level of fatigue) and the external factor (*e.g.* age) as main effects. Uniform DIF is then for the main effect of gender (and there is another for class interval) and non-uniform DIF is the interaction between class interval and (*e.g.* age).
7\) Unidimensionality by independent *t*-test at the person level showing less than 5% of tests to be significant (or the lower bound of the binomial confidence interval to overlap 5%, where required) \[[@B27],[@B28]\].
8\) Pearson correlation coefficients between item residuals less than 0.3 (local independence).
For Rasch analysis, a sample size of 243 will provide accurate estimates of item and person locations irrespective of the scale targeting \[[@B29]\]. The Rasch analysis was performed using the RUMM 2020 computer software (<http://www.rummlab.com>). The unrestricted (partial credit) Rasch polytomous model was used with a conditional pair-wise parameter estimation \[[@B30]\]. Failure of items to fit Rasch model expectations led to an iterative procedure using techniques for collapsing response categories, item deletion, and adjusting for DIF where necessary.
If data from a scale fit the Rasch model, then the summed ordinal raw scores can be considered to be sufficient to determine the level of fatigue in an individual. However, calculation of change scores can only be done with interval level data and so a conversion table of the raw ordinal score to the interval level metric, for any resultant scale, would be provided.
### External comparison
Comparisons of the person locations from the final scale to the summed raw scores of the FSS, VAS, and SIS were made by Spearman correlation (assuming the raw scores were non-parametric), with the expectation that these would be mild to moderate (*i.e.* rho between 0.3 and 0.7) in each case \[[@B31]\].
Results
=======
Qualitative analysis
--------------------
Five of the six patients recruited for the qualitative interviews were female. The mean age of participants was 51.3 years (SD 13.4, range 34--68), the mean duration since last stroke was 22 months (SD 39.3 range 3--108). The interview data were analysed in the context of a 'framework' of standard symptom description. Complete thematic equivalence of the stroke data was observed when compared to the existing MS data. In other words, no new features, specific to post-stroke fatigue, were identified when compared to MS fatigue (Table [1](#T1){ref-type="table"}).
######
Some examples of the features of post-stroke fatigue, as described by patients, grouped according to the thematic framework derived from MS
**MS Framework** **Stroke quotations**
------------------------------------------------------------------------------------------------------------------------------------------------------------------------- --------------------------------------------------------------------------------------------------------------------------------------------------------------
Subjective experience *Basically just tiredness to the point where you're worn out. Tired. Done in.*
*Tiredness all the time... Its just tiredness, constant tiredness*
*Shattered. No energy. Whacked out. Weary.*
motor *It just takes it out your body. You just want to lie down and you're drained.*
*If I do anything, you know, anything physical. Or go to the shops. Really shatters me.*
cognitive *I'm still not reading...I can't concentrate on it.*
*It feels, sort, of, my eyes start going cos I've got to concentrate on the story.*
motivation, energy and need to rest *If I know I've got something to do, I'm quite happy to get on and do it. But if I know its not that day then I'm tired and I can't be bothered doing it...*
*But my fatigue is, when I get home here in my bedroom, I sort of give in then*
*ll take my son to school, get back in the car and go home and I'll go straight back to bed for a few hours. But if I'm busy of a morning, I'll go to bed at lunchtime*
Sleep and behavioural response *I'm really tired and just want to go to bed and sleep and not bother with anything*
*Yes, well then the tiredness takes over. Basically I want to stay sitting down then and I'm weary*
*Sometimes you feel like when you do get up you're tired more'*
Quantitative analysis
---------------------
### Subjects and non-responder analysis
284 packs were returned and two were discarded because of evidence of substantial visual field defect. This gave a 28.2% (282/999) response, sufficient for the Rasch analyses. The demographic details and disease characteristics are given in Table [2](#T2){ref-type="table"}. Indication of the functional consequence of stroke is also given. The median SIS score was 17 which equates to a Modified Rankin Score \[[@B32]\] of 2 (slight disability) \[[@B22]\], but a full range of disability was observed, for instance subjects with severe disability of both upper and lower limb function were represented (see Table [2](#T2){ref-type="table"} for frequencies). The VAS fatigue scores were normally distributed (skewness -0.25, kurtosis -0.39) with median and modal values both of 5 cm. The distribution of the FSS revealed a substantial ceiling effect of 7.1%. Histograms of the VAS, FSS and SIS can be found in the [supplemental material](#S1){ref-type="supplementary-material"}.
######
Characteristics of subjects both completing the pack and non-responders
**responders** **non-responders**
------------------------------------------------------------ --------------------- ----------------------
n 282 717
mean age at questionnaire completion (SD, range) 67.3 (13.4, 18--95) --
mean age at onset of last stroke (SD, range) 66.5 (12.4, 18--93) 70.9 (12.8, 16--102)
male (%) 61.3 45.8
mean months post stroke (SD, range) 17.2 (11.4, 2--50) --
previous stroke (%) 9.6 21.1
previous TIA (%) 11.6 16.6
ischaemic stroke (%) 78.7 75.3
working (%) 16.5 --
median Stroke Impact Scale score (range) 17 (0--64) --
very difficult or unable to climb one flight of stairs (%) 25.9 --
very difficult or unable to dress top half of body (%) 10.1 --
very difficult or unable to control bladder (%) 8.3 --
very difficult or unable to transfer from bed to chair (%) 5.2 --
Ages are in years. SD-standard deviation.
76 records were available for the retest analysis.
The non-responders, when compared to the responders, were slightly older with a mean age difference of 4.4 year (95% CI 2.6--6.2, p \< 0.001). A greater proportion were female (53% vs. 38%, p \< 0.001) and had suffered more than one stroke (21% vs. 10%, p \< 0.001) but there was no difference in previous TIA (p = 0.062) or type of stroke (p = 0.334). None of the differences were considered to be extreme or confound the validity of the Rasch analysis.
### Rasch analysis
Data from the four individual subscales, and the summary scale of the NFI were then fitted to the Rasch measurement model. The findings, related to the analysis of each domain, are given in Table [3](#T3){ref-type="table"}. All thresholds were ordered in all subscales. Data from the Physical subscale satisfied Rasch model expectations, were unidimensional, free of DIF and local dependency (Table [3](#T3){ref-type="table"}, Analysis 1). Likewise, data from the Cognitive subscale also satisfied model expectations and were free of DIF and local dependency, although the reliability (PSI) was only consistent with group use and the summary item residual standard deviation was a little high, and one item (coordination gets worse) had a slightly high negative residual at -2.75 (Table [3](#T3){ref-type="table"}, Analysis 2). The summary scale also satisfied model expectations and was unidimensional and free of DIF and local dependency (Table [3](#T3){ref-type="table"}, Analysis 3). Overall, respondents had a slightly higher level of fatigue (1.04 logits) than the average of the scale (0.0 logits) (Figure [1](#F1){ref-type="fig"}). These three scales could now be called NFI-Stroke.
######
Summary Fit Statistics for Rasch analyses
**Analysis Number** **Analysis Name** **Item Residual** **Person Residual** **Chi-Square** **PSI** **Unidimensional**
--------------------- ------------------------------ ------------------- --------------------- ---------------- ------------- -------------------- ----------------- -------------- --------------------------
1 Physical (8 items) -0.379 1.011 -0.547 1.415 21.4 0.922 0.89 5.98%
(3.2-8.8)
2 Cognitive (4 items) -0.043 1.977 -0.597 1.224 18.9 0.092 0.78 3.29%
(0.4-6.2)
3 Summary (10 items) -0.357 1.156 -0.622 1.551 52.8 0.085 0.89 5.0%
(2.2-7.8)
4 Diurnal -- Initial (6 items) -0.423 1.862 -0.683 1.351 50.5 0.001 0.70 7.72%
(5.1-10.4)
5 Diurnal -- Final (5 items) -0.842 1.783 -0.648 1.174 18.9 0.219 0.69 5.56%
(2.8-8.3)
6 Nocturnal Sleep (5 items) 0.229 1.451 -0.563 1.501 31.5 0.008 0.69 4.22%
(1.4-7.0)
***Acceptable Values*** ***0*** ***\<1.4*** ***0*** ***\<1.4*** ***\>0.05***^a^ ***\>0.85*** ***\< 5.0% (Lower CI)***
^a^ Bonferroni adjusted alpha level.
{#F1}
The sleep scales were more problematic. The Diurnal sleep scale failed to meet model expectations (Table [3](#T3){ref-type="table"}, Analysis 4). The item 'I try to get everything done in the morning' showed misfit, with a significant Chi-Square statistic, and the item set displayed multidimensionality (7.72%; CI 5.1-10.4%). Removal of this item improved fit, and resulted in a unidimensional scale (Table [3](#T3){ref-type="table"}, Analysis 5). However, the item residual standard deviation, at 1.783, indicated some continuing misfit caused by another item with a high negative fit residual, indicating redundancy. The Nocturnal sleep subscale failed to fit the model (Table [3](#T3){ref-type="table"}, Analysis 6), and no solution could be found.
### External construct validity
Comparison of the person locations from the Physical, Cognitive and Summary Scales to the FSS gave Spearman correlation coefficients of 0.604, 0.509 and 0.622 respectively. Likewise, to the VAS, 0.556, 0.385 and 0.534 respectively, and to the SIS of 0.615, 0.532 and 0.628 respectively.
### Test-retest
Test-retest correlation coefficients of the Physical, Cognitive and Summary Scales were 0.903, 0.786 and 0.896 respectively. There were no significant differences in the mean scores at the two time points. Analysis of DIF by time showed that the Physical, Cognitive and Summary scales were invariant between the initial and retest time points.
### Raw score to interval scale conversion
Given fit to the Rasch model of the Physical, Cognitive and Summary Scales, a straightforward conversion is available between the raw score for each scale, and the interval scale estimate of the latent trait of fatigue provided by the model (Table [4](#T4){ref-type="table"}). This can be used when data are complete.
######
Raw score to interval scale conversion table for the scales
**Raw** **Summary** **Physical** **Cognitive**
--------- ------------- -------------- ---------------
0 0.00 0.00 0.00
1 2.34 1.97 1.46
2 4.05 3.43 2.69
3 5.29 4.51 3.73
4 6.31 5.42 4.70
5 7.22 6.24 5.60
6 8.05 7.00 6.43
7 8.83 7.73 7.21
8 9.59 8.45 7.98
9 10.32 9.16 8.78
10 11.05 9.88 9.65
11 11.77 10.61 10.71
12 12.49 11.35 12.00
13 13.22 12.13
14 13.96 12.94
15 14.70 13.78
16 15.46 14.65
17 16.24 15.53
18 17.03 16.41
19 17.83 17.29
20 18.64 18.21
21 19.44 19.21
22 20.25 20.38
23 21.06 21.93
24 21.89 24.00
25 22.75
26 23.67
27 24.71
28 25.95
29 27.66
30 30.00
N.B. The conversions only remain valid if there are no missing data. The transformation can only occur when data are complete because, for example, a score of say 6 from complete data is not the same as a score of 6 from incomplete data. The latter is likely to represent a higher level of the attribute being measured.
Discussion
==========
Qualitative construct validation in adapting scales to a different diagnosis is novel. By testing the construct equivalence from the current sample against the original qualitative analysis from people with MS, the manifestation of post-stroke fatigue appeared to be qualitatively similar to that of MS fatigue, including, for example, features associated with physical and cognitive aspects \[[@B14]\]. The NFI-Stroke reflects these components, and provides a simple scale of fatigue that satisfies the strictest measurement standards, supporting the internal construct validity of the scale. The substantive correlations (\>0.5) with the comparator measures provide strong evidence of the external validity of the scale, with slightly stronger correlations with the physically orientated domains of the FSS and SIS. The lower correlation of the cognitive scale with the VAS possibly reflects the choice of anchors for the latter which were necessarily concise and may not have conveyed the nuances of cognitive fatigue.
The qualitative similarity between post-stroke fatigue and MS fatigue and indeed the facility with which fatigue in the two conditions could be measured on a common metric is notable given the obvious differences in pathophysiology between the two diseases. This paradox may be a potentially important starting point for future pathophysiological enquiry.
The raw score from the NFI-Stroke components is a sufficient statistic such that a simple summed score can provide an ordinal estimate of the persons (component) level of fatigue. It also can provide a straightforward ordinal to interval scale transformation, courtesy of a special property of the Rasch model \[[@B24],[@B33]\].
There were a number of limitations to the study. For example, the sample was restricted to those with a disease duration of 4 years from their most recent stroke; this was mainly determined by the age of the stroke register from which the sample was drawn. Fatigue seems to increase in the first twelve months \[[@B34]\] following stroke but is known to persist for more than two years \[[@B6]\] and so four years was felt to be an adequate disease duration for the current sample.
Subjects had to be cognitively able to interpret and respond to the scale. Further validation of the NFI-Stroke might involve clinician administration to patients with cognitive deficits.
The qualitative stroke sample was predominantly female and it could be argued that equivalence of the thematic structure to MS was confounded by sex bias. However, no sex differences were found in the MS qualitative analysis \[[@B14]\] and in the current Rasch analysis, all items were free from DIF by sex.
The non-response level of the study was also high and older patients with multiple strokes appeared to have been underrepresented. Respondents with low levels of disability were well represented. However some respondents had very high SIS scores suggesting that those with higher disability were not wholly excluded. In addition, the VAS scores were normally distributed suggesting those with extreme levels of fatigue (both low and high) were captured. Nevertheless, representativeness is not a requirement for Rasch analysis as, for example, item difficulty estimates are independent of the distribution of the sample of persons \[[@B23]\]. The fact that the Summary Scale was adequately targeted to the sample, and that the sample covered a wide range of those with low to high fatigue is more important with respect to the construction of a measure than the sample's representative nature. It should be remembered that patients were being presented with a whole pack of different scales and demographic questions which may have contributed to inflation of the non-response rate. The NFI-Stroke is a brief scale with straightforward and concise items. This format is in-keeping with other self-report scales used in neurologic disease, including stroke, and so there was minimal concern that non-response was due to some deterrent intrinsic to the structure of the NFI-Stroke.
There was one item in the Cognitive scale (coordination gets worse) with a slightly high negative fit residual which indicated a degree of redundancy and accounted for the inflated overall item residual standard deviation. The scale was not discarded because all other fit statistics were acceptable and the retention of a comparable cognitive fatigue scale between stroke and MS was felt to be desirable. Additionally, the same item had satisfactory fit within the Summary scale, albeit with the lowest chi square probability.
The sleep scales were found to be less than optimal. This was also the case in the context of MS, and thus remains a challenge for measurement. Both sets of diagnostic-specific qualitative analysis supported the importance of sleep (or its disturbance) and therefore further work is required to develop the NFI sleep scales for these populations. Whether, or not, relief by diurnal sleep or rest is adaptive or consequent, remains to be determined. It is possible that diurnal sleep represents an inherent part of the pathophysiology of fatigue, but it could also be a secondary behavioural response. The Diurnal sleep scale overall fit statistics only showed a high item residual standard deviation, comparable to the Cognitive scale. However, because, even after some modification, there was persistent individual item misfit the scale was discarded. Unlike MS, the Abnormalities of Nocturnal Sleep scale could not be resolved for the current diagnostic group.
Given the interval scaling of the NFI-Stroke, the potential now exists to model the antecedent and consequent factors associated with fatigue, and the associations within the broader biopsychosocial model, using path analysis or other appropriate multivariate techniques.
Conclusion
==========
The NFI-Stroke provides a brief (12 item) and easy-to-use tool for measurement of a clearly defined concept of fatigue. The scale satisfies strict Rasch model measurement requirements and, as a result, interval level scaling is available for when change scores need to be calculated. The scales have specific validation for stroke and can be used on patients of, amongst other factors, any age, or sex.
It is suggested that the scale would be useful in both a clinical setting and as an outcome measure in clinical trials. The NFI-Stroke is free for use by all state-funded healthcare organisations and not-for-profit agencies, and can be obtained, after appropriate registration, from <http://www.leeds.ac.uk/medicine/rehabmed/psychometric/Scales1.htm> or by contact of the authors.
Competing Interests
===================
The authors declare that they have no competing interests.
Author's Contributions
======================
RJM, CAY, MK, SD and AS contributed to the design, implementation, and analysis of the study. JFP and AT contributed to the analysis of the study. All authors contributed to the writing of the manuscript, and all approved the final version.
Supplementary Material
======================
###### Additional file 1
Histograms showing the distribution of the visual analogue scale (VAS), Fatigue Severity Scale (FSS) and Stroke impact Scale (SIS).
######
Click here for file
Acknowledgements
================
The authors would like to thank all the interviewees and respondents for their willingness in taking part in this study and Dave Watling and the staff of the Clinical Trials Unit, WCNN for their assistance with the mailout.
| {
"pile_set_name": "PubMed Central"
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INTRODUCTION {#sec1-1}
============
> Try not to become a man of success, but rather try to become a man of value.
>
> --Albert Einstein
Few dermatologic problems carry as much emotional overtones as the complaint of hair loss, especially in the female patient. Adding to the patient\'s worry may be prior frustrating experiences with physicians, who tend to trivialize complaints of hair loss. This attitude on the part of physicians may result form a lack of knowledge making them feel uncomfortable in dealing with the complaint of hair loss, which is then readily discarded as a negligible medical problem. Even if the complaint may seem disproportionate to the extent of recognizable hair loss, the proportion of women suffering of true imaginary hair loss is small. Moreover, hair loss may be a symptom of a more general medical problem that needs to be investigated appropriately.
A detailed patient history focussing on chronology of events, examination of the scalp and pattern of hair loss, a simple pull test, dermoscopy of the scalp and hair, few pertinent screening blood tests and a scalp biopsy in selected cases will usually establish a specific diagnosis.\[[@ref1]\]
Once a diagnosis is certain, appropriate treatment is likely to control hair loss, though with limitations in terms of indications and efficacy. Therefore, patient education about the basics of the hair cycle, what may be expected from treatment and why considerable patience is required for effective cosmetic recovery is paramount.
PREREQUISITES FOR SUCCESSFUL MANAGEMENT OF HAIR LOSS {#sec1-2}
====================================================
> *It is easier to write a prescription than to come to an understanding with the patient*.
>
> --Franz Kafka, A Country Doctor
Prerequisites for successful management of hair loss are twofold: On the technical and on the psychological level.
On the technical level, prerequisites for success are: A specific diagnosis, a profound understanding of the underlying pathophysiology, the best available evidence gained from the scientific method for clinical decision making and regular follow-up of the patient combining standardized global photographic assessments and epiluminiscence microscopic photography with or without computer-assisted image analysis. With respect to the diagnosis one must remain open minded for the possibility of a multitude of cause-relationships underlying hair loss and therefore also for the possibility of combined treatments and multitargeted approaches to hair loss. With respect to the practice of evidence-based medicine, one must remain aware of the fact that good medical practice means integrating individual clinical expertise with the best available external evidence from evidence-based medicine.
On the psychological level, for a successful encounter at an office visit, one must be sure that the patient\'s key concerns have been directly and specifically solicited and addressed: Acknowledge the patient\'s perspective on the hair loss problem, explore patient\'s expectations from treatment and educate patients into the basics of the hair cycle and why patience is required for effective cosmetic recovery. One must recognize the psychological impact of hair loss.\[[@ref2][@ref3][@ref4][@ref5]\] Physicians should recognize that alopecia goes well beyond the simple physical aspects of hair loss. Patients' psychological reactions to hair loss are less related to physicians' ratings than to patients' own perceptions. Some patients have difficulties adjusting to hair loss. The best way to alleviate the emotional distress is to eliminate the hair disorder that is causing it. Only a minority of patients suffer from true imaginary hair loss. These have varied underlying mental disorders ranging from overvalued ideas \[[Figure 1](#F1){ref-type="fig"}\] to delusional disorder. In these cases, one must aim at making a specific psychopathologic diagnosis.\[[@ref6]\]
{#F1}
The difficult patient can be defined as one who impedes the clinician\'s ability to establish a therapeutic relationship.\[[@ref7]\] Data from physician surveys suggest that nearly one out of six out-patient visits are considered as difficult.
The recent past has seen an increase in the study of the difficult patient, with the literature warning against viewing the patient as the only cause of the problem. It suggests, rather, that the clinician-patient relationship constitutes the proper focus for understanding and managing difficult patient encounters. Therefore, communication between clinician and patient is a key factor in understanding and caring for patients who are perceived to be difficult.\[[@ref8][@ref9][@ref10]\]
Probably, the most frequent cause for difficult patient encounters are prior negative patient experiences with physicians, others are specific psychopathological disorders related to the somatic complaint, that again have to be identified as such.
COMMUNICATION SKILLS {#sec1-3}
====================
> The man of breed shuns a loud speech and a fast pace.
>
> --Confucius, Analects
Communication is an important part of patient care and has a significant impact on the patient\'s well-being: Successful communication is the main reason for patient satisfaction and treatment success, whereas failed communication is the main reason for patient dissatisfaction, irrespective of treatment success.
Communication skills are not a question of talent. Communication skills can be improved through training and through experience, though traditionally, communication in medical school curricula is incorporated informally as part of rounds and faculty feedback but without a specific focus on skills of communication. Basically, communication skills require a genuine interest in the problem of hair loss (on the technical level) and a genuine interest in the patient (on the psychological level).
Communication with the patient has to include:
Listening to the patientUnderstanding the patientInforming the patient on: Diagnostic procedures, diagnosis. therapeutic considerations and prognosisConvincing the patientGiving the patient hopeLeading the patient to take personal responsibilityJointly rejoicing over therapeutic progress.
Avoid:
Precipitance/hecticnessA personal dominant behaviorStereotype prejudices.
PSYCHOPATHOLOGICAL DISORDERS {#sec1-4}
============================
> The educated among the physicians make an effort into an understanding of the mind.
>
> --Aristotle, Nicomachic Ethics
There remain a number of psychopathological conditions related to the complaint of hair loss. The clinician must keep in mind that the distress the patient feels from having a hair disease can be handled both dermatologically and psychologically.
The most frequent mental disorder related to hair loss are the adjustment disorders, either with depressed mood, anxiety and/or disturbance of conduct, somatic and/or sexual dysfunction and feelings of guilt and/or obsession. From a psychopathological point of view, adjustment disorders result form the stressful event of hair loss, depending on its acuity, extent and prognosis. The intensity of the distress that the patient feels should be part of the clinician\'s formula in deciding how aggressively to treat the hair disorder, irrespective of the clinical recognizable extent of hair loss.
The more challenging psychopathological disorders that may be related to difficult patients encounters are the somatoform disorders (conversion disorder, somatoform pain disorder, hypochondriacal disorder and body dysmorphic disorder) and the personality disorders (anxious, negativistic, histrionic and paranoid).
In imaginary hair loss or psychogenic pseudoeffluvium, patients are frightened of the possibility of going bald, without any objective findings of hair loss. These patients only make up for a minority of female patients complaining of hair loss, but the spectrum of underlying psychopathology is wide, with a majority at the neurotic end of the spectrum with merely overvalued ideas about their hair, whereas a minority of patients suffer from true delusional disorder. The most common underlying psychiatric disorders are: Depressive disorder and body dysmorphic disorder. Differential diagnosis is particularly challenging, since there is considerable overlap between hair loss and psychological problems, with lower self-confidence, higher depression scores, greater introversion, higher neuroticism and feelings of being unattractive.
The patient with hypochondriacal disorder has no real illness but is overly obsessed over normal bodily functions. They read into the sensations of these normal bodily functions the presence of a feared illness. Due to misinterpreting bodily symptoms, then become preoccupied with ideas or fear of serious illness, whereas appropriate medical investigation and reassurance do not relieve these ideas. These ideas cause distress that is clinically important or impairs work, social, or personal functioning. The disorder usually develops in middle age or later and tends to run a chronic course.
Patients with body dysmorphic disorder probably represent the most difficult of patients for the dermatologist in practice.\[[@ref11]\] The patient becomes preoccupied with a non-existent or minimal cosmetic defect and persistently seeks medical attention to correct it. This preoccupation again causes distress that is clinically important or impairs work, social, or personal functioning. The disorder tends to occur in younger adults. One of the various theories attempting to make the disorder understandable is the self-discrepancy theory, in which affected patients present conflicting self-beliefs with discrepancies between their actual and desired self. Media-induced factors are considered to predispose to the disorder by establishing role models for beauty and attractiveness.
Although Thersites complex is but another term used synonymously for body dysmorphic disorder, named after Thersites who was the ugliest soldier in Odysseus' army, according to Homer, the more recently described Dorian Gray syndrome probably represents a variant of the body dysmorphic disorder, in which patients wish to remain forever young and seek life-style drugs (including hair growth promoting agents) and surgery to deter the natural aging process, named after Dorian Gray, the protagonist of the respective Oscar Wilde novel.\[[@ref12]\]
Finally, patients with a personality disorder may have a negative impact on the clinician\'s ability to establish a therapeutic relationship. In the case of an anxious, negativistic, histrionic, or paranoid patient, it is important never to be judgemental or scolding because this may rapidly close down communication. The patient gains therapeutic benefit just from venting concerns in a safe environment with a caring physician.
NOCEBO REACTION {#sec1-5}
===============
In a strict sense, a nocebo reaction refers to undesirable effects subject experiences after receiving an inert dummy drug or placebo.\[[@ref13]\] Nocebo reactions are not chemically generated and are due only to the subject\'s pessimistic belief and expectation that treatment will produce negative consequences. In a wider sense, the term is being increasingly used for unexpected negative reactions to an active drug.
The influence of the prescribing physician should be kept in mind, since inspiring confidence versus scepticism and fear clearly impacts the outcome of treatment. Nevertheless, some patients with somatoform disorder and specific personality disorders, e.g. anxious, negativistic, histrionic, or paranoid, are more prone to nocebo reactions and should be recognized as such.
In general, nocebo reactions are observed more frequently in women and the older age group.
PATIENT NON-COMPLIANCE {#sec1-6}
======================
Treatment success relies on patient compliance that, on its part, relies on confidence and motivation. Non-compliance is a major obstacle to the delivery of effective hair loss treatment. More often than being a failure of the patient, patient non-compliance results from failure of the physician to ensure that essential confidence and motivation for successful treatment. Patient compliance and therapeutic success rely on comprehension of treatment benefit, confidence and motivation.\[[@ref14][@ref15][@ref16]\]
Major barriers to patient compliance are:
Denial of the problemLack of comprehension of treatment benefitsOccurrence or fear of side effectsCost of the treatmentComplexity of treatment regimenPoor previous experiencePoor communication and lack of trustNeglect and forgetfulness.
Recommendations for improvement of patient compliance are:
Only recommending treatments that are effective in circumstances they are requiredPrescribing the minimum number of different medications, for example, combining active ingredients into a single compoundSimplifying dosage regimen by selecting different treatment or using a preparation that needs fewer doses during the daySelecting treatment with lower levels of side effects or fewer concerns for long-term risksDiscussing possible side-effects and whether it is important to continue medication regardless of those effectsAdvising on minimizing or coping with side effectsRegular follow-up for reassurance on drug safety and treatment benefitsDeveloping trust so patients don't fear embarrassment or anger if unable to take a particular drug, allowing the doctor to propose a more acceptable alternative.
A positive physician-patient relationship and regular follow-up visits are the most important factors in determining the degree of patient compliance. The overall goal is to gain short-term compliance as a prerequisite to long-term adherence to treatment:\[[@ref17]\]
Short-term compliance issues addressed by the physician within the first 3 months of therapy are: Winning the patient\'s confidence in the diagnosis and treatment plan and detecting problems relating to the prescribed treatment regimen, or drug tolerance.
Long-term compliance issues addressed at 6, 12 months of follow-up and thereafter yearly are: Treatment efficacy and sustainability, long-term toxicities and treatment costs.
CONCLUDING REMARKS {#sec1-7}
==================
> Tuto, celeriter, iucunde (Latin for: safely, swiftly, gladly)
>
> --Asclepiades of Bithynia
Asclepiades of Bithynia (124-56 BC) was the personal physician and near friend of notable personalities of Ancient Rome, such as Cicero and Marc Anthony.\[[@ref18]\] While the foreign Greek physicians were originally encountered with distrust by the Romans and especially its aristocracy, Asclepiades managed to convince through his high learning, brilliant medical achievements and worldly wisdom. Above all, he was attentive and sympathetic to the individual needs of his patients. Ultimately, Asclepiades advocated humane treatment of mental disorders. The same way Asclepiades won the Roman populace and aristocracy for his cause, the physician caring for patients complaining of hair loss must advance to build his reputation and to secure the confidence of his patients. A liaison with patients, respect for their individuality and professional expertise are preliminary toward creating an atmosphere of trust, which, on one side, enables physician\'s professional contribution to the somatic healing process and on the other side, assists patients to draw also from their own mental healing capacities.
**Source of Support:** Nil
**Conflict of Interest:** None declared.
| {
"pile_set_name": "PubMed Central"
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The role of microbes during the DWH oil spill {#s1}
=============================================
The DWH oil rig exploded in the Gulf of Mexico in April 2010. The spill lasted for approximately 84 d, which was recorded the largest oil spill in United States to date (Crone and Tolstoy, [@B22]). The accident yielded an unremitting flow of oil that ultimately totaled 4.9 million barrels (779 million liters, ±10%) (Lehr et al., [@B61]). An oil plume that stretched for more than 35 km was observed (Camilli et al., [@B12]; Hazen et al., [@B38]). Crude oils are composed of thousands of chemicals, which mainly include saturated hydrocarbons, aromatic hydrocarbons, and the more polar, non-hydrocarbon components in resins and asphaltenes (Head et al., [@B40]). These four main components have different levels of toxicity to marine organisms and vary in proportion with different types of crude oil. Exposure to large amounts of oil in marine environments can cause significant negative impacts on resident organisms and human health (Grattan et al., [@B34]). Oil spills can lead to acute and chronic environmental damage, and they can have toxic effects on the aquatic ecosystems (Campagna et al., [@B13]; Hu et al., [@B43]; Whitehead et al., [@B107]).
By 20 August 2010, 78% of the oil from the DWH oil spill was disposed by either human intervention (direct recovery, *in situ* burning, skimming, and dispersal) or natural processes (naturally dispersed, evaporated, and dissolved) (Ramseur, [@B83]), whereas the fate of the remaining 22% of the oil spill was uncertain. Microorganisms, especially bacteria, are believed to act as the key players in bioremediation of residual oil (Head et al., [@B40]; Wang et al., [@B105]). The ability to degrade hydrocarbon components of crude oil has been widely detected among different bacteria (Head et al., [@B40]). Considering the large amount of oil released from the DWH oil spill and specific features of the rig (the broken riser pipe was at a depth of 1,500 m below the surface), the responses and roles of bacteria following the spill have drawn significant attention from scientists (Kimes et al., [@B50]). Results from different research groups indicated that indigenous bacterial communities in different habitats (seawater, deep-sea sediment, marshes, and beach sands) responded rapidly to the spilled oil and that some bacterial groups might play significant roles in reducing the environmental contamination (Table [1](#T1){ref-type="table"}). Moreover, sunlight and temperature were found to be the key factors which influenced the composition of bacterial communities and selected for oil-degrading bacteria in surface water and bottom water, respectively (Bacosa et al., [@B6]; Liu et al., [@B65]).
######
Responses of bacterial communities to the Deepwater Horizon oil spill and various microbiological methods applied in different studies.
**Habits** **Sampling time** **Elapsed time (months)** **Dominant bacterial phylotypes** **Technologies** **References**
----------------- ------------------------------------------------------------ --------------------------- ------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------ -----------------------------------------------------------------------------------------------------
Sediments 09/2010--10/2010 5--6 Uncultured γ-*proteobacteria* and *Colwellia* 16S rRNA gene-based clone library, metagenomics, and SIP Mason et al., [@B72]
12/2010 8 Uncultured γ-*proteobacteria* and δ-*Proteobacteria* (*Desulfobacterales, Desulfuromonadales*, and *Desulfarculales*) 16S rRNA gene-based clone library, and Illumina amplicon sequencing Simister et al., [@B95]
11/2011 19 *Oceanospirillales* and *Colwellia* Metagenomics and metatranscriptomics Yergeau et al., [@B111]
Deep water Before spill α-*proteobacteria, Pelagibacter* and *Actinobacteria* PhyloChip, 16S rRNA gene-based clone library, and SIP Hazen et al., [@B38]
05/2010 1 γ-*proteobacteria* (*Oceanospirillales, Alteromonadales, Cycloclasticus*, 454 pyrosequencing Joye et al., [@B46]
05/2010 06/2010 1--2 γ-*proteobacteria* (*Oceanospirillales, Oleispira, Spongiispira, Cycloclasticus, Oleiphilus, Thalassolituus*, and *Colwellia*) PhyloChip, 16S rRNA gene-based clone library, and SIP Hazen et al., [@B38]; Valentine et al., [@B102]; Redmond and Valentine, [@B85]; King et al., [@B51]
09/2010 5 Methylotrophic bacteria (*Methylococcaceae, Methylophaga*, and *Methylophilaceae*) 16S rRNA gene-based clone library Kessler et al., [@B48]
11/2011 19 γ-*proteobacteria* Metagenomics and metatranscriptomics Yergeau et al., [@B111]
Surface water Before spill α-*proteobacteria, Pelagibacter*, and *Actinobacteria*. PhyloChip, 16S rRNA gene-based clone library, and SIP Hazen et al., [@B38]
05/2010 1 *Oceanospirillales* and *Rhodospirillales* Metagenomics and SIP Dombrowski et al., [@B26]
05/2010 1 *Alcanivorax, Marinobacter, Alteromonas, Cycloclasticus*, and *Colwellia)-* SIP Gutierrez et al., [@B37]
06/2010 2 *Cyanobacteria* and α-*proteobacteria* (SAR11 clade, *Rhodobacterales*, and *Rhodospirillales*) 16S rRNA gene-based clone library Chakraborty et al., [@B19]
09/2010 5 γ-*proteobacteria* (*Pseudoalteromonas, Pseudomonas, Vibrio, Acinetobacter*, and *Alteromonas*) PhyloChip, 16S rRNA gene-based clone library, and SIP Redmond and Valentine, [@B85]
11/2011 19 *Cyanobacteria* Metagenomics and metatranscriptomics Yergeau et al., [@B111]
Marsh sediments 06/2010, 07/2010, and 09/2010 2--5 *Proteobacteria, Bacteroidetes, Actinobacteria*, and *Firmicutes* (*Bacilli* and *Clostridia*) PhyloChip and GeoChip Beazley et al., [@B8]
05/2010, 09/2010, 06/2011, 09/2011, and 08/2013 1--40 *Proteobacteria, Firmicutes, Bacteroidetes*, and/or *Chloroflexi* phyla GS FLX amplicon pyrosequencing Engel et al., [@B30]
The fall of 2011, the spring of 2012, and the fall of 2013 18--36 *Desulfococcus, Marinobacter*, and *Mycobacterium* Metagenomics Atlas et al., [@B4]
07/2012 24 *Nitrosopumilus* cluster TRFLP Analysis Bernhard et al., [@B10]
Beach sands 07/2010 3 α-*proteobacteria* (*Rhodobacteraceae*) and γ-*proteobacteria* (*Alcanivorax, Marinobacter, Pseudomonas*, and *Acinetobacter*) Pure culture, automated ribosomal intergenic spacer analysis (ARISA), microarray, and SSU rRNA pyro sequence libraries Kostka et al., [@B57]
06/2011 Over 12 α-*proteobacteria* (*Hyphomonas, Parvibaculum*, and *Micavibrio*) and γ-*proteobacteria* (*Alcanivorax, Pseudomonas*, and *Marinobacter*) Metagenomics and 16S rRNA gene-based clone libraries Rodriguez-R et al., [@B87]
The DWH oil spill provides a unique opportunity to understand the significant roles that microbes play in the recovery process at oil-polluted environments and to compare different methods for microbial ecological research. In this review, we focus on analyzing and summarizing various methods applied in investigating the responses of microbial communities to the DWH oil spill. These include both culture-dependent methods (i.e., mainly pure culture) and culture-independent methods \[i.e., classical molecular biological methods and several advanced methods, including stable isotope probing (SIP), microarray, metagenomics, metatranscriptomics, and single-cell sequencing\]. Additionally, we propose some prevalent methods \[e.g., microbial ecosystems metabolic networks (MEMNs) and microbial molecular ecological networks\] that exhibit great potential for the future studies.
Methods for microbiological analysis {#s2}
====================================
Cultivation-dependent methods
-----------------------------
Traditional methods, such as isolation and purification of bacterial strains from various environments are essential as exemplified in microbiological studies of the DWH spill. The isolation of microbes in pure culture can provide valuable information on the functions, metabolic pathways, and physiological characteristics of bacteria that respond to an oil spill. During the DWH spill, different bacterial strains (e.g., *Alcanivorax, Alteromonas, Cycloclasticus, Halomonas, Marinobacter*, and *Pseudoalteromonas*) with hydrocarbon-degrading abilities were isolated from surface slicks and deep oil plumes (Gutierrez et al., [@B37]). With the addition of oil (or the oil dispersant Corexit) as the sole carbon source, Bælum et al. ([@B7]) successfully isolated one hydrocarbon-degrading strain (belonging to the family *Colwelliaceae*), which were the dominant microorganism in the oil plume.
Despite the successful isolation of some oil-degrading bacteria, most of the indigenous bacterial strains could not be isolated by conventional culture-based methods. This might be attributed to the fact that artificial culture conditions are different from the *in situ* environment, due to the absence of symbiotic organisms and specific nutrient levels (Zengler et al., [@B113]). Furthermore, specific environmental conditions in deep sea (low temperature, O~2~ restriction and hydrostatic pressure) are always neglected or difficult to setup in the *ex situ* experiments, which limit the isolation of oil-degrading bacteria (Scoma et al., [@B92]). Recently, some previously uncultured microorganisms were successively grown in pure culture using newly-developed culturing techniques, including (1) supplying various chemical compounds found in their natural environment (Kaeberlein et al., [@B47]; Rappé et al., [@B84]; Zengler et al., [@B113]); (2) lengthening the period of incubation and protecting cells from exogenous peroxides (Stevenson et al., [@B97]); (3) isolating uncultived microorganisms in a simulated natural environment with specific devices (Kaeberlein et al., [@B47]); (4) high-throughput dilution to extinction culturing technology (HTC) (Rappé et al., [@B84]; Stingl et al., [@B98]). The above microbiological cultivating technologies have not been used to isolate bacteria from the DWH oil spill-contaminated environment, and could be considered in microbiological investigations of future oil spills. Considering the particularity of the deep-sea environment, the method based on the simulated/real natural environment (Kaeberlein et al., [@B47]) can be applied in future studies. Briefly, surface sediments with *in situ* microorganism can be mixed with warmed agar and sealed in a diffusion chamber sealed by two membranes which not only allow exchange of chemicals between the chamber and the environment, but also restrict the movement of cells. The sealed chambers can be cultivated in a marine aquarium with simulated natural environment (low temperature, O~2~ restriction and hydrostatic pressure) or *in situ* in the deep-sea environment. The development of novel cultivating technologies to isolate previously uncultured bacteria could permit a more comprehensive understanding of unexplored bacterial communities, including their metabolic pathways, and functions.
Classical molecular biological methods
--------------------------------------
The majority (\>99%) of bacterioplankton in marine environments remains largely recalcitrant to cultivation. Applications of molecular biological methods such as 16S rRNA clone library, terminal restriction fragment length polymorphism (T-RFLP), denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), fluorescence *in situ* hybridization (FISH), and real-time quantitative polymerase chain reaction (RT-PCR) are useful techniques in investigating the composition and potential functions of indigenous bacterial communities in a specific environment.
### 16S rRNA gene clone library
The 16S rRNA gene is well-known as a marker gene for the reconstruction of prokaryotic (bacterial and archaeal) phylogenies and for the investigation of microbial diversity (Woese and Fox, [@B108]; Klindworth et al., [@B54]). The construction of clone libraries based on 16S rRNA genes has extensively been used to investigate bacterial communities until the advent of next generation sequencing (NGS). 16S rRNA gene-based clone library analysis provides a useful, though somewhat limited, tool for capturing the diversity and composition of bacterial communities (Kumar et al., [@B59]; Schäfer et al., [@B90]). In the literature on the DWH oil spill, 16S rRNA gene-based clone library analysis was one of the most popular techniques for investigating microbial communities. Several studies using 16S rRNA gene clone libraries revealed the microbial community responses to the contamination caused by the deep-sea plumes of hydrocarbons. These communities exhibited significant differences in different habitats (plume and non-plume water, surface and deep-sea water, sediments, and beach sands; see Table [1](#T1){ref-type="table"}) and presented dramatic changes over the duration of the spill (Hazen et al., [@B38]; Valentine et al., [@B102]; Kessler et al., [@B48]; Redmond and Valentine, [@B85]; Yang et al., [@B110]). For instance, Hazen et al. ([@B38]) identified members of the order *Oceanospirillales* in the γ*-Proteobacteria* class as dominant in deep-sea plumes. *Oceanospirillales* were not detected in most of the plume samples in June, while *Cycloclasticus* and *Colwellia* were dominant (more than 95% of the sequences). This indicates that the DWH *Oceanospirillales* bloomed initially, followed by *Colwellia* and *Cycloclasticus* (Redmond and Valentine, [@B85]; Yang et al., [@B110]).
Construction and analysis of 16S rRNA gene clone libraries provided useful information on the structures and responses of bacterial communities during the first stage after the spill occurred. However, the 16S rRNA gene clone library technique became less prevalent after NGS was used to investigate microbial communities.
### Gene fingerprinting techniques
Denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) are two commonly used gene fingerprinting techniques that separate different DNA/RNA in the same sample via chemical or temperature gradients. Muyzer et al. ([@B77]) first introduced DGGE into the field of microbial ecology to describe the composition of bacterial communities. Different bands on the gel indicate different bacterial phylotypes and band intensity shows the relative abundance of certain bacterial taxa. By cutting DGGE/TGGE strips, building clone libraries, and sequencing, the information related to a band can be further interpreted. Although the information provided by these two techniques are thought to be insufficient (as there are always dozens of bands for one sample) and NGS can generate more data, DGGE/TGGE techniques are still widely used to study microbial communities as they can intuitively display the differences between different samples via gel profiles within short time periods (1--2 d for an experimental cycle).
After the DWH oil spills, DGGE provided some of the first information on naturally occurring microbial communities in the sediment from shorelines along the northern coast of the Gulf of Mexico (Lisle, [@B64]). Some active bacterial species exhibited increases in biomass during the degradation process. Bacosa et al. ([@B5]) used 16S rRNA gene-based DGGE to detect microbial community dynamics and composition with the stressor of Light Louisiana Sweet (LLS) crude oil under dark and natural light conditions. The results demonstrated that different bacterial groups were involved in the degradation of n-alkane under the dark and light conditions.
Like DGGE/TGGE, TRFLP, or sometimes T-RLP is another gene fingerprinting technique. By generating a fingerprint for pattern comparison and then complementing the data with a clone library, resolving peaks using a database, and performing multivariate analysis, TRFLP can reveal the differences in unknown microbial communities in various environmental samples (Liu et al., [@B66]). T-RFLP was applied to investigate the composition and succession of bacterial communities after the oil spill and successfully revealed the relatively higher contribution of the bacterial group *Flavobacteria*, which is a secondary consumer of methane, oil, or cellular decay products (Redmond and Valentine, [@B85]). Profiles generated by TRFLP indicated that up to 56% of the total bacteria in plume samples collected in September 2010 were affiliated with *Flavobacteria*, whereas this number was only 10--30% when 16S rRNA gene-based clone library techniques were applied (Redmond and Valentine, [@B85]). In a recent study, the TRFLP analysis for community composition of ammonia oxidizers in salt marshes after the DWH oil spill revealed that exposure to oil, even 2 years post-spill, could contribute to subtle changes in population dynamics of bacterial communities (Bernhard et al., [@B10]). Although T-RFLP could provide more authentic data compared to clone libraries, it could still underestimate community diversity, especially when complex and large amounts of data were applied. In the abovementioned study (Redmond and Valentine, [@B85]), some terminal restriction fragments could be assigned to multiple groups. Due to the significant overlaps between the *methanotrophs, methylotrophs*, and some other γ-*proteobacteria*, it was not possible to distinguish the δ-*proteobacteria, Actinobacteria*, and SAR406 clades from each other. Nevertheless, some groups, such as *Flavobacteria*, α-*proteobacteria*, and γ-*proteobacteria* could be readily differentiated.
### Other classic molecular methods
In addition to the aforementioned molecular methods, other molecular tools were also used to study indigenous microbial communities, such as FISH and real-time quantitative polymerase chain reaction (qPCR). The FISH method has been widely used to investigate composition of bacterial communities in recent years. It can provide fluorescent images indicating the existence and relative abundance of target genes combing with special fluorescent probes. 16S rRNA genes or functional genes can be detected by this approach without PCR amplification (DeLong et al., [@B24]; Amann and Fuchs, [@B2]). Catalyzed reporter deposition in combination with FISH (CARD--FISH) were performed to detect the microbial aggregate formations in a microcosm study using deep plume seawater with addition of nutrients (Kleindienst et al., [@B53]). The results revealed that the potential dispersant-degrader, *Colwellia* was one of the dominant bacterial phylotypes in microaggregate which indicated that this bacterial group could play an important role in marine oil snow formation. By developing two new 16S rRNA-targeted oligonucleotide probes (Mrb-0625-aandMrb-0625-b) for the FISH assay, McKay et al. ([@B73]) successfully monitored the rapid increase of the oil-degrading bacterium, *Marinobacter*, after amending n-hexadecane in an enrichment experiment with a deep-sea oil plume water sample. Compared to FISH, qPCR can quantify targeted functional bacterial phyla more accurately. It provides relatively accurate data via real-time monitoring of the fluorescent signals generated by fluorescent probes (Heo et al., [@B41]; Yuan et al., [@B112]). Because probes and primes are necessary for both FISH and RT-qPCR detection, only bacterial phylotypes with probes/primers can be detected and quantified, whereas some unknown groups without specific probes/primers are difficult to be detected using these methods.
Advanced molecular techniques
-----------------------------
Traditional approaches applied in ecological microbiology have promoted developments in this field for decades and have provided general information on the existence (FISH), composition (Clone Library, DGGE/TGGE, FISH, and T-RTLP), and abundance (Clone Library and RT-qPCR) of microbes. Nevertheless, the information generated by these approaches is finite, and the unit cost (the cost per sequence) is relatively expensive. For instance, only dozens of DGGE bands or clones could be identified by combining the techniques of DGGE and clone libraries which could only generate limited information and hardly reflected on the real composition of bacterial communities. Furthermore, the unit cost for a single sequence can be much higher by Sanger sequencing than NGS. With the development of new approaches, such as DNA/RNA microarrays, NGS, and SIP, ecological microbiology research is faced with new opportunities. These currently prevalent techniques can generate massive data sets for detailed investigation on the composition and diversity of bacterial communities in different habitats or following particular events. Some new techniques that provide impressive information have been used in investigating the succession and function of the microbial communities after the DWH oil spill.
### DNA microarray
DNA microarrays are collections of microscopic DNA spots, representing single target genes, that are attached to a chemical matrix and arrayed on a solid surface. By DNA-DNA or DNA-RNA hybridization, DNA microarrays can provide qualitative or quantitative measurements of microbial diversity and functional gene expression (Loy et al., [@B67]; Bodrossy et al., [@B11]). This method has been used for nearly 20 years and has experienced several generations of improvement. By hybridizing target genes with probes attached to a specific microchip, DNA microarrays can provide a deep understanding of indigenous microbial communities because of its advantages of specificity, sensitivity, quantitative analysis, and high-throughput methods (Gentry et al., [@B33]). Two different DNA microarray methods (PhyloChip and GeoChip) have been extensively used in the field of environmental microbiology. PhyloChip can be used to analyze the diversity of microbial communities, while the GeoChip method can be used to study the activity of functional microbes by targeting functional genes. These two different microarrays can be used together to study and compare microbial communities from different treatments or environments.
Both PhyloChip and GeoChip were applied to investigate the succession of bacterial communities and reveal the functional genes in the biodegradation of oil pollution during the DWH oil spill (Bodrossy et al., [@B11]; Hazen et al., [@B38]; Beazley et al., [@B8]). The PhyloChip analysis of 16S rRNA microarray conducted by Hazen et al. ([@B38]) suggested that the oil plume significantly altered the microbial community composition and structure. In addition, the functional gene-based GeoChip microarray analysis revealed significant increases in the expression of more than 1,600 genes involved in hydrocarbon degradation (BTEX, alkane, cycloalkanes, and PAH) compared to the background non-plume samples (Hazen et al., [@B38]). In another study, the microbial communities in the Gulf of Mexico\'s coastal marshes during and after the oil spill were determined via both GeoChip and PhyloChip (Beazley et al., [@B8]). A total of 12,018 operation taxonomy units (OTUs) were successfully identified by PhyloChip microarray-based analysis, and the result demonstrated that bacterial phyla with potential genes for oil degradation exhibited significant increases after the oil spill. For instance, bacterial phyla affiliated to *Actinomycetaceae, Dietziaceae, Nocardioidaceae, Erythrobacteraceae*, and others, which have been shown to be capable of degrading alkanes and polycyclic aromatic hydrocarbons (PAHs), dramatically increased during the oil infiltration of sediments. In contrast, bacterial phyla of *Verrucomicrobia, Cyanobacteria*, and *Planctomycete* showed obvious decreases due to their relatively low tolerance to oil pollution. GeoChip microarray-based analysis was used to identify 16,383 unique functional genes. The results indicated that a relative abundance of functional genes involved in hydrocarbon degradation (e.g., *alkB, alkH, phaB, dsrA/B*, and others) were significantly increased between June and July in the inlet sediments (Beazley et al., [@B8]).
A single chip can be used to analyze thousands of genes in one assay. Therefore, microarrays are becoming one of the major culture-independent identification/quantification methods. However, the microarray technique faces the challenges of natural sequence diversity and potential cross-hybridization in complex environmental bioaerosols (Li and Huang, [@B63]). Lack of a probe sequence and the complexity of the gene chip\'s interactive hybridization have been shown to induce low specificity and poor sensitivity of experimental results (Zhou and Thompson, [@B117]; Gentry et al., [@B33]).
### 16S rRNA gene based high-throughput sequencing
The 16S rRNA gene has been applied to study the diversity of bacterial communities via cultivation, cloning, T-RFLP, and other traditional microbiological approaches. While the information generated from these approaches can preliminarily illustrate the composition of bacterial communities, their application has been restricted by limited information, high cost, and omission of "rare biospheres." The NGS techniques, e.g., Roche\'s 454 GS20 pyrosequencing (Margulies et al., [@B68]), Illumina Hiseq/Miseq sequencing (Bennett, [@B9]), ion torrent sequencing (Rothberg et al., [@B88]), and "single-molecule real-time" (SMRT) sequencing (Eid et al., [@B29]), allowed the generation of massive data sets at a much lower cost (Medini et al., [@B74]; Armougom and Raoult, [@B3]; Klindworth et al., [@B54]).
The DWH oil spill occurred in 2010, when the use of high-throughput sequencing was on the rise. The new techniques of microbial ecology were comprehensively applied after the event to reveal the impacts of the oil spill on bacterial communities and the responses of different bacterial phyla (Gutierrez, [@B36]). 454 pyrosequencing of the 16S rRNA gene was the first NGS technique applied in this event (Bælum et al., [@B7]), providing information on the succession of bacterial communities. The results, based on the NGS data, specifically showed the increase in the bacterial phyla *Colwelliaceae* and *Oceanospirillales*, which was consistent with the results from 16S rRNA gene-based clone library and PhyloChip (Hazen et al., [@B38]; Redmond and Valentine, [@B85]). The results were based on the analysis of 148,276 quality-filtered reads from 16 samples, whereas the first study after the spill which applied 16S rRNA gene based clone library only generated 250 high qualitied sequences from three samples (Hazen et al., [@B38]). The great amount of sequencing could produce more useful information for revealing the bacterial responses to the oil spill. Illumina sequencing was also used in later studies (Mason et al., [@B70], [@B72]; Simister et al., [@B95]). For instance, by analyzing massive amounts of data generated by Illumina sequencing, Simister et al. ([@B95]) found that the dominant bacteria in both sediment and floc samples were *Proteobacteria* (55--64%). They also found that the bacterial composition of the floc samples (mostly aerobic or facultative aerobic phylotypes including *Rhizobiales, Rhodobacterales, Sphingomonadales, Rickettsiales, Alteromonadales*, and *Pseudomonadales*) was different from those in the sediment samples (aforementioned aerobic species and anaerobic phylotypes such as *Desulfobacterales, Desulfuromonadales*, and *Desulfarculales*).
### Metagenomics
Metagenomics is a genetic analysis aimed at studying the genetic composition and community structure of microorganisms by directly analyzing DNA mixtures from microbial communities in different environmental samples (Warnecke and Hess, [@B106]; Coyotzi et al., [@B21]). Metagenomic approaches have revolutionized our ability to explore the microbial world by producing results that conventional cloning and sequencing methods have not been able to achieve (Gutierrez, [@B36]). 16S rRNA gene based analysis mainly characterize a bacterial community by defining its composition, succession and the relative abundance of different groups, while metagenomics analysis can afford useful metabolic information for investigating microbe functions (Scholz et al., [@B91]). It is helpful to fully understand uncultured microbes and provide a complete understanding of microbial activity at community levels. Specifically, metagenomic approaches enable a mechanistic understanding of the bioremediating processes and can provide some hints for optimizing the efficiency of bioremediation (Techtmann and Hazen, [@B101]). For instance, by analyzing metagenomic sequence data, some overlooked but important functional executor and functional genes can be explored (Jackson et al., [@B45]; Campeão et al., [@B14]).
Before NGS became prevalent, metagenomic analysis based on shotgun sequencing data was constrained by the limited data on individual genomes and environmental genetic data. The high-throughput, short runtime, and low-cost attributes of NGS have altered our overview of microbial metagenomics (Scholz et al., [@B91]). By applying metagenomic analysis, (Mason et al. ([@B70], [@B72])) investigated the responses of bacterial communities in deep sea oil plumes and sediment. Metagenomic data from these two studies demonstrated that the indigenous microbiota contributed an important ecosystem service of oil remediation in the Gulf of Mexico. Functional genes encoding for the degradation of aliphatic and simple aromatics hydrocarbon were significantly enriched after the oil spill. Nevertheless, no dramatic changes were found in the abundance of genes involved in the degradation of PAHs. This indicated that recalcitrant compounds could not be actively degraded at the sampling time and that these compounds might need a longer period for biodegradation. Another study based on metagenomic analysis found that a greater number of δ*-proteobacteria* and anaerobic functional genes were present in sediments closer to the DWH blowout site, which indicated that δ*-proteobacteria* might play a dominant role in anaerobic hydrocarbon degradation (Kimes et al., [@B49]).
### Metatranscriptomics
Metagenomics can provide useful information on the genomic potential of a microbial community, whereas metatranscriptomics can be used to investigate genes that are transcribed under certain environmental conditions. It can assess microbial gene expression in a special environment or under unusual conditions via pyrosequencing of total RNA directly extracted from natural microbial assemblages (Shi et al., [@B94]; Warnecke and Hess, [@B106]). Metatranscriptomic data takes into account the dynamic state of RNA levels and thereby overcomes the constraint of metagenomics which relies on DNA for the reconstruction of metabolic models to improve prediction accuracy. Thus, metatranscriptomic analysis could provide supporting data or more convincing data in comparison to metagenomic analysis. In addition to metagenomic analysis, Mason et al. ([@B70]) conducted metatranscriptomic analysis to determine the expressed functional genes in the active microbial community in the oil plume. Metatranscriptomic data revealed more pronounced differences in the relative abundance of active genes that have the potential for biodegradation compared to DNA-based analysis. These active genes were mostly associated with aliphatic hydrocarbon degradation but not PAH degradation, which is consistent with metagenomic analysis. In a study that considered the microbial communities of methane degradation (Lesniewski et al., [@B62]), data generated from metatranscriptomics revealed that the bacterial communities of both the plume and background samples were dominated by the same groups of methanotrophs and chemolithoautotrophs despite marked increases in plume total RNA concentrations (3--4 times) and microbial-mediated manganese oxidation rates (15--125 times background levels).
### Single-cell sequencing
Single-cell sequencing is a newly developed technique to obtain genomes of individual microorganisms. This technique was first reported by Raghunathan et al. ([@B82]) and was selected as the method of the year in 2013 by "Nature Publishing Group." Single cells can be first isolated from various environments (soil, seawater, marine sediments, and human gastrointestinal tracts) by micro manipulation, flow cytometry or microfluidics. The whole genomes of selected single cells are then amplified by multiple displacement amplification (MDA) to generate sufficient amounts of genetic material for sequencing. Genomes can be sequenced by NGS and analyzed by new methods in bioinformatics (SPAdes) (Woyke et al., [@B109]). By generating genomic information on previously inaccessible species from different environments, the structure of microbial communities and the physiology of single cells can be deeply investigated. Mason et al. ([@B70]) successfully obtained two single-cell genomes of the genus *Oceanospirillales*, which have been shown to be dominant phylotypes after the DWH oil spill (Hazen et al., [@B38]; Redmond and Valentine, [@B85]). Single-cell sequencing revealed that both cells possessed genes encoding for n-alkane and cycloalkane degradation. Furthermore, the genomic information helped in the reconstruction of the near-complete pathway for cyclohexane oxidation in the two single cells. This provided powerful evidence proving that bacteria in the order *Oceanospirillales* were responsible for the biodegradation of aliphatic hydrocarbons in the deep sea (Mason et al., [@B70]). Thus, with the support of metagenomic sequencing data, single-cell sequencing analysis (e.g., the genomic properties of *Colwellia*) could extend our understanding of the successional changes of the dominant microbial players (Mason et al., [@B69]) and their specific metabolic activities in biochemical cycle (Musat et al., [@B76]).
Bioinformatic analysis
----------------------
Methods based on NGS mentioned above and DNA microarrays revolutionized microbial studies with vast quantities of data at a relatively low cost. However, the analysis of enormous amounts of data and interpretation of their biological meaning are still a great challenge. A large number of software tools (ST) and databases (DB) have been produced for bioinformatic analysis, including Greengenes (DB; DeSantis et al., [@B25]), Ribosomal Database Project (RDP, DB; Cole et al., [@B20]), SILVA (DB; Quast et al., [@B81]), MEGA6 (ST; Tamura et al., [@B99]), and QIIME (ST; Caporaso et al., [@B17]) for 16S rRNA gene data analysis; and Kyoto Encyclopedia of Genes and Genomes (KEGG, DB; Ogata et al., [@B79]), The Carbohydrate-Active EnZymes (CAZY, DB; Cantarel et al., [@B15]), Clusters of Orthologous Groups of proteins (COGs, DB; Tatusov et al., [@B100]), MetaCyc (DB; Caspi et al., [@B18]), and RefSeq (DB; Pruitt et al., [@B80]), Transporter Classification Database (TCDB, DB; Saier et al., [@B89]), MetaPathways (ST; Konwar et al., [@B56]), RAxML version 8 (ST; Stamatakis, [@B96]), and Circos (ST; Krzywinski et al., [@B58]) for analyzing metagenomics, metatranscriptomics, and single genome data. A series of visualization tools have also been developed in recent years, including Tablet (Milne et al., [@B75]), Integrative Genomics Viewer (IGV) (Robinson et al., [@B86]), Sequence Annotation, Visualization, and ANalysis Tool (Savant) Genome Browser (Fiume et al., [@B32]), MagicViewer (Hou et al., [@B42]), and Cytoscape (Kohl et al., [@B55]). In the microbiological studies on the DWH oil spill, these ST and databases were broadly used for analyzing DNA- and RNA-based data. Moreover, Mason et al. ([@B70]) applied multiple ST and databases to interpret biological meaning from a mass of metagenome, metatranscriptome, and single-cell sequencing data. For instance, the GeoChip database (He et al., [@B39]) was applied to blast proteins involved in hydrocarbon degradation based on raw metagenomic, metatranscriptomic and single-cell reads, while the database of COGs was used to estimate genome sequence completeness. Reads from single cells were assembled using Velvet (Zerbino and Birney, [@B114]). Unassembled metatranscriptomic reads were mapped to the single-cell draft genome using the CLC Genomics Workbench (CLC bio). Assembled single-cell data were annotated using CAMERA (v2.0.6.2) (Seshadri et al., [@B93]). Clustered regularly interspaced short palindromic repeat regions were identified in the draft genome using CRISPRFinder (Grissa et al., [@B35]). Although bioinformatic tools have been vastly applied and greatly stimulated the data analysis, there is urgent need for the establishment of more comprehensive databases or big data sets. Meanwhile, equipment simplification, standardization methods and more user-friendly bioinformatic tools need to be developed (Capobianchi et al., [@B16]).
Perspectives {#s3}
============
What kind of methods should be used in future studies?
------------------------------------------------------
The culture-dependent and culture-independent methods discussed in this review can partially reveal the responses of indigenous bacterial communities to the DWH oil spill, whereas a full map of bacterial community composition and activity cannot be described by a single method (Figure [1](#F1){ref-type="fig"}). Biodegradation efficiency and metabolite mechanisms can be adequately researched by obtaining pure cultures of bacterial strains. However, the uncultivability of bacteria from marine environments (\>99% of microorganisms resist cultivation in the laboratory Kaeberlein et al., [@B47]) is a dramatic obstacle for revealing the real executors of *in situ* biodegradation. The 16S rRNA gene-based culture-independent methods, including DGGE, clone libraries, and NGS, can be comprehensively used to identify the bacterial composition in oil spill sites, but these methods provide limited information on metabolism of bacterial communities. Metagenomics and metatranscriptomics based on NGS can provide detailed information on a broad overview of the metabolic potentiality of bacterial communities in the polluted environment at the DNA and RNA levels, respectively. Nevertheless, these two meta-omics techniques provide limited information on the specificity of a metabolic process or the differences due to physiological or environmental conditions. The amplification bias is another obstacle in the application of these two meta-omics techniques (Lasken, [@B60]). Single-cell sequencing can provide genomic information on uncultivable bacteria, and it can help predict the potential pathways of biodegradation performed by the studied cell. However, single cell DNA/RNA sequences usually have high amplification bias. This is usually caused by some genomic regions being amplified more than others (Ning et al., [@B78]). Furthermore, analysis of single cell sequencing data always needs support from meta-omics data for additional analysis. In addition, the determination of metabolic products is also necessary for unraveling of environmental PAH biodegradation processes and also to describe microbial interactions effecting the biodegradation process (Vila et al., [@B103]). Therefore, a combination of different approaches is necessary for a full understanding of the composition and activity of bacterial communities. Application of integrated approaches could: (1) deeply describe the components of a complex community, including existing organisms, functions, and their activities at a given time point (Zimmerman et al., [@B118]); (2) identify key players, sometimes diverse, rare taxa instead of majority groups (Kleindienst et al., [@B52]); (3) reveal more complicated microbial interactions by determining the connections of both cultured and currently uncultured microorganisms (Abraham, [@B1]); (4) predict functions of the whole community with the help of different bioinformatic tools which can improve prevention and remediation of potential perturbations (Ivshina et al., [@B44]).
{#F1}
In the first microbial study after the oil spill, Mason et al. ([@B71]) applied the PhyloChip 16S ribosomal RNA (rRNA) microarray and clone library to study the bacterial community composition in the oil plume. The authors successfully isolated oil-degrading bacteria by a convenient cultivation method. Additionally, multivariate analysis of phospholipid fatty acid (PLFA) was applied in this study to analyze the composition of bacterial communities by identifying different assemblages of PLFA. Mason et al. ([@B70]) investigated the role of the indigenous microbial community by combining different molecular methods including 16S rRNA gene sequencing, metagenomics, metatranscriptomics, and single-cell genomics. These methods based on NGS are currently prevalent in the field of microbial ecology, and they can provide abundant information for thorough analysis of the composition and behavior of the bacterial communities.
Analysis based on SIP is a typical strategy for bacterial community study that combines culture-dependent and culture-independent approaches. First, cultivation is necessary to obtain molecular markers (DNA, RNA, proteins, or PLFA) that are labeled with a stable isotopic element (e.g., ^13^C, ^15^N, and ^18^O). The labeled molecular markers can then be analyzed by 16S rRNA gene sequencing (clone library or NGS), metagenomic analysis, metatranscriptomic analysis, and mass spectrometer (MS) analysis. These methods can be used to generate data to reveal the bacterial community and activity. SIP analysis is a powerful tool for linking microbial species and metabolism (Dumont and Murrell, [@B27]). By using DNA-SIP in combination with 16S rRNA gene clone libraries and T-RFLP analysis, Redmond and Valentine ([@B85]) demonstrated that bacteria in the genus *Colwellia* were active in ethane and propane oxidation in oil polluted deep-sea water plumes. Mason et al. ([@B72]) used surface sediments polluted by the DWH oil spill and ^14^C-labeled hydrocarbons to perform mineralization experiments. They successfully found that the key hydrocarbon degradation pathway by sediment microbes was as follows: propylene glycol, dodecane, toluene, and phenanthrene. By monitoring the biodegradation efficiency of ^13^C-labeled different hydrocarbons (aliphatic and aromatic), Gutierrez et al. ([@B37]) applied 16S rRNA gene-based NGS and qPCR detection to reveal the activity of bacterial communities. The results indicated that the different bacterial phyla were responsible for the degradation of different hydrocarbons. For instance, bacteria in the genera *Alcanivorax* and *Marinobacter* were found to be enriched in the experimental setups with the addition of aliphatic hydrocarbons, whereas *Alteromonas, Cycloclasticus*, and *Colwellia* dominated the experimental setups supplemented with polycyclic aromatic hydrocarbon.
How to illustrate the cooperation of different bacteria?
--------------------------------------------------------
A global perspective calls for a deep understanding of the composition and activity of the bacterial communities in a relevant environmental context (Faust and Raes, [@B31]). We should not only focus on functional bacterial communities that are directly degrading pollutants, but also on the other biological components involved in the biodegradation or bioremediation processes via chemical and biological interactions (Head et al., [@B40]). For instance, microorganisms without oil-degrading capability could also be involved in the biodegradation process by consuming the metabolites released by oil-degrading bacteria. Some non-oil-degrading microorganisms can either provide limiting nutrients or produce surfactant or emulsification of oil and water for the primary oil degraders (Head et al., [@B40]). Reconstructing the microbial network might be an efficient method for investigating the complicated interactions among indigenous bacteria. Zhou et al. ([@B115]; [@B116]) developed a random matrix theory (RMT)-based conceptual framework to reconstruct functional molecular ecological networks based on metagenomics and genechip data. This approach was applied to evaluate the responses of bacterial communities under long-term, free-air CO~2~ enrichment (Zhou et al., [@B115], [@B116]). The reconstructed networks revealed dramatic changes in the interactions among different phylogenetic groups/populations. The changes in network structure were significantly correlated with nutrient contents. In our previous study, we investigated the responses and activities of bacterial communities under stresses from hydrocarbons with different structures by reconstructing microbial networks and predicting the individual roles of functional bacteria in the key nodes of the networks. The interactions of different bacteria in the networks helped microbes co-acclimate to the changing environment and initiate biodegradation (Wang et al., [@B104]). In all, the microbial networks can provide additional information besides the composition of bacterial communities. Most importantly, the key functional bacterial phyla, including the degrading conductor and tightly associated non-degrading bacteria, could be identified (key nodes in the network). Based on this information, the bacterial communities in polluted sites can be manipulated for enhancing the abundances of beneficial species and functions for bioremediation (Faust and Raes, [@B31]). The key strategies for applications based on network reconstruction and analysis is engineering a whole community instead of a single species (Curtis et al., [@B23]; Dunham, [@B28]).
Conclusions {#s4}
===========
The impacts of the DWH oil spill are gradually fading, hence the research on its impacts on indigenous microbial communities is decreasing. However, current literatures adequately address the approaches for investigating the composition and activity of microbial communities. In this review, we summarized various culture-dependent and culture-independent techniques applied in the study of microbes after the DWH oil spill. Conventional methods, such as pure culture and classical molecular biological methods (e.g., Clone Library, TRFLP, TGGE/DGGE, and RT-qPCR), provide qualitative impressions about the functional bacteria conducting biodegradation. Prevalent microbiological molecular techniques (e.g., microarrays, NGS, metagenomics, metatranscriptomics, and single-cell sequencing) have also generated a great amount data on bacterial composition and predicted potential pathways for hydrocarbon degradation. For future investigation, we recommend an informed application of prevalent NGS-based genomic technologies and microbial network reconstruction in conjunction with conventional culture-dependent approaches. Especially, integrated application of different approaches could afford a more comprehensive view of indigenous bacterial communities and their potential functions in polluted sites.
Author contributions {#s5}
====================
SZ, ZH, and HW provided substantial contributions to the conception and design of the work. SZ and HW drafted the work. SZ, ZH and HW agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.
Conflict of interest statement
------------------------------
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We thank Taylor & Francis Editing Services and Dr. Mary Otieno from International Center for Tropical Agriculture (CIAT) for their linguistic assistance during the preparation of this manuscript.
**Funding.** Funding for this study was provided by National Natural Science Foundation of China (31500096, 31670117), the Guangdong Science and Technology Department \[SAIL Foundation for Distinguished Scholars (HW)\] and the Department of Education of Guangdong Province \[Pearl River Scholar for Young Investigator (HW)\].
[^1]: Edited by: Michail M. Yakimov, Consiglio Nazionale Delle Ricerche (CNR), Italy
[^2]: Reviewed by: Martin Krüger, Federal Institute for Geosciences and Natural Resources, Germany; Tony Gutierrez, Heriot-Watt University, United Kingdom
[^3]: This article was submitted to Microbiotechnology, Ecotoxicology and Bioremediation, a section of the journal Frontiers in Microbiology
| {
"pile_set_name": "PubMed Central"
} |
Array-CGH data have been deposited in NCBI's Gene Expression Omnibus (GEO accession number GSE63216; <http://www.ncbi.nlm.nih.gov/geo>).
Introduction {#sec005}
============
Cancer is caused by genomic aberrations that drive tumor initiation and progression. Oncogene activation and tumor suppressor gene inactivation can be caused by several classes of somatic DNA aberrations, including non-synonymous (point) mutations, chromosomal copy number aberrations (CNAs) and structural variants (SVs) \[[@pone.0138141.ref001]\]. SVs represent deletions, insertions, inversions, and intra- and inter-chromosomal translocations, all of which involve chromosomal breaks \[[@pone.0138141.ref002]\]. Interestingly, while point mutations and DNA copy number changes have been examined extensively, the effects of genes with chromosomal breaks are poorly characterized. Taking colorectal cancer (CRC) as an example, to date several in-frame fusion genes have been reported including *VTI1A-TCF7L2*, *NAV2-TCF7L1* and the R-spondin fusions *PTPRK-RSPO3* and *EIF3E-RSPO2* \[[@pone.0138141.ref003]--[@pone.0138141.ref005]\]. The R-spondin fusions activate the Wnt signaling pathway and are mutually exclusive with *APC* mutations, indicating that these translocations cause gain-of-function protein alterations. Alternatively, SVs can also cause loss-of-function alterations. For example, deletion of the stop codon of the *EPCAM* gene results in a transcriptional read-through that causes hypermethylation and consequently silencing of the adjacent mismatch repair gene *MSH2* \[[@pone.0138141.ref006]\]. However, despite these intriguing examples, thorough investigation of SVs in CRC has been hampered by the lack of whole genome deep sequencing information on large series of samples. Consequently, the putative impact of SVs in (colorectal) tumor development is probably highly underestimated.
We previously performed high-resolution array-comparative genomic hybridization (array-CGH) analysis on a series of approximately 350 primary CRC samples from patients who had developed metastatic disease and participated in the CAIRO and CAIRO2 phase III clinical trials \[[@pone.0138141.ref007]--[@pone.0138141.ref009]\]. In the present study we used these data to determine the genomic positions of chromosomal breakpoints, based on the assumption that intra-chromosomal changes in CNA-status can only be explained by mechanisms that involve chromosomal breaks. Although such an analysis does not provide a comprehensive overview of SVs in the cancer genome, we hypothesized that this analysis would identify a substantial subset of SVs at a sufficient resolution to allocate chromosomal breaks to gene positions. Moreover, we anticipated that non-random recurrent events among CRC samples would reveal genes that enhance tumor development. Here, we show that this approach revealed 748 recurrent breakpoint genes and demonstrate their impact on CRC classification.
Materials and Methods {#sec006}
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Copy number aberration-associated chromosomal breakpoint detection {#sec007}
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Patients selected for the current study participated in either of the two multicentre phase III trials of the Dutch Colorectal Cancer Group (DCCG), namely CAIRO (CKTO 2002--07, ClinicalTrials.gov; NCT00312000) and CAIRO2 (CKTO 2005--02, ClinicalTrials.gov; NCT00208546). The two randomized clinical trials were approved by the Committee on Human-Related Research Arnhem---Nijmegen and by the local institutional review boards. The written informed consent required for all patients before study entry also included translational research on tumour tissue. CNA-associated chromosomal breakpoint detection was performed across 352 CRC samples. Array-CGH data were previously obtained using DNA isolated from formalin-fixed paraffin-embedded (FFPE) primary tumors and patient-matched normal tissue pairs \[[@pone.0138141.ref009]\]. The 4548 probes that had been added to enrich for the coverage of 238 Cancer Census genes were now excluded for chromosomal breakpoint analysis, leaving 168823 probes that were evenly distributed across the genome at approximately 17kb intervals ([S1 Table](#pone.0138141.s002){ref-type="supplementary-material"}). Genomic probe positions were based on human genome NCBI Build36/hg18. Array-CGH data have been deposited in NCBI's Gene Expression Omnibus (GEO accession number GSE63216). DNA copy number segments were defined by the R-package "DNAcopy" (version 1.36.0) and were demarcated by the first and last probe of the segment \[[@pone.0138141.ref010]\]. Chromosomal breakpoints were defined by the genomic start positions of DNA copy number segments ([Fig 1B](#pone.0138141.g001){ref-type="fig"}) with the exception of the first DNA segment of each chromosome and of breakpoints between two copy number neutral regions as defined by the R-package "CGHcall" (version 2.17.6) \[[@pone.0138141.ref011]\]. To detect genes that were affected by CNA-associated chromosomal breaks, breakpoints were mapped to gene annotations based on human reference genome hg18/Ensembl54.
{#pone.0138141.g001}
Statistical analysis of chromosomal breakpoint detection {#sec008}
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A dedicated statistical significance analysis was devised for the gene-based chromosomal breakpoint analysis, consisting of three steps. First, per array-CGH profile the baseline probability of a breakpoint occurring in a gene at random was determined, accounting for the number of breakpoints in a profile, gene length by gene-associated probe coverage and the number of gene-associated probes using a logistic regression. Second, the test statistic was defined as the number of profiles with at least one breakpoint in a given gene. Then, a *P*-value was computed from the null-distribution of the test statistic. This null-distribution was a convolution (over independent profiles) of Bernoulli random variables with a gene- and profile-specific 'success (= breakpoint) probability'. Third, to all *P*-values of the candidate breakpoint genes, multiple testing was applied by a dedicated Benjamini-Hochberg-type FDR correction \[[@pone.0138141.ref012]\]. This correction accounts for the discreteness of the null-distribution. The probe-based chromosomal breakpoint statistical analysis was performed under the assumption that per array-CGH profile the probability to be a CNA-associated breakpoint probe is equal across probes. In this case the dedicated Benjamini-Hochberg-type FDR correction is equivalent to the standard Benjamini-Hochberg FDR correction, because, unlike for the genes, all probes correspond to the same null-distribution. FDR less than 0.1 was considered significant.
Gene mutation analysis {#sec009}
----------------------
*APC*, *TP53*, *KRAS*, *PIK3CA*, *FBXW7*, *SMAD4*, *BRAF* and *NRAS* are genes with a published mutation prevalence in CRC of approximately 3% or more \[[@pone.0138141.ref004]\]. FFPE DNA samples were analyzed by next generation sequencing using the TruSeq Amplicon Cancer Panel (TSACP; Illumina Inc, San Diego, CA USA). Gene mutation status was determined using the variant caller pipeline "Falco" \[[@pone.0138141.ref013]\]. Reads were aligned to the human reference genome (NCBI Build37/hg19) and variants were annotated to dbSNP entries (build 137). Mutations were called when the annotated variant was observed in at least 20% of the reads and was designated as a non-synonymous aberration.
Network Based Stratification {#sec010}
----------------------------
NBS was used to cluster CRC samples, while including information from gene breakpoint and gene mutation molecular interactions \[[@pone.0138141.ref014]\]. The baseline clinicopathological characteristics of the CRC samples (n = 203, see [S5 Table](#pone.0138141.s006){ref-type="supplementary-material"}) were similar to the series analyzed by Haan et al. \[[@pone.0138141.ref009]\]. The *SMAD4* gene acquired both breakpoints and mutations, which were merged for NBS analysis. For the network propagation step the predefined STRING human protein interaction network was used as supplied with the NBS distribution. NBS parameters were set to their default values except for *k* that was set to 4. Using the sample-similarity matrix from NBS, samples were assigned to CRC subtypes by average linkage hierarchical clustering. CRC patients were clustered into four CRC subtypes and OS rates were visualized by Kaplan-Meier curves and corresponding *P*-values were calculated by log-rank testing.
CRC subtype-associated genes {#sec011}
----------------------------
NBS does not provide network-based gene aberration scores as standard output. Therefore, to determine what genes were significantly associated with a specific CRC subtype, the network-based gene aberration scores for each gene per sample were extracted as follows. First, for every NBS iteration *i* of in total *n* iterations (n = 1000) the input matrices *V* ~*i*~ were reconstructed from the factor matrices *W* ~*i*~ and *H* ~*i*~ that were obtained during the non-negative matrix factorization procedure: $$V_{i} = \, W_{i}H_{i}$$
The matrices, *V* ~*i*~, represent the data used by NBS to determine the sample clustering for every iteration. An averaged vector of network-based gene aberration scores, *R* ~*s*~ was now obtained for every sample *s* by averaging over the input matrices *V* ~*i*~ across all *n* iterations: $$R_{s} = \frac{1}{C_{s}}\left\lbrack {\sum\limits_{i = 1}^{n}V_{is}} \right\rbrack$$
Here, *V* ~*is*~ is the row from *V* ~*s*~ that corresponds to sample *s* in iteration *i*. *C* ~*s*~ is a normalization factor, defined as the number of iterations in which a sample *s* was selected for clustering. Note that if a sample was not selected during clustering all *V* ~*is*~ values are set to zero. Mann-Whitney U tests were performed over the averaged network-based gene-aberration scores to test if a specific gene contributed to the formation of a CRC subtype. For every gene these *R* ~*s*~ scores were grouped according to the CRC subtype to determine the *P*-values, indicating whether a gene contributed significantly to the formation of a specific CRC subtype.
Multi-Dendrix {#sec012}
-------------
Data input for Multi-Dendrix analysis was identical to the data input used for NBS analysis, except for genes that shared the same breakpoints, which were now grouped in "pools" ([S8 Table](#pone.0138141.s009){ref-type="supplementary-material"}). Multi-Dendrix parameters were set to k7t7s11 \[[@pone.0138141.ref015]\].
Results {#sec013}
=======
Detection of recurrent breakpoint genes {#sec014}
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Chromosomal CNA status of 352 primary advanced CRC samples was determined using 180K Agilent arrays that cover the genome with an average probe spacing of approximately 17kb, as previously described \[[@pone.0138141.ref009]\]. Following DNA copy number segmentation, the genomic locations of changes in DNA copy number status were now used to estimate the position of CNA-associated chromosomal breakpoints ([Fig 1A and 1B](#pone.0138141.g001){ref-type="fig"}). Statistical evaluation yielded 1605 genomic breakpoint locations with recurrences in multiple CRC samples (FDR\<0.1; [S1 Table](#pone.0138141.s002){ref-type="supplementary-material"}), indicating that the position of CNA-associated breaks is often non-random. When grouping breakpoints by gene affected, a total of 748 recurrent breakpoint genes were identified (FDR\<0.1; [S2 Table](#pone.0138141.s003){ref-type="supplementary-material"}). The genome distribution and prevalence of chromosomal breakpoints at the q-arm of chromosome 13 is provided in [Fig 1C](#pone.0138141.g001){ref-type="fig"} and for all other chromosomes in [S1 Fig](#pone.0138141.s001){ref-type="supplementary-material"}.
The gene with highest prevalence of chromosomal breakpoints was *MACROD2*, which was affected in 40.9% of CRC samples. Another 169 recurrent breakpoint genes were affected in \>3% of advanced CRC samples, similar to the mutation frequencies of commonly affected oncogenes and tumor suppressor genes ([Fig 2](#pone.0138141.g002){ref-type="fig"}).
{#pone.0138141.g002}
Clinical relevance of recurrent breakpoint genes {#sec015}
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Recurrent breakpoint genes may represent genomic regions that are vulnerable to chromosomal breaks, *i*.*e*. an epiphenomenon associated with CNAs. Alternatively, recurrent breakpoint genes may drive cancer and undergo positive selection during tumorigenesis, and consequently affect clinical outcome such as patient overall survival (OS). Therefore, for each of the recurrent breakpoint genes that was identified, the OS of the subgroup of patients with that specific gene breakpoint was compared to the subgroup of patients without that breakpoint. None of the individual recurrent breakpoint genes was significantly associated with OS (log-rank *P*-values followed by Bonferroni correction for multiple testing, data not shown).
Cancer-related biological processes are complex and controlled by the concerted action of multiple genes. For that reason we performed a combined analysis of the 170 most prevalent (\>3%) recurrent breakpoint genes described above and gene mutation status of key cancer genes. Using DNA isolated from formalin-fixed paraffin-embedded archival tissue, mutation status of *TP53*, *APC*, *KRAS*, *PIK3CA*, *FBXW7*, *SMAD4*, *BRAF* and *NRAS* was determined by targeted next generation sequencing, which succeeded for 204 CRC samples ([Fig 2](#pone.0138141.g002){ref-type="fig"}, [S3](#pone.0138141.s004){ref-type="supplementary-material"} and [S4](#pone.0138141.s005){ref-type="supplementary-material"} Tables). As one case lacked breakpoints and mutations for the selected genes, 203 cases were available to provide both gene breakpoint and gene mutation data as input for Network Based Stratification (NBS) \[[@pone.0138141.ref014]\]. NBS was applied to propagate sparse gene breakpoint and gene mutation events to the predefined protein interaction network STRING followed by clustering of patients into CRC subtypes based on the affected sub-networks \[[@pone.0138141.ref014]\]. This analysis revealed four CRC subtypes ([Fig 3A](#pone.0138141.g003){ref-type="fig"} and [S6 Table](#pone.0138141.s007){ref-type="supplementary-material"}). Baseline clinicopathological patient characteristics were highly comparable across the four CRC subtypes (one-side Fisher Exact test; [S5 Table](#pone.0138141.s006){ref-type="supplementary-material"}). OS analysis revealed significant differences among these subtypes (log-rank *P* = 0.001; [Fig 3B](#pone.0138141.g003){ref-type="fig"}), with CRC subtype 3 having a significantly poorer OS than the other three CRC subtypes (HR = 2.17; log-rank *P* = 0.0002; [Fig 3C](#pone.0138141.g003){ref-type="fig"}), with 218 days difference in median overall survival.
{#pone.0138141.g003}
When further exploring the networks associated with this CRC classification, most of the contributing genes turned out to be recurrent breakpoint genes supplemented with some of the commonly mutated CRC oncogenes and tumor suppressor genes ([S7 Table](#pone.0138141.s008){ref-type="supplementary-material"}). At an individual gene level within the identified CRC subtype-associated genes, the poor prognostic CRC subtype 3 was *a*.*o*. enriched for gene point mutations in *BRAF* (two-sided Fisher Exact test: *P*\<0.0001) and *FBXW7* (*P* = 0.01), and for gene breakpoints in *WWOX* (*P*\<0.0001), *FHIT* (*P*\<0.0001), and *PIBF1* (*P* = 0.03). Because mutations in *BRAF* are often associated with microsatellite instable (MSI) tumors \[[@pone.0138141.ref016]\], we examined the distribution of MSI samples across the four CRC subtypes. Interestingly, eight out of ten MSI samples in this group of 203 CRCs were in subtype 3 (two-sided Fisher Exact test: *P*\<0.0001; [S5 Table](#pone.0138141.s006){ref-type="supplementary-material"}). Taken together, these data indicate that CNA-associated recurrent breakpoint genes are clinically relevant as they contribute to CRC classification of subtypes with prognostic value.
Biological relevance of recurrent breakpoint genes {#sec016}
--------------------------------------------------
To further investigate whether recurrent breakpoint genes may drive CRC development, the Multi-Dendrix algorithm was applied to identify oncogenic pathways or gene modules based on two criteria, namely: 1) the events within one module must be mutually exclusive, and 2) these events should cover nearly all cancer samples studied \[[@pone.0138141.ref015]\]. The input data for this analysis was identical to that for NBS, *i*.*e*. the gene mutation status of eight commonly affected CRC genes and the breakpoint status of the 170 most prevalent (\>3%) recurrent breakpoint genes from 203 CRC samples. This analysis revealed four distinct gene modules, three modules containing both gene mutations and gene breakpoints and one module being entirely composed of recurrent breakpoint genes ([Fig 4](#pone.0138141.g004){ref-type="fig"}). The strongest mutual exclusivity was observed between *TP53* mutations and *PIBF1* breakpoints, a gene whose breakpoints were most prevalent in the CRC subtype 3 that showed poorest prognosis. The genomic location of *PIBF1* on the q-arm of chromosome 13 is highlighted in [Fig 1D](#pone.0138141.g001){ref-type="fig"}, illustrating enrichment of gene breakpoints in the distal part of this gene. Moreover, also *MACROD2*, *PPP1R12B*, *AKAP13*, *ERGIC1*, *PTPRT*, *SLC22A5*, *HIST1H1A*, *ASNS*, and *ROCK1* breakpoint genes were represented in one of the gene modules, and contributed to CRC subtype classification ([Fig 4](#pone.0138141.g004){ref-type="fig"} and [S7 Table](#pone.0138141.s008){ref-type="supplementary-material"}). These data imply that multiple recurrent breakpoint genes play an important biological role in CRC development.
{#pone.0138141.g004}
Discussion {#sec017}
==========
Molecular characterization of somatic DNA aberrations is a helpful strategy to aid prognosis and therapy prediction of individual patients. While the analysis of non-synonymous point mutations in commonly mutated cancer genes and determination of chromosomal CNAs have become standard of practice for characterizing tumor samples, large-scale genome-wide detailed analysis of SVs is still in its infancy. We here demonstrated that CNA profiles allow to detect genes whose function may be affected by CNA-associated chromosomal breaks. In total 748 recurrent breakpoint genes were identified based on the analysis of a large series (n = 352) of high-resolution array-CGH samples of primary tumors from patients who participated in two phase III clinical trials in metastatic CRC. In addition to their abundance also the prevalence of recurrent breakpoint genes was relatively high, with 170 genes being affected by chromosomal breaks in more than 3% of cancer cases. As such, the prevalence of genes affected by CNA-associated chromosomal breaks is comparable to the prevalence of point mutations in well-known and commonly affected CRC oncogenes and tumor suppressor genes ([Fig 2](#pone.0138141.g002){ref-type="fig"}).
One of the key questions we aimed to address is whether chromosomal breaks within genes are just an epiphenomenon associated with chromosomal instability or whether recurrent breakpoint genes represent cancer drivers with biological and clinical relevance. While none of the individual recurrent breakpoint genes exhibited significant effects on OS, propagation of the 170 most prevalent breakpoint genes in combination with eight commonly mutated genes onto a predefined network allowed to classify CRC into four subtypes by NBS. One of the CRC subtypes, CRC subtype 3, had a significantly poorer prognosis than the others ([Fig 3](#pone.0138141.g003){ref-type="fig"}), indicating that clinically distinct subtypes could be identified. In a multivariate analysis (data not shown) the factors 'WHO performance status', 'LDH at randomization', 'prior adjuvant therapy', 'tumor stage primary tumor', 'number of affected organs' and 'MSI status' were retained. Because genomic mutations are causal for tumorigenesis and dictate tumor behavior, it is very well possible that phenotypic factors, which ultimately are a result of underlying biology, mask the prognostic effect of the genomic CRC subtypes. Such a dependency between clinicopathological prognostic parameters and the CRC subtypes as described here therefore does not refute univariate prognostic value of this classification.
CRC subtype 3 turned out to be enriched for tumors with *BRAF* mutations (33% of cases *versus* 5% in the other CRC samples; [S7 Table](#pone.0138141.s008){ref-type="supplementary-material"}) and MSI tumors (30% of cases *versus* 1% in the other CRC samples). *BRAF* is mutated at much higher frequencies in MSI tumors than in chromosomal instable tumors, and within MSI tumors, *BRAF* mutation is known to be associated with poor prognosis \[[@pone.0138141.ref016]\]. Although MSI tumors have a relatively good prognosis in early stage disease, they also are associated with explicitly poor prognosis in metastatic CRC \[[@pone.0138141.ref017]\]. MSI tumors often have a lower frequency of CNA aberrations than tumors that are microsatellite stable (MSS) and therefore have less CNA-associated chromosomal breakpoints than MSS tumors. This suggests that MSI tumors may become clustered into one distinct CRC subtype irrespective of (alterations in) function of recurrent breakpoint genes. Following this line of reasoning, one would predict that the poor prognosis CRC subtype 3 lacks recurrent breakpoint genes with increased mutation frequencies compared to the other CRC subtypes. However, our data showed otherwise ([S7 Table](#pone.0138141.s008){ref-type="supplementary-material"}), with significant enrichment of breakpoint frequencies in CRC subtype 3 *versus* the other CRC samples for *WWOX* (33% *versus* 5%), *FHIT* (59% *versus* 13%), and *PIBF1* (15% *versus* 3%). These data emphasize that recurrent breakpoint genes contribute significantly to clinically relevant CRC classification.
As our data support clinical relevance of recurrent breakpoint genes, it is expected that chromosomal breaks within these genes somehow result in positive selection of cancer cells and stimulate tumor development. Functional analysis of recurrent breakpoint genes to understand their biological effects was beyond the scope of the current study. However, for many of these a role in tumorigenesis has been described in literature. *WWOX* and *FHIT* have long been known to reside at common fragile sites and have been demonstrated to act as suppressors of tumor development by gene knockout mouse models \[[@pone.0138141.ref018]\]. Moreover, *WWOX* overexpression was shown to promote the immune response in a glioma model \[[@pone.0138141.ref019]\] while *FHIT* positively regulates expression of MHC class I molecules on cancer cells \[[@pone.0138141.ref020]\]. These data suggest that loss of function of *WWOX* and *FHIT* help to escape immunosurveillance. Likewise, the progesterone immunomodulatory binding factor *PIBF1* was identified as a secreted factor that can prevent pregnancy loss by dampening the immune response. Considering that *PIBF1* breakpoints were predominantly observed in the distal part of the gene ([Fig 1D](#pone.0138141.g001){ref-type="fig"}) it is tempting to speculate that *PIBF1* gene breakpoints disrupt its nuclear localization signal upon which it becomes a secreted protein with anti-tumor immune-suppressing capabilities \[[@pone.0138141.ref021]\]. MSI tumors are thought to evoke an anti-tumor immune response that prevents metastatic spread, however, once circumvented these tumors become very aggressive \[[@pone.0138141.ref016]\]. In this respect it is of interest to note that the breakpoint genes that contributed most to the classification of the poor prognosis CRC subtype 3, *i*.*e*. *WWOX*, *FHIT*, and *PIBF1*, all have been implicated to modulate immune responses.
*MACROD2* was the most prevalent recurrent breakpoint gene in our cohort, being affected in 41% of CRC cases. This gene was also one of the most frequently observed rearranged genes through a focal deletion in other studies \[[@pone.0138141.ref003],[@pone.0138141.ref022]\]. *MACROD2* is able to hydrolyze endogenous mono-ADP-ribosyl groups, a reversible post-translational modification moiety, from target proteins such as *GSK3B*. This restores the function of *GSK3B*, which is a key inhibitor of the Wnt signaling pathway. Hence, the absence of functional *MACROD2* may decrease the kinase activity of *GSK3B* and thereby promote Wnt signaling \[[@pone.0138141.ref023]--[@pone.0138141.ref025]\]. Moreover, *MACROD2* may play a role in modulating the function of histone proteins and is recruited in case of DNA damage response \[[@pone.0138141.ref023],[@pone.0138141.ref025]\].
To further address the biological relevance of recurrent breakpoint genes we tried to construct modules of putative cancer driving genes, using Multi-Dendrix. On the one hand, this analysis can reveal oncogenic pathways by looking for mutually exclusive gene mutation patterns that cover nearly all CRC samples. On the other hand, if an apparently homogeneous group of tumors consists of distinct tumor subtypes, mutual exclusivity between genes can also reflect the presence of (previously unrecognized) cancer subtypes. The strongest mutual exclusivity was observed between *TP53* mutations and *PIBF1* breakpoints ([Fig 4](#pone.0138141.g004){ref-type="fig"}). Loss of *TP53* function is a critical step towards chromosomal instability while *PIBF1* breakpoints were enriched in CRC subtype 3, which harbored the majority of MSI tumors. Therefore, the *TP53*---*PIBF1* module may represent genes that drive genomic instability, comprising distinct chromosomal instable and microsatellite instable CRC subtypes. Another gene module contained *APC*, *KRAS* and *BRAF* mutations, *i*.*e*. somatic alterations that are known to occur early in tumor development compared to *TP53* aberrations. This module supports the known mutual exclusivity between *KRAS* and *BRAF* mutations. Moreover, it included *ROCK1* breakpoints, which are enriched in CRC subtype 4 ([S7 Table](#pone.0138141.s008){ref-type="supplementary-material"}). Interestingly, inhibitors of *ROCK1* improve the success rate of bringing embryonic stem cells into culture \[[@pone.0138141.ref026]\] and are supplemented to the medium for *in vitro* cultures of human colon epithelial organoids, indicating that inhibition of *ROCK1* supports early stages of tumor development. With the exception of *ROCK1*, there appeared to be limited overlap between recurrent breakpoint genes in the Multi-Dendrix modules and the currently well-studied signal transduction pathways in cancer, suggesting that recurrent breakpoint genes may affect carcinogenesis through mechanisms that require further research.
*MACROD2* was part of a module together with four other recurrent breakpoint genes, *i*.*e*. *TRIM38*, *MTA1*, *PTPRT* and *ASNS*. The fact that these genes appear in one module suggests that they may act together in one pathway. Although extensive knowledge about the function of these genes is not available, it appears that most of them are (in)directly capable to affect Wnt signaling. As discussed, loss of *MACROD2* can promote Wnt signaling through decreasing *GSK3B* kinase activity \[[@pone.0138141.ref023]--[@pone.0138141.ref025]\]. Also *MTA1* has been described to affect Wnt signaling through modulation of *GSK3B* activity \[[@pone.0138141.ref027]\]. *PTPRT* is able to dephosphorylate *STAT3* \[[@pone.0138141.ref028]\], which in turn can interact with and modulate the function of the WNT pathway mediator β-catenin \[[@pone.0138141.ref029]\]. The canonical Wnt pathway may also be activated by elevation of NF-κB signaling, which results in cell dedifferentiation towards tumor-initiating cells \[[@pone.0138141.ref030]\]. *TRIM38* may counteract NF-κB activity \[[@pone.0138141.ref031]\], suggesting that loss of *TRIM38* might enhance tumorigenesis. Taken together, this module comprises genes whose function may directly or indirectly affect different aspects of the Wnt pathway, a hypothesis that needs further investigation by functional analysis of (combinations of) these genes.
Technically, the genome-wide detection of SVs at nucleotide resolution is currently possible by next generation sequencing. However, in practice genome-wide analyses of SVs of large sample series with well-documented patient survival and other clinical information are scarce. In contrast, DNA CNA profiles of primary tumors are abundantly available in public archives and are still being generated for molecular characterization of cancer samples. In our study, the molecular profile of the primary tumors was used to characterize metastatic disease, because DNA aberrations of metastases show high concordance with the patient-matched primary tumor counterpart \[[@pone.0138141.ref032],[@pone.0138141.ref033]\]. We now demonstrated that these commonly and widely available DNA CNA profiles allow detection of a significant subset of SVs, *i*.*e*. genes with CNA-associated chromosomal breaks. Importantly, we showed that recurrent breakpoint genes are highly prevalent and clinically relevant, emphasizing the need to characterize larger sample series from more tumor types to fully appreciate their impact. We therefore argue that, in addition to gene mutation and gene copy number analyses, molecular characterization of cancer samples should also comprise the detection of recurrent breakpoint genes.
Supporting Information {#sec018}
======================
###### Chromosomal breakpoint frequencies and their distribution over chromosomes.
(PDF)
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Click here for additional data file.
###### Chromosomal breakpoints, probe-level.
(XLSX)
######
Click here for additional data file.
###### Chromosomal breakpoints, gene-level.
(XLSX)
######
Click here for additional data file.
###### Gene mutation matrix, per CRC sample.
(XLSX)
######
Click here for additional data file.
###### Gene mutation frequencies, summary.
(XLSX)
######
Click here for additional data file.
###### Baseline patient characteristics.
(XLSX)
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Click here for additional data file.
###### CRC subtype and OS data.
(XLSX)
######
Click here for additional data file.
###### CRC subtype-associated genes.
(XLSX)
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Click here for additional data file.
###### Pools of genes that share probe(s) associated with chromosomal breakpoints.
(XLSX)
######
Click here for additional data file.
The authors thank Marianne Tijssen, Sandra Mongera, Dirk van Essen, Francois Rustenburg and Danielle Heideman (VU University medical center, The Netherlands) for expert assistance with the TSACP gene mutation analysis.
[^1]: **Competing Interests:**The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: EB GAM RJAF. Analyzed the data: EB MJJD OK DS JCH JJHT MAW SA GAM RJAF. Contributed reagents/materials/analysis tools: JCH IDN CJAP BC BY GAM. Wrote the paper: EB MJJD OK DS JCH JJHT MAW IDN CJAP BC BY SA GAM RJAF.
[^3]: Current Address: Netherlands Cancer Institute, Amsterdam, The Netherlands
| {
"pile_set_name": "PubMed Central"
} |
INTRODUCTION {#sec1-1}
============
Head and neck malignancy and its treatment might adversly influence the patient\'s activities of daily living These patients have issues with regards to speech capacity, sustenance, and appearance (Deleyiannis, et al, 1997, Gellrich, et al, 2002).\[[@ref1][@ref2]\]
Longitudinal information uncovers personal satisfaction of treatment even when swallowing and speech troubles proceed to exist (Dholam *et al*., 2013, Mady *et al*., 2003).\[[@ref3][@ref4]\] Patients with oral and oropharyngeal malignancy are particularly prone to speech difficulties. Speech outcome depends upon flexibility of structures in the oral cavity and oropharynx.\[[@ref5]\]
Sufficient control of lips, tongue, and soft palate is important for production of speech. Any impairment in the range of movement, strength, and adaptability of these dynamic articulators might influence the capacity to make exact individual speech movements and coarticulations required in connected speech.
The impacts of cancer on speech rely on the area and size of the growth. For instance, a sore or lump on the lips might restrict movement. This could bring about unclear production of speech sounds made with the lips, for example, p, b and m. Cancer of the tongue can bring about issues with many sounds, for example, if lesion is in the anterior portion of the tongue, or in the posterior aspect of the tongue, articulation is affected.
The outcomes after surgical treatment depends on the region and length of the cancerous growth. Other essential factors consist of quantity of tissue removed in surgical operation, frequency of speech/swallowing treatment, and initiative of the patient.\[[@ref6][@ref7]\]
The routines used for speech evaluation are heterogeneous, and they go from assessment of single phoneme\[[@ref8]\] or word\[[@ref9]\] on semiqualitative scales and identification of the spoken word by testing of communicative intelligibility in questions, descriptions,\[[@ref10]\] or longer text passages.\[[@ref8]\] They range from assessment of single phoneme or word on semiqualitative scales and communicative intelligibility of the spoken words are evaluated in questions, descriptions, or longer text passages.
Speech outcome is evaluated by the utilization of pointers of speech production (oral capacity and verbalization tests, air motion facilitating, and acoustical breaks down), speech recognition (coherence and adequacy), and self-reported speech adequacy in ordinary life circumstances (surveys). Various methodological contrasts exist between studies on speech nature of patients treated for oral or oropharyngeal malignancy. By and by, it can be reasoned that discourse challenges are exceedingly reliant on tumor size and site. Eagerly, patients experiencing resection of bigger tumors have more speech troubles. After resection of oral carcinomas, patients experience verbalization issues due to tissue misfortune, structure modification, or tongue mobility impairment. Target sounds may be misshaped, substituted, or discarded, prompting diminished comprehensibility. Speech production issues of patients with oropharyngeal deformities incorporate nasal reverberation issues in view of velopharyngeal insufficiency. On account of tissue misfortune or versatility disability, air will escape through the nose, vowels sound nasal, and inadequate weight can be developed in the oral cavity to deliver stops and fricatives. On account of proceeded with velopharyngeal conclusion, the air stream cannot escape through the nose, and the nasal consonants are denasalized.
Research has demonstrated that speech results can be a real determinant of the tolerant postoperative personal satisfaction (Radford, *et al*., 2004).\[[@ref11]\] In any case, the surgical variables focus postoperative speech and tongue capacity are not totally comprehended and not one or the other is the particular attribute of glossectomy speech (Bressmann, *et al*., 2004).\[[@ref12]\] Specialists have contended that degree of the resection, Rentschler 1980,\[[@ref13]\] deformity site (Logemann *et al*., 1993, Michiwaki, *et al*., 1993),\[[@ref14][@ref15]\] and reproduction system for the imperfection (Konstantinovic and Dimic, 1998)\[[@ref16]\] are the pivotal variables deciding the postoperative discourse results. There likewise has been exchange whether the leftover tongue ought to be kept adaptable, at the cost of lessening it in volume (Imai and Michi, 1992),\[[@ref17]\] or whether cumbersome, raised folds ought to be utilized to supplant lost tissue (Yanai *et al*., 2008,\[[@ref18]\] Kimata *et al*., 2003).\[[@ref19]\] Pauloski *et al*., 1998\[[@ref20][@ref21]\] evaluated the speech of 142 incomplete glossectomy patients and found that the degree of the resection related with the reduction in articulatory exactness.
Nicoletti *et al*.\[[@ref22]\] utilized a robotized speech analyzer to survey the creation of select fricative sounds, for example,/s/,/∫/,/f/, and/è/in 196 patients. The results from the program analyzer were joined with a general measure of speech worthiness (\"conversational understandability\"). Bressmann *et al*.\[[@ref23]\] said that the most frequently distorted target consonant was/g/, followed by/s/in preoperatively; after surgery, the total number of articulatory distortions observed was increased and the most frequently distorted target consonant was/d/, followed by/k/,/r/,/s/, and/t∫/.
In a late study utilizing ultrasound imaging, Rastadmehr, *et al*., 2008,\[[@ref24]\] found that the inverse was the situation in a gathering of ten patients with little- to medium-sized deformities. In spite of desires, the glossectomy patients expanded the tallness and the pace of their midsagittal tongue development in the postoperative talking condition. This impact was seen in all patients, paying little heed to the system of imperfection recreation. It is conceivable that glossectomy patients energetically adjust for a loss of lingual tissue by making more extensive and quicker developments with the lingering tongue, Fletcher 1988.\[[@ref25]\]
Aim {#sec2-1}
---
The aim of this study was to examine speech outcome difficulties by method for a multidimensional speech evaluation in pre- and post-operative patients with oral cancer and speech challenges in connection to tumor site.
METHODOLOGY {#sec1-2}
===========
Participants {#sec2-2}
------------
Twelve pre- and post-operative patients (totally, 24) in the age range between 20 and 60 years with oral cancers (buccal and tongue cancer patients) were included in the study. In preoperative patients, five were with buccal cancers (buccal mucosa carcinoma) and seven were with tongue cancers (tongue lateral border carcinoma). In postoperative patients, five were with buccal cancers and seven were with tongue cancers. The buccal cancer patients were operated by composite resection and pectoralis major myocutaneous (PMMC) flap reconstruction. The tongue cancer patients were operated by hemiglossectomy (for five patients) and composite resection and PMMC flap reconstruction (for two patients).
Assessment of phoneme production {#sec2-3}
--------------------------------
For articulation evaluation, "Tamil articulation test" was used. The words that contain target phoneme were first presented by a clinician and the patient has to repeat the words. Each sound was marked as normal or as distorted, substituted, added, and omitted.
Assessment of speech intelligibility {#sec2-4}
------------------------------------
Overall, intelligibility was calculated by percentage of number of words perceived intelligible divided by total number of words spoken. For this, the patient has to speak about any general topic and his/her speech were recorded and analyzed by a clinician who has the same native language of patients.
RESULTS {#sec1-3}
=======
Phoneme production {#sec2-5}
------------------
In buccal cancer patients, preoperatively, no articulatory errors were found. In postoperative evaluation of phonemes, substitution and distortion errors were observed in linguadental, linguopalatal, and linguavelar sounds. Most frequently distorted consonant was/t/, followed by/ʃ/,/ț/,/g/, and/r/. Furthermore, intraoral pressure was reduced in some postoperative patients.
In tongue cancer patients, preoperatively, substitution and distortion errors were noted in lingua-alveolar and linguopalatal sounds. In postoperative patients, substitution, distortion, and omission errors were noted in bilabial, lingua-alveolar, and linguopalatal sounds and also in patients who underwent hemiglossectomy have less error than who underwent flap reconstruction. In tongue cancer patients prior to surgery, the most frequently distorted target consonant was/r/,/t/, followed by/s/,/t∫/,/dʒ/, and/l/. Postoperatively, the most frequently distorted target consonant was/t/, followed by/d/,/t∫/,/r/, and/b/. Some substitution errors are also noted for consonants/k/and/ʃ/.
Speech intelligibility {#sec2-6}
----------------------
Preoperatively, speech intelligibility for buccal cancer patients is above 80% and up to 100%, with an average of 93.07%, and postoperatively, it ranges from 78--96%, with an average of 91.4%.
In tongue cancer patients, preoperatively, the intelligibility score ranges from 30% to 100%, an average of 74.28%, and postoperatively, it ranges from 20--96%, average of 73.42%.
DISCUSSION {#sec1-4}
==========
This study evaluated the speech characteristics of a small, convenience-sampled group of oral cancer patients. After surgery, voice quality and resonance are compromised because of changes in oral cavity volume, and articulation is affected because the tongue is unable to assume the normal position to provide valuing action needed for precise articulation. The results showed that the speech outcome depends on the site of lesion as tongue cancer patients have more articulation errors and less speech intelligibility. The observation showed that even before the surgery, patients had reduced speech intelligibility. In both groups, speech intelligibility worsened significantly after the surgery. It also was found that the patients with larger defects and flap reconstructions had poorer articulation ability than the patients with smaller defects and local reconstructions (hemiglossectomy).
Since there were relatively few consonant distortions observed, the hierarchy of consonant distortions may be of limited transferability to other groups of glossectomy patients. As reported by Bloomer and Hawk,\[[@ref26]\] Kalfuss\[[@ref27]\] evaluated the speech of 22 glossectomy patients and noted distortions of the vowel/i/and of the consonants/l/,/v/,/k/,/g/,/ɵ/,/s/,/z/,/∫/,/t∫/, and/dƷ/. Beck *et al*.^(1998)[@ref28]^ noted distortions of/r/,/l/,/s/,/z/,/∫/,/t∫/, and/dƷ/in five patients with floor of mouth resections and/r/,/j/,/l/,/s/,/k/, and/∫/in five patients with resections of the dorsum of the tongue. The rank order found in our study slightly the same from these previous studies. The differences are probably explained by differences in the defect sizes and locations as well as the reconstructive techniques employed by the surgeons.
The articulation intelligibility was better in patients not receiving flap than in those receiving flap. Reconstruction with flaps, which may additionally intrude with the flexibility and mobility of the tongue, may make contributions to articulatory impairment (Su WF *et al*., 2002).\[[@ref29]\]
Rehabilitation of speech is the most important aspect in preventing severe speech problems and re-establishing interpersonal communication. The rehabilitation for oral cancer patients depended upon the assessment of their postoperative articulation level, education, job, age, family, and motivation. In rehabilitation, one should consider the benefits with use of oral facilitative exercises, direct articulation techniques, compensatory techniques, surgical procedure, and prosthetic appliances (Kirita and Omura 2015).\[[@ref30]\]
Oral facilitative exercises are generally prescribed to improve the strength and range of movement of the articulators. Direct articulation techniques are recommended mostly to improve the production of single or group of phonemes. Compensatory techniques may need to be considered when patient fails to make the correct placement of the target phoneme or group of sounds. Prosthetic procedures are preferred usually than surgical procedures. For example, velopharyngeal incompetence after surgery is generally treated with speech bulb or palatal lift than palatal reconstruction surgeries.
CONCLUSION {#sec1-5}
==========
The perceptual analysis of oral cancer patients showed specific articulation issues and reduced intelligibility of speech in regards to site of lesion and type of reconstruction surgery. However, there is no one to one correlation between articulatory errors in oral cancer patients. Because the ability of the articulation changes based on various factors such as compensatory ability and motivation of the patient, it also varies depend on type of surgery for removal of tumor. After free construction, the patient showed better articulation ability than flap construction. If speech is the outcome of interest, flap reconstruction may not be beneficial with hemiglossectomy or other partial (minor) glossectomy within the hemitongue. A comprehensive speech evaluation and analysis of error patterns would help us in planning the rehabilitative measures of speech which is the most important factor in re-establishing interpersonal communication and well-being of the individual.
Financial support and sponsorship {#sec2-7}
---------------------------------
Nil.
Conflicts of interest {#sec2-8}
---------------------
There are no conflicts of interest.
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1. Introduction {#sec1-materials-10-01167}
===============
The Mg-Li alloy possesses many impressive advantages, such as a low density, high specific elastic modulus, high specific strength, good electromagnetic shielding, and damping property \[[@B1-materials-10-01167],[@B2-materials-10-01167],[@B3-materials-10-01167],[@B4-materials-10-01167]\]. Therefore, it has been widely applied in the fields of weapon, automobile, aerospace, aviation, electronics, and military industries, etc. \[[@B5-materials-10-01167],[@B6-materials-10-01167],[@B7-materials-10-01167]\]. It has been reported that the addition of an Li element could change the Mg crystal structure by reducing the c/a ratio of the hexagonal lattice \[[@B8-materials-10-01167]\]. When the Li content is less than 5.7%, the alloy matrix structure will reveal the hexagonal close-packed (hcp) lattice \[[@B9-materials-10-01167]\]. When the Li content ranges from 5.7--11.3%, the hexagonal close-packed (hcp) structure of Mg will be transformed into a hexagonal close-packed (hcp)+body-centered cubic (bcc) dual-matrix \[[@B10-materials-10-01167]\]. When the Li content is more than 11.3%, the single body-centered cubic (bcc) structure will be presented wholly in the alloy \[[@B11-materials-10-01167]\]. Nevertheless, with an increasing Li content, the mechanical properties of the alloy, corrosion resistance, and high temperature resistance will decline \[[@B12-materials-10-01167]\]. These drawbacks will restrain its wide application in the national economy. Many researchers have found that studies should focus on its strength improvement. Hence, previous researchers have adopted many positive approaches to improve the strength of the Mg-Li alloy and have gained some good results \[[@B13-materials-10-01167],[@B14-materials-10-01167],[@B15-materials-10-01167],[@B16-materials-10-01167],[@B17-materials-10-01167]\]. Their methods involved alloying element addition (Al, Zn, Ca, Sr, and so on), rare earth element addition (Ce, Y, Nd, La, and so on), ageing and solid solution processes, as well as equal channel angular pressing, etc. However, there are few reports about cold rolling and annealing studies on an Mg-Li alloy sheet. Moreover, quantitative analyses and mathematical relationship modeling on the Mg-Li alloy have been rarely reported.
In this paper, the microstructural evolution, mechanical properties, and mathematical relationship of an α, α + β, and β phase Mg-Li alloy during the cold rolling and annealing process were investigated.
2. Experiments {#sec2-materials-10-01167}
==============
The Mg-Li alloys in this investigation involved Mg-5Li-3Al-2Zn-0.2Y, Mg-8Li-3Al-2Zn-0.2Y, and Mg-11Li-3Al-2Zn-0.2Y. The experimental materials were commercial pure Mg ingot, pure Li ingot, pure Al ingot, pure Zn ingot, and Mg-25%Y master alloy ingot. The ingots were melted in an iron crucible under the atmosphere of SF~6~, and simultaneously the flux mixture was used to keep the melt away from the air. Then, the melt was poured into the permanent mould to gain an as-cast alloy. The received cast alloys were then rolled into the sheets during the multi-pass process. After each pass rolling, the sheet was heated at 523 K for 15 min and then rolled in the next pass. The rolling reduction was set as 3%. In the end, a sheet with a thickness of 2 mm was obtained. The completed sheets were heat-treated at different temperatures for 24 h (473 K--573 K) and were then quenched into the cold water. The rolling flow chart of the Mg-Li alloy sheet is shown in [Figure 1](#materials-10-01167-f001){ref-type="fig"}.
Metallographic specimens were polished mechanically and etched with a solution of 5 vol. % nitric acid alcohol. The microstructural observation was examined by an optical microscope (OM) and a scanning electron microscope (SEM, Oxford Instruments (China), Shanghai, China) equipped with an Oxford energy dispersive spectroscope (EDS, Oxford Instruments (China), Shanghai, China). The phase identification was performed with X-ray diffraction (XRD, PANalytical B.V. (China), Beijing, China). The mechanical properties of the alloys were investigated at room temperature on the universal testing machine with a strain rate of 1.0 × 10^−3^ s^−1^.
3. Results and Discussion {#sec3-materials-10-01167}
=========================
3.1. Microstructural Observation {#sec3dot1-materials-10-01167}
--------------------------------
[Figure 2](#materials-10-01167-f002){ref-type="fig"} shows the XRD patterns of the as-cast alloys. It indicates that a transformation between the lattice structures took place with increasing Li content. When the Li content was increased to 5%, the Mg-5Li-3Al-2Zn-0.2Y alloy was mainly composed of α-Mg (hcp) phase, as shown in [Figure 2](#materials-10-01167-f002){ref-type="fig"}a. However, when the Li addition increased to 8%, some Li matrix peaks with relatively high intensity emerged, implying that the Mg-8Li-3Al-2Zn-0.2Y alloy was mainly composed of α-Mg+β-Li dual-phase, as shown in [Figure 2](#materials-10-01167-f002){ref-type="fig"}b. When the Li content was further increased to 11%, the previous α-Mg peak was mostly replaced by the β-Li peak, deducing that the Mg-11Li-3Al-2Zn-0.2Y alloy primarily consisted of β-Li phase, as shown in [Figure 2](#materials-10-01167-f002){ref-type="fig"}c. [Figure 3](#materials-10-01167-f003){ref-type="fig"} shows the microstructures of the as-cast alloys. Based on the previous XRD patterns, the microstructural evolution confirmed that the increasing Li element transformed the Mg lattice structure from hcp to bcc.
[Figure 4](#materials-10-01167-f004){ref-type="fig"} shows the microstructures of the as-rolled alloys. In Mg-5Li-3Al-2Zn-0.2Y (see [Figure 4](#materials-10-01167-f004){ref-type="fig"}a), the α-Mg (white zone) and mixed secondary (dark gray zone) phases were both elongated and well distributed along the rolling direction. Moreover, some shear bands aligned with the rolling direction were presented in the α-Mg matrix, indicating that the α-Mg alloy revealed the poor ability of plastic deformation at low temperature. This shear deformation resulted from the incomplete slip between the crystal lattices during the deformation process. The basal slip was the predominant slip mode during the early deformation. However, the normal slip deformation could not continue after basal slip finishing as the non-basal slips were difficult to activate at low temperature. Hence, the shear deformation emerged and then took over the primary deformation mode in the later deformation process.
In Mg-8Li-3Al-2Zn-0.2Y (see [Figure 4](#materials-10-01167-f004){ref-type="fig"}b), the β-Li phase (black zone) was obviously elongated along the rolling direction and shear bands were not found. It indicated that the plasticity of the β-Li matrix was much better than that of the α-Mg matrix. The difference between them was ascribed to the characteristics of both crystal structures. The β-Li with the bcc structure possessed many more slip systems and a symmetrical crystal structure compared with α-Mg. Hence, the coordinating deformation and dislocation movements between grains in the β-Li matrix are an advantage compared to the α-Mg matrix. Throughout the rolling process, no shear deformation could be observed in the α-Mg phase. It illustrated that the increasing Li element gradually improved the plasticity of the α-Mg matrix. The reason for this improvement was that the Li addition effectively reduced the parameter c/a axial ratio of the Mg crystal structure, promoting the non-basal slips, such as {10-10} prismatic and {10-12} pyramidal slips \[[@B2-materials-10-01167]\]. Thereby, the deformation of the α-Mg matrix would be relatively amenable in the rolling process. In Mg-11Li-3Al-2Zn-0.2Y (see [Figure 4](#materials-10-01167-f004){ref-type="fig"}c), the markedly elongated β-Li microstructure was well distributed along the rolling direction. The results confirmed that the β-Li phase possessed a relatively outstanding plasticity.
[Figure 5](#materials-10-01167-f005){ref-type="fig"} shows the microstructural evolution of the as-rolled alloys during annealing at different temperatures for 24 h (473 K--573 K). In Mg-5Li-3Al-2Zn-0.2Y, a small number of fine equiaxed grains at 473 K were presented in the matrix, indicating that static recrystallization behavior occurred at approximately 473 K. As indicated in [Figure 5](#materials-10-01167-f005){ref-type="fig"}a, the deformed microstructure still occupied the most area in the matrix. At 498 K, a great number of small equiaxed grains emerged from the matrix and the mean grain size was measured as 3.1 μm. Additionally, the previous deformed microstructure was apparently substituted by the recrystallized microstructure, illustrating that the recrystallization behavior had taken place, as shown in [Figure 5](#materials-10-01167-f005){ref-type="fig"}d. With the annealing temperature increasing to 523 K, the grain growth phenomenon was gradually manifested, in which the mean grain size was measured as 8.9 μm, as shown in [Figure 5](#materials-10-01167-f005){ref-type="fig"}g. In the range of 548 K--573 K (see [Figure 5](#materials-10-01167-f005){ref-type="fig"}j,m), the coarse grains and clear grain boundaries emerged, wherein the mean grain size at 548 K and 573 K was 13.5 μm and 20.3 μm, respectively.
Considering the recrystallization and grain growth, the grain (nuclei) growth speed could be characterized by Equation (1). As indicated in Equation (1), the grain (nuclei) growth speed was directly proportional to deformation energy. It illustrated that serious deformation would contribute to accelerating grain growth at the same condition, which could be explained by the fact that the deformed zone with high energy would provide a fast channel for the atomic diffusion to promote highly frequent nucleation. Equation (1) could be further simplified as Equation (2). From the Equation (2), the decreasing growth activation energy would accelerate the grain (nuclei) growth, which could be explained by the fact that the relatively high dislocation density could reduce barriers over which the atoms would stride in order to diffuse thoroughly. Hence, the relatively low activation energy would be in favor of grain growth. The mechanisms of recrystallization and grain growth behaviors should be attributed to the transition process from high free energy to low free energy. $$\left\{ \begin{array}{l}
{V = \frac{D_{B}}{KT} \cdot \frac{E_{S}}{\lambda}} \\
{D_{B} = D_{0}\exp( - \frac{Q_{g}}{RT})} \\
\end{array} \right.$$ where $V$ is the grain (nuclei) growth speed, $D_{B}$ is the diffusion coefficient at the grain boundary, $\lambda$ is the interface width, $K$ is a constant, $E_{S}$ is the deformation energy, $R$ is the gas constant, and $T$ is the temperature. $$V = V_{0}\exp( - \frac{Q_{g}}{RT})$$ where $Q_{g}$ is the growth activation energy.
In Mg-8Li-3Al-2Zn-0.2Y, in the annealing period of 473 K--498 K, the two matrices did not generate clear microstructural evolution, wherein the elongated α and β phases were still distributed along the rolling direction, as shown in [Figure 5](#materials-10-01167-f005){ref-type="fig"}b,e. However, a little variation was gradually exhibited around the β phase edge where the small β grains with a granule shape emerged at 523 K, as shown in [Figure 5](#materials-10-01167-f005){ref-type="fig"}h. It indicated that the recrystallization behavior in the β matrix might take place at approximately 523 K. With the temperature increasing to 548 K, the recrystallized β grains became more numerous than before, as shown in [Figure 5](#materials-10-01167-f005){ref-type="fig"}k. When the alloy was annealed at 573 K, the mean grain size of β was measured as 15.6 μm, as shown in [Figure 5](#materials-10-01167-f005){ref-type="fig"}n. The whole recrystallization process in the β matrix should be ascribed to a recrystallization mechanism where the combination between the subgrains and dislocation absorbing was the main evolution process. In addition, the above--mentioned combination resulted from the transformation among the grain boundary category from a low angle to high angle \[[@B18-materials-10-01167]\].
In Mg-11Li-3Al-2Zn-0.2Y, no obvious microstructural variation could be found below 498 K, as shown in [Figure 5](#materials-10-01167-f005){ref-type="fig"}c,f. When annealing treatment was set at 523 K (see [Figure 5](#materials-10-01167-f005){ref-type="fig"}i), a certain number of tremendous grains emerged around the fine grain zone, which were almost 100 times as big as the fine grains. With the increased annealing temperature (548 K), this phenomenon became more apparent, as shown in [Figure 5](#materials-10-01167-f005){ref-type="fig"}l. With annealing at 573 K, the coarse grains were predominant, and simultaneously a small number of residual fine grains were presented in the matrix, as shown in [Figure 5](#materials-10-01167-f005){ref-type="fig"}o. This behavior mentioned above belonged to the abnormal grain growth whose activation could be characterized by the driving force during the abnormal grain growth (see Equation (3)). As indicated in Equation (3), the activation of abnormal grain growth was the result of an interfacial energy difference between grains. Furthermore, the grain size in the matrix was closely associated with the interfacial energy. Obviously, the size of advantageous grain was coarser than that of fine grain. Hence, *p* \> 0 and the abnormal grain growth would be activated. $$\left\{ \begin{array}{l}
{p = \Delta\gamma} \\
{\Delta\gamma = a\gamma(\frac{1}{\overline{D}} - \frac{1}{D})} \\
\end{array} \right.$$ where $p$ the is driving force, $\Delta\gamma$ is the interfacial energy difference, $\overline{D}$ is the mean grain diameter of fine grain, $D$ is the grain diameter of advantageous grain, and $a$ is a constant.
In the stable recrystallized matrix, a small number of advantageous grains were treated as the nuclei for abnormal grain growth. These advantageous grains could grow preferentially because of the inconsistently dissolved secondary phases. Moreover, their morphology category almost belonged to the polyhedron (\>6) whose surface was revealed as concave in favor of grain boundary diffusion. In the growing process, the existence of a particular orientation difference contributed to enhancing the migration velocity to effectively promote the abnormal grain growth.
[Figure 6](#materials-10-01167-f006){ref-type="fig"} shows the XRD patterns of the as-rolled alloys during annealing for 24 h (473 K--573 K). In Mg-5Li-3Al-2Zn-0.2Y (see [Figure 6](#materials-10-01167-f006){ref-type="fig"}a), with increasing temperature, no evident phase evolution could be observed. In Mg-8Li-3Al-2Zn-0.2Y (see [Figure 6](#materials-10-01167-f006){ref-type="fig"}b), the peak altitude of AlLi phase manifested a little fluctuation with increasing temperature. However, the other phases remained stable. In Mg-11Li-3Al-2Zn-0.2Y (see [Figure 6](#materials-10-01167-f006){ref-type="fig"}c), the AlLi peak variation was consistent with that in Mg-8Li-3Al-2Zn-0.2Y, indicating that the elevating temperature changed the peak of AlLi phase. The above-mentioned results illustrated that the annealing temperature might influence the AlLi phase solubility in the matrix.
[Figure 7](#materials-10-01167-f007){ref-type="fig"} shows the SEM results of the as-rolled alloys during annealing for 24 h (473 K--573 K). In Mg-5Li-3Al-2Zn-0.2Y, the crushed Al~2~Y phase was steadily distributed in the matrix with increasing temperature. Meanwhile, no other obvious change was observed in the matrix, as shown in [Figure 7](#materials-10-01167-f007){ref-type="fig"}a,d,g,j,m. The EDS results were obtained to make a better analysis for Al~2~Y phase, as shown in [Figure 8](#materials-10-01167-f008){ref-type="fig"}. The Al~2~Y stability that would not be affected by the annealing temperature should be attributed to its special crystal structure. The Al~2~Y crystal structure and its electronic density difference (De) of the (111) plane are shown in [Figure 9](#materials-10-01167-f009){ref-type="fig"}a,b \[[@B19-materials-10-01167]\]. The stability mechanism resulted from an intense interaction between the valence electron orbits of the Al and Y atoms, in which a sort of Laves phase structure was made. Additionally, the metallic, covalent, and ionic bonds involved in the Al~2~Y lattice structure also played a very important role. The crystal structure parameters of Al~2~Y are listed in [Table 1](#materials-10-01167-t001){ref-type="table"} \[[@B19-materials-10-01167]\].
In Mg-8Li-3Al-2Zn-0.2Y, a great many dispersed white granules were well distributed in the β matrix at 473 K (see [Figure 7](#materials-10-01167-f007){ref-type="fig"}b). With annealing at 498 K, these dispersed granules became less numerous. In the annealing period of 523--548 K (see [Figure 7](#materials-10-01167-f007){ref-type="fig"}h,k), the number of white granules apparently decreased, implying that they decomposed and dissolved into the β matrix. According to the previous XRD analysis, these dissolved granules were considered as AlLi phase. At 573 K (see [Figure 7](#materials-10-01167-f007){ref-type="fig"}n), a small amount of residue was only maintained in the microstructure. Furthermore, a similar dissolution law occurred in the Mg-11Li-3Al-2Zn-0.2Y alloy, as shown in [Figure 7](#materials-10-01167-f007){ref-type="fig"}c,f,i,l,o.
The atomic solid solubility in the matrix could be analyzed by the Hume-Rothery empirical rule, which was named the "15%" rule (see Equation (4)). As indicated in Equation (4), when *δ* is more than 15%, the solubility of the dissolved atom in the matrix is extremely low because the relatively big radius difference limits the atomic dissolution, promoting the formation of an intermetallic compound. According to the calculation, *δ* on Li-Al was only 5.9%. Thereby, Al solubility in the β matrix was relatively high. In addition, the bonding energy between the Al and Li atoms in the intermetallic compound was relatively low. Therefore, based on the above-mentioned analysis, the increased temperature would promote the decomposition and dissolution of AlLi phase during the annealing process. The solid solution law with the annealing process is shown in [Figure 10](#materials-10-01167-f010){ref-type="fig"}. It described the microstructural evolution law in the α and β matrices. In this evolution, the recrystallization phenomenon gradually emerged in the two matrices during annealing treatment. Meanwhile, the amount of AlLi phase in the β matrix decreased gradually, indicating that the solid solution behavior in the β matrix could be gradually activated by an increasing temperature. The recrystallization behavior should be ascribed to the dislocation density reduction and grain boundary migration. In addition, the increasing temperature accelerated the atomic diffusion and promoted the solid solution process. $$\delta = (\frac{\left| {D_{a} - D_{b}} \right|}{D_{a}}) \times 100\% > 15\%$$ where $D_{a}$ is the matrix atom diameter and $D_{b}$ is the dissolved atom diameter.
3.2. Mechanical Properties {#sec3dot2-materials-10-01167}
--------------------------
[Figure 11](#materials-10-01167-f011){ref-type="fig"} shows the mechanical properties of the three alloys at different conditions. In Mg-5Li-3Al-2Zn-0.2Y (see [Figure 11](#materials-10-01167-f011){ref-type="fig"}a), during annealing (473 K--573 K), the strength revealed the declining trend with increasing temperature. The ultimate tensile strength decreased from the original value of 274.3 MPa (at room temperature) to 157.8 MPa (at 573 K). In contrast, the elongation increased from 6.7% to 23.8%. This variation should be ascribed to the recrystallization softening effect. In the recrystallization process, the large number of dislocations was absorbed to effectively promote the grain boundary diffusion in which no strengthening factor was generated simultaneously. Furthermore, the reduction of dislocation density also contributed to enhancing the crystal slips. Thereby, the tensile properties of the Mg-5Li-3Al-2Zn-0.2Y alloy exhibited the weakening law during annealing treatment.
In Mg-8Li-3Al-2Zn-0.2Y (see [Figure 11](#materials-10-01167-f011){ref-type="fig"}b), the tensile properties of the alloy were strengthened by increasing the temperature during annealing. The ultimate tensile strength increased from the original value of 202.2 MPa (at room temperature) to 251.6 MPa (at 573 K). At the same time, the elongation decreased from 26.5% to 16.8%. There existed a similar variation law arising in the tensile properties of the Mg-11Li-3Al-2Zn-0.2Y alloy (see [Figure 11](#materials-10-01167-f011){ref-type="fig"}c), wherein the ultimate tensile strength increased from the original value of 182.3 MPa (at room temperature) to 265.3 MPa (at 573 K), and simultaneously the elongation decreased from 25.6% to 4.5%.
This phenomenon whereby the annealing process improved the properties of the alloy should be ascribed to the effect of solid solution strengthening. This solid solution mechanism could be measured by the interaction energy between the dissolved atom and dislocation, as shown in Equation (5). Equation (5) could be further simplified as Equation (6). $$\left\{ \begin{array}{l}
{E = - p \cdot \Delta V} \\
{p = \frac{1}{3}\left( {\sigma_{xx} + \sigma_{zz} + \sigma_{yy}} \right) = - \frac{1}{3} \cdot \frac{1 + n}{1 - n} \cdot \frac{Gb}{\pi} \cdot \frac{y}{x^{2} + y^{2}}} \\
{r^{2} = x^{2} + y^{2}} \\
{\Delta V = 4\pi R_{0}^{3}\varepsilon} \\
{\varepsilon = \frac{R - R_{0}}{R}} \\
\end{array} \right.$$ $$E = \frac{4}{3} \cdot \frac{1 + n}{1 - n} \cdot GbR_{0}^{3}\varepsilon \cdot \frac{\sin\theta}{r}$$ where $E$ is the interaction energy, $p$ is the normal stress on the dislocation, $n$ is the poisson ratio, $G$ is the shear modulus, $b$ is the burgers vector, $r$ is the distance between the point defect and dislocation, $\Delta V$ is the lattice volume change, $R_{0}$ is the radius of the matrix atom, $R$ is the radius of the dissolved atom, and $\sin\theta$ is *y*/*r*.
As indicated by Equations (5) and (6), the attraction between the dissolved atoms and dislocations will occur when *E* is positive. In contrast, the repulsion between the dissolved atoms and dislocations will occur when *E* is negative. Furthermore, *E* was inversely proportional to *r*, indicating that the smaller the distance between the point defect and dislocation, the higher the value of \|*E*\|. In addition, the strengthening mechanism can be explained by the dissolved atoms, which had a "pinning" effect on the dislocations. The theory was that the dissolved atoms were segregated around the dislocation line because of the elastic interaction between the dissolved atoms and dislocations. Thereby, the dislocation movements changed the equilibrium position of dissolved atoms, which gave rise to enhancing the system energy and restraining dislocation movements. This restraining extent could be defined by pinning stress (*τ*), as shown in Equations (7) and (8). $$\left\{ \begin{array}{l}
{\tau = \frac{f_{\max}}{b}} \\
{f_{\max} = \frac{3\sqrt{3}A}{8br_{0}^{2}}} \\
{A = \frac{4}{3}(\frac{1 + n}{1 - n})GbR_{0}^{3}\varepsilon = const} \\
\end{array} \right.$$ $$\tau = \frac{3\sqrt{3}}{8b^{2}r_{0}^{2}} \cdot \frac{4}{3}(\frac{1 + n}{1 - n})GbR_{0}^{3}\varepsilon$$ where $\tau$ is the pinning stress, $f_{\max}$ is the highest force on the dislocation, and $r_{0}$ is the radius of edge dislocation.
3.3. Establishing Mathematical Relationship {#sec3dot3-materials-10-01167}
-------------------------------------------
To quantitatively measure the strengthening effect, the relationship between the microstructure and mechanical properties should be proposed and determined. Hence, the fitting method with a high accuracy is used to determine their relationship models. The fitting accuracy is measured in terms of the relative error (*RE*), the mean relative error (*MRE*), and the mean square error (*MS*), as shown in Equation (9). The strength and AlLi volume fraction at different temperatures are listed in [Table 2](#materials-10-01167-t002){ref-type="table"}. $$\left\{ \begin{array}{l}
{RE = \frac{\left| {q_{i} - Q_{i}} \right|}{Q_{i}} \times 100\%} \\
{MRE = \frac{1}{n}{\sum\limits_{i = 1}^{n}{\frac{\left| {q_{i} - Q_{i}} \right|}{Q_{i}} \times 100\%}}} \\
{MS = \sqrt{\frac{1}{n}{\sum\limits_{i = 1}^{n}{(q_{i} - Q_{i})}^{2}}}} \\
\end{array} \right.$$ where $Q_{i}$ is the experimental value, $q_{i}$ is the calculated value from the model, and $n$ is the experimental number.
[Figure 12](#materials-10-01167-f012){ref-type="fig"} shows the fitting relationship results of Mg-8Li-3Al-2Zn-0.2Y and Mg-11Li-3Al-2Zn-0.2Y. As indicated in [Figure 12](#materials-10-01167-f012){ref-type="fig"}a, based on the evolution characteristic between the AlLi volume fraction and annealing temperature, the fitting method adopted the linear fitting and quadratic polynomial fitting, respectively. The fitting equations of Mg-8Li-3Al-2Zn-0.2Y and Mg-11Li-3Al-2Zn-0.2Y are shown in Equation (10). According to the fitting accuracy (see [Figure 12](#materials-10-01167-f012){ref-type="fig"}b), the mean relative error for Mg-8Li-3Al-2Zn-0.2Y and Mg-11Li-3Al-2Zn-0.2Y was 7.38% and 5.412%, respectively. The fitting results indicated that the effect of the annealing temperature on the dissolution number of AlLi phase in Mg-8Li-3Al-2Zn-0.2Y was relatively more remarkable than that in Mg-11Li-3Al-2Zn-0.2Y. Based on the analysis, α-Mg matrix occupied a certain amount of area in the Mg-8Li-3Al-2Zn-0.2Y alloy. Furthermore, a large amount of AlLi phase was distributed in the β-Li matrix instead of the α-Mg matrix. Thereby, compared with Mg-11Li-3Al-2Zn-0.2Y, the AlLi phase dissolution in Mg-8Li-3Al-2Zn-0.2Y was more sensitive to the annealing temperature. [Figure 12](#materials-10-01167-f012){ref-type="fig"}c shows the fitting results between the ultimate tensile strength and AlLi volume fraction, wherein the fitting equations are shown in Equation (11). The corresponding mean relative error for Mg-8Li-3Al-2Zn-0.2Y and Mg-11Li-3Al-2Zn-0.2Y was 0.94% and 3.7%, respectively, as shown in [Figure 12](#materials-10-01167-f012){ref-type="fig"}d. Compared with the previous fitting results, the mean relative error for Mg-8Li-3Al-2Zn-0.2Y and Mg-11Li-3Al-2Zn-0.2Y decreased by 6.44% and 1.712%, respectively, indicating that the fitting accuracy increased. It also indicated that the relationship between the strength and dissolution number was much closer. $$AVF = \begin{cases}
{46.39 - 0.77T} & {Mg - 8Li - 3Al - 2Zn - 0.2Y} \\
{- 240.03 + 1.072T - 0.0011T^{2}} & {Mg - 11Li - 3Al - 2Zn - 0.2Y} \\
\end{cases}$$ $$UTS = \begin{cases}
{278.27 - 14.62AVF + 0.684AVF^{2}} & {Mg - 8Li - 3Al - 2Zn - 0.2Y} \\
{299.507 - 8.927AVF} & {Mg - 11Li - 3Al - 2Zn - 0.2Y} \\
\end{cases}$$
To accurately make a quantitative analysis for the strength on multi-condition, the relationship between the ultimate tensile strength, dissolution number, and annealing temperature should be proposed and determined. The fitting map of ultimate tensile strength under different volume fractions and temperatures is shown in [Figure 13](#materials-10-01167-f013){ref-type="fig"}. The corresponding fitting equations are shown in Equation (12). Considering the fitting accuracy, the mean square error (*MS*) was calculated by Equation (9). The result manifested that the mean square error for Mg-8Li-3Al-2Zn-0.2Y and Mg-11Li-3Al-2Zn-0.2Y was 0.33 MPa and 1.62 MPa, respectively. Thereby, the fitting accuracy of their relationship models was relatively high. [Figure 14](#materials-10-01167-f014){ref-type="fig"} shows the comparison between the experimental value and calculated value of Equation (12). The comparison results also verified the relatively high fitting accuracy on their relationship models. $$UTS(473K - 573K) = \begin{cases}
{- 507.3 + 1.312T + 69.68AVF - 0.11T \cdot AVF - 0.77AVF^{2}} & {Mg - 8Li - 3Al - 2Zn - 0.2Y} \\
{- 6946 + 26.61T + 69.3AVF - 0.15T \cdot AVF + 0.024T^{2}} & {Mg - 11Li - 3Al - 2Zn - 0.2Y} \\
\end{cases}$$
4. Conclusions {#sec4-materials-10-01167}
==============
With increasing Li content, an α-Mg matrix was gradually transformed to a β-Li matrix. Meanwhile, the alloy plasticity was obviously enhanced due to decreasing the c/a axis ratio of Mg, as well as activating other non-basal slips. During annealing, the rolled microstructure (high energy zone) in Mg-5Li-3Al-2Zn-0.2Y and Mg-8Li-3Al-2Zn-0.2Y was gradually substituted by the recrystallized microstructure. Furthermore, a kind of abnormal grain growth was observed in Mg-11Li-3Al-2Zn-0.2Y, but not detected in Mg-5Li-3Al-2Zn-0.2Y and Mg-8Li-3Al-2Zn-0.2Y. In addition, a kind of solid solution in the β-Li matrix gradually strengthened the properties of the alloy. To quantitatively analyze this strengthening effect, mathematical modeling was used to determine the relationship between strength and multiple factors.
This research was financially supported by the National Key Research Development Program of China (2016YFB0301104).
Qichi Le and Yan Tang conceived and designed the experiments; Yan Tang performed the experiments, analyzed the data, and wrote the paper; Tong Wang and Xingrui Chen contributed the materials and analysis tools; Yan Tang and Xingrui Chen reviewed the paper.
The authors declare no conflict of interest.
{#materials-10-01167-f001}
{#materials-10-01167-f002}
{#materials-10-01167-f003}
{#materials-10-01167-f004}
{#materials-10-01167-f005}
{#materials-10-01167-f006}
{#materials-10-01167-f007}
{ref-type="fig"}.](materials-10-01167-g008){#materials-10-01167-f008}
{#materials-10-01167-f009}
{#materials-10-01167-f010}
{#materials-10-01167-f011}
{#materials-10-01167-f012}
{#materials-10-01167-f013}
{#materials-10-01167-f014}
materials-10-01167-t001_Table 1
######
Crystal structure parameters of Al~2~Y.
----------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Space Group Atom Number in Primitive Cell Atom Site Pearson Sign Equilibrium Crystal Parameters (nm) Unit Cell Volume (nm^3^) Density (g/cm^3^)
------------- ------------------------------- -------------------------- -------------- ------------------------------------- -------------------------- -------------------
Fd 3m (227) 6 Al: (0.625,0.625,0.625)\ cF24 0.554 0.124 3.838
Y: (0,0,0)
----------------------------------------------------------------------------------------------------------------------------------------------------------------------------
materials-10-01167-t002_Table 2
######
Strength and AlLi volume fraction at different temperatures.
Designed Alloy Annealing Temperature (K)/T AlLi Volume Fraction (%)/AVF Ultimate Tensile Stress (MPa)/UTS
---------------------- ----------------------------- ------------------------------ -----------------------------------
Mg-8Li-3Al-2Zn-0.2Y 473 K 9.2 198.6
498 K 8.9 205.6
523 K 6.1 215.6
548 K 3.8 230.5
573 K 2.1 251.6
Mg-11Li-3Al-2Zn-0.2Y 473 K 13.9 161.2
498 K 14.1 182.3
523 K 11.7 200.5
548 K 7.2 238.3
573 K 3.5 265.3
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The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.
Introduction {#s1}
============
While gastric cancer is the fourth most common cancer in the world, it is the second leading cause of death. [@pone.0111693-Jemal1] Its incidence is significantly higher in Asian countries, including Korea, where it is the second most common cancer. [@pone.0111693-Shin1] Recently, several targeted therapeutics for gastric cancer have been discovered, which provide additional options for physicians and patients [@pone.0111693-Smyth1]--[@pone.0111693-Nadauld1].
In the era of targeted therapy, mutation profiling of the causative cancer is crucial for therapeutic decisions. Attempts to profile mutations have been made using traditional Sanger sequencing; however, it is not an optimal method in clinical settings due to the cost, time and labor required. Moreover, Sanger sequencing requires substantial amounts of DNA; evaluating small amounts of specimen for several genes at the same time is not possible. [Introduction](#s1){ref-type="sec"} of next generation sequencing (NGS) methods has resolved this problem by multiplex, high-throughput sequencing of many samples for multiple genes simultaneously. [@pone.0111693-Metzker1], [@pone.0111693-MacConaill1] One of the NGS platforms, the Ion Torrent AmpliSeq Cancer Panel, relies on non-optical detection of hydrogen ions in a semiconductor device, [@pone.0111693-Rothberg1] and is able to detect 2,855 oncogenic mutations in 50 commonly mutated genes ([Table S1](#pone.0111693.s003){ref-type="supplementary-material"}). It is superior to other mass spectroscopy-based sequencing methods, providing sequencing results faster and at lower cost. [@pone.0111693-Rothberg1] It is applicable in formalin-fixed paraffin-embedded (FFPE) tissue specimens with small amounts of DNA. Because it ensures high sensitivity in screening known oncogenic mutations, [@pone.0111693-Singh1], [@pone.0111693-Beadling1] the Ion Torrent AmpliSeq Cancer Panel is the choice of 5 major cancer centers in the United States for molecular diagnostics in targeted therapy [@pone.0111693-Simon1].
Amplification of oncogenes is a major mechanism for gene overexpression and contributes to tumor development. [@pone.0111693-Dai1] Examples include amplification of *HER2*, *MET*, *FGFR2* and *KRAS* genes in gastric cancers. [@pone.0111693-Wu1], [@pone.0111693-Deng1] In the detection of copy number variations (CNVs) in clinical samples, fluorescence in situ hybridization (FISH) and/or immunohistochemistry (IHC) has been widely used. However, high costs and small sample sizes of biopsy materials limit the application of these methods, and there is still a need for further high-throughput technology with easy accessibility, high sensitivity and low costs. nCounter CNV CodeSets (Nanostring technologies, Life Sciences, Seattle, WA) provide superior accuracy and reproducibility for studies of all sizes and produce better, faster results with substantially less effort than with real-time quantitative polymerase chain reaction (qPCR) or CNV arrays [@pone.0111693-Geiss1].
Better-tailored cancer treatment may improve patient outcome. Patient tumor samples will be required in order to characterize cancer at a molecular level and identify the disease subgroups that should receive different treatments. The use of FFPE tissue is important for enabling such studies. [@pone.0111693-AustinTanney1] Here we tested AmpliSeq and nCounter custom CNV panels in FFPE gastric cancer samples to determine if they are applicable in archival clinical samples for personalized targeted therapies.
Materials and Methods {#s2}
=====================
Samples {#s2a}
-------
Tumor cell percentage with more than 75% were dissected under microscopy from 4 mm unstained sections by comparison with a H&E stained slide, and genomic DNA was extracted using a Qiagen DNA FFPE Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions from 96 patients with advanced gastric cancer. After extraction, we measured concentration as well as 260/280 and 260/230 nm ratio by spectrophotometer (ND1000, Nanodrop Technologies, ThermoFisher Scientific, MA, USA). Each sample was then quantified with the Qubit fluorometer (Life Technologies, Carlsbad, California). Genomic DNA with \>10 ng measured by Qubit fluorometer was subjected to library preparation and seven samples failed to construct libraries and were excluded from this study. Finally, 89 cases were finally analyzed and included 31 female and 58 male patients. [Table 1](#pone-0111693-t001){ref-type="table"} lists the clinical and pathologic features of the patients in this study. Recurrence or metastasis developed in 11 patients with median follow-up period of 76 months (range 5.5--149.3). The study was approved by the institutional review board (IRB) at Samsung Medical Center. All clinical investigation was conducted according to the principles expressed in the Declaration of Helsinki. The written informed consent was waived by the IRB due to retrospective analysis and anonymous data. Samples were collected as part of a routine medical procedure and were collected by the authors for this study. Samples from deceased patients or live patients were all de-identified, including removal of any and all demographic information, prior to analysis and informed consent form was waived by the IRB.
10.1371/journal.pone.0111693.t001
###### Clinical and pathological characteristics of 89 patients with gastric cancer.
{#pone-0111693-t001-1}
Number of cases (n = 89)
------------------------------------- -------------------------- -----------------
Gender F 31
M 58
Age Mean 53
Median 55
Lauren's classification Intestinal 27
Diffuse 60
Mixed 2
Location Upper 1/3 11
Mid 1/3 32
Lower 1/3 46
pT stage T1, 2 23
T3 51
T4 15
pN stage N1 41
N2 44
N3 4
AJCC/UICC stage (7^th^ ed) I 1
II 22
III 66
Recurrence and/or distantmetastasis present 11
absent 78
Follow up period (months) Median (range) 76 (5.5--149.3)
Ion AmpliSeq cancer panel v2 {#s2b}
----------------------------
We used the Ion AmpliSeq Cancer Panel v2 (Ion Torrent) to detect frequent somatic mutations that were selected based on literature review. It examines 2855 mutations in 50 commonly mutated oncogenes and tumor suppressor genes ([Table S1](#pone.0111693.s003){ref-type="supplementary-material"}). First, 10 ng of DNA from each of 89 FFPE tumor samples underwent single-tube, multiplex PCR amplification using the Ion AmpliSeqCancer Primer Pool and the Ion AmpliSeqKit reagents (Life Technologies). Treatment of the resulting amplicons with FuPa Reagent partially digested the primers and phosphorylated the amplicons. The phosphorylated amplicons were ligated to Ion Adapters and purified. For barcoded library preparation, we substituted barcoded adapters from the Ion Xpress Barcode Adapters 1--96 Kit for the non-barcoded adapter mix supplied in the Ion AmpliSeq Library Kit. The ligated DNA underwent nick-translation and amplification to complete the linkage between adapters and amplicons and to generate sufficient material for downstream template preparation. Two rounds of Agencourt AMPure XP Reagent binding at 0.6 and 1.2 bead-to-sample volume ratios removed input DNA and unincorporated primers from the amplicons. The final library molecules were 125∼300 bp in size. We then transferred the libraries to the Ion OneTouch System for automated template preparation. Sequencing was performed on the Ion PGM sequencer according to the manufacturer's instructions. We used IonTorrent Software for automated data analysis.
To measure the sensitivity and specificity of the Ion AmpliSeq cancer panel, whole exome sequencing results from 4 gastric cancer samples with known mutation status were used [@pone.0111693-Kang1].
nCounter Copy Number Variation CodeSets {#s2c}
---------------------------------------
For detection of CNV, nCounter Copy Number Variation CodeSets were used with 300 ng purified genomic DNA extracted from 2--3 sections of 4-µm-thick FFPE representative tumor blocks using QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany). DNA was fragmented via AluI digestion and denatured at 95°C. Fragmented DNA was hybridized with the codeset of 86 genes in the nCounter Cancer CN Assay Kit (Nanostring Technologies) for 18 hours at 65°C and processed according to the manufacturer's instructions. The nCounter Digital Analyzer counted and tabulated the signals of reporter probes and average count numbers of \>3 were called and confirmed by IHC, FISH or real-time PCR.
IHC for HER2, EGFR (HER1) and CCNE1 {#s2d}
-----------------------------------
For validation of CNV results obtained from nCounter, we performed IHC for HER2 in all cases, and EGFR and CCNE1 in selected cases. After deparaffinization and rehydration, 4 mm sections on silane-coated slides were immunostained for HER2. The HercepTest (Dako, Glostrup, Denmark) was used according to the manufacturer's guidelines as previously described. [@pone.0111693-Cho1] For EGFR we used anti-NCL-L-EGFR-384 mouse monoclonal primary antibody (1∶100 dilution; Novocastra/Vision Biosystems, Newcastle, UK) and for CCNE1 we used anti-CCNE1/Cyclin E1 Antibody (clone HE12; 1∶200 dilution; Thermo Fisher Scientific, MA). The Ventana BenchMark XT automated slide-processing system was used according to the manufacturer's protocol. An expert pathologist (KMK) evaluated the results.
FISH for HER2 {#s2e}
-------------
FISH was performed using dual-color DNA-specific probes from PathVision™ (Abbott/Vysis: LSI HER2 SpectrumOrange™ and CEP 17 SpectrumGreen™) as previously described in cases with equivocal HER2 overexpression. [@pone.0111693-Cho2] We counted the hybridization signals in 20 nuclei per sample under a fluorescent microscope (Zeiss Axioskop) using filter sets recommended by Vysis (DAPI/Spectrum Orange dual bandpass, DAPI/Spectrum Green dual bandpass). All overlapping nuclei were excluded, and only nuclei with a distinct nuclear border were evaluated. *HER2* gene was considered amplified when the FISH signal ratio of *HER2/CEP17* was greater than or equal to 2.0 [@pone.0111693-Ruschoff1].
Real-time PCR for KRAS and MET amplification {#s2f}
--------------------------------------------
We used DNAs obtained from FFPE gastric carcinoma tumor tissues. The reaction mixture contained 2 uL genomic DNA template, 10 uL of Taqman universal PCR master mixture (Applied Biosystems Inc, Foster City, CA) and 0.2 uM of each primer. For accurate detection of CN alterations, we analyzed three different regions of the *KRAS* gene: a region within intron 1 (TaqMan Copy Number Assay Hs06943812_cn), a region within intron 2 (Hs002534878_cn), and a region within exon 6 (Hs02739788_cn). For *MET* gene, we used the primers as previously described [@pone.0111693-Ha1].
We measured copy number gain using the following profile: 2 min at 50°C, denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. We determined relative quantification using the 7900 HT fast real-time PCR system in quadruplicate. An RNaseP assay kit (Applied Biosystems) was used as a control. After amplification, we imported the experiment results containing threshold-cycle values for the copy number and reference assay into the CopyCaller Software (Applied Biosystems) for post-PCR data analysis as previously described. [@pone.0111693-Graziano1] We assigned the CN gain status and the number of *KRAS* copies based on the concordance of the results in at least two of the three probes.
Analytical methods {#s2g}
------------------
We excluded all synonymous changes after an automated mutation-calling algorithm was used to detect supposed mutations. Recurrent calls in more than 10 of 89 samples were regarded as false positive and were excluded. We used cutoff values of more than 6% variant frequency and more than X100 coverage to detect true mutational changes in accordance with previous studies and our own experience. [@pone.0111693-Singh1], [@pone.0111693-Beadling1] We filtered out single-nucleotide polymorphisms after manual review of each polymorphism in the Catalogue of Somatic Mutations in Cancer (COSMIC, <http://cancer.sanger.ac.uk/cancergenome/projects/cosmic>) ([Figure 1](#pone-0111693-g001){ref-type="fig"}). For well-known genes mutated in gastric carcinomas (*TP53*, *APC*, *PIK3CA*, *STK11*, *CDKN2A*, *KRAS*, *HRAS*, *BRAF* and *CTNNB1*), a manual review of automated calling results was performed to catch deleterious mutations with slightly low-variant frequency.
{#pone-0111693-g001}
Results {#s3}
=======
Results of the Ion AmpliSeq cancer panel {#s3a}
----------------------------------------
The concentrations of DNAs, their concentration fold, average coverage of the samples, total numbers of bases, \>Q20 bases, reads, mean read length, mapped reads, on-target rate (%), mean depth and uniformity of the results are described in [Table S2](#pone.0111693.s004){ref-type="supplementary-material"}. In total we obtained 8178 variant calls from 89 samples, among them 3554 calls were non-synonymous changes. After filtering out recurrent calls, \<6% of variant-allele frequency, \<100X coverage and those in intron region, 65 variant calls were selected. Additionally, we reviewed the automated calls in well-known mutations such as *BRAF*, *KRAS* and *PIK3CA* and could save two variant calls, which were excluded during the filtering processes. Thirty-nine of the 89 samples (43.8%) harbored at least one mutation ([Figure 1](#pone-0111693-g001){ref-type="fig"}). Two cases showed 22 and 5 mutations, respectively. The latter case harbored *MLH1* somatic mutation \[missense mutation in exon 20: c.1147A\>G(p.M383 V)\] and *MLH1* promoter hypermethylation with MLH1 protein losses by IHC using the previously described methods, [@pone.0111693-Lee1] suggesting hypermutated tumor. However, although the mutations found in the former case passed variant frequency and coverage cut offs, those mutations were not confirmed by Sanger sequencing, suggesting false positive in this case due to poor quality of DNA. So, this case was excluded from final analyses of the results. Frequently detected somatic mutations included *TP53* (24 cases, 27.0%), *APC* (9 cases, 10.1%), *PIK3CA* (5 cases, 5.6%), %), *KRAS* (3 cases, 3.4%), *SMO* (4 cases, 4.5%), *STK11* (3 cases, 3.4%), *CDKN2A* (3 cases, 3.4%), and *SMAD4* (3 cases, 3.4%) as shown in [Table 2](#pone-0111693-t002){ref-type="table"}. [Table 2](#pone-0111693-t002){ref-type="table"} also summarized the amino acid changes in frequently mutated genes. We identified 19 patients (21.3%) with two or more unique and concomitant somatic mutations.
10.1371/journal.pone.0111693.t002
###### Frequency of mutations and amino acid changes in 89 gastric carcinomas.
{#pone-0111693-t002-2}
Gene N[\*](#nt101){ref-type="table-fn"} \% Amino acid change (N, %)
----------- ------------------------------------ ------ ---------------------------------------------------------
*TP53* 24 27.0 R248Q (N = 3, 3.3%)^§^
R248W (N = 1, 1.1%)
R213fs[\*](#nt101){ref-type="table-fn"}34 (N = 1, 1.1%)
R213[\*](#nt101){ref-type="table-fn"} (N = 1, 1.1%)
R273H (N = 1, 1.1%)
R273C (N = 1, 1.1%)
R175H (N = 2, 2.2%)
R185R (N = 1, 1.1%)
R342[\*](#nt101){ref-type="table-fn"} (N = 1, 1.1%)
C135C (N = 1, 1.1%)
C135fs[\*](#nt101){ref-type="table-fn"}35 (N = 2, 2.2%)
C176S (N = 1, 1.1%)
D208V (N = 1, 1.1%)
G245R (N = 1, 1.1%)
T626C (N = 1, 1.1%)
L206fs[\*](#nt101){ref-type="table-fn"}41 (N = 1, 1.1%)
V173A (N = 1, 1.1%)
Y236C (N = 1, 1.1%)
*APC* 9 10.1 K1359E (N = 1, 1.1%)^§^
K1363E (N = 1, 1.1%)
P1433L (N = 1, 1.1%)
*PIK3CA* 5 5.6 E545K (N = 2, 2.2%)
N1044K (N = 1, 1.1%)
E1037K (N = 1, 1.1%)
H1047R (N = 1, 1.1%)
*KRAS* 3 3.4 G13V (N = 2, 2.2%)
G12V (N = 1, 1.1%)
*SMO* 3 3.4 E518K (N = 1, 1.1%)
E208K (N = 1, 1.1%)
R512H (N = 1, 1.1%)
*STK11* 3 3.4 S31F (N = 1, 1.1%)^§^
T32I (N = 1, 1.1%)
*CDKN2A* 3 3.4 T79I (N = 1, 1.1%)
H66R (N = 1, 1.1%)
C315A (N = 1, 1.1%)
*SMAD4* 3 3.4 R361H (N = 1, 1.1%)
M447I (N = 1, 1.1%)
Q448X (N = 1, 1.1%)
*CDH1* 2 2.2 L343P (N = 1, 1.1%)
G382D (N = 1, 1.1%)
*FBXW7* 2 2.2 R393[\*](#nt101){ref-type="table-fn"} (N = 1, 1.1%)^§^
*ATM* 1 1.1 Y861H (N = 1, 1.1%)
*CTNNB1* 1 1.1 T41A (N = 1, 1.1%)
*ERBB2* 1 1.1 V842I (N = 1, 1.1%)
*FGFR2* 1 1.1 G906A (N = 1, 1.1%)
*FGFR3* 1 1.1 A369A (N = 1, 1.1%)
*KDR* 1 1.1 W1143X (N = 1, 1.1%)
*MLH1* 1 1.1 A424G (N = 1, 1.1%)
*PTEN* 1 1.1 R15S (N = 1, 1.1%)
*SMARCB1* 1 1.1 H177Y (N = 1, 1.1%)
*RET* 1 1.1 A641T (N = 1, 1.1%)
\*N, total number of samples with mutation, ^§^INS/DEL in the remaining cases.
In four gastric cancer samples with known mutation frequencies determined by whole exome sequencing and confirmed by Sanger sequencing, we identified somatic mutations in *TP53*, *ERBB4* and *CTNNB1* with no false-positive calls in other genes ([Table S3](#pone.0111693.s005){ref-type="supplementary-material"}).
Amplification by nCounter and validation by IHC, FISH or real-time PCR {#s3b}
----------------------------------------------------------------------
Amplifications of *HER2*, *CCNE1*, *MYC*, *KRAS* and *EGFR* genes were observed in 8 (8.9%), 4 (4.5%), 2 (2.2%), 1 (1.1%) and 1 (1.1%) cases, respectively ([Table 3](#pone-0111693-t003){ref-type="table"}). We did not observe amplification of *MET*, *FGFR2*, *CDK4* and CDK6 in any of the cases. In cases with amplification, IHC for HER2, EGFR and CCNE1 showed overexpression of proteins in the tumor cells ([Figure 2A, B and C](#pone-0111693-g002){ref-type="fig"}). In one case with HER2 2+ by HercepTest, FISH showed heterogeneous amplification of *HER2* genes ([Figure 2D](#pone-0111693-g002){ref-type="fig"}).
{#pone-0111693-g002}
10.1371/journal.pone.0111693.t003
###### Gastric cancers with copy number variation (CNV) detected by nCounter.
{#pone-0111693-t003-3}
Gene Number of samples with CNV Range of CNV (mean)
------------------------------------------- ---------------------------- ---------------------
*ERBB2* [\*](#nt102){ref-type="table-fn"} 8 (8.9%) 9--62 (31.9)
*CCNE1* 4 (4.5%) 8--22 (12.8)
*MYC* 2 (2.2%) 13--38 (25.5)
*EGFR* 1 (1.1%) 7
*KRAS* 1 (1.1%) 18
\*All samples have proven to show positivity in immunohistochemical staining (8 samples: 3+; 1 sample: 2+; 1 sample: 1+).
In real-time PCR for *KRAS*, one case with amplification showed increased copy numbers (36, 37 and 49); in cases that were negative for *KRAS* amplification, there was no increase of copy numbers (0.9 to 2.4, mean 1.4).
For *MET* gene, we find no positive case because of their rarity [@pone.0111693-Ha1]. Therefore, we used additional ten (five each amplified and non-amplified) gastric cancer samples and *MET* amplified gastric cancer cell lines (MKN45 and SNU5) with known copy numbers and mRNA amounts. CNVs detected by nCounter correlated well with copy numbers detected by real-time PCR ([Table S4](#pone.0111693.s006){ref-type="supplementary-material"}) and mRNA levels of *MET* gene (Pearson's correlation test; r = 0.874, p = 0.001) ([Figure S2](#pone.0111693.s002){ref-type="supplementary-material"}).
Discussion {#s4}
==========
By using Ion AmpliSeq v2 we found that 39 out of 89 advanced gastric adenocarcinoma samples contained somatic mutations, demonstrating that this platform is easily applicable in archival FFPE tissue samples. *TP53* was the most frequently found mutation, followed by *APC*, *PIK3CA* and *KRAS*. Moreover, our custom CNV panel successfully detected CN increases of *HER2*, *CCNE1*, *MYC*, *EGFR* and *KRAS* genes, which we confirmed by IHC and real-time PCR.
Mutational frequencies in the COSMIC database reveal substantial similarities to the data we obtained from this study: *TP53* (32%), *PIK3CA* (10%), *KRAS* (6%), *APC* (6%), *CTNNB1* (5%), *CDKN2A* (5%), *FBXW7* (5%), *SMO* (4%), *ERBB2* (2%) and *STK11* (2%). Recent whole exome sequencing studies on gastric adenocarcinoma showed somewhat higher frequencies of *TP53* (36% and 73%) and *PIK3CA* (14% and 20%) mutations compared to our results. [@pone.0111693-Wang1], [@pone.0111693-Zang1] Although our mutation frequencies were lower when compared to exome sequencing results, there was a significant increase when compared to our previous data on mass spectrometry-based OncoMap v4. [@pone.0111693-Lee2] Both AmpliSeq and OncoMap detect mutations in hotspot regions, which explain findings of less frequent mutation in some oncogenes and tumor suppressor genes. [Figure S1](#pone.0111693.s001){ref-type="supplementary-material"} compares AmpliSeq v2 and OncoMap v4 in detectable mutational profiles. Previous OncoMap tests in 237 gastric adenocarcinomas revealed that *PIK3CA* mutations were frequent in advanced stages of disease (5.1% in Stage IV; 6.4% in stage II/III; 2.4% in stage IB). [@pone.0111693-Lee2] In this study we observed three *PIK3CA* mutations in patients with stage III and two in stage II disease, supporting their biological role in tumor progression. We also observed *HER2* (*ERBB2*) c.2524G\>A (V842I) mutation in a case of gastric cancer. In preclinical studies, cell lines harboring the V842I mutation were resistant to trastuzumab, but were sensitive to irreversible HER2 inhibitor, neratinib [@pone.0111693-Bose1].
Semiconductor-based sequencing has fundamental differences in sensing and signal transduction compared to mass spectrometry-based sequencing. Instead of using optical methods to detect nucleotide changes, the semiconductor-based technique senses pH changes by release of protons (H^+^) when nucleotides integrate into the growing DNA strand. [@pone.0111693-Rothberg1] Therefore, there is a significant reduction in the cost and the time required for data processing compared to other NGS platforms. Providing fast and accurate information on mutations at low cost is crucial for patients with highly aggressive cancers, including gastric cancer.
In this study, we manually reviewed the automated calls in well-known mutations after applying cutoff values of frequency and coverage, subsequently adding two calls with low-variant coverage. Although their coverage values did not reach our initial criteria, their frequencies exceeded our first setting (6%) and the quality of the data was good. This emphasizes the importance of manual review after automated screening. Recently published results using AmpliSeq as the analyzing platform also emphasize compensation of screening data with manual review [@pone.0111693-Singh1], [@pone.0111693-Beadling1].
Personalized targeted therapy for advanced cancers primarily relies on the concept of "oncogene addiction," in which multiple genetic abnormalities are addicted to one or a few genes for tumor cell maintenance and survival. [@pone.0111693-Ma1] An open-label, international, phase 3, randomized controlled ToGA (Trastuzumab for Gastric Cancer) trial indicated that trastuzumab in combination with chemotherapy is a new standard option for patients with HER2-positive advanced gastric or gastro-esophageal junction cancer. [@pone.0111693-Bang1] A preclinical trial showed that a subset of gastric cancers with *EGFR* or *MET* amplification and overexpression respond to cetuximab or MET receptor tyrosine kinase inhibitor therapy. [@pone.0111693-Zhang1] Additionally, amplifications of cell cycle mediator *CCNE1* suggest the potential for therapeutic inhibition of cyclin-dependent kinases in gastric cancers. [@pone.0111693-Network1] Screening amplified genes for targeted therapy with high-throughput technology is very important. Traditional methods such as FISH and array comparative genomic hybridization suffer from low resolution of genomic regions, high cost and are labor- and time-consuming. [@pone.0111693-Duan1] In this first study on nCounter CNV analyses, we found that this technology is applicable in FFPE clinical samples and we validated the results by IHC, FISH and real-time PCR. Although we did not validate all the genes used in the custom primers, validation results in several selected genes were remarkable.
In summary, we successfully performed semiconductor-based sequencing and nCounter CNV analyses in FFPE tissue specimens from 89 gastric adenocarcinomas. High-throughput sequencing and CNV screening in archival clinical samples enables faster, more accurate and cost-effective detection of hotspot mutations and CNV in genes. In the era of personalized genomic medicine, we plan to use these tools to screen for gastric cancer patients who may benefit from targeted therapies.
Supporting Information {#s5}
======================
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**Comparison of coverage of Ion AmpliSeq v2 cancer panel versus Oncomap v4.**
(TIF)
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Click here for additional data file.
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**Plots of correlation between** ***MET*** **CNVs detected by nCounter and mRNA levels of** ***MET*** **gene by real-time PCR.**
(TIF)
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Click here for additional data file.
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**The Gene List for the Ion Torrent AmpliSeq Cancer Panel.**
(XLS)
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Click here for additional data file.
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**The concentrations of DNAs, their concentration fold, average coverage of the samples, total numbers of bases, \>Q20 bases, reads, mean read length, mapped reads, on-target rate (%), mean depth and uniformity.**
(XLS)
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Click here for additional data file.
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**Somatic mutations in** ***TP53*** **,** ***ERBB4*** **and** ***CTNNB1*** **.**
(XLS)
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Click here for additional data file.
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**CNVs detected by nCounter correlated well with copy numbers detected by real-time PCR.**
(XLSX)
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Click here for additional data file.
[^1]: **Competing Interests:**The authors have the following interests: This study was funded in part by Samsung Biomedical Research Institute and Samsung Medical Center. Co-authors Seokhwi Kim, Jeeyun Lee, Min Eui Hong, In-Gu Do, So Young Kang, Sang Yun Ha, Seung Tae Kim, Se Hoon Park, Won Ki Kang, Min-Gew Choi, Jun Ho Lee, Tae Sung Sohn, Jae Moon Bae, Sung Kim and Kyoung-Mee Kim are employed by Samsung Medical Center. Co-author Duk-Hwan Kim is employed by Samsung Biomedical Research Institute. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.
[^2]: Conceived and designed the experiments: JL KMK. Performed the experiments: JL KMK. Analyzed the data: Seokhwi Kim JL IGD SYK DHK SYH KMK. Contributed reagents/materials/analysis tools: SHP WKK MGC JHL TSS JMB Sung Kim DHK JL MEH STK. Contributed to the writing of the manuscript: KMK Seokhwi Kim JL.
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#Sec1}
============
Children with developmental coordination disorder (DCD) have significant difficulty in acquiring and executing the essential, coordinated motor skills involved in self-care (e.g., dressing), recreational activities (e.g., ball skills), and academic performance (e.g., handwriting) compared to their typically developing counterparts^[@CR1]^. DCD is estimated to affect around 6% of children^[@CR1]^ and can have a significant impact on their socio-emotional wellbeing^[@CR2]^ and future health status^[@CR3]^. As such, there is a need for carefully designed and executed randomised control trials (RCT) to investigate the efficacy of interventions for children with DCD^[@CR4],[@CR5]^. Additionally, given that children with DCD represent a very heterogeneous population; in terms of the range and variability of impairment, and the influence of co-occurring disorders, it is important that RCT outcome measures are sensitive to subtle and individual changes in coordination^[@CR6]^. The current paper applies recently proposed statistical learning techniques to explore how children with DCD might find individualised, self-organising movement solutions following a group-based training intervention.
The intervention itself is grounded in research that has demonstrated that the quiet eye (QE)^[@CR7]^ - an objective measure of visuomotor control in targeting and interception tasks - can be trained, with significant benefits for performance^[@CR8],[@CR9]^. Wilson *et al*. were first to determine that the QE mediated performance differences between children of varying motor coordination abilities in a throw and catch task^[@CR10]^. Highly proficient children revealed longer QE pursuit tracking durations -- locating the ball more quickly and tracking it for longer - prior to more accurate catch attempts. This finding was not wholly surprising, as a body of evidence has linked DCD to significant impairments in general visuomotor control and the processing of task-relevant, visual information^[@CR11]^; the ability to use predictive information to guide action^[@CR12]^; and the pursuit tracking of objects^[@CR13]^. However, particular strengths of the study^[@CR10]^ were the use of a 'real world' throw and catch task that represents the building blocks for sport and playground games, and the collection of eye movement videos (from mobile eye trackers) of expert performers that could be used as feed-forward models for subsequent training interventions. Specifically, QE training videos were created that showed the expert eye movements from a first-person perspective alongside an auditory commentary that identified the key targets to be attended (see^[@CR14]^ supplementary files, or <http://see2learn.co.uk/videos/> for example videos).
Two separate RCTs subsequently showed that while children with DCD do have impairments in visual control -- as evidenced by later and shorter QE durations on the incoming ball - this could be improved via QE training. Importantly, these improvements in gaze control (longer QE durations) also translated into performance improvements^[@CR14],[@CR15]^. In comparison, a control group who received typical movement-focused video instructions (Technical Training; TT), revealed no improvement in QE or catching technique after training. The authors concluded that QE training served to improve the attentional control of these children, providing earlier information with which to prepare the interceptive catch attempt.
While group-based changes in measures of QE and performance quality were evident in both these studies, examining the movement patterns underpinning an improvement in performance provides important information from the perspective of motor control in DCD. Specifically, identifying if there were common or unique characteristics in technique change would help inform knowledge of how movement coordination emerges, and what training might be useful to underpin learning. Indeed, it is possible that children found different motor solutions to the catching problem. It has been suggested that by focusing on an external target, QE training allows the body to self-organise to meet the end point goal of getting the hands into position at the right time and place to make catching possible^[@CR15],[@CR16]^. Research to date has not been able to test this hypothesis for both methodological and theoretical reasons. First, the focus in RCT studies is the detection of common improvements in the treatment group compared to the control group. Any variability in response to treatment is seen as a limitation of the generalizability of the intervention, as opposed to potential individualised solutions to the problem. This limitation is in turn related to the fact that few studies take a dynamical systems perspective to the self-organisation of movement under constraints^[@CR6],[@CR17]--[@CR19]^.
The dynamical systems perspective has its roots in biological systems theory and contends that the timing and coordination of movement are emergent properties of the individual physical system in its interaction with the environment^[@CR17],[@CR18]^. Specifically, according to Newell's^[@CR17]^ model of constraints, movement coordination emerges as a consequence of the interaction of organismic, environmental and task constraints on the system. Optimal patterns of coordination and control therefore emerge from the unique confluence of constraints impinging on individual neuro-musculoskeletal systems through a process referred to as 'self-organizing optimality'^[@CR17]--[@CR20]^. As such, a key principle of a dynamical systems approach to skill acquisition is that there is no single optimal technique for a goal-directed action (like catching a ball)^[@CR21]^, which may be further exaggerated in the heterogeneous population of children with DCD^[@CR6]^. While *end point* variability may be evidence of poor task performance, *coordinative* variability is associated with multiple ways of achieving the task goal via exploration of the perceptual-motor space^[@CR22],[@CR23]^.
This dynamical systems perspective is under-represented in the DCD literature^[@CR6]^ and has implications for how research into DCD is conducted. First, it calls into question how useful comparisons of endpoint variability between groups of typically developing (TD) children and children with DCD may be for understanding the particular constraints acting on an individual^[@CR6],[@CR24]^. Recent reviews have lamented that while such comparisons are plentiful, they do not address either the 'developmental' or 'coordination' aspects of the disorder^[@CR6],[@CR24],[@CR25]^. It is therefore important for research to assess intra-individual changes in coordination over time. The current study answers these calls by examining how children with DCD might learn to adopt different coordination solutions following a four-week intervention designed to improve throw and catch performance. Specifically, to measure the complexity inherent in biological systems, we applied methodology inspired by Kerkman *et al*.^[@CR26]^ and used tools routinely applied in neuroscience and data mining^[@CR27]--[@CR29]^. Namely, we quantified coordination patterns using covariance matrices and Riemannian geometry and visualised the relationships between the patterns using multidimensional scaling.
The current study therefore has two main aims: (1) to provide additional support for a novel intervention^[@CR14]^ and (2) to examine the emergence of coordination from a dynamical systems perspective, using novel measures. We have previously reported a training advantage for this intervention in terms of a subjective rating of 'end point' performance quality^[@CR14]^ so wanted to examine if mathematically derived coordination measures would reveal similar group differences. Specifically, we hypothesized that (1) the QET participants would reveal increased coordination (movements occurring concurrently rather than subsequently), as evidenced by increased covariance of the kinematic measures following training, compared to their TT counterparts. Additionally, if the QE training intervention supported the creation of self-organizing solutions, we would hypothesize that (2) there would be high variability in the post training coordination patterns between and within individuals.
Results {#Sec2}
=======
We first checked if covariances between pairs of kinematic measures increased after the intervention. We chose to use covariances rather than correlations, because they capture not only coordination between the measures but also inform us about the range of motion. In the QE trained group, we found a significant increase in absolute values of covariance (medians over trials) in 10 out of the 15 pairs of measures, while there was no such increase in the technique-trained (TT) control group (see Table [1](#Tab1){ref-type="table"}). We interpret an increase in the absolute value of covariance as an increase in coordination; in practice it might for example mean that left and right arm were moving together or that movement of an arm was 'smoother' (e.g., elbow and shoulder were moving concurrently).Table 1Differences of median pair-wise absolute covariance values between the kinematic measures in the gaze (QET) and technique (TT) trained groups.Pair of measuresQETFDRTTFDR1: Left elbow flexion,**216.60.0342**231.70.73412: Right elbow flexion1: Left elbow flexion,**111.70.048**0.70.79353: Left shoulder total flexion1: Left elbow flexion,104.70.2789−37.60.85134: Right shoulder total flexion1: Left elbow flexion,**220.90.0386**26.10.79355: Left shoulder flexion1: Left elbow flexion,55.80.285437.80.73416: Right shoulder flexion2: Right elbow flexion,**114.20.0423**−20.30.79353: Left shoulder total flexion2: Right elbow flexion,**131.90.0480**57.60.73414: Right shoulder total flexion2: Right elbow flexion,120.40.116242.60.73415: Left shoulder flexion2: Right elbow flexion,115.20.11041320.70436: Right shoulder flexion3: Left shoulder total flexion,**159.20.0386**17.60.79354: Right shoulder total flexion3: Left shoulder total flexion,**287.40.0386**45.60.79355: Left shoulder flexion3: Left shoulder total flexion,**142.40.0423**−18.20.85136: Right shoulder flexion4: Right shoulder total flexion,**189.60.0165**43.20.79355: Left shoulder flexion4: Right shoulder total flexion,175.80.157537.50.79356: Right shoulder flexion5: Left shoulder flexion,**160.90.039**−144.90.85136: Right shoulder flexionStatistical test: one-sided Mann-Whitney-Wilcoxon test with Benjamini Hochberg false discovery ratio (FDR) correction for 15 tests in each group. In bold FDR \< 0.05.
We then checked to what extent change in the *total* coordination - defined as sum of absolute values of medians (over trials) of covariances between the 15 pairs of the 6 kinematic measures - explained improvement in catching performance. Figure [1](#Fig1){ref-type="fig"} depicts the relationship between post-intervention change in total coordination, ∆~*TOTAL\ COORDINATION*~, for each participant against their change in catching score, ∆~SCR~. The change in total coordination predicted 39% of the variance in change of catching performance (Pearson's correlation, *p* = 0.004); the greater the total coordination, the greater the improvement in catching performance. Overall, the QE trained group revealed an increase in total coordination after training; median ∆~*TOTAL\ COORDINATION*~ = 2463.2, whereas the total coordination of the technique-trained control group decreased; median ∆~*TOTAL\ COORDINATION*~ = −230.1, (p = 0.0175 one-sided Mann-Whitney-Wilcoxon test).Figure 1Plot of the change in total coordination (normalized), ∆~*TOTAL\ COORIDNATION*~, versus ∆~*SCR*~, the differences between the medians of catching scores of the participants (Pearson's correlation coefficient R = 0.6279, p = 0.004). Green diamonds and red brown dots represent participants in QET and TT groups respectively and the grey line represents the regression line, y = 3.4834*x* + 2.0215.
However, this basic analysis of the total coordination only provides a group-level understanding. To analyse changes in individual coordination *patterns*, we computed Riemannian distance between all the pairs of covariance matrices for all participants in all baseline and retention trials. We then used multidimensional scaling (MDS) to represent the matrices as points in an abstract geometric space given by two first principal dimension of the MDS. The two first principal dimensions represented 78% of the relations encoded in the raw Riemannian distances, meaning that they are suitable for visualization and analysis of the data^[@CR29]^.
Figure [2](#Fig2){ref-type="fig"} shows intra- and inter-personal variability. Points corresponding to all of the participants' trials in a given condition form a cluster in the MDS space that we encircled with an ellipse representing a bivariate normal distribution fitted to the cluster of points on the plane (see^[@CR30]^ for details of methodology for computing the ellipse). Interestingly, the QET participants showed a greater change in the coordination patterns than the TT participants but the nature of each change was individual specific. To measure the change in coordination patterns, we computed overlap ω between baseline and retention ellipses, with ω = 0 meaning that the ellipses did not overlap at all (i.e. the movements are completely different), and ω = 1 reflecting complete overlap (i.e. the movements did not change); median ω~QET~ = 0.2, median ω~TT~ = 0.31 (p = 0.0564 one-sided Mann-Whitney-Wilcoxon test).Figure 2Plot of the changes in coordination patterns of individual participants, illustrated with two principal dimensions of multidimensional scaling. (**a**--**j**) Data for individual QET participants (green), (**k**--**s**) data for individual TT participants (brown-red). Small dots represent individual covariance matrices of each participant (grey -- baseline, colour -- retention); the dots are encircled by ellipses that represents 0.7 of the mass of fitted bivariate normal distribution. To show how individual participants compare with all others, the two large ellipses indicate the entire baseline (grey) and the entire retention (black) data; they represent 0.8 of the mass of fitted bivariate normal distribution. (**t**) Shows overlap between ellipses representing the covariance matrices of the retention trials in which on average participants achieved improvement ∆~SCR~ ≥ 1. Title shows participant's identifier, median retention catching score, median baseline catching score and ∆~SCR~.
It is also evident that the variability of the coordination pattern does not change in a consistent way. For example, Fig. [2(a)](#Fig2){ref-type="fig"} or (d) show less variability after QE training (the green ellipse is smaller than the grey), while Fig. [2(b)](#Fig2){ref-type="fig"} or (h) show higher variability after QE training (the green ellipse is larger than the grey). In order to further examine variability in coordination, we attempted to control for performance. First, we compared the retention test coordination patterns of all participants who improved performance after training (i.e. with *∆*~*SCR*~ ≥ 1). Figure [2(t)](#Fig2){ref-type="fig"} shows that while there is some similarity between these coordination patterns - there is a large degree of overlap between ellipses - the overlap is not complete, meaning that there are inter-personal differences.
To further investigate variability in the coordination patterns underpinning successful trial performance, we analysed covariance matrices from trials where participants achieved a catching score of 8 or higher (all trials where the ball was caught, even if not cleanly caught on the first attempt -- see Methods). There were 29 such trials in the baseline condition, 30 among the retention trials of the TT group and 66 among the retention trials of the QET group. Figure [3(a)](#Fig3){ref-type="fig"} shows that there were a number of successful coordination patterns from the retention trials of the QET group (green diamonds) that were not similar to any of the baseline trials (11 green diamonds are outside of the big grey ellipse). It also shows that the successful coordination patterns from the retention trials of the TT group (red-brown circles) were always similar to the successful coordination patterns from the baseline trials (blue squares). Overall, this demonstrates that gaze training allowed for self-organization and emergence of individual coordination patterns that were different from all the movements in the baseline trials.Figure 3Plot of the changes in coordination patterns of trials in which participants achieved scores ≥ 8, illustrated with two principal dimensions of the multidimensional scaling. (**a**) Markers represent individual covariance matrices from all the baseline trials (grey crosses), 29 baseline trials with score ≥ 8 (blue squares), 30 retention trials from TT group with score ≥ 8 (red-brown circles), 66 retention trials from QET group with score ≥ 8 (red circles). The markers are encircled by ellipses that represents 0.7 of the mass of fitted bivariate normal distribution. The large grey ellipse encircles all the baseline markers and represents 0.8 of the mass of fitted bivariate normal distribution. (**b**--**e**) are representative examples of the covariance matrices; values in the matrices are colour coded. Markers in panel (**a**) that correspond to these exemplar matrices are indicated with a black circle and corresponding label.
Discussion {#Sec3}
==========
The main aim of this study was to investigate a dynamical systems approach to examining changes in coordination following training of children with DCD. This approach considers the role of self-organization under multiple constraints present at various levels within the child-task-environment interaction^[@CR6]^. Both experimental and robotics fields have identified that an epistemological shift towards understanding the dynamics of a system within constraints - where redundancy and variability of the system are used to satisfy collective dynamics - may be mathematically, theoretically, and practically more fruitful^[@CR31]^. It may also be particularly relevant for the study of children with DCD, who are a heterogeneous population in themselves^[@CR6]^. The current study therefore addresses recent calls^[@CR6],[@CR24],[@CR25]^ for this approach to be applied to better understand the coordination deficits inherent in DCD. Additionally, a dynamical systems approach enables us to explore the hypothesis that gaze (quiet eye) training might guide children with DCD to find individual coordination solutions via exploration of the perceptual-motor space, rather than creating a single 'optimal' pattern of coordination.
Our findings were supportive of both hypotheses. First, we found that the QE trained group had significantly higher coordination (as indexed by covariance values between the pairs of kinematic measures) than the technique-trained control group (Table [1](#Tab1){ref-type="table"}) and that change in total coordination predicted 39% of the variance in change of catching performance (Fig. [1](#Fig1){ref-type="fig"}). In this way we validated our measure of coordination against a measure of end point performance and supported previous research claiming benefits of QE training for children with DCD^[@CR14],[@CR15]^. Second, we showed that this group performance benefit occurred with significant intra- and inter person variability in the coordination patterns, as predicted (Fig. [2](#Fig2){ref-type="fig"}). In other words, while the coordination patterns of the QET participants changed more than their TT counterparts, this was not in a consistent manner (Fig. [2(a--j)](#Fig2){ref-type="fig"}). Indeed, we showed that the coordination patterns of some QET participants became more variable after training, while others became less variable.
Even when we controlled for performance, individual differences in coordination were evident. First, we compared the post-training coordination patterns of all participants who improved their catching performance from baseline to retention. While there was a large degree of overlap between ellipses (i.e. coordination patterns were similar), individual variability meant that this overlap was not complete (see Fig. [2(t)](#Fig2){ref-type="fig"}). Second, we compared coordination patterns for all trials in which participants managed to catch the ball. Again, there was evidence of individual variability in coordination patterns despite all reflecting successful (end point) performance (Fig. [3](#Fig3){ref-type="fig"}). Importantly - and in support of our second hypothesis - QE training allowed for self-organization and emergence of individual coordination patterns that were different from all patterns in the baseline trials. The successful coordination patterns from the retention trials of the TT group on the other hand, were always similar to those from baseline trials (Fig. [3](#Fig3){ref-type="fig"}).
The overall message is that while higher levels of covariance may be related to better performance, the distinct patterns of this coordination emerged in an individualised manner, depending on the unique constraints impinging on individual neuro-musculoskeletal systems^[@CR17]^. Within this framework, individualized solutions are bound to exist as the specific environmental, individual, and task constraints will vary on a case-by-case (and throw by throw) basis^[@CR32]^. As such, these findings support the benefit of developing a dynamical systems model of coordination in DCD and its applicability for further investigation of therapeutic training responses at the level of an individual. From this perspective, variability in movement systems is omnipresent and unavoidable due to the distinct constraints that shape each individual's behaviour. Rather than being seen as something to limit, variability in movement systems may help individuals adapt to the unique constraints (personal, task and environmental) impinging on them across different timescales^[@CR17],[@CR33]^.
The consideration of timescale is important when we consider the 'developmental' aspect of DCD. The pattern of coordination and control produced by the neuromusculoskeletal system is only optimised in relation to the immediately imposed constraints. Since the constraints imposed on an individual dynamical movement system (in this case a child with DCD) fluctuate continuously over time, the emergent pattern of coordination and control for any given motor activity will also change accordingly^[@CR33]^. This is why research like the current study is important -- in that it considers both the 'developmental' (change over time) and 'coordination' (interacting dynamics of the movement patterns) elements of DCD^[@CR6],[@CR24],[@CR25]^. The 'disorder' element of DCD is also considered in this approach, in that it is recognised that while coordination patterns may be momentarily self-optimised, performance may still be lower than that of their typically developing peers^[@CR34]^.
Interventions therefore need to be able to provide benefits within this changing landscape and thus the current study has some important implications for therapy. It is important to understand the different compensatory strategies that children with DCD might adopt to find adequate -- even if not optimal -- solutions to motor problems, based on their unique constraints, if interventions are to be personalized for their recipients. If initial system conditions are different -- and constantly changing -- it is unlikely that standardized interventions designed to coach specific movement solutions will be appropriate for children with DCD. A dynamical systems approach to understanding the deficits in perceptual-motor coordination of children with DCD provides a useful framework for designing personalized interventions that consider the individual constraints under which a child operates^[@CR35]--[@CR37]^.
This paper demonstrates one of the first interventions to show that allowing for self-organisation is actually better than prescribing technique in a DCD population. Additionally, while the gaze instructions themselves were standardised, they appeared to provide such a launchpad for self-organisation. We propose that this is because instructions relate to the perception component (quiet eye) of the perception-action coupling, rather than the action (specific technique cues) component. Key task relevant information is prioritised, but the means to achieving the task goal is via exploration of the perceptual-motor space^[@CR37]^. These findings also support previous research that suggests that QE training provides a more implicit form of learning, relying less on conscious motor control than when providing explicit technical training instructions^[@CR38]^. Future research should seek to employ similar mathematical approaches as adopted in the current study to explore the coordination between ongoing gaze behaviour (e.g., QE) and kinematic measures to further develop QE theory^[@CR39]^.
From a practical, therapeutic perspective, the data also suggest that while children with DCD may reveal greater spatial variability than their TD counterparts^[@CR34]^, reducing variability should not be the sole focus of any intervention designed to improve movement outcomes for this group. Therapeutic sessions may then be best structured around guiding DCD children to utilize task-specific sources of information rather than attempting to guide movement effectiveness directly, particularly through explicit instruction^[@CR40]^. Support for this contention is also underlined by previous research that has suggested that the deficits associated with DCD are linked with a child's persistence with ineffective strategies rather than a generalized inability to learn motor movements^[@CR41]^. We have now shown that -- when guided appropriately via targeted videos - children with DCD can learn to optimise effective gaze control^[@CR14],[@CR15]^ and, in doing so, become more coordinated. While^[@CR14]^ provided some indication (from parental reports) that QE training had a positive impact on subsequent sporting participation, future research needs to explore whether this newly learned strategy can transfer to other skills, and impact on ongoing and extended physical activity and social integration^[@CR2],[@CR3]^.
To conclude, the current study reveals the potential advantages of applying data science techniques to assess trial by trial variability in movement patterns for intervention studies involving clinical groups; in this case, children with DCD. We provide additional support for the efficacy of gaze training interventions, as evidenced by increased coordination between kinematic measures following training. Despite these group differences in coordination, there was considerable variability in the individual coordination patterns within and between groups (and trials). Evidence was therefore found for a self-organization approach, where improved post-training movement coordination emerged from the unique constraints impinging on individuals. These findings suggest that interventions that train the perception element of a visually guided movement task (e.g., QE Training) do not enforce a particular, rigid movement solution, but instead provide perceptual information by which individuals develop their own individual solutions.
Methods {#Sec4}
=======
Data Collection {#Sec5}
---------------
The participants were twenty-one children (aged 7--10) who scored below the 5th percentile on the Movement Assessment Battery for Children-2 (MABC-2)^[@CR42]^. In line with the additional DSM-5 criteria for DCD^[@CR1]^, all children were classified as of 'normal' intelligence based on their teacher/parent reports and scored below the cut-off (98^th^ percentile) on parental reports of the Attention Deficit/Hyperactivity Disorder (ADHD) Rating Scale-VI^[@CR43]^. NHS ethical approval (15/NW/0279) was granted by the RES Committee North West-Greater Manchester South, before any testing was carried out, and parents and children provided written informed consent before taking part. All methods were carried out in accordance with these ethical guidelines and regulations. Figure [4](#Fig4){ref-type="fig"} shows a CONSORT flow diagram outlining participant recruitment and analysis (The CONSORT checklist is available as supplementary data in^[@CR14]^). The trial was registered on the 19^th^ September 2016 (see <https://clinicaltrials.gov/ct2/show/NCT02904980>) and more detail on the full trial protocol is provided in Supplementary Methods.Figure 4Consort 2010 Flow diagram for participant selection into the trial.
Participants performed 50 trials of the throw and catch task from the MABC-2 at three time points: before (baseline) and after training (retention), and at a 6-week delayed retention test. The throw and catch task required children to throw a ball against a wall two meters away and try to catch it on its return -- without letting the ball bounce - using both hands^[@CR42]^. Based on baseline measures, participants were pseudo-randomly divided into either Quiet Eye training (QET) (8 male 3 female, mean age of 8.6 years (*SD* = 1.04); mean MABC-2% of 2.6 (*SD* = 2.09); mean ADHD% of 87.9 (*SD* = 17.1)) or technical training (TT) groups (7 male 3 female, mean age of 8.6 years (*SD* = 1.84); mean MABC-2% of 1.9 (*SD* = 2.19); mean ADHD% of 90.5 (*SD* = 14.7)).
Training consisted of a 4-week group therapy intervention, involving a combination of observational learning via videos, and team games/exercises designed to reinforce the learning points (see 10.1371/journal.pone.0171782.t002 and supplementary data for details). Week 1 of training focused on accurate throwing, week 2 on effective catching, week 3 on linking the throw and catch, and week 4 served as a summary week in which children selected their favourite activities from the previous three weeks. The TT group were given movement-related instructions via video, relating to the throw and catch phases; specifically, by training them to adopt a smooth arm swing during the throw, and to ready themselves and use soft hands to catch. The QET group's video instructed them how to adopt expert-like gaze control; specifically, by training them to fixate a target location on the wall prior to the throw and to track the ball prior to the catch (videos available as supplementary files in^[@CR14]^). The training games (e.g., throwing to targets, catching on the move) were the same for each group but the instructions provided related to the instructions provided in the training videos.
Data Acquisition {#Sec6}
----------------
A 3D motion analysis system (MyoMotion Research Pro, Noraxon Inc., Scottsdale, AZ, USA) was used to collect kinematic data from six upper limb 3D inertial motion capture sensors fitted according to the Noraxon standard manual (Noraxon Inc., Scottsdale, AZ, USA). Two sensors were located on each upper and lower arm, and one sensor on both the pelvis and cervical spine, allowing shoulder and elbow angles and range of motion (ROM, degrees) to be determined for both arms (sampling frequency of 100 Hz). In total, six kinematic measures were computed by the Noraxon software; left and right elbow flexion, shoulder total flexion (taking into account flexion and abduction around the shoulder), and shoulder flexion. Calibration was performed in the standing position to define the 0° of ROM.
Kinematic data were only collected for the first 10 of the 50 trials at each time point, as (1) pilot testing showed that participants frequently struggled to not interfere with the sensors and the eye tracker when worn for too long, and (2) our previous studies had only used 10 trials in each condition^[@CR10],[@CR14],[@CR15]^. Due to calibration problems, we only had complete kinematic data for 19 participants (9 TT and 10 QET) and in this analysis we focus on pre-post intervention data (i.e. baseline to immediate retention) in order to access immediate individual differences due to training.
Data Processing and Analysis {#Sec7}
----------------------------
Data were recorded as time series continuously during the task, so each time series contains ten task trials for each participant. First, we divided the time series into trials (time series segments) by detecting the throwing and catching periods. In our analysis, we focus on the "catching period" (from ball release to catch), which is expected to have highest variability between participants and can be more closely compared to a measure of catching performance.
### Coordination Patterns Evaluation {#Sec8}
Figure [5(a)](#Fig5){ref-type="fig"} provides a summary of the three-step approach adopted to compare coordination before and after training. To consider the temporal relationships (coordination) between kinematic measures, we first computed the covariance between them. The covariance between two data sets *A* and *D* is defined as:$$\documentclass[12pt]{minimal}
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\begin{document}$$cov(A,D)=\frac{1}{N-1}\sum _{i=1}^{N}({A}_{i}-{\mu }_{A})({D}_{i}-{\mu }_{D})$$\end{document}$$where *μ*~*A*~ and *μ*~*D*~ are the mean values of the sets *A* and *D* respectively, and *N* is the number of samples in the sets. Figure [5(b,d)](#Fig5){ref-type="fig"} shows exemplar time series data of the catching period of two different trials. Covariances between pairs of the time series were next saved in a matrix; Fig. [5(c,e)](#Fig5){ref-type="fig"}.Figure 5(**a**) Data processing and analysis pipeline. (**b**,**d**) Time series of kinematic measures: 1: Left elbow flexion, 2: Right elbow flexion, 3: Left shoulder total flexion, 4: Right shoulder total flexion, 5: Left shoulder flexion, 6: Right shoulder flexion. (**c**,**e**) Their covariance matrices CM~1~ and CM~2~, covariance values are colour coded. (**f**) Two first principal dimensions of the multidimensional scaling. Each dot represents a covariance matrix from a single trial. Dots representing covariance matrices CM~1~ and CM~2~ are indicated with black circles. Distances between the dots on the plane of the principal MDS dimensions (black line) are an approximation of the Riemannian distance, δ(CM~1~,CM~2~), between the covariance matrices.
Second, after computing the covariance matrices for all trials for all participants, we then estimated the distance between these covariance matrices by applying a Riemannian geometry approach^[@CR27],[@CR28]^. Riemannian geometry allows us to analyze data that lie in a curved space, where we can no longer apply Euclidian space operators, which is the case for covariance matrices^[@CR27]^. The Riemannian distance between two covariance matrices *CM*~1~ and *CM*~2~ is given by:$$\documentclass[12pt]{minimal}
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\begin{document}$$\delta (C{M}_{1},C{M}_{2})=\sqrt{\sum _{n=1}^{N}lo{g}^{2}{\lambda }_{n}}$$\end{document}$$where *λ*~*n*~ are the N eigenvalues of matrix $\documentclass[12pt]{minimal}
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\begin{document}$$C{M}_{1}^{-1/2}C{M}_{2}C{{M}_{1}}^{-1/2}$$\end{document}$ (or equivalently $\documentclass[12pt]{minimal}
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\begin{document}$$C{M}_{1}^{-1}C{M}_{2}$$\end{document}$)^[@CR28]^.
Third, once the distances between all the pairs of the covariance matrices were calculated, we used Multidimensional Scaling (MDS) to analyse and visualise the differences and similarities between coordination patterns. MDS is a data analysis technique widely applied to visualise the similarities/ differences between data sets^[@CR29]^ (see^[@CR30]^ for an example in movement analysis). MDS allows us to represent the covariance matrix of each participant as a dot in an abstract geometric space. For visualisation purposes we used the first two (i.e. most significant) dimensions of this abstract space. Figure [5(f)](#Fig5){ref-type="fig"} shows a visualisation of all the covariance matrices from all trials using two first principal dimension of the MDS of all the Riemannian distances between pairs of the covariance matrices from the current study.
### Catching Score {#Sec9}
The catching performance scale^[@CR14],[@CR15]^ was used to assess the quality of each attempted catch on an 11-point scale, between '0' (Makes no move towards the ball as it comes back) and '10' (The catch is made exclusively with the palms and fingers). The assessment was made by a researcher - blinded to training group status -- from video recordings taken of all catching attempts. Each number on the scale has an associated description (e.g., '6' -- Ball hits body and is trapped with arms but not hands -- see <https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0171782>). A mean value was then computed for baseline and retention conditions and used in subsequent analyses. We have previously reported a significant difference in training effect for catching score for the participants of this trial^[@CR14]^: The control group did not significantly improve (Bonferroni-corrected *p* = 0.028) from baseline (M = 3.73, *SD* = 2.02) to retention (M = 5.45, *SD* = 2.30), whereas the QE Trained group did (M = 4.10, *SD* = 1.58, to M = 6.54, *SD* = 2.06; Bonferroni-corrected *p* \< 0.001).
### Statistical and Computational Methods {#Sec10}
To test statistical significance of our findings we used non-parametric Mann-Whitney-Wilcoxon test as implemented in Matlab with command ranksum^[@CR44]^. Additionally, where appropriate we control for multiple comparison using Benjamini Hochberg false discovery ratio method^[@CR45]^ as implemented in Matlab with command mafdr (...,'BHFDR', 1). To assess correlations we used Pearson R coefficient of linear dependence \[R, p\] = corr(x,y, 'type', 'Pearson'). Covariance matrices and distances between them were computed in Matlab using cov and distance_riemann, commands respectively. The function distance_riemann computes the Riemannian distances, and can be found in a freely available toolbox called *Covariance Toolbox (*<https://github.com/alexandrebarachant/covariancetoolbox>*)*. The Matlab command for MDS is cmdscale.
Detailed methods and preliminary performance and qualitative data from this trial (ClinicalTrials.gov NCT02904980, 19th September 2016) were published in Wood *et al*. (2017) PLoS ONE 12(2): e0171782. doi:10.1371/journal. pone.0171782.
Supplementary information
=========================
{#Sec11}
Supplementary Methods
**Publisher's note:** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Piotr Słowiński and Harun Baldemir contributed equally.
Electronic supplementary material
=================================
**Supplementary information** accompanies this paper at 10.1038/s41598-018-38204-z.
This research was funded by grants from Liverpool Hope University (HEIF5 Programme 2011--2015); The Waterloo Foundation (1119/1603); and the EPSRC Doctoral College, University of Exeter. KTA gratefully acknowledges the financial support of the EPSRC via grant EP/N014391/1 and PS was generously supported by the Wellcome Trust Institutional Strategic Support Award (204904/Z/16/Z. The funders were not involved in the study design, data collection, data analysis, manuscript preparation, nor publication decisions.
Trial was designed by G.W., M.W. and S.V. Study design was generated by M.W., K.T.A., H.B. and P.S. Data were collected and initially processed by G.W., O.A. and G.C. Data and statistical analyses were performed by P.S. and H.B. M.W., P.S., G.W., S.V., Gen. W. and K.T.A. wrote the paper. All authors discussed the results and provided critical evaluation and comments on the manuscript. Funding for the study was provided by G.W. and M.W.
The datasets generated and analysed during the current study are available at the University of Exeter online repository; DOI:10.24378/exe.783.
Competing Interests {#FPar1}
===================
The authors declare no competing interests.
| {
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Pembrolizumab is a monoclonal antibody against the programmed cell death 1 (PD-1) receptor, and is widely used for the treatment of various malignancies, most commonly malignant melanoma. Here we report the first documented case of Organizing Pneumonia complicating treatment with Pembrolizumab.
1. Case report {#sec1}
==============
A 73 year old patient was admitted to our hospital due to non-resolving pneumonia. He complained of progressive shortness of breath and fever (38.5 °C) that started ten days prior to his admission. Chest x-ray showed the presence of an alveolar infiltrate in the right upper lung field. He was treated with oral ciprofloxacin and cefuroxime, for suspected pneumonia.
Past medical history was significant for recurrent metastatic melanoma which had been initially diagnosed on his forearm more than 30 years ago and was treated by local excision. Recurrent metastatic disease was diagnosed 8 years prior to his admission, and was treated with recurrent local excision, axillary lymph node dissection, partial liver resection, local radiotherapy and whole cell tumor vaccines. Recently an adrenal mass was identified on PET-CT. He was given three courses of ipilimumab, but was switched to pembrolizumab four months prior to his admission due to disease progression. The last dose of treatment with pembrolizumab was two weeks prior to his admission. No known lung metastasis were evident. Other past medical conditions were ischemic heart disease, hypertension, hypercholesterolemia, benign prostatic hyperplasia and depression. Chronic medications included bisoprolol, atorvastatin, valsartan, hydrochlorothiazide, esomeprazole, ezetimibe, mianserin, risperidone, lorazepam, tamsulosine, escitalopram and aspirin. None of these has been started recently.
On examination the patient was febrile with a temperature of 38.2, in no obvious respiratory distress and with an oxygen saturation of 93% while breathing ambient air. He had decreased breath sounds over the right lung with coarse crackles.
Laboratory workup was unremarkable aside from hyponatremia (128 mmole/L). Chest CT revealed a new large consolidation in the right upper lobe, with air-bronchogram ([Fig. 1](#fig1){ref-type="fig"}).
The patient was started on intravenous cefuroxime and ciprofloxacin, which was later changed to piperacillin/tazobactam due to persistent fever andh no improvement of his symptoms. Blood cultures, serum CMV and EBV PCR, urinary testing for Legionella and Pneumococcal antigens and throat PCR-swabs for mycoplasma and respiratory viral antigens were all negative.
Flexible bronchoscopy was performed, and cytopathologic and microbiologic analysis were negative for infectious causes. Trans-bronchial biopsies demonstrated organizing pneumonia ([Fig. 2](#fig2){ref-type="fig"}). He was consequently started on treatment with high dose corticosteroids (I.V. hydrocortisone 100mg TID) with prompt resolution of his fever and marked improvement of his shortness of breath. Repeat chest CT was done 12 weeks after his admission showing marked improvement of his lung infiltrates ([Fig. 3](#fig3){ref-type="fig"}).
2. Discussion {#sec2}
=============
Discovered in 1992, programmed death receptor 1 (PD-1) is a member of the B7-CD28 superfamily [@bib1]. It is expressed on activated T (CD8^+^ and CD4^+^) cells, B cells, monocytes, natural killer T-cells and antigen-presenting cells (APC). Inflammation-induced cytokines produced as a result of infection or tumor formation induce the expression of programmed death receptor ligand 1 (PD-L1) on various cell types, and programmed death receptor ligand 2 (PD-L2) on APC. The PD-1/PD-L1/PD-L2 interaction negatively affects the function of T and B cells, leading to decreased cytokine production and antibody formation, thereby inhibiting autoimmunity, anti-tumor and anti-infectious immunity [@bib2].
Pembrolizumab (previously known as MK-3475 and lambrolizumab) is a humanized IgG-4 monoclonal antibody against PD-1. This blockade enhances the functional activity of the target lymphocytes to facilitate tumor regression and ultimately immune rejection [@bib3]. It is the first anti--PD-1 agent to be approved by the US Food and Drug Administration (FDA) for the treatment of melanoma. It is approved for patients with metastatic melanoma who have failed Ipilimumab treatment and, if *BRAF* mutation positive, also for patients who have failed treatment with a BRAF inhibitor [@bib4]. It is also currently approved for use in melanoma in several additional countries, including recent approval in the European Union [@bib2].
Survival results reported to date in melanoma following this treatment are highly encouraging [@bib5], [@bib6] and further investigation in other malignancies is currently ongoing (e.g. in advanced non-small cell lung cancer) [@bib7]. However, this agent is not free from toxicity and the novel mechanism of action of pembrolizumab is associated with a risk of side effects. These immune-related adverse events are reversible and easily manageable in most cases [@bib8]. However, some of them can be potentially life-threatening and their diagnosis and treatment require experience and the involvement of not only oncologists but also other specialties such as pulmonologists and endocrinologists.
The most frequently reported side effects in the KEYNOTE-002 trial (phase 2 trial of pembrolizumab including 540 patients) were fatigue (30%), pruritus (21%), diarrhea (20%), myalgia (12%), aspartate aminotransferase (AST) increase (10%), nausea (10%), headache (10%) and asthenia (10%). The most common grade 3--4 treatment related side effects reported were hypopituitarism, colitis, diarrhoea, decreased appetite, hyponatremia, and pneumonitis (about 1% of patients each) [@bib6]. Other immune-related adverse events of pembrolizumab reported subsequently in the literature are skin reactions (in up to 42% of patients) [@bib9], [@bib10], uveitis, arthritis, myositis, pancreatitis, hemolytic anemia, partial seizures arising in a patient with inflammatory foci in brain parenchyma, adrenal insufficiency, myasthenic syndrome, optic neuritis, and rhabdomyolysis. These are usually mild and in general are managed with corticosteroids and when essential, interruption of treatment [@bib15].
The prevalence of pneumonitis secondary to anti-PD-1 therapy is variable and may be as high as 4% [@bib10], [@bib12], [@bib13], [@bib14], however data is lacking regarding the definition of pneumonitis used, and tissue confirmed diagnosis. Most cases reported are mild in nature. Workup for these patients includes chest imaging, yet there are no characteristic radiographic findings that will rule in or rule out pneumonitis. In patients with pulmonary metastases or cardiopulmonary comorbidities, evaluation can be particularly challenging. Tumor progression (e.g. lymphangitic spread), pseudoprogression (i.e. inflammation of an existing metastasis), exacerbations of chronic obstructive pulmonary disease, congestive heart failure, diffuse alveolar hemorrhage, and pulmonary embolism are often possible confounding diagnoses [@bib13].
Organizing pneumonia is a known manifestation of drug induced lung injury. It is a histopathologic reaction to a nonspecific inflammatory insult and can occur after exposure to a number of drugs including many anti neoplastic agents. Symptoms may include nonproductive cough and shortness of breath with bilateral crackles. Imaging shows patchy airspace infiltrates, peribronchial or subpleural in location, with air trapping. The histopathologic changes seen in organizing pneumonia include intraluminal buds of granulation tissue with preserved lung architecture [@bib16].
In this paper we report the first biopsy-proven case of organizing pneumonia in a patient treated with pembrolizumab. Our patient presented with signs of lung inflammation while extensive microbiologic workup did not reveal any evidence of active pulmonary infection. His prompt clinical response to corticosteroid treatment is also suggestive of an inflammatory process.
There has been one report of organizing pneumonia in a patient receiving ipilimumab (a cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) blocker) [@bib11] which our patient had received previously, however his symptoms and lung findings began four months after he had stopped this treatment with no pulmonary symptoms, and began treatment with pembrolizumab.
This report highlights the importance of recognizing immune related adverse events, specifically pulmonary inflammation, in patients receiving treatment with novel immune-modulating agents.
Authors contribution {#sec3}
====================
Patient follow up, data collection and manuscript drafting -- RK, ML, AA, UL, NB and ZGF.
Pathology interpretation -- TN.
Radiology interpretation -- EMB.
Conflicts of interest {#sec4}
=====================
All authors declare no conflict of interest regarding this publication.
{#fig1}
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| {
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1. Introduction {#sec0005}
===============
Medicinal plants have been used in the prevention and cure of various diseases based on traditional knowledge for a long time. These plants are often considered safe because of the absence of adverse events. However, many of these plants have not been subjected to toxicological tests to establish their safety profiles \[[@bib0005],[@bib0010]\].
*Kaempferia parviflora* Wall. ex Baker (KP), commonly known as Krachai-dam in Thailand, is a member of the Zingiberaceae family. Its rhizomes are used to treat a variety of gastrointestinal disorders \[[@bib0015]\], improve blood flow \[[@bib0020],[@bib0025]\], and as a traditional treatment for inflammatory and allergic disorders \[[@bib0030],[@bib0035]\]. An alcoholic extract of KP rhizomes is also used by communities in Northern Thailand to treat inflammation \[[@bib0040],[@bib0045]\], as a spasmolytic \[[@bib0050]\], and to treat gastric ulcers \[[@bib0055]\]. Recently, a standardized extract of KP was reported to suppress body weight increase, body fat accumulation, and glucose intolerance in obese mice \[[@bib0060], [@bib0065], [@bib0070]\]. Other reports have suggested that oral intake of standardized KP extract increases energy expenditure and fat utilization \[[@bib0075],[@bib0080]\]. Moreover, dietary intake of standardized KP extract reportedly decreased body fat (both visceral and subcutaneous) in overweight and pre-obese individuals \[[@bib0085]\]. Collectively, these findings suggest that KP extract could reduce body fat in humans.
KP extract has been widely used as a functional food and dietary supplement in various forms over the centuries. KP extract is considered safe as there have been no reports of adverse effects related to its use. A previous study examining daily administration of KP extract for 6 months in rats reported no toxicological effects \[[@bib0090]\]. Other studies reported that daily intake of KP extract for 12 weeks had no adverse effects on blood chemistry parameters \[[@bib0085],[@bib0095],[@bib0100]\]. However, the safety profiles of KP extract are still unavailable and more data are needed. This study was carried out to address this gap. Standardization of the hydroalcoholic KP extract used in this study was ensured via implementation of strict in-process controls during manufacturing. We assessed the mutagenicity and sub-chronic toxicity profiles of standardized KP extract using an *in vitro* bacterial reverse mutation test and an *in vivo* 90-day repeated oral toxicity experimental model.
2. Materials and methods {#sec0010}
========================
2.1. Materials {#sec0015}
--------------
Dried KP rhizomes were received from a selected farm in Thailand. An authentic sample of KP extract (KPFORCE^™^, lot no: 61121028; Maruzen Pharmaceuticals Co., Ltd., Hiroshima, Japan), prepared via hydroethanolic extraction followed by spray drying, was obtained by implementing strict in-process manufacture control along with a detailed certificate of analysis indicating its phytochemical and nutritional compositions, microbiological status, and heavy metal content (quality assurance criteria of KPFORCE^™^ listed in Supplementary Table 1). KPFORCE^™^ contained the same amounts of dextrin,γ-cyclodextrin and KP extract (i.e. 100 mg of KP extract, 100 mg of dextrin and 100 mg of γ-cyclodextrin in 300 mg of KPFORCE^™^). KPFORCE^™^ standardized polymethoxyflavone (8.0--12.0%), and the following six compounds identified via high-performance liquid chromatography: 5,7-dimethoxyflavone, 3,5,7-trimethoxyflavone, 5,7,4′-trimethoxyflavone, 3,5,7,4′-tetramethoxyflavone, 5,7,3′,4′-tetramethoxyflavone, and 3,5,7,3′,4′-pentamethoxyflavone.
2.2. Genotoxicity studies (bacterial reverse mutation test) {#sec0020}
-----------------------------------------------------------
Bacterial reverse mutation tests were conducted at the BoZo Research Center Inc. (Tokyo, Japan). The ability of KPFORCE^™^ to induce reverse mutations at the histidine loci of *Salmonella typhimurium* TA98, TA100, TA1535, and TA1537 (Division of Genetics and Mutagenesis, National Institute of Health Sciences, Japan) and *Escherichia coli* WP2 *uvrA* strains (National Institute of Technology and Evaluation, Japan) was evaluated according to standard procedures \[[@bib0105]\]. The mutagenicity of KPFORCE^™^ was assessed via a preincubation method with and without the exogenous S9 metabolism activation system (Kikkoman Biochemifa Co., Tokyo, Japan). A total of six concentrations (39.1, 78.1, 156, 313, 625, and 1250 μg/plate) were evaluated for *S. typhimurium* TA100, TA1535, and TA1537 in the absence of metabolic activation. In addition, six concentrations (156, 313, 625, 1250, 2500, and 5000 μg/plate) were evaluated for *S. typhimurium* TA98 without metabolic activation and for *S. typhimurium* TA98, TA100, TA1535, and TA1537 with metabolic activation. Five concentrations (313, 625, 1250, 2500, and 5000 μg/plate) of *E. coli* WP2 *uvrA* were evaluated, irrespective of metabolic activation, as no growth inhibition was observed in the preliminary dose selection test.
Sterile water (Otsuka Pharmaceutical Factory, Inc., Tokushima, Japan) was used as the solvent control. The following standard mutagens were used as positive controls: 2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide (AF-2, Wako Pure Chemical Industries, Ltd., Japan), sodium azide (NaN~3~, Wako Pure Chemical Industries, Ltd.), 2-methoxy-6-chloro-9-\[3-(2-chloroethyl)-aminopropylamino\]acridine-2HCl (ICR-191, Polysciences, Inc., Warrington, PA, USA), 2-aminoanthracene (2AA, Wako Pure Chemical Industries, Ltd.), and benzo-alpha-pyrene (BαP, Wako Pure Chemical Industries, Ltd.).
For the bacterial reverse mutation test, 0.1 mL test sample, 0.1 mL bacterial suspension, and 0.5 mL S9 mixture or phosphate buffer (pH 7.4) were added to 2.0 mL top agar containing trace amounts of histidine and biotin and maintained at 45 °C. After careful mixing, the sample was layered on a minimal glucose agar plate, which was inverted after solidification of the top agar and incubation at 37 °C for 48 h. After incubation, revertant colonies were counted. The test results were considered positive if an increase ≥ 2-fold was observed in the number of revertant colonies compared with that observed in the solvent control.
2.3. 90-Day repeated (sub-chronic) oral toxicity study {#sec0025}
------------------------------------------------------
### 2.3.1. Animals {#sec0030}
Healthy Sprague-Dawley rats (Crl:CD, 4 weeks old) were obtained from Charles River Laboratories Japan, Inc. (Hino Breeding Center, Shiga, Japan). After 1 week acclimation, 40 rats (weighing approximately 146--166 g for males and 128--150 g for females) were assigned randomly to four groups comprising five males and five females each. The four groups included one control group and three treatment groups administered 25, 125, and 249 mg/kg bw/day KP extract, respectively, equivalent to 75, 375, and 747 mg KPFORCE^™^/kg bw/day, respectively. According to previous reports \[[@bib0080],[@bib0085]\], the recommended daily KP extract intake is equivalent to 150 mg/day KPFORCE^™^. The safety factors at the maximum, medium, and lowest doses in this 90-day study were approximately 300, 150, and 30, respectively, assuming an average human body weight of 60 kg. In this 90-day toxicity study, in order to reduce the volume administered to animals, we used KP extract with the excipients removed from KPFORCE^™^.
Rats were housed individually in stainless steel wire cages with 12-h light/dark cycles. Rats were provided radiation-sterilized solid food (FR-2; Funabashi Farm Co., Ltd., Chiba, Japan) and tap water (Kaizu City, Gifu, Japan) *ad libitum*. The temperature was maintained at 22--24 °C with 40--70% relative humidity and a ventilation frequency of 10--15 air changes/h.
### 2.3.2. Study design {#sec0035}
The use of animals was approved by the Ethics Committee on Animal Use (Approval no: JBS-07-ROSA-494, Center of Japan Biological Chemistry Co., Ltd.) and standard procedures described in the "Guidelines on Repeat Dose Toxicity Studies" were followed (PFSB, Notification-655, Japan) \[[@bib0110],[@bib0115]\].
### 2.3.3. General observations {#sec0040}
General observations were recorded daily. Body weights as well as food and water consumption were recorded weekly. Blood samples were obtained at the end of the study for hematological and biochemical analyses. All animals were euthanized via intraperitoneal injection of 3% sodium pentobarbital for necropsy at the end of the study, and selected organs were removed and weighed. Histological examinations were performed on all tissues.
### 2.3.4. Hematology and biochemistry {#sec0045}
At the end of the 90-day treatment period, the rats were fasted for 16--18 h, anesthetized via intraperitoneal injection of 3% sodium pentobarbital solution, and blood was collected from the jugular vein. Blood samples for hematological analyses were collected in tubes containing ethylenediaminetetraacetic acid. The following parameters were assessed using an XT-2000i multi-item automated hematology analyzer (Sysmex Corp., Hyogo, Japan): red blood cell count, hemoglobin, hematocrit, platelet count, white blood cell count, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, and leukocyte differential counts.
For biochemical analyses, blood was collected in tubes without anticoagulant and centrifuged to obtain serum. The following serum chemistry parameters were determined using a Hitachi 7170 automated clinical analyzer (Hitachi Ltd., Tokyo, Japan): serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total protein, albumin, blood urea nitrogen, creatinine, total bilirubin, glucose, total cholesterol, and triglycerides.
### 2.3.5. Necropsy and histopathology {#sec0050}
All rats were sacrificed and complete necropsies was performed. The brain, pituitary gland, thyroid, thymus, lungs, heart, liver, spleen, adrenal glands, kidneys, testes, prostate, ovaries, and uterus were weighed. For the thyroid, adrenal glands, kidneys, testes, ovaries, and salivary glands, paired organs were weighed together. Organ to body weight ratios (relative weights) were also calculated. In addition, the following tissue samples were obtained and fixed in 10% neutral-buffered formalin: seminal vesicles, spinal marrow, harderian gland, mesenteric lymph nodes, submandibular lymph nodes, submandibular glands, sublingual glands, pancreas, tongue, trachea, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, bladder, epididymides, vagina, skin, skeletal muscle, sternum, and femur. The testicles and eyes were fixed in Bouin's and Davidson's solutions, respectively. Stored organs and tissue samples from each animal in the control and high-dose groups were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined under an optical microscope. Macroscopic lesions observed during necropsy were also examined from each animal in the other dose groups.
### 2.3.6. Statistical analyses {#sec0055}
Statistical analyses were performed using StatLight^®^ software (Yukms Co., Ltd., Kanagawa, Japan). Data regarding body weight, food and water consumption, urine volume, hematological and biochemical parameters, and organ weights were compared using Bartlett's test for homogeneous variance (*p* \< 0.05). Normally distributed data were compared using one-way analysis of variance and significant differences were further evaluated using Dunnett's test for multiple comparisons (*p* \< 0.05, two-tailed). Non-normally distributed data were analyzed using the nonparametric Dunnett's method (*p* \< 0.05, two-tailed).
3. Results {#sec0060}
==========
3.1. Bacterial reverse mutation test {#sec0065}
------------------------------------
As shown in [Table 1](#tbl0005){ref-type="table"}, the positive controls showed a greater number of revertant colonies than the negative control. However, KP extract treatment did not increase the number of revertants in any of the tested strains in either the absence or presence of S9 metabolic activation. A dose-dependent increase in the number of revertant colonies was observed in the case of *S. typhimurium* TA1537 with S9, although this increase was \< 2-fold relative to the negative control. Similar results were obtained in further confirmation tests (data not shown). No increases in the number of revertant colonies \> 2-fold relative to the negative control were observed with the other test strains and no dose-dependent changes were observed.Table 1Effects of *Kaempferia parviflora* (KP) extract (KPFORCE^™^) on bacterial reverse mutations.Table 1TreatmentDose (μg/plate)TA100TA1535WP2 *uvrA*TA98TA1537−S9+S9−S9+S9−S9+S9−S9+S9−S9+S9Negative control091, 114139, 1329, 138, 1323, 2424, 2419, 1131, 267, 510, 11KPFORCE^TM^39.1108, 123－7, 8－－－－－7, 778.193, 97－4, 5－－－－－8, 4156104, 97165, 16911, 68, 6－－16, 1341, 264, 515, 1531398, 107162, 1705, 712, 1126, 2425, 3520, 1750, 344, 510, 1362575, 83152, 1765[\*](#tblfn0005){ref-type="table-fn"}, 7[\*](#tblfn0005){ref-type="table-fn"}10, 620, 1536, 2915, 1836, 333[\*](#tblfn0005){ref-type="table-fn"}, 6[\*](#tblfn0005){ref-type="table-fn"}14, 18125044[\*](#tblfn0005){ref-type="table-fn"}, 68[\*](#tblfn0005){ref-type="table-fn"}161, 1744[\*](#tblfn0005){ref-type="table-fn"}, 3[\*](#tblfn0005){ref-type="table-fn"}8, 1016, 1725, 2817, 1233, 281[\*](#tblfn0005){ref-type="table-fn"}, 1[\*](#tblfn0005){ref-type="table-fn"}18, 202500－171, 160－4[\*](#tblfn0005){ref-type="table-fn"}, 5[\*](#tblfn0005){ref-type="table-fn"}16, 1527, 1919[\*](#tblfn0005){ref-type="table-fn"}, 19[\*](#tblfn0005){ref-type="table-fn"}27[\*](#tblfn0005){ref-type="table-fn"}, 25[\*](#tblfn0005){ref-type="table-fn"}－7[\*](#tblfn0005){ref-type="table-fn"}, 9[\*](#tblfn0005){ref-type="table-fn"}5000－95[\*](#tblfn0005){ref-type="table-fn"}, 110[\*](#tblfn0005){ref-type="table-fn"}－4[\*](#tblfn0005){ref-type="table-fn"}, 3[\*](#tblfn0005){ref-type="table-fn"}18, 1619, 1519[\*](#tblfn0005){ref-type="table-fn"}, 12[\*](#tblfn0005){ref-type="table-fn"}35[\*](#tblfn0005){ref-type="table-fn"}, 25[\*](#tblfn0005){ref-type="table-fn"}－6[\*](#tblfn0005){ref-type="table-fn"}, 5[\*](#tblfn0005){ref-type="table-fn"}AF-20.01571, 588－－－67, 62－－－－－0.1－－－－－－321, 327－－－NaN~3~0.5－－257, 273－－－－－－－ICR-1911.0－－－－－－－－1070, 1052－BαP5.0－892, 946－－－－－391, 427－95, 822AA2.0－－－305, 278－－－－－－10.0－－－－－739, 878－－－－[^1][^2][^3]
3.2. 90-Day repeated oral toxicity study {#sec0070}
----------------------------------------
### 3.2.1. General observations {#sec0075}
No animals in any of the treatment groups died during the study. Increased salivation was observed in two male and three female rats in the medium-dose group (125 mg/kg bw/day KP extract), and in four male and four female rats in the high-dose group (249 mg/kg bw/day KP extract). However, the increased salivation was mild in severity, occurred approximately 1--3 min post dosing, and only persisted for approximately 10 min in all of the affected animals.
No significant differences between the treatment groups and the control group were observed with regard to weekly body weight, food consumption, or water consumption (Supplementary Tables 2 and 3).
### 3.2.2. Hematological and biochemical parameters {#sec0080}
After 90 days of KP extract administration, sporadic and generally statistically non-significant changes in hematological parameters were observed ([Table 2](#tbl0010){ref-type="table"}). Platelet counts in female rats in the low-dose group (25 mg/kg bw/day KP extract) were significantly higher than those in control group rats. However, no statistically significant differences were observed in the medium-dose (125 mg/kg bw/day KP extract) or high-dose (249 mg/kg bw/day KP extract) groups, indicating the absence of any dose-dependent trend. Evaluation of the biochemical parameters shown in [Table 3](#tbl0015){ref-type="table"} revealed no significant KP extract-associated changes in animals of either sex in any dose group compared with their respective controls.Table 2Effects of KP extract on hematological parameters in rats treated orally for 90 days.Table 2ParameterGroup (females)Group (males)ControlLow-doseMedium-doseHigh-doseControlLow-doseMedium-doseHigh-doseWBC (10^9^/L)54.5 ± 10.963.2 ± 12.442.1 ± 9.047.5 ± 8.696.5 ± 12.891.7 ± 17.497.8 ± 32.3106.0 ± 24.1RBC (10^12^/L)724.4 ± 21.9752.6 ± 30.1708.0 ± 10.2724.0 ± 21.6788.6 ± 41.2788.8 ± 32.4791.8 ± 35.7770.4 ± 11.5HG (g/L)14.3 ± 0.214.5 ± 0.614.0 ± 0.414.1 ± 0.214.7 ± 0.615.0 ± 0.514.7 ± 0.814.5 ± 0.2HC (%)39.3 ± 0.839.4 ± 1.137.9 ± 1.138.6 ± 0.940.4 ± 1.241.3 ± 1.240.3 ± 2.240.0 ± 0.5PLT (10^12^/L)82.8 ± 9.1100.4 ± 12.5[\*](#tblfn0010){ref-type="table-fn"}88.6 ± 10.383.3 ± 5.796.2 ± 3.493.3 ± 11.7104.4 ± 13.796.7 ± 9.9MCV (fL)54.3 ± 1.352.3 ± 1.053.6 ± 1.653.4 ± 1.251.3 ± 1.552.4 ± 2.250.9 ± 2.352.0 ± 1.3MCH (pg)19.8 ± 0.619.2 ± 0.419.8 ± 0.619.5 ± 0.518.7 ± 0.519.0 ± 0.518.5 ± 0.818.8 ± 0.5MCHC (g/dL)36.5 ± 0.436.8 ± 0.637.0 ± 0.236.5 ± 0.536.5 ± 0.436.3 ± 0.636.4 ± 0.436.1 ± 0.4Lymphocytes (%)78.3 ± 8.582.4 ± 8.280.8 ± 6.085.3 ± 1.379.3 ± 4.081.9 ± 4.782.4 ± 2.083.5 ± 3.5Neutrophils (%)16.8 ± 8.112.9 ± 8.314.1 ± 4.910.0 ± 0.415.3 ± 3.412.8 ± 4.112.7 ± 1.312.1 ± 2.7Other (%)4.9 ± 1.14.7 ± 2.15.1 ± 1.34.7 ± 1.55.4 ± 0.75.3 ± 1.94.9 ± 1.34.4 ± 1.6[^4][^5][^6][^7]Table 3Effects of KP extract on biochemical parameters in rats treated orally for 90 days.Table 3ParameterGroup (females)Group (males)ControlLow-doseMedium-doseHigh-doseControlLow-doseMedium-doseHigh-doseAST (U/L)64.2 ± 2.969.4 ± 9.859.4 ± 5.467.4 ± 27.394.2 ± 24.871.6 ± 15.887.8 ± 31.883.2 ± 17.6ALT (U/L)26.6 ± 4.831.6 ± 5.526.2 ± 3.428.4 ± 12.140.4 ± 18.329.4 ± 5.934.4 ± 11.835.4 ± 7.2ALP (U/L)110.4 ± 23.2121.6 ± 34.4147.4 ± 45.1139.0 ± 88.1248.2 ± 56.3246.2 ± 65.9236.4 ± 13.3291.8 ± 63.4Total protein (g/L)61.0 ± 4.061.8 ± 4.064.2 ± 4.862.6 ± 2.956.2 ± 1.357.0 ± 2.453.8 ± 2.855.4 ± 3.5Albumin (g/L)30.2 ± 2.831.0 ± 2.332.4 ± 3.131.0 ± 2.124.0 ± 1.023.8 ± 0.822.8 ± 1.324.2 ± 1.6BUN (mmol/L)6.44 ± 0.486.45 ± 1.155.38 ± 0.846.13 ± 1.255.79 ± 0.375.88 ± 0.296.26 ± 0.545.91 ± 0.58CRE (mmol/L)33.59 ± 7.4033.59 ± 3.9531.82 ± 4.8426.52 ± 0.0030.06 ± 4.8426.52 ± 6.2530.06 ± 4.8424.75 ± 3.95T-BIL (μmol/L)1.0 ± 0.91.0 ± 0.91.0 ± 1.51.4 ± 0.80.7 ± 0.90.7 ± 0.90.0 ± 0.00.0 ± 0.0Glucose (mmol/L)81.38 ± 10.0682.82 ± 9.7781.04 ± 6.7979.93 ± 5.3084.71 ± 10.0685.04 ± 13.3980.71 ± 8.8185.37 ± 12.35T-Cho (mmol/L)1.83 ± 0.331.69 ± 0.182.16 ± 0.732.08 ± 0.131.72 ± 0.271.83 ± 0.431.74 ± 0.292.03 ± 0.44TG (mmol/L)0.48 ± 0.100.61 ± 0.300.99 ± 0.550.74 ± 0.240.72 ± 0.340.84 ± 0.340.84 ± 0.611.03 ± 0.37[^8][^9][^10]
### 3.2.3. Pathology {#sec0085}
Organ weight data are presented in [Table 4](#tbl0020){ref-type="table"}. Compared with the control group, male rats in the low-dose group (25 mg/kg bw/day KP extract) exhibited significantly higher relative weights of the thymus and heart, and female rats in the high-dose group (249 mg/kg bw/day KP extract) showed significantly lower relative adrenal gland weight. However, these changes were not dose-dependent, and all were within the ranges of the testing institution's historical records. As such, these changes were not considered to be biologically significant.Table 4Effects of KP extract on relative organ weights in rats treated orally for 90 days.Table 4ParameterGroup (females)Group (males)ControlLow-doseMedium-doseHigh-doseControlLow-doseMedium-doseHigh-doseLiver(g/100 g)2.55 ± 0.132.57 ± 0.272.66 ± 0.092.68 ± 0.132.88 ± 0.213.03 ± 0.332.69 ± 0.103.03 ± 0.25Kidneys(g/100 g)0.60 ± 0.020.57 ± 0.050.62 ± 0.040.61 ± 0.040.58 ± 0.070.56 ± 0.070.59 ± 0.050.61 ± 0.07Spleen(g/100 g)0.20 ± 0.030.17 ± 0.010.19 ± 0.020.17 ± 0.030.16 ± 0.020.18 ± 0.020.18 ± 0.040.17 ± 0.03Thymus(g/100 g)0.13 ± 0.010.12 ± 0.030.12 ± 0.010.14 ± 0.030.08 ± 0.010.10 ± 0.02[\*](#tblfn0015){ref-type="table-fn"}0.09 ± 0.000.09 ± 0.02Heart(g/100 g)0.33 ± 0.050.35 ± 0.030.32 ± 0.030.33 ± 0.020.27 ± 0.030.32 ± 0.04[\*](#tblfn0015){ref-type="table-fn"}0.27 ± 0.010.28 ± 0.02Brain(g/100 g)0.63 ± 0.050.67 ± 0.060.60 ± 0.060.60 ± 0.080.38 ± 0.020.38 ± 0.030.36 ± 0.030.39 ± 0.04Lungs(g/100 g)0.37 ± 0.030.36 ± 0.030.36 ± 0.020.34 ± 0.030.27 ± 0.010.26 ± 0.030.26 ± 0.020.27 ± 0.02Pituitary(mg/100 g)5.4 ± 1.56.0 ± 1.25.8 ± 1.35.0 ± 1.02.4 ± 0.52.4 ± 0.91.8 ± 0.42.4 ± 0.5Thyroid(mg/100 g)5.8 ± 1.05.9 ± 0.85.9 ± 1.35.4 ± 0.93.7 ± 0.64.2 ± 0.44.3 ± 0.64.6 ± 0.5Adrenal gland(mg/100 g)25.5 ± 6.123.3 ± 5.723.3 ± 3.118.3 ± 2.4[\*](#tblfn0015){ref-type="table-fn"}11.1 ± 2.110.9 ± 3.211.5 ± 2.911.0 ± 2.7Testes(g/100 g)－－－－0.61 ± 0.080.61 ± 0.030.54 ± 0.030.60 ± 0.06Prostate(g/100 g)－－－－0.25 ± 0.060.21 ± 0.120.25 ± 0.050.27 ± 0.10Uterus(g/100 g)0.24 ± 0.040.24 ± 0.050.27 ± 0.100.17 ± 0.01[\*](#tblfn0015){ref-type="table-fn"}－－－－Ovaries(mg/100 g)29.7 ± 7.124.3 ± 5.326.2 ± 6.025.1 ± 5.5－－－－[^11][^12][^13]
Necropsy and histopathological examinations were performed on rats in the control and high-dose (249 mg/kg bw/day of KP extract) groups. As shown in Supplementary [Table 4](#tbl0020){ref-type="table"}, histopathological examinations showed no apparent difference with regard to the severity or frequency of findings between the two groups. During necropsy, testicular edema and small epididymal size (unilateral and right, respectively) were noted in one male animal in the low-dose group (25 mg/kg bw/day KP extract). However, as these changes were sporadic, they were considered unrelated to KP extract treatment.
4. Discussion {#sec0090}
=============
KP extract has long been used to treat gastrointestinal disorders \[[@bib0015]\], improve blood flow \[[@bib0020],[@bib0025]\], and as a remedy for inflammatory and allergic disorders \[[@bib0030],[@bib0035]\]. KP extract is also used in commercially prepared functional foods because of its anti-oxidative, anti-inflammatory, and anti-obesity properties \[[@bib0030],[@bib0065],[@bib0085],[@bib0120]\]. However, few studies have provided toxicological evaluations of KP extract. A previous study examining daily administration of KP extract for 6 months in rats reported no toxicological effects \[[@bib0090]\]. Other studies reported that daily intake of KP extract for 12 weeks had no adverse effects on blood chemistry parameters [@bib0085],[@bib0095],[@bib0100]\]. Moreover, a previous toxicology report of dietary ingredients including KP extract revealed no adverse effects \[[@bib0125]\]. However, safety profiles for KP extract are still unavailable and more data are needed. In this study, therefore, we investigated the toxicity profiles of KP extract via genotoxicity and sub-chronic oral toxicity studies. The results of these studies indicate that KP extract had no significant adverse effects. These data suggest the safety of KP extract and support its potential use in functional foods and/or dietary supplements.
A previous acute toxicity study in mice reported a mean lethal dose of KP rhizomes of \>13.3 g/kg \[[@bib0130]\]. Through an acute toxicity study, we determined that the mean lethal dose of KP extract used in this trial was \>2000 mg/kg (data not shown). Moreover, toxicity analyses of 6 months dietary intake of 500 mg/kg bw/day KP ethanolic extract in rats indicated no adverse effects \[[@bib0090]\]. Similarly, the results of the present study revealed no genotoxicity or sub-chronic toxicity related to polymethoxyflavone-standardized KP extract treatment. The statistically significant changes in platelet count and organ weight observed in some cases were not considered toxicologically relevant, as these changes were non-severe and parameters remained within the historical control ranges of the testing laboratory and/or exhibited no clear dose-response relationships. These data suggest that the KP extract is safe; however, synegistic effects have been reported for toxic stimuli \[[@bib0135]\], thus it is necessary to conduct additional safety profile studies for various combinations in the future. Furthermore, assessing the maternal toxicity and embryo/fetal development following oral administration in pregnant animals is also necessary \[[@bib0140]\].
In summary, the results of these 90-day toxicity and genotoxicity studies suggest that KP extract is safe for dietary consumption as either a functional food or dietary supplement.
5. Conclusions {#sec0095}
==============
In the present study, KP extract did not induce gene mutations in bacteria or exhibit toxicity in male or female rats after 90 days of repeated oral administration at high dose (249 mg/kg bw/day, equivalent to 747 mg KPFORCE^™^/kg bw/day). These results indicate that KP extract is not genotoxic and that its no-observed-adverse-effect level in rats is \>249 mg/kg bw/day.
This study was conducted at BoZo Research Center Inc. and Center of Japan Biological Chemistry Co., Ltd., an independent contract research laboratory. We would like to thank Editage ([www.editage.jp](http://www.editage.jp){#intr0005}) for English language editing.
[^1]: Results are shown as means from two plates as indicated by the number of revertant colonies (n = 2).
[^2]: Abbreviations: TA100, *Salmonella typhimurium* TA100; TA1535, *Salmonella typhimurium* TA1535; WP2 uvrA, *Escherichia coli* WP2 uvrA; TA98, *Salmonella typhimurium* TA98; TA1537, *Salmonella typhimurium* TA1537; AF-2, 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide; ICR-191, 2-methoxy-6-chloro-9-\[3-(2-chloroethyl)aminopropylamino\]-acridine dihydrochloride; BαP, benzo-\[α\]-pyrene; 2AA, 2-aminoanthracene.
[^3]: Growth inhibition was observed.
[^4]: Results are shown as means ± standard deviation (n = 5).
[^5]: Low dose: 25 mg/kg bw/day; medium dose: 125 mg/kg bw/day; high dose: 249 mg/kg bw/day.
[^6]: Abbreviations: WBC, white blood cell count; RBC, red blood cell count; HG, hemoglobin; HC, hematocrit; PLT, platelet count; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration.
[^7]: Statistically significant difference relative to the control (*p* \< 0.05).
[^8]: Results are shown as means ± standard deviation (n = 5).
[^9]: Low dose: 25 mg/kg bw/day; medium dose: 125 mg/kg bw/day; high dose: 249 mg/kg bw/day.
[^10]: Abbreviations: AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase; BUN, blood urea nitrogen; CRE, creatinine; T-BIL, total bilirubin; T-Cho, total cholesterol; TG, triglycerides.
[^11]: Results are shown as means ± standard deviation (n = 5).
[^12]: Low dose: 25 mg/kg bw/day; medium dose: 125 mg/kg bw/day; high dose: 249 mg/kg bw/day.
[^13]: Statistically significant difference relative to the control (*p* \< 0.05).
| {
"pile_set_name": "PubMed Central"
} |
INTRODUCTION {#sec1-1}
============
Maternal death is defined as death during pregnancy or up to 42 days after termination of pregnancy, irrespective of the duration and site of pregnancy, resulting from any relevant cause or as a result of care provided during pregnancy, excluding accidental death as a cause. The maternal mortality ratio (MMR) is defined as the ratio of maternal deaths to live births within a given time period. Various methods are used to determine MMR, which may be classified as "empirical" or "analytical" according to the goals and sources of information available.\[[@ref1]\] Individual countries generally use empirical methods, while global estimates use modeling methods. In developing countries, the most commonly used method is based on surveys that directly and indirectly measure MMR.\[[@ref2][@ref3][@ref4]\] Various studies have explored the best method for determining MMR.\[[@ref2][@ref5][@ref6][@ref7]\] The maternal mortality surveillance system (MMSS) was established in countries such as Iran to determine the exact MMR and consequently to control and prevent maternal mortality resulting from complications arising during pregnancy.
Because MMR is one of the main indicators of development and one of the millennium development goals (MDGs), its estimation indicates a country\'s progress through the MDGs.
The capture-recapture (CRC) method is an analytical method,\[[@ref1]\] in which different data sources are used that separately register a single outcome. Taking into account the number of cases registered by each source, the number of cases common among the sources and the number of cases unregistered, eventually the overall numbers of incident cases of an outcome are estimated in a population in this method. This method is used in estimating health outcomes in situations as diverse as AIDS,\[[@ref8]\] tuberculosis,\[[@ref9]\] diabetes,\[[@ref10]\] meningitis,\[[@ref11]\] occupational disabilities,\[[@ref12]\] genetic diseases such as Down\'s syndrome,\[[@ref13]\] traffic accidents\[[@ref14][@ref15]\] and is even used in showing the sensitivity of databases for finding articles.\[[@ref16]\]
To examine the validity of CRC, the results of this study is compared with MMR estimates obtained in other studies through other methods. In estimating MMR, the Inter-Agency Group (Inter-Agency Maternal Mortality Estimate Group; World health Organization, United Nations Children\'s Fund, United Nations Population Fund and the World Bank), classifies the country according to the available data and uses different methods for estimating MMR in each class. In the 2007 estimation, Iran was in Group F (using census data) and MMR was estimated using the reported proportion of maternal deaths among all female deaths and the estimated deaths among those of fertile age.\[[@ref17]\] In the 2010 estimations, Iran was assigned to Group B and MMR estimates were modeled using available country data from the MMSS;\[[@ref18]\] Hogan *et al*. estimated MMR using data available from various countries regarding maternal deaths, overall deaths and the adult death rate.\[[@ref19]\] The objective of this study is to evaluate the applicability, firstly by means of feasibility in conducting the calculation with data sources from a developing country and secondly agreement with other estimates, of CRC as an analytical method to estimate MMR in countries.
METHODS {#sec1-2}
=======
The MMSS and National Death Registry (NDR) databases were used to estimate maternal mortality.
Sources of data {#sec2-1}
---------------
### Iranian MMSS {#sec3-1}
All maternal deaths in Iran are registered in the MMSS, using the definitions of the Ninth International Classification of Diseases (ICD-9). The sources of information for the MMSS are as follows: A list of the deaths of married women aged 10-49 years who died of any cause except accidents and provided by the district death registry officer; sudden maternal death that occurred in hospital, reported by the hospital supervisor to the district health network manager; and telephoned or personal reports to the nearest health-care unit for women not covered by the healthcare system. If a death occurs at home or on the way to the health-care unit, a team of investigators is sent to visit the deceased\'s house and the centers where she has received her maternal care and the appropriate questionnaires are completed. All the collected data are examined by the District Health Committee and the causes of death and their relevancy to pregnancy are assessed.\[[@ref20]\]
### Iranian NDR {#sec3-2}
The NDR registers and collects information regarding all deaths in Iran, along with their causes. The sources of information for the NDR are as follows: Hospitals, official cemeteries, medico-legal organizations and all basic health units and rural health centers. The 2004 data from three provinces and the 2005 data from one province are not complete in the NDR. Therefore, all estimates were calculated twice: Including and excluding these incomplete provinces. We estimated the missing data using the number of maternal deaths registered in the MMSS with the assumption that the sensitivity of the registration system in the MMSS was similar throughout the country.
Capture re capture method {#sec2-2}
-------------------------
When it is not possible to identify and obtain a correct consensus of the individuals of a community with common characteristics (e.g. a specific disease), the CRC method is used to estimate the population. In this situation different sources that have registered the concerned population are used. In fact, in this method, the overall population is estimated by the number of individuals that have not been identified by different sources \[[Figure 1](#F1){ref-type="fig"}\]. The figure and the relevant text have been added to the article. Based on the number of maternal deaths in each data source, the CRC method estimates the overall number of maternal deaths (including all cases not registered by the data sources). If *n*~1~ is the number of maternal deaths in the NDR, *n*~2~ is the number of cases registered in the MMSS, and *d* is the number of cases common to the two data sources, then the total number of maternal deaths is calculated as follows:
*N* = \[(*n*~1~ + 1) (*n*~2~ + 1)/(*d* + 1)\] - 1,
and its variance is calculated using the equation\[[@ref21]\]
Var (*N*) = (*n*~1~ + 1) (*n*~2~ + 1) (*n*~1~ - *d*) (*n*~2~ - *d*)/(*d* + 1)^2^ (*d* + 2).\[[@ref21]\]
{#F1}
In applying the CRC method, we must assume that the population under study was closed, the individuals were homogenous, complete matching between the data sources is possible and that the data sources were independent in data-collection. In the present study, the first two assumptions are acceptable; the other assumptions were examined as follows.
Matching between two databases (determining the number of cases common to the two banks) {#sec2-3}
----------------------------------------------------------------------------------------
Prior to performing CRC, it is necessary to determine the number of deceased that are common to the two databases and the requisites of matching must be clearly defined. The similarity of individuals was defined at three levels, taking into account problems in the documented registration of data, differences in computer data entry between the two databases and differences in the database officers' opinions:
### Perfect matching {#sec3-3}
When the following criteria are all the same: name and surname (without taking into account the prefix and suffix), age (±5 years), address and time of death (±1 month).
### Semi-perfect matching {#sec3-4}
Despite small differences in name and surname (by altering or omitting a few letters), two individuals are considered the same when all other criteria are similar.
### Non-perfect matching {#sec3-5}
When the name and surname are worded differently, the address is the same, but the ages are different (±5 years) or months of death are different (difference of 1-2 months), the two individuals are still considered the same. The degree of rigidity in determining the similarity of the two databases is reduced from level one to level three.
### Matching process {#sec3-6}
Because the NDR includes all the deaths, we separated maternal deaths (according to the ICD coding) in the beginning and then compared them with the MMSS data. Two databases were imported to one data file and because the number of cases was few, we handled the matching process manually. We used deterministic approach in each matching level for estimating the MMR.\[[@ref22]\]
Examining the independence of the databases {#sec2-4}
-------------------------------------------
The assumption of independence of databases is most important in estimating the desired outcome in the CRC method. The term "independence of databases" means that the presence of an individual in one database will not influence the presence or absence of the same individual in another database. No analytical method can test for independence if only two sources of data are used for estimation. One of the sources of data used to identify cases of death in the MMSS is the NDR list of deaths in the past 3 months; this step in the MMSS can lead to dependence of the MMSS on the NDR. Therefore, two procedures were performed to examine the independence of the data-collection procedure: We examined the source of information regarding the occurrence of maternal death for cases registered in the MMSS and performed a sensitivity analysis on the dependency of the two databases.
In the first procedure, all provinces were requested to report to the Family Health Unit of the "Ministry of Health and Medical Education" the source of information for each case of maternal death recorded in the MMSS. The response rate was 84%. These data showed that 46% of maternal mortalities were reported from hospitals by supervisors, while the rest were reported through other routes to the district Family Health Unit. The data also revealed that among cases common to the two databases in 2005, the source of information was the hospital in 60% of cases. Information regarding maternal mortalities is actively sent from the hospital to the district health network by the hospital supervisor, while information regarding hospital deaths is sent passively to the coordination affairs office of the district health network once every 3 months in NDR. Therefore, at least 60% of the data in each database is collected independently. Under the most pessimistic conditions, the degree of dependency between the two databases is 40%.
In sensitivity analysis, if dependency is assumed to be 40% between the two databases for MMR, then the MMR would increase (the number of cases estimated to be registered by only one database is equal to the product of "number of cases currently registered by that database" and "the ratio of cases common in the other database").
*N*~2~= (*n*~2~ - *d*) × (1/(\[*d*/*n*~1~\] - dependency)).
Hence, the numbers of cases in the MMSS databank have been calculated after a certain rate of dependency; they have then been added to the other databank\'s cases. The total number has then been calculated.
We assumed that the two databases were independent; however, if they are in fact dependent, then the present estimates are lower than the real ratios and the ratios obtained in the present study are the lowest possible.
Examining the validity of cause of death in the NDR {#sec2-5}
---------------------------------------------------
Some of the cases registered in the NDR as maternal deaths are not recorded in the MMSS. To improve the accuracy of the MMR calculation, we inspected all such cases for accuracy by extracting information from the NDR database and by visiting the recorded street addresses.
RESULTS {#sec1-3}
=======
An analysis of cases common to the two databases, based on the three matching levels revealed that the number of common cases increased as the matching level increased \[[Table 1](#T1){ref-type="table"}\]. As seen in [Table 1](#T1){ref-type="table"}, 100 more cases are reported each year by the NDR than by the MMSS. [Table 2](#T2){ref-type="table"} shows the results of MMR, calculated using the CRC method for each level of matching after excluding provinces with incomplete documentation.
######
Cases of registered maternal death and common cases in the two databases after matching (excluding provinces with incomplete data)
######
MMR calculated for various matching levels before and after correction of NDR data and assuming complete independence or 40% dependency, for 2004 and 2005
A review of the cases of maternal death present in the NDR but absent in the MMSS (thus causing major changes in the MMR) revealed 40 such cases in 2004, with the real cause of death assessable in 27 cases: Of these, the cause of maternal death was pregnancy-related in only seven. In 2005, 30 such cases were present and the real cause of death was assessable in 18 cases; of these, the cause of maternal death was pregnancy-related in only three.
By "best-case" scenario, we are referring to the MMR calculated for 100% independence of two databanks and under circumstances where the calculations have been done in spite of the provinces' incomplete data. By "worst-case" scenario, we are referring to a state where there is 40% dependency between the databanks and the provinces' data have not been entered in completely. According to the CRC method, MMR in 2004 and 2005 were 33 and 25 in the best-case scenarios respectively and 86 and 59 in the worst-case scenarios respectively.
DISCUSSION {#sec1-4}
==========
We used the CRC method to estimate the Iranian MMR for 2004 and 2005. According to our study, MMR could range between 25 (under the conditions of 100% independence of databases, including provinces with incomplete data, with matching level three and after correcting the NDR) and 59 (under the conditions of 40% dependency of databases, perfect matching level and excluding provinces with incomplete data, perfect matching).
Three main infrastructures are required to do CRC and obtain accurate estimates: Having valid databases, having common variables for homogeneity and the pre-assumptions of CRC.
The number and quality of variables available for comparison is a strong predictor of estimate accuracy in record linkage methods.\[[@ref23]\] Correct registration of cause of death in databases is the first stand to achieve accurate estimates by CRC. In this study the correction of causes of death in one database, i.e. NDR has significantly changed the MMR estimation.
All methods for determining, the number of maternal deaths are influenced by two major errors: Identification of deaths and determining the relevance of the cause of death to pregnancy.\[[@ref1]\] In the present study, dependency between the databases existed only in identification of deaths. Because all of the cases reported in the MMSS are examined for the true cause of death, there was no dependence between the two databases in determining the cause of death.
There are various ways of managing the disadvantage of being unable to analytically test the independence of the two databases: The source of information for each individual can be accurately registered in both databases and three databases could be used to estimate MMR in the future. Currently, a third database does not exist in Iran, but data available from public and private insurance firms could possibly be modified for this purpose. Another solution is directly using the "data sources" of NDR rather than using NDR data directly (e.g. using data from cemeteries and the National Organization for Civil Registration along with the MMSS). However, how close is the MMR rate calculated by CRC to the true estimate? To investigate this issue, we can use the agreement between estimates made at international level by Hogan and WHO \[[Table 3](#T3){ref-type="table"}\]. As illustrated, the CRC estimates are close to the estimates made by Hogan and WHO in 2010, but widely differ from the estimates made by international organizations in 2007.
######
Comparison of various estimates of the Iranian MMR
To examine the validity of CRC, the results of this study were compared with MMR estimates obtained in other studies through other methods \[[Table 3](#T3){ref-type="table"}\]. As illustrated in [Figure 2](#F2){ref-type="fig"}, the CRC estimates for 2005 are close to the estimates for the same year made by Hogan and WHO (published in 2010), but widely differ from the estimates made by international organizations in published in 2007. Even though, the uncertainty of CRC is high, it implies that the international estimations in 2007 are far from the truth. Therefore, at this point, CRC is a cost-effective method for estimating health related indicators. Since every database has been designed according to its own specific goals and this method is an optimal method of using available data, upgrading the validity of data in databases should be kept in mind. Therefore, quality control should be executed regularly.
{#F2}
Limitation {#sec2-6}
----------
One of the greatest challenges in the present study was the matching procedure. As we can see, MMR estimation dependent on matching levels are different. Inaccurate registration of case\'s attributes will damage the estimates. Numerical variables (such as National ID) could be used to identify individuals in all data registration systems, to increase the flow and validity of the matching process.
As mentioned previously, the most important assumption in using the CRC method is that the data-collection process of the databases is independent. Because only two sources of data were used in the present study, it was not possible to test for independence using analytical tests. Previous similar studies were either certain of the independence of the two databases and provided no proof for this assumption,\[[@ref24][@ref25]\] or examined dependency through a questionnaire to the data-collection sites of both sources.\[[@ref26]\] Another limitation of the study is that databases were not accessible at the appropriate time; the NDR data was not accessible for the years following 2005. There are two expansions in this regard; the first is conducting new calculation with most recent data to get more reliable estimates of MMR and the second is conducting other methods such as "sisterhood within a 5-year census" along the country to validate the MMR from another perspective.
CONCLUSIONS {#sec1-5}
===========
In developing countries using available data is a good way of estimating health rates. Therefore, it is vital for part of health systems' activities to focus on promoting the quality of registering the diseases. In order to improve the MMR estimation, it\'s suggested to add some questions in the survey, which is carried out every 5 years in Iran. In order to increase the feasibility of the CRC, doing studies for improving the quality of NDR and use of three sources for CRC is suggested.
The authors acknowledge the Maternal Mortality Surveillance System and Death Registry System staff for their intensive work in order to producing these data banks.
**Source of Support:** This work was supported by the Ministry of Health and Medical Edcation and by Tehran University of Medical Sciences (grant number 132/12142)
**Conflict of Interest:** None declared.
| {
"pile_set_name": "PubMed Central"
} |
See related research article <http://arthritis-research.com/content/7/5/R1063>
In daily clinical practice, the Disease Activity Score using 28 joint counts (DAS28) is used to monitor the disease activity of rheumatoid arthritis patients treated with disease-modifying anti-rheumatic drugs (DMARDs) and biological agents. This is useful to inform the rheumatologist about whether the treatment is producing the expected effects in an appropriate period of time or whether the treatment should be more intensified.
In an article in the present issue, Vander Cruyssen and colleagues investigated which variables can best be measured to evaluate the effect of therapy and the remaining disease activity in daily clinical practice \[[@B1]\]. This study was based on a cohort of 511 patients with active refractory rheumatoid arthritis who were treated with infliximab \[[@B2]\]. Patients who were judged by their physicians to have an insufficient response at week 22 received a dose increase at week 30. According to the authors, the decision to increase the dose was based on clinical judgement, without knowledge of outcome measures such as the DAS28. In their study, the authors found that the DAS28 as a continuous composite index correlated best with the decision to give a dose increase of infliximab, which was used as a surrogate measure of insufficient response. The discriminative capacity of the DAS28 could only slightly be improved by the inclusion of supplemental variables in the regression model. Recalculation of the DAS28 coefficients in a discriminative function obtained similar coefficients and the same discriminative capacity as the original DAS28. For a better understanding of these results, it is informative to know how the Disease Activity Score and the DAS28 were developed back in the 1990s.
The DAS28 was developed in a similar way to the Disease Activity Score, but the DAS28 contains reduced, ungraded, joint counts and has different weights \[[@B3],[@B4]\]. The DAS28 was developed in a cohort from an outpatient clinic, using the data from 227 early rheumatoid arthritis patients that were followed-up for 9 years between 1985 and 1994. Because no gold standard for disease activity is available, decisions on DMARD therapy were used as an external standard of \'high\' and \'low\' disease activity in the development of the DAS28. The DAS28 formula optimally discriminated between these two clinically relevant states. The validity of the DAS28 was tested using a similar cohort from another clinic. Since their development, the Disease Activity Score and the DAS28 have extensively been validated \[[@B5]\].
An interesting finding from the study of Vander Cruyssen and colleagues is that they also used decisions to change (infliximab) treatment as a proxy for the underlying disease activity, and produced the same DAS28 as found 20 years earlier in a cohort in which only conventional DMARDs were used, without a need to change its content or form. This means that the DAS28 is able to discriminate between clinically relevant states of disease activity, rather than discriminating a \'readiness\' to change treatment (from physicians and patients) to start, to stop or to continue DMARD treatment. This enforces the validity and generalisability of the DAS28.
The authors reached their conclusion based on a series of analyses comparing the performance of multiple measures in several ways. The authors used receiver-operating characteristic curves and sensitivity, specificity and predictive values to rank the measures in order of their performance. As the authors state, these statistics for diagnostics may be used to rank measures in a study, but it is difficult to generalise the values for sensitivity, specificity, and so on, beyond the study. This difficulty occurs because all values for these statistics heavily depend on the distributions found in the study (see Figure 1 in \[[@B1]\]). Moreover, the use of sensitivity, specificity, and so on, does not reflect the way the DAS28 is used, as one would not use the DAS28 to \'diagnose\' physician opinion on whether or not to increase the infliximab dose.
However, the results of Vander Cruyssen and colleagues can best be understood when looking at Figure 1 in their article \[[@B1]\], depicting the differences in disease activity measures between both groups of patients. Two lessons can be learned from this figure.
First, higher scores of the DAS28 and the other disease activity measures are found in patients in which a decision was made to increase the dose of infliximab. Only a few other studies used external criteria for high and low disease activity to study the validity of the Disease Activity Score and the DAS28. In a study performed in Italy in the late 1990s, it was found that the Disease Activity Score was the best measure to discriminate between predefined states of low and high disease activity, in a sample of 202 patients \[[@B6]\]. A recent study used a different, opinion-based, approach, with expert rating (*n*= 35) of a sample of clinical profiles that were categorised into remission, low disease activity, moderate disease activity and high disease activity \[[@B7]\]. Interestingly, the cut-off criteria for the DAS28 that were found in this way were only slightly different from the established cut-off points for the DAS28, which can therefore be regarded as confirmation.
The second interesting finding from Vander Cruyssen and colleagues\' study, which was not highlighted in the article, is that more than 50% of the patients in which the infliximab dose was not increased had DAS28 \>3.2, which means \'moderate\' or \'high\' disease activity. One may ask whether a dose increase would also have been indicated in these patients, as the aim is to reach low disease activity or even remission. This illustrates that the target of anti-rheumatic treatment is moving in time. It is therefore an extra advantage to use a continuous measure with absolute values to measure disease activity in daily clinical practice and clinical trials.
Conclusion
==========
The study of Vander Cruyssen and colleagues confirms that the DAS28 is a valid measure to monitor disease activity and to titrate treatment with biologicals \[[@B8]\].
Abbreviations
=============
DAS28 = Disease Activity Score using 28 joint counts; DMARD = disease-modifying anti-rheumatic drug.
Competing interests
===================
The author(s) declare that they have no competing interests.
| {
"pile_set_name": "PubMed Central"
} |
All relevant data are within the manuscript.
Introduction {#sec004}
============
Dengue virus (DENV), an enveloped virus with a single-stranded, positive sense RNA genome, belongs to the genus flavivirus and comprises mainly four serotypes (DENV-1, -2, -3, and -4). DENV infection causes a wide range of clinical signs in humans, from asymptomatic to acute febrile illness (dengue fever, DF), to severe hemorrhagic fever/dengue shock syndromes (DHF/DSS) \[[@pone.0214328.ref001]\]. Dengue disease is a major public health problem in developing tropical countries and has being continuously spreading to new geographical areas \[[@pone.0214328.ref002], [@pone.0214328.ref003]\]. Frequent international travel to dengue endemic or epidemic regions has contributed to the escalating numbers of imported dengue cases in temperate region. Outbreaks of the four DENV serotypes have been increasing reported in the tropics and sub-tropics mainly in Asia, South America, and the Caribbean; multiple virus serotypes have been found co-circulating in the hyperendemic regions in Southeast Asia and Pacific \[[@pone.0214328.ref002]\]. Taiwan, located in the tropical-subtropical region of the Northern Hemisphere, has seen many DENV outbreaks since the first half of 20th century. Since 2006, southern Taiwan has faced dengue outbreaks of different scales every year; relatively large outbreaks occurred in 2014 and 2015, with DENV-1 and -2 being the major serotype, respectively \[[@pone.0214328.ref004]\].
Diagnosis of DENV infection cannot rely solely on clinical signs and symptoms as the majority of the infected individuals are either asymptomatic or present with symptoms similar to those of other febrile-episode-inducing diseases \[[@pone.0214328.ref005]\]. DENV serotyping is important for disease management and public health surveillance. Several reports have indicated that DENV-2 and DENV-3 may cause more severe diseases and that DENV-4 is responsible for a milder illness than the other serotypes \[[@pone.0214328.ref006]\]. In addition, antibody-mediated enhancement (ADE) of DENV infection further complicates disease severity \[[@pone.0214328.ref007]\]. Chances for developing DHF-DSS is elevated when infection with one of the four serotypes is followed by a heterotypic serotype; the replacement of DENV-3 by DENV-1 in Sri Lanka in 2009, was associated with a wave of severe dengue epidemic in Sri Lanka \[[@pone.0214328.ref008]--[@pone.0214328.ref010]\].
DENV is transmitted to humans by mosquitoes (*Aedes aegypti* and *A*. *albopictus)*. *A*. *aegypti* can pick up DENV from people showing no symptoms or oligosymptom, resulting in silent transmission \[[@pone.0214328.ref011]\]. A positive association was established between DENV infection in humans and mosquitoes at very fine spatiotemporal scales in the natural setting; specifically, human cases were reported at about one week after positive *A*. *aegypti* in one study \[[@pone.0214328.ref012], [@pone.0214328.ref013]\].
Timely on-site detection and serotyping of DENV in human and mosquito can potentially alert front-line health professionals invasion of a new or long time absent serotype, allowing timely implementation of intervention strategies focuses in those areas to help mitigate disease outbreaks in human \[[@pone.0214328.ref002], [@pone.0214328.ref014]\]. Current methods to aid diagnosis of DENV infection include virus isolation (e.g. antigen detection immunofluorescence assay), nucleic acid amplification tests (NATs; e.g. reverse transcription-polymerase chain reaction \[RT-PCR\], real-time RT-PCR \[qRT-PCR\]), and serological assays (e.g. NS1 antigen detection, plaque reduction neutralization titers (PRNT), and enzyme linked immunosorbent assay \[ELISA\]) \[[@pone.0214328.ref015]\]. Although a number of NS1 rapid diagnostic tests are commercially available to detect NS1 antigen during the first few days of fever, they do not provide serotype information \[[@pone.0214328.ref016]\]. PRNT, antigen detection immunofluorescence assay are able to determine DENV serotypes \[[@pone.0214328.ref017]\], but they are both time-consuming, expensive, laborious, and feasible only in well-equipped laboratories. With relatively high specificity and sensitivity, NATs were recommended for the detection of DENV RNA by the World Health Organization \[[@pone.0214328.ref001]\]. Several multiplex qRT-PCR methods capable of serotyping have been reported \[[@pone.0214328.ref018]--[@pone.0214328.ref020]\]. However, performance of qRT-PCR tests requires skilled technicians and relatively expensive equipment that are not available to remote areas or developing countries; transportation of specimens is another major obstacle. In order to bring early serotyping of DENV to points of need (PON), a rapid, easy, mobile, NAT method of high sensitivity and specificity is needed.
Recently, the portable, simple and compact POCKIT Nucleic Acid Analyzer (POCKIT, GeneReach, Taichung, Taiwan) which can automatically detect and interpret PCR results within one hour became available in mobile PCR laboratory formats \[[@pone.0214328.ref021]--[@pone.0214328.ref023]\]. A lightweight hand-held model, POCKIT Micro Plus Nucleic Acid Analyzer (POCKIT Micro Plus), that works with a built-in rechargeable battery is also available. In this system, insulated isothermal PCR (iiPCR) is achieved consistently in a capillary tube (R-tube, GeneReach) in a simple, specially designed insulated heater and relies on fluorescent probe hydrolysis for signal detection \[[@pone.0214328.ref023], [@pone.0214328.ref024]\]. Various iiPCR/RT-iiPCR-based reagents, available commercially in a lyophilized format to facilitate long-term storage and easy shipping, have been validated to have analytical and clinical performance comparable to reference methods (real time PCR, nested PCR, virus isolation) for different important microbial pathogen hosts \[[@pone.0214328.ref025]--[@pone.0214328.ref031]\]. The POCKIT device has been bundled with easy field-deployable methods for nucleic acid extraction, with potential to serve as a flexible mobile PON tool for rapid DENV detection in human and mosquito.
A pan-DENV-specific RT-iiPCR assay was validated recently on the POCKIT system to have clinical performance equivalent to that of a laboratory qRT-PCR for the detection of DENV-1--4 serotypes in human plasma and serum \[[@pone.0214328.ref032], [@pone.0214328.ref033]\]. Four singleplex DENV serotyping RT-iiPCR reagents have become available recently for the identification of DENV-1, 2, 3, and 4 serotypes separately on the POCKIT system, allowing DENV serotyping near patients and soon after the mosquitoes are trapped even at low-resource settings. In this study, we evaluated the performance of the four DENV serotyping reagents on the POCKIT and POCKIT Micro Plus PCR devices for the detection of the respective target DENV serotypes in human serum and mosquito samples.
Materials and methods {#sec005}
=====================
Ethics statement {#sec006}
----------------
Serum samples were collected from clinically suspected dengue patients for routine diagnosis using RT-PCR methods \[[@pone.0214328.ref034]\] at the Tropical Medicine Center, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan in 2012. The use of retrospective clinical specimens in this study was approved by the Kaohsiung Medical University Hospital Institutional Review Board (KMUHIRB-F(I)-20180009); waiver of informed consent was obtained. All collected samples were anonymized.
Virus and mosquito samples {#sec007}
--------------------------
Tissue culture fluids containing DENV-1 (Hawaii strain), -2 (NGC strain), -3 (DN8700829A strain), or -4 (DN9000475A Strain) were collected after the viruses were propagated in the mosquito C6/36 cell line (*A*. *albopictus*). Zika virus strains (MR766, PRVABC59) were from the American Type Culture Collection, Manassas, VA, USA. Chikungunya virus (CK9500004) was from the Taiwan Center of Disease Control, Taipei, Taiwan.
The *A*. *aegypti* (UGAL) mosquito strain was used in this study and infected with DENV-1, DENV-2, DENV-3 or DENV-4 by micro-injection (nanoinjector) into the thoracic cavity. Adult female mosquitoes, aged 7--8 days, were cold anesthetized and inoculated using a microcapillary needle that had been pulled to a point with needle puller. The 4 serotype of dengue virus stocks were standardized to 2x10^6^ PFU/ml, and 0.2 μl was injected into each mosquito (approximately 400 PFU/mosquito). Infected mosquitoes were maintained in cages at 28 ± 1°C and 70% ± 5% relative humidity with 12h/12h light-dark cycle and fed with 10% sucrose solution. Three infected mosquitoes were collected every other day for the 7-day incubation period to determine the virus titers. The remaining mosquitoes were frozen and stored at −80°C until further use.
Nucleic acid extraction {#sec008}
-----------------------
Nucleic acid extraction was performed by using the taco Preloaded DNA/RNA Extraction Kit (GeneReach) on a taco mini Automatic Nucleic Acid Extraction System (taco mini; GeneReach) according to the manufacturer's instructions. Briefly, sample (200 μl) were added into the first well of the extraction plate and the automatic extraction steps were performed. All nucleic acids were subjected subsequently to the respective serotyping RT-iiPCR and the reference qRT-PCR systems in parallel. For mosquito samples, before taco mini extraction, each mosquito was homogenized in 250 μl PBS with a disposable grinder and centrifuged briefly. Subsequently, 200 μl of the upper aqueous sample were transferred into the first well of the preloaded extraction plate before starting the extraction program.
DENV serotyping reverse transcription-insulated isothermal polymerase chain reaction {#sec009}
------------------------------------------------------------------------------------
The four DENV serotyping RT-iiPCR reagents (POCKIT Dengue Virus Serotype 1 Reagent Set, POCKIT Dengue Virus Serotype 2 Reagent Set, POCKIT Dengue Virus Serotype 3 Reagent Set, POCKIT Dengue Virus Serotype 4 Reagent Set; GeneReach) were performed as described in their user manuals. Briefly, lyophilized RT-iiPCR reagent was rehydrated with 50 μl of Premix buffer (GeneReach), and 5 μl of sample was added to the mixture. Subsequently, 50 μl of the final mixture were transferred to an R-tube (GeneReach), which was spun briefly in a cubee mini centrifuge (GeneReach), The R-tubes were placed into the reaction chamber of a POCKIT Nucleic Acid Analyzer or a hand-held POCKIT Micro Plus Nucleic Acid Analyzer, and a run was initiated. The default program, including an RT step at 50°C for 10 min and an iiPCR step at 95°C for about 30 min, was completed in less than 1 h. Results based on signal-to-noise (S/N) ratios according to the default S/N thresholds used by the built-in algorithm \[[@pone.0214328.ref035]\] were shown on the display screen at the end of the program.
CDC DENV-1--4 real-time reverse transcription-polymerase chain reaction {#sec010}
-----------------------------------------------------------------------
To evaluate the performance of the four DENV serotyping RT-iiPCR reagents in detecting DENV in serum samples, side-by-side comparison with the multiplex CDC DENV-1-4 Real Time RT-PCR Assay (reference qRT-PCR) \[[@pone.0214328.ref036]\] was performed. The reference qRT-PCR assay includes 4 sets of oligonucleotide primers and 4 dually labeled 5' fluorescent TaqMan probes to differentiate the four serotypes. The reaction was performed with a SuperScript III Platinum One-Step qRT-PCR kit (Invitrogen, Carlsbad, CA, USA) without 6-carboxy-X-rhodamine in a Magnetic Induction Cycler (MIC, Bio Molecular System, Upper Coomera, Queensland, Australia). Each reaction included 5 μl of the sample nucleic acid. Signals from the DENV-1, -2, -3, and -4 probes were collected using the 6-carboxyfluorescein, hexachlorofluorescein, Texas red, and Cy5 channels, respectively. The thermocycling program included an RT step at 50°C for 30 min, followed by 95°C for 2 min and 45 cycles of denaturation at 95°C for 15 s and annealing at 60°C for 1 min. Samples generating a threshold cycle (*CT*) value were considered positive.
Statistical analysis {#sec011}
--------------------
The degree of agreement between two assays was assessed by calculating Cohen's kappa values.
Results {#sec012}
=======
Analytical sensitivity of DENV-1, -2, -3, -4 serotyping RT-iiPCR {#sec013}
----------------------------------------------------------------
The detection endpoints of the DENV-1, -2, -3, -4 serotyping RT-iiPCR reagents were evaluated by side-by-side comparison with the reference multiplex qRT-PCR by using their respective target DENV serotypes. 10-fold serial dilutions (100, 10, 1, 0.1, and 0.01 PFU/ml) of each isolate were made in DENV-negative human serum and each was subjected to nucleic acid extraction in triplicate. The results are summarized in [Table 1](#pone.0214328.t001){ref-type="table"}. The 100% detection endpoints were found at 10 and 1 PFU/ml DENV-1 with the reference qRT-PCR and DENV-1 RT-iiPCR, respectively, at 1 PFU/ml DENV-2 with both qRT-PCR and DENV-2 RT-iiPCR, at 1 PFU/ml DENV-3 with both qRT-PCR and DENV-3 RT-iiPCR, and at 10 and 1 PFU/ml DENV-4 with the qRT-PCR and DENV-4 RT-iiPCR, respectively. All data indicated that the four DENV-1, -2, -3, and -4 RT-iiPCR had analytical sensitivity comparable to that of the reference qRT-PCR in detecting their target DENV serotypes.
10.1371/journal.pone.0214328.t001
###### Analytical Sensitivity of dengue virus serotype 1-, 2-, 3-, and 4-specific RT-iiPCR reagents.
{#pone.0214328.t001g}
---------------------------------------------------------------------------
RT-iiPCR Strain Titer\ Detection rate\
(PFU/ml) (no. positive/no. total)
---------- --------------- --------------- -------------------------- -----
DENV-1 DENV-1 10^3^ 3/3 3/3
10^2^ 3/3 3/3
10^1^ $\boxed{3/3}$ 3/3
10^0^ 0/3 $\boxed{3/3}$
10^−1^ 0/3 2/3
10^−2^ 0/3 0/3
DENV-2 DENV-2 10^3^ 3/3 3/3
10^2^ 3/3 3/3
10^1^ 3/3 3/3
10^0^ $\boxed{3/3}$ $\boxed{3/3}$
10^−1^ 2/3 0/3
10^−2^ 0/3 0/3
DENV-3 DENV-3 10^3^ 3/3 3/3
10^2^ 3/3 3/3
10^1^ 3/3 3/3
10^0^ $\boxed{3/3}$ $\boxed{3/3}$
10^−1^ 0/3 1/3
10^−2^ 0/3 0/3
DENV-4 DENV-4 10^3^ 3/3 3/3
10^2^ 3/3 3/3
10^1^ $\boxed{3/3}$ 3/3
10^0^ 0/3 $\boxed{3/3}$
10^−1^ 0/3 1/3
10^−2^ 0/3 0/3
---------------------------------------------------------------------------
DENV, dengue virus; RT-iiPCR, reverse transcription-insulated isothermal polymerase chain reaction; PFU, plaque forming unit; qRT-PCR, real-time reverse transcription-polymerase chain reaction; boxed, 100% detection end point.
Analytical specificity of DENV-1, -2, -3, -4 serotyping RT-iiPCR {#sec014}
----------------------------------------------------------------
To assess the specificity of each DENV serotyping RT-iiPCR reagent, the four DENV serotypes and two other viruses (Zika virus MR766, Zika virus PRVABC59, and chikungunya virus CK9500004) known to cause febrile illness or skin rash illness were tested. All four singleplex DENV serotyping RT-iiPCR reagents did not react with the other three non-targeted dengue virus serotypes, Zika virus and chikungunya virus in the exclusivity test panel ([Table 2](#pone.0214328.t002){ref-type="table"}), indicating that the reagents had excellent specificity for their target DENV serotypes.
10.1371/journal.pone.0214328.t002
###### Analytical specificity of dengue virus serotype 1-, 2-, 3-, and 4-specific RT-iiPCR reagents.
{#pone.0214328.t002g}
-----------------------------------------------------------------------------------------------------------------
Pathogen Titer\ RT-iiPCR
(PFU/ml)
--------------- ------------- -------------------- -------------------- -------------------- --------------------
DENV1 8/6 1.8 x 10^3^ $\boxed{positive}$ negative negative negative
DENV2 99.8.30 2.1 x 10^6^ negative $\boxed{positive}$ negative negative
DENV3 5/12 1.0 x 10^6^ negative negative $\boxed{positive}$ negative
DENV4 1021021 1.9 x 10^5^ negative negative negative $\boxed{positive}$
ZIKV PRVABC59 2.5 x 10^4^ negative negative negative negative
ZIKV MR766 1.2 x 10^5^ negative negative negative negative
CHIKV 1.4 x 10^5^ negative negative negative negative
-----------------------------------------------------------------------------------------------------------------
DENV, dengue virus; ZIKV, Zika virus; CHIKV, chikungunya virus; PFU, plaque forming unit; RT-iiPCR, reverse transcription-insulated isothermal polymerase chain reaction.
Clinical performance of DENV-1, -2, -3, -4 serotyping RT-iiPCR {#sec015}
--------------------------------------------------------------
To evaluate the clinical performance of each DENV serotyping RT-iiPCR reagent, 40 serum samples (about 20 DENV serotype-positive and 20 DENV-negative) were tested for each respective reagent. For this purpose, 20 DENV-1, 20 DENV-2, 20 DENV-3, and 20 DENV-negative samples previously identified by a real-time PCR \[[@pone.0214328.ref034]\] were used. Due to the lack of DENV-4 positive clinical samples in the region, DENV-4 samples were prepared by spiking 20 DENV-negative human serum specimens with different concentrations of the DENV-4 DN9000475A stock (1.9 x 10^5^ PFU/ml). The samples were subjected directly to nucleic acid extraction by the taco mini method. Nucleic acid extracts were tested by the respective DENV serotyping RT-iiPCR reagents and the reference multiplex qRT-PCR in parallel. Completely matched results were found for DENV-1 detection (20 positive and 20 negative) between the DENV-1 RT-iiPCR and the reference qRT-PCR, as well as for DENV-4 detection (20 positive and 20 negative) between the DENV-4 RT-iiPCR and the qRT-PCR ([Table 3](#pone.0214328.t003){ref-type="table"}). 20 and 19 samples were DENV-2 positive and negative, respectively, by both the DENV-2 RT-iiPCR and the reference assays; whereas one sample was DENV-2 negative by the index assay but positive by the reference assay (Ct = 44.49, [Table 3](#pone.0214328.t003){ref-type="table"}). Similarly, 20 and 19 samples were determined to be DENV-3 positive and negative, respectively, by both the DENV-3 RT-iiPCR and reference assays; one sample was DENV-3 negative by the qRT-PCR but positive by the RT-iiPCR ([Table 3](#pone.0214328.t003){ref-type="table"}). Therefore, compared to the reference qRT-PCR, the DENV-1 RT-iiPCR and DENV-4 RT-iiPCR had 100% overall agreement (CI95%, 97.3--100%), the DENV-2 RT-iiPCR had 97% overall agreement (CI95%, 89.8--97.5%) and the DENV-3 RT-iiPCR had 97% overall agreement (CI95%, 89.8--97.5%), indicating that the four DENV serotyping RT-iiPCR reagents on the POCKIT system had clinical performance comparable to those of the reference qRT-PCR to detect their target DENV serotypes in serum samples.
10.1371/journal.pone.0214328.t003
###### Clinical performance characteristics of dengue virus serotypes 1-, 2-, 3-, and 4-specific RT-iiPCR reagents on POCKIT nucleic acid analyzer for the detection of dengue virus in human serum.
{#pone.0214328.t003g}
-------------------------------------------------------------------------------------------------------------------
Assay and Result Reference qRT-PCR \% Specificity\ \% Sensitivity\ \% Agreement\
(95% CI) (95% CI) (95% CI)
--------------------- ------------------- --------------------- ---------------------- ---------------------- -- --
**DENV-1 RT-iiPCR** DENV-1 100% (88 \~ 100%) 100% (88 \~ 100%) 100% (93.7 \~ 100%)
Positive 20 0 20
Negative 0 20 20
Total 20 20 40
**DENV-2 RT-iiPCR** DENV-2 100% (87.5 \~ 100%) 95.2% (81.5 \~ 100%) 97.5% (89.8 \~ 100%)
Positive 20 0 20
Negative 1 19 20
Total 21 19 40
**DENV-3 RT-iiPCR** DENV-3 95% (80.6 \~ 100%) 100% (88.1 \~ 100%) 97.5% (89.8 \~ 100%)
Positive 20 1 21
Negative 0 19 19
Total 20 20 40
**DENV-4 RT-iiPCR** DENV-4 100% (88 \~ 100%) 100% (88 \~ 100%) 100% (93.7 \~ 100%)
Positive 20 0 20
Negative 0 20 20
Total 20 20 40
-------------------------------------------------------------------------------------------------------------------
DENV, dengue virus; RT-iiPCR, reverse transcription-insulated isothermal polymerase chain reaction; qRT-PCR, real-time reverse transcription-polymerase chain reaction; CI, confidence interval.
Serotype-specific detection of dengue virus serotypes 1--4 in mosquitos {#sec016}
-----------------------------------------------------------------------
The combination of the hand-held POCKIT Micro Plus and the compact taco mini is available in a suitcase for pathogen surveillance at points of need. With the four serotyping RT-iiPCR reagents, it will be possible to performed DENV serotyping soon after the mosquitoes are trapped on site even at settings of limited resources. Here, we evaluated preliminarily the performance of the four serotyping RT-iiPCR reagents to detect their target DENV serotypes in mosquito specimens on the hand-held POCKIT Micro Plus PCR system. Nucleic acids extracted from female *A*. *aegypti* mosquitoes experimentally infected with DENV-1, -2, -3, and -4 serotype were subjected to PCR testing by the POCKIT DENV-1, -2, -3, or -4 RT-iiPCR. The results showed that the DENV-1, -2, -3, and -4 RT-iiPCR can detect their target DENV serotypes but not the serotypes to be excluded ([Table 4](#pone.0214328.t004){ref-type="table"}), indicating excellent specificity for DENV serotypes in mosquito sample matrix.
10.1371/journal.pone.0214328.t004
###### Detection of dengue virus serotypes 1, 2, 3, and 4 in mosquito samples by dengue virus serotype 1-, 2-, 3-, and 4-specific RT-iiPCR reagents on POCKIT nucleic acid analyzer.
{#pone.0214328.t004g}
DENV-infected mosquitos (serotype) RT-iiPCR
------------------------------------ -------------------- -------------------- -------------------- --------------------
DENV-1 $\boxed{positive}$ negative negative negative
DENV-2 negative $\boxed{positive}$ negative negative
DENV-3 negative negative $\boxed{positive}$ negative
DENV-4 negative negative negative $\boxed{positive}$
DENV, dengue virus; RT-iiPCR, reverse transcription-insulated isothermal polymerase chain reaction; qRT-PCR.
Discussion {#sec017}
==========
There is an urgent need for better surveillance and control of DENV spread to help mitigate the global spread of epidemic dengue. Accurate and rapid detection and serotyping of DENV can help improve the recognition of severe dengue warning signs in patients suspected of DENV infection. Timely reporting on the detection and serotyping status of DENV in mosquito can serve as an alert to people who fall ill to consider the possibility of dengue infection and seek medical assistant in time, and to initiate timely community and vector control programs to help mitigate the spread of DENV infection.
In this study, tests with clinical serum samples showed that the four singleplex serotyping RT-iiPCR reagents had clinical performance comparable to that of the reference qRT-PCR for the detection of their target serotypes in human serum. There was one qRT-PCR-positive/RT-iiPCR-negative sample for DENV-2 and qRT-PCR-negative/RT-iiPCR-positive for DENV-3. The discrepancy in detection between the two assays was likely due to low viral loads in these samples; one supporting observation was that the one qRT-PCR-positive/RT-iiPCR-negative sample had a Ct of 44.49 in qRT-PCR. The four index reagents offered excellent analytical sensitivity and specificity to detect their target DENV serotypes in human serum on the compact field-deployable POCKIT device, and also had great analytical specificity in mosquito samples on the hand-held POCKIT Micro Plus.
In DENV diagnosis, DENV serotyping is also important since DENV-2 and DENV-3 are more often associated with severe diseases than the other serotypes \[[@pone.0214328.ref006]\]. Furthermore, when patients with previous DENV infection were infected with a heterotypic serotype, the chances for them to develop DHF-DSS were elevated \[[@pone.0214328.ref007]\]. The pan-DENV RT-iiPCR/POCKIT system validated previously for the detection of all four DENV serotypes in human plasma and serum \[[@pone.0214328.ref032], [@pone.0214328.ref033]\] is useful in aiding the identification of acute DENV infection, especially for remote regions with high burdens of DENV infection. However, this system could not differentiate between different DENV serotypes. In this study, we showed that the four new RT-PCR reagents for DENV serotyping can work on the same field-deployable PCR system to serve as tools to allow timely near-patient serotyping of DENV in human and mosquitos to facilitate efficient disease management and public health surveillance.
As shown in [Table 1](#pone.0214328.t001){ref-type="table"}, similar to that of the reference qRT-PCR for all four serotypes, the sensitivity of the RT-iiPCR system was at biological titers of around 10^0^ PFU/ml. This was consistent to the performance of other molecular detection methods for DENV \[[@pone.0214328.ref037]--[@pone.0214328.ref039]\]. As reported previously, RNA copy numbers were likely significantly higher than PUFs, due to defective virus particles or viral RNA freed from infected cells in the sample matrix \[[@pone.0214328.ref040]--[@pone.0214328.ref042]\].
To aid laboratory confirmation of DENV infection during the first 5 to 6 days after symptomatic onset, detection methods for DENV RNA have been recommended by WHO \[[@pone.0214328.ref001]\]. Among them, real-time RT-PCR allows serotyping of DENV. However, this technology is in general not available at most PONs to provide timely serotyping results in regions with threats of epidemic dengue; RNA degradation during the shipping process to the central laboratories is also a concern \[[@pone.0214328.ref043]\]. The POCKIT or POCKIT Micro Plus device has been bundled with the field-deployable taco mini extraction system in a durable suitcase (POCKIT combo, POCKIT Micro combo, respectively; GeneReach) to meet the needs of PON applications at different settings. The equipment can be operated with a car battery or a rechargeable battery and only a few simple steps are needed from sample to results with this mobile PCR system.
Current commercially available NS1 immunological test products are rapid and do not require trained personnel to operate. They have been shown to have great performance for detecting DENV in both human and mosquitoes \[[@pone.0214328.ref044]\]. However, they do not provide serotype information. In addition, its sensitivity was relatively low on days 1 and 2 and after day 5 post-symptomatic onset in human, compared to that seen with the-RT-PCR methods \[[@pone.0214328.ref016]\].
In conclusion, performed on the portable POCKIT system, the four POCKIT singleplex serotyping RT-iiPCR reagents have potential to serve as a relatively inexpensive, rapid, and simple PON tool for early detection and serotyping of DENV in viremic patients as well as in infected mosquitoes, enabling timely management and control of dengue disease in underserved communities. Studies to verify and validate further the performance of these reagents on the mobile PCR laboratory system for DENV subtyping in both human and mosquitoes are underway.
We thank Ms. Ying-Hui Wang for her administrative assistance.
[^1]: **Competing Interests:**The authors declare the following potential conflicts of interest with respect to the research, Cover Letter authorship, and/or publication of this article: P.-H.C., F.-C.L., C.-F.P. and P.-Y.A.L. are employed by GeneReach Biotechnology, Taichung, Taiwan. This does not alter our adherence to PLOS ONE policies on sharing data and materials. The rest of us declare no conflicting interests with respect to our authorship or the publication of this article.
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"pile_set_name": "PubMed Central"
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1. Introduction {#s0005}
===============
Over the last 10 years, concern has been mounting over rapid rises in the prevalence of non-communicable diseases (NCDs) in the global South and the health and economic burden they represent. This has been driven, in part, by the World Health Organisation (WHO) which has published a number of reports on the topic and most recently adopted a *Global Action Plan for the Prevention and Control of Noncommunicable Diseases 2013--2020* ([@bib127], [@bib128]). The World Bank and the United Nations Development Programme (UNDP) -- two of the leading organisations in international development -- have also been active and issued discussion and policy papers about 'the mounting danger of chronic diseases' for emerging economies ([@bib133], [@bib114]). Governments, too, have expressed their alarm over this rising threat, recently passing a *Political Declaration on the Prevention and Control of Non-Communicable Diseases* at a high-level meeting of the United Nations\' General Assembly, the second of only two such meetings held about health ([@bib115]). Of course, this attention to chronic diseases in the global South has not been the sole preserve of international organisations and governments. Public health and medical experts have long called for more attention to be paid to NCDs in this region. For example, one of the leading voices in the global health community, *The Lancet*, has published regular special issues with research on the epidemiological, economic and clinical aspects of chronic diseases since 2005 (e.g. [@bib64]; [@bib8]; [@bib55]). Likewise, civil society and the private sector are showing a growing interest in the subject. Most significantly, in 2010, over two thousand health charities and patient organisations including the American Cancer Society and the World Heart Federation established, with support from the pharmaceutical industry, the NCD Alliance to lobby for and make chronic diseases a global health and development priority ([@bib60]).
As these different actors have repeatedly argued, NCDs -- defined in this context as comprising four conditions (cardiovascular disease, cancer, diabetes and chronic respiratory disorders) overwhelmingly caused by four behavioural risk factors (diet, physical activity, smoking and alcohol) -- have become a critical issue for low and middle income countries (LMICs). Drawing on sophisticated epidemiological data, they point out that more than 60% of deaths worldwide are NCD-related and nearly 80% of these deaths occur in LMICs ([@bib127], [@bib114]). Indeed, in most countries across South America and Asia, chronic diseases are now the leading cause of death. Only in the African region are there more deaths from infectious diseases and even that is predicted to change over the next 15 years. This high prevalence of NCDs across the global South, these actors argue, constitutes 'one of the major challenges for development in the 21st century' ([@bib115], p.1). As they explain, the relationship between chronic diseases and development is two-fold ([@bib133], [@bib1], [@bib114]). On the one hand, the growing prevalence of NCDs in emerging economies is viewed as a negative consequence of socio-economic development, with economic growth and rapid urbanisation associated with a rise in 'modern' lifestyles (drinking, smoking, unhealthy diets, and physical inactivity) and an ageing population. On the other hand, the chronic disease epidemic in the global South is understood to be a serious threat to the sustainability of development through both its negative impact on the productivity of working age populations and the double burden of disease it places on health systems already overstretched by infectious, maternal and perinatal diseases. Predictably perhaps, many of the solutions put forward by these actors are health strategies successfully used in North America and Europe and which are deemed commensurate with the economic context of LMICs ([@bib136], [@bib73], [@bib3], [@bib128]). They include tools such as epidemiological surveillance systems as well as public health and clinical interventions that are 'highly cost-effective cheap, feasible and culturally acceptable' such as tobacco taxation, media campaigns for healthy diets and multidrug regimens for people at risk of cardiovascular diseases ([@bib119], p.47).
There has been no lack of academic attention given to the issue of NCDs in the global South from the public health community ([@bib2], [@bib25], [@bib84], [@bib109]). In contrast, critical social science engagements are comparatively rare, although interesting work has recently begun to emerge. For example, political scientists have examined the reasons behind the relative neglect of NCDs in global health policy and funding compared to issues like AIDS, pointing to the expert and advocacy networks involved and the ways issues are framed (e.g. [@bib81]; [@bib67]; cf. also [@bib108]). Similarly, historians and others have started to explore the globalisation of chronic diseases and particular public health strategies like tobacco taxes over the last 50 years (e.g. [@bib17]; [@bib103]; [@bib123]). Another important part of this emerging body of work is the research carried out by anthropologists and geographers into the way ideas and practices associated with NCDs have been translated, resisted and re-appropriated when travelling to the global South. To illustrate, [@bib77], [@bib78] has pointed to the absence of pain relief medication and the very different understandings of pain in cancer wards in Botswana; while [@bib72]; cf. also [@bib62]) have shown how alcohol control policies in Cape Town have been recast as an instrument to fight criminality rather than improve health. Others have looked into how the ideas and practices associated with NCDs have transformed subjectivities and notions of patienthood in the global South (e.g. [@bib21]; [@bib132]; [@bib124]; cf. also [@bib104]).
While this emerging body of critical studies on NCDs in the global South is a step in the right direction, much more needs to be done before we can start making sense of current initiatives to problematise and govern the chronic disease epidemic in emerging economies. So, for example, while the role of expert networks and discursive framings in problematising NCDs in the global South needs to be further scrutinised, we also need to explore the technologies and materialities like epidemiological maps and models that make it possible to view chronic diseases as a development issue. Likewise, while the influence of the tobacco, alcohol and food companies in globalising risk factors associated with NCDs is at risk of being over-analysed (e.g. [@bib137]; [@bib110]), we know very little about the role of the pharmaceutical industry and philanthropic foundations in creating new markets for vaccines and drugs to treat chronic diseases in the region (e.g. [@bib119]; [@bib113]; cf. also [@bib97]). It would also be helpful to know more about the complex relationships that exist between current initiatives to tackle NCDs and ideas and traditions that have been critical to the field of health and medicine such as post-colonialism, neoliberalism and securitisation ([@bib26], [@bib34], [@bib4]). Last but not least, despite the efforts of some anthropologists (e.g. [@bib75], [@bib76]), we still understand very little about the impact of NCD-related interventions on existing inequalities and the everyday lives of the poor in the global South ([@bib39]). More generally, then, there is a need to know more about the types of places that produce chronic diseases in the global South and, in turn, the ways in which the politics of NCDs reform and reshape places and people in the name of risk management and disease control.
The contributions in this special issue are an attempt to begin addressing these and other similar questions and themes. To locate these contributions within the broader critical social science literature on global health (e.g. [@bib26]; [@bib34]; [@bib121]; [@bib110]; [@bib42]; [@bib40]; [@bib12]; [@bib4]), this introduction outlines three themes that, we believe, are central to a critical engagement with the politics of NCDs in the global South. First, an attention to 'problematisation' is an opportunity of examine and reflect on the conditions -- intellectual categories, moral principles, geopolitical models, medical practices and other techniques -- that make it possible to think about chronic diseases as a problem for developing countries today. Second, a concern with 'care' can help analyse and question contemporary NCD policies in the global South and their consequences by drawing on a tradition of social justice and human rights. Third, a focus on 'culture' offers a grid of analysis to explore and make sense of how both unhealthy lifestyles and public health policies associated with NCDs are translated, resisted and transformed when they travel from the global North to the global South. The six papers in this special issue represent important contributions to these themes while also highlighting the incredibly broad range, depth and complexity of concerns that the politics of NCDs invoke. These concerns then signpost future research agendas across the social sciences that are attuned to the current tenor of policy debates as well as shifts in the landscape of global health funding and programmatic priorities that will be drawn out at the end of this introduction.
2. The problematisation of chronic disease in the global South {#s0010}
==============================================================
For the most part, public health experts are concerned with confirming that NCDs are an issue for LMICs and articulating possible solutions. In contrast, more critically minded researchers are interested in exploring the conceptual, political and material conditions that make it possible to identify and think about chronic diseases as a problem of development today. Such an approach draws on the notion of 'problematisation' developed by [@bib46], [@bib47] and others (e.g. [@bib102]; [@bib57]; [@bib85]; [@bib71]). For these scholars, 'problems are not pre-given, laying there waiting to be revealed', but 'have to be constructed and made visible' through 'a complex and often slow process' ([@bib85], p.14). It is this 'process of problematisation' that they are keen to analyse in order to understand 'how and why certain things (behaviour, phenomena, processes) became a problem' ([@bib46], p.17). This means exploring the 'ensemble of discursive and non-discursive practices that make something enter into the play of true and false and constitute it as an object of thought, whether in the form of moral reflection, scientific knowledge or political analysis' (Foucault cited in [@bib102], p.xviii-xix). Put differently, it involves studying the progressive development and assemblage of the scientific, moral and political rationalities, institutions, practices and techniques that make it possible to think of certain things as a problem today.
Taking such an approach, then, is to argue that chronic diseases in the global South are not a pre-given reality waiting to be revealed through ever more sophisticated epidemiological investigations but a problem that has been made thinkable through the progressive articulation of a complex assemblage of geopolitical categories, modernisation theories, biomedical practices and international networks of experts in health and development ([@bib95]). We sketch here a tentative genealogy of some of these rationalities, practices and networks that make it possible to conceive of chronic diseases as a problem of development today. An important, early moment in such a genealogy has to be the elaboration of the notion of 'health development' in the post-World War II period ([@bib120]). This was a period marked by the dismantling of the old colonial empires and the birth of the 'Third World' articulated through the theories and practices of development ([@bib35]). At first, the new development experts did not attach much importance to health. Indeed, for them, development was about economic growth and physical capital like roads, railways and industries. It is only from the 1960s onwards that they began to recognise that development was also about poverty alleviation and human capital. For the most part, this meant investing in education and healthcare systems so as to improve the quality and quantity of the labour force and bolster national productivity ([@bib44]).
Another, critical step in the framing of chronic disease as a development issue was the articulation of the concept of chronic disease itself. As [@bib6] has argued, this concept only came to prominence in the postwar period (cf. also [@bib122]). The elaboration of this concept made it possible to bring together and view disorders such as cancer and heart ailments -- which until then had been thought to be the product of the natural process of ageing and, as such, outside the realm of medicine -- as part of a new diagnostic category: diseases with an aetiology of multiple, lifestyle-related risk factors that had a lasting impact on someone\'s capacity to function normally. As [@bib5] also shows, this new diagnostic category came together with a new model of medicine -- surveillance medicine -- that progressively displaced pathological medicine from the 1950s onwards. Pathological medicine was about investigating the physiological lesion in the body of the patient in the hospital through clinical examinations, laboratory analyses and post-mortems ([@bib45]). In contrast, surveillance medicine was concerned with identifying possible risk factors of future illness through regular medico-social surveys and screening programmes of everyone in the community, both the ill and the seemingly healthy. Unlike pathological medicine, it also assumed a responsible patient who actively engaged in his or her surveillance, education and care, which comprised healthy lifestyles promotion campaigns, screening tests and life-long drug regimens ([@bib96]).
For most public health experts, chronic diseases and the developing world were long thought to be mutually exclusive, with chronic diseases deemed to be the preserve of the rich, industrialised countries of the North while the major concern for the South was infectious diseases and malnutrition ([@bib20], [@bib16]). In the minds of these experts, these differences in disease patterns were closely related with the demographic and socio-economic changes associated with modernisation. Perhaps the most influential account of this relationship between disease and modernity was Abdel Omran\'s notion of epidemiological transition. In Omran\'s terms, so-called 'developed countries' had undergone an epidemiological transition and entered the 'Age of Degenerative and Man-Made Diseases', which was not only characterised by chronic diseases but also by: low fertility, high life expectancy and ageing populations; economies articulated around technology and mass consumption; as well as rationality, nuclear families and high living standards ([@bib94], p.516--517). In contrast, 'undeveloped' societies, he posited, had yet to complete this transition and were still in the 'Age of Pestilence and Famine' defined not only by infectious diseases and malnutrition but also by: high fertility, high mortality and young populations; economies mixing subsistence farming with early industrialisation; as well as traditional values, extended families and poor, unsanitary living conditions (ibid.).
These different disease patterns and development levels were further associated with differing healthcare systems. While surveillance medicine was the dominant paradigm in North America and Europe, the notion of primary health care (PHC) enshrined in the Declaration of Alma Ata prevailed across the Third World ([@bib41]; [@bib30]). After independence, developing countries quickly realised that the healthcare systems inherited from colonial times and based around the hospital and eradication campaigns against tropical diseases were not appropriate to their situation: hospitals, usually located in cities, were not accessible to the rural poor that made up most of their population; eradication campaigns were associated with authoritarian practices that jarred with the spirit of decolonisation; and Western medical technologies were too expensive. PHC was developed as an alternative model of healthcare tailored to the specific needs of the Third World. It promised to offer essential healthcare made accessible to all citizens via a network of rural health workers and centres and characterised by community participation, an emphasis on prevention and simple, cheap technologies. While the programmes put in place to operationalise the PHC ideal varied across the developing world, they tended to concentrate on communicable diseases and child and maternal health issues, including: oral rehydration therapy for diarhoea; family planning; nutrition; and mass immunisations against major infectious diseases like measles and diphtheria ([@bib90]).
This way of thinking, which deemed chronic diseases and the developing world as mutually exclusive and associated the latter with infectious diseases, maternal and child health, malnutrition and PHC remained predominant until the turn of the century. The Millennium Development Goals, for example, owed a lot to this style of reasoning, not least by viewing health as critical to development and by constraining its health-related efforts to maternal and child health, infectious diseases and malnutrition. But, from the late 1970s onwards, an increasing number of reports from physicians and mostly small, hospital-based epidemiological surveys in LMICs showing a growth in the number of patients suffering from NCDs began to challenge this way of thinking ([@bib98], [@bib103]). Unsurprisingly, this gradually led to efforts to construe chronic diseases as a development issue. Of course, the WHO did some work on chronic diseases in the Third World, launching its Integrated Programme for Community Health in Non-Communicable Diseases in a small number of developing countries in the 1980s ([@bib122]). But, it was the efforts of economists and epidemiologists at the World Bank -- especially Dean Jamison\'s Health Sector Priorities Review, Richard Feachem\'s work on the Health of Adults in the Developing World and Christopher Murray\'s Global Burden of Disease Project -- that would prove to be the most influential in identifying chronic disease as an issue for the global South and reconfiguring the relationship between development levels, disease patterns and healthcare models ([@bib43], [@bib66], [@bib91]).
There were many reasons for why the Bank\'s efforts proved to be so influential. First, this was a time when the Bank\'s investment in health-related projects grew exponentially, making it the world\'s premier health institution and pushing the WHO to the sidelines ([@bib19], [@bib24]). Second, the Bank\'s experts articulated a new understanding of the relation between development and disease that made it possible to think NCDs as an issue for LMICs. They suggested that one should stop classifying all developing countries together and recognise instead their growing economic and epidemiological diversity ([@bib49], [@bib65]). Specifically, complexifying Omran\'s model, they recommended distinguishing between two groups of developing countries: (i) low-income, usually African or South Asian, countries typified by infectious diseases and malnutrition; and (ii) middle-income, mostly East Asian or Latin American, countries characterised by a double burden of both infectious and chronic diseases ([@bib65]). It was this second group -- whose emergence was due to the success of existing PHC programmes at reducing infant mortality and changing patterns of risk such as unhealthy lifestyles generated by rapid urbanisation and rising incomes -- that was the novelty and allowed the Bank\'s specialists to associate developing countries and chronic diseases for the first time ([@bib14], [@bib66], [@bib88]). Third, the claims about changing patterns of disease and development made by the Bank's experts seemed to be supported by the new, allegedly more rigorous estimates of worldwide mortality and morbidity generated by Murray's Global Burden of Disease project, something which was critical at a time when evidenced-based approaches were becoming all the rage ([@bib91]; Reubi, this issue). Fourth, the Bank\'s experts ensured that the problem of NCDs in the global South gained traction by linking it with a question that came to dominate the political agenda in most developing countries after the energy crises and global recession of the 1970s: how to finance healthcare systems in the face of mounting national debts and budgetary restrictions? ([@bib105], [@bib103]). They did so through the notion of double burden of disease burden characteristic of the new, second group of developing countries, arguing that it would substantively add to the financial strain already impacting these countries' healthcare systems ([@bib49], [@bib66]). Fifth, unlike the WHO, the Bank was not wedded to PHC and was able to outline alternative healthcare models ([@bib24]). In particular, it argued that PHC programmes, with their focus on rural populations, infectious diseases and child and maternal health, had become too limited and called for a new healthcare model articulated around rational policies, epidemiological surveillance, cost-effective interventions focused on prevention and, sometimes, privatisation ([@bib89], [@bib13]).
Over the last fifteen years, the Bank has shown less interest in chronic disease and development, leaving the WHO and other organisations like the NCD Alliance and *The Lancet* to take the lead in this field ([@bib123]). As mentioned at the start of this introduction, the numerous reports, action plans and scientific papers published by these organisations have further consolidated and propagated the ideas of NCDs as a development issue. Of course, these organisations have brought some of their own concepts and idiosyncrasies -- like the WHO\'s addition of a reworked and weakened notion of PHC -- to the way they frame this issue. But, overall, the way they conceive chronic diseases in the global South is strongly influenced by the analyses and ideas articulated by the World Bank\'s experts during the 1980s and 1990s. To illustrate, most of the documents on the topic published by these organisations share the Bank\'s understanding that the relationship between NCDs and development is a two-way process, with economic growth generating unhealthy lifestyles and reducing chronic disease prevalence critical to improving productivity (e.g. [@bib127]; [@bib114]). Likewise, most of these documents, echoing the Bank, express the significance of the NCD epidemic in the global South through rigorous epidemiological data and emphasise the importance of using cost-effective health interventions and public-private partnerships (e.g. [@bib73]; [@bib128]).
3. Chronic diseases and the politics of care {#s0015}
============================================
A focus on problematisation is, of course, not the only critical approach that can be used to make sense of current efforts to tackle NCDs in the global South. Another, important lens through which to explore these efforts is a critique characterised by a concern with social justice and human rights ([@bib11], [@bib10], [@bib70], [@bib118]). This frame points to the political importance of care to the ways in which we approach NCDs across a number of domains. Specifically, the invocation of social justice and human rights acts as a critique of current approaches to NCDs in two ways. First, of the global health community\'s selective deployment of the tools, techniques, funds and interventions that permits the care of people. Second, of the ability of the state to ensure the adequate care of its citizens. If the first critique calls the contemporary architecture of global health into question ([@bib40], [@bib53]), then the second scrutinises the ability of this architecture to deliver sustainable, effective and equitable health improvements on the ground ([@bib10], [@bib118]). The politics of NCDs in the global South are thus bound into and directly shaped by the nature, delivery and critique of care by a variety of actors. The ability and will to care, in turn, is shaped by the complex, multi-scalar politics and resource flows that condition so much of the global health enterprise. Care implies a need for empathy, responsibility and duty just as much as it does the fair distribution of medical services and resources and the capacity to access and make use of these ([@bib69]). It is therefore an essential -- if under-acknowledged -- component of the politics of NCDs in countries of the global South.
The capacity to care is constrained by a number of factors that warrant further scrutiny. In the first place, current efforts to address chronic diseases in LMICs are indubitably limited by the very delineation of NCDs themselves. This, in turn, draws attention to the ways in which the global health enterprise is so often enacted within a number of specific and siloed realms, with little structural capacity to deal with the implications of the complex porosity of definitional categories. For example, 'the boundaries between communicable and non-communicable diseases are often indistinct' ([@bib40], p. 321). So, it could be argued that, with the development of antiretroviral therapies, AIDS has become a chronic disease that can be managed through life-long drug regimens and changes in one\'s lifestyle. Similarly, some have argued that cervical cancer, a current priority of the global health community, is more akin to a communicable disease given that it is triggered by the sexually-transmitted Human Papilloma Virus (HPV) and is now preventable through a vaccine ([@bib77]). There are, moreover, similar boundary problems with mental health issues like depression that are excluded from the official NCD definition but yet seem to fit the notion of a disease that has a lasting impact on someone\'s capacity to function in society ([@bib127]). The notion of NCD also partakes in the 'mistake of pitting one set of pathologies against another' for attention and funding from the global health community, instead of promoting an approach to public health policy and practice that is intersectoral and holistic ([@bib40], p.322). Interestingly, this division and fragmentation is also encouraged by the focus on discrete, cost-effective health interventions developed by the World Bank at the end of the 20th century and taken over by the WHO and others over the last 15 years.
Another issue relates to the capacity of the state to provide adequate care for its citizens and, moreover, the consequences of this for the sustainability of global health programmes ([@bib82]). Failure in this domain is both deflected and reinforced by the lack of focus on the social determinants of NCDs and the role of mounting inequalities in entrenching these. Moreover, the twin phenomena of globalisation ([@bib9], [@bib135]) and rapid urbanisation ([@bib87], [@bib130]) have unsettled the assumptions inherent within the epidemiological transition model. Now, households are gripped not just by the 'double burden' of disease ([@bib22]), but in some cases, a 'triple' or even 'quadruple burden' that also includes injuries and violence, as well as perinatal and maternal diseases ([@bib15]). Crucially, the characteristics of these burdens vary not just between countries, but also within them and at ever-finer geographic scales. Even within one household, for example, there might be underweight, malnourished family members living alongside equally malnourished obese relatives ([@bib31]). It is important then to consider the social determinants of this complicated and multi-layered disease burden: poverty, inequality, quality of housing, access to sanitation, unemployment, education, transport, food security, the nature of healthcare provision and environmental degradation. It is these structural, economic, political and social drivers that largely condition the dynamics of the four main risk factors for chronic disease: diet, exercise, alcohol and tobacco ([@bib126]). Yet, while the proportion of people living in poverty may have fallen ([@bib116]), rates of both inequality and, perhaps even more importantly, inequity, within many countries is accelerating ([@bib93]). This means that while advances in medical science remain essential to reducing mortality and morbidity, there is also an absolute imperative for 'economic and social policies that would improve basic living conditions' for all household members in LMICs ([@bib11], p. 110). Moreover, it must also be acknowledged that while urbanisation creates new behavioural risks for those living in cities, in many LMICs, it also produces a profound care gap in which older family members are left in rural areas without either adequate health infrastructure or family networks to care for them in times of illness ([@bib74]). These transitions, in turn, test the capacity of the state just as much as the current machinery of global health.
With its overwhelming focus on single diseases and technocratic solutions, global health does offer a model of care, but it is one that can often be problematically short-lived and partial ([@bib54]). It can also be outcomes rather than process-orientated. This raises the question of the type of care global health endeavours to provide and for whom. Indeed, the degree to which the mechanisms of global health penetrate broader social structures and, as a result, the determinants of health, is a question that is infrequently asked and nowhere near being solved. Under conditions where global health activities have supplanted the responsibilities of the state, there is the danger that this may start to precipitate 'a striking culture of indifference to affliction present in areas of extreme inequality', which, in turn, 'facilitates a pathogenic biosocial spiral of socioeconomic exclusion and deteriorating health' ([@bib92], 448; see also [@bib39]; [@bib101]). Thus, while many countries of the global South have witnessed meteoric climbs in their middle classes, the gulf between rich and poor has only widened. Across the global South, there are also mounting inequities between state and private healthcare provision, the distribution of essential medical technologies, drugs and expertise as medical professionals seek employment in the global North, the cities of Asia and the Gulf ([@bib86]; [@bib465]). This necessarily means a situation where 'the rich, although increasingly shielded from most disease threats, are able to purchase better health' ([@bib92], 449) and may actually only rarely come into direct contact with global health programmes. Moreover, when the richest can access healthcare elsewhere, this does little to either inculcate a broader ethic of care or to bolster support for efforts to address the wider social determinants of health ([@bib58]). As a result, social justice and human rights remain a persistent absence in the politics of NCDs in the global South.
This absence is further reinforced by the fracturing of the social solidarities that have traditionally underpinned an ethic of care in the face of global change. This in turn reveals a further, painful irony at work in efforts to tackle NCDs in the global South. NCDs require not only the care of others, but also necessitate care of the self, especially in relation to the four major lifestyle risk factors. This need is occurring just as the traditional state-centred mechanisms of care and the will to care may be being eroded, not least because of the reforms associated with structural adjustment policies ([@bib105]). NCDs require adherence to both prevention and treatment regimes. Both are amenable to some degree of individual control (such as not smoking or drinking in moderation), but are equally often determined by the structural factors underpinning the distal pathogenic effects of inequality. These can erode real choices as well as the capacity to make reasoned choices. Further complicating care, treatments for NCDs, even if available, may be expensive or their supply intermittent. Treatment regimes may require lifetime adherence (e.g. statins for high cholesterol), certain levels of competence (e.g. diabetes blood sugar testing and insulin therapy), expensive technologies or complex surgical techniques (e.g. MRI scanners, laser surgery) or basic palliative medications such as analgesics that are unavailable ([@bib7], 1442). Moreover, where infectious disease and NCDs coexist, as they so often do in the global South, existing poor health, compromised immunity or episodic illness may undermine the capacity to undertake either prevention or treatment activities. Not only may this ignite conditions under which the rhetoric of individual blame may be invoked, but it also ensures, as [@bib75], [@bib76], [@bib77] exemplary work in Botswana has explored, that people need more care, often earlier in their lives and the consequences of illness can be catastrophic in terms of economic and social disenfranchisement.
4. Chronic diseases and the politics of culture {#s0020}
===============================================
Another, third way to critically examine the politics of NCDs in the global South is through the lens of 'culture'. This immediately begs the question: what is meant here by culture? Do we mean culture in the normative sense, as a 'thing' (for example, a set of health-related practices, beliefs or behaviours) that is shared by a specific cultural group or within a geographical space that is argued to have dominant cultural norms ([@bib33])? It is certainly the case that such a normative perspective has been mobilised in analyses of non-communicable diseases in the global South as elsewhere; especially those that highlight the importance of lifestyle risk factors. Such analyses are often framed by a different transition model to the ones we have already discussed; to the epidemiologic and health transition models we can add the idea of the nutrition transition ([@bib100]). This latter model emphasises the relationship between levels of economic growth or development and patterns of dietary behaviour. As [@bib32] explain, diets at one time primarily associated with the rich industrialised nations of the global North -- the so-called 'western' diet, which is high in fats, especially meat and milk products, saturated fats and sugars -- are no longer regarded as being spatially fixed. Put simply, relatively early studies into the structure of global diets in the 1960s and 1970s suggested that as GNP per capita rises within nations so too does the consumption of foods associated with the western diet. More recent analyses, such as that offered by [@bib32], [@bib99], [@bib68], add further layers of understanding to this fairly simplistic model by suggesting that a host of other factors, including urbanisation, global food advertising and marketing and associated shifts in socio-cultural practice, also play an important role in this transition ([@bib59]).
Of particular concern here is the question of the rapidity of the nutrition transition and the importance of culture to it; as [@bib23], p. 954; cf. also: [@bib125]; [@bib129]) remarked, '\[a\]larm has been expressed about the rapid spread of the fast food culture, perhaps exemplified most visibly by McDonald\'s'. There is a tendency in analyses that draw on culture in this way to treat it as a 'thing' that invades or colonises other, often by implication indigenous or 'traditional', cultural practices; as [@bib117] explain, 'the diffusion and adoption of Western culture in other places is often termed "Westernisation", whereby societies and individuals adopt particular ideas and practices from more economically developed and commercialised countries'. So, for example, studies such as theirs point to the replacing of 'indigenous' foods with 'western' ones: rice, fish and vegetables for eggs, dairy and meat. This surprisingly imperialist vision of an invasive western culture mirrors long-standing concern with the impact of acculturation on the food habits of migrants; as shown, for example, in relatively early studies of migration, dietary change and chronic heart disease in 1960s USA (cf. [@bib111]; [@bib83]).
Such analyses of cultural transition, here relating to dietary behaviours, highlight the disruptive tendencies of social change brought about by processes such as rapid urbanisation or the globalisation of cultural practices and their often negative influence on population health ([@bib112]). This is certainly an important area for further academic enquiry especially in the many and diverse countries that make up the global South. Indeed, as [@bib131] notes, most 'cross-cultural' studies of this kind have been carried out in the multi-ethnic settings of high-income countries in the global North. However, it is not the only way in which we might approach the question of culture as it relates to the politics of NCDs. [@bib28] influential essay on cultural approaches to health is useful here. As he argues, the body is a 'cultural object'; one that provides a 'powerful medium through which we interpret and give expression to our individual and social experience' ([@bib28], p. 60; see also [@bib79]). Bodies are differentially constituted as healthy, diseased, risky and so on across a range of media and with consequences that are felt at different spatial scales as well as at the level of individual bodies. While this is perhaps especially so with regards infectious or contagious bodies, all bodies that are understood as out of control or outside of socially constituted notions of normality -- obese ones, depressed ones, cancerous ones, psychotic ones, intoxicated ones -- are those upon which political and ideological optics are focused ([@bib27], [@bib18]).
A further illustration of the importance of engaging critically with the politics of culture and health that emerge here comes from the discourse of the contemporary obesity epidemic. Across the social sciences, there is a lively and often contentious debate relating to Foucault\'s concepts of biopower and biopolitics and how they might be drawn upon in critical analyses of non-communicable diseases in general and obesity more specifically (e.g. [@bib134]; [@bib80]). The participants in this debate not only contest the science of obesity (e.g. [@bib52]; [@bib56]) but, more importantly here, they critique the pathologisation of fatness and an associated governmental impulse that prioritises the production of bodies that conform to cultural norms regarding size and shape as well as to contemporary public health imperatives relating to individual and population health (e.g. [@bib36]; [@bib38]; [@bib51]; [@bib61]). As Bethan Evans and Rachel Colls argue, such an impulse is *biopolitical* in that individuals are the subjects of 'surveillance, punishment and training' and relates to Foucault\'s broader understanding of *biopower* because the discourse surrounding obesity is directed at 'man-as-species' and is concerned more generally with the health of populations (Foucault cited in [@bib38], p. 1055).
[@bib37] extends this reading of the biopolitics of obesity in a subsequent essay discussing notions of threat and pre-emptive politics as they relate to the body, the population and the nation. As she argues, the 'war' on obesity that has developed in the high-income countries of the global North, which is a war on specific types of bodies as much as it is a war on the environments that help to produce them ([@bib56]), is concerned primarily with the threat that the '*matter* of bodies' pose in the future ([@bib37], p. 22). Evans distinguishes here between the public health logics of *prevention* and *pre-emption* and focuses on interventions directed at the 'bodies of the future': children ([@bib37], 30). Her argument is that where the western tradition of public health has in the past concerned itself with the prevention of known and calculable risks to health, it is now more focused with taking pre-emptive action in the face of futures that are less certain, less knowable. As she argues, obesity policy is 'reliant on the temporal gap between onset of risk factor and onset (or not) of ill-health. This gap provides an opportunity for pre-emptive action...' ([@bib37], p. 30).
There are two key points to take from the above discussion. Firstly, if we only treat culture normatively in our analyses of NCDs in the global South, as there has been a tendency to do, we risk obscuring the political contestation that arises around specific bodies and the (western) practices that have rendered them problematic. [@bib136] suggest there is a threat inherent in the importation of western medical responses; for them, it relates to the pharmaceuticalisation of public health as well as to the reliance on procedures such as bariatric surgery (cf. [@bib124]). Arguing from a health economics perspective and for more emphasis on evidence-based prevention strategies, they suggest such interventions risk the vitality of entire health systems as money is diverted to expensive and unaffordable treatments. We would argue this is not the only 'threat' that needs critical attention. To it we would add the threat posed by neoliberal ideologies that have underpinned the response to NCDs in the global North and which see care for certain bodies not only as an 'excessive cost' in the present but as an unacceptable burden on the future ([@bib56], 54). Secondly, and more briefly, the above discussion challenges us to consider more seriously and much more critically the emerging preventive and pre-emptive strategies that are being put in place in the global South to assure against the apparent threat posed by western cultural practices.
5. An outline of the special issue {#s0025}
==================================
The six papers of this special issue help shed light, in varying ways, on our respective concerns with problematisation, care and culture within the politics of NCDs in the global South. [@bib460] paper explores how epidemiological models used to problematise smoking in developing countries are building on notions of time and space associated with postwar theories about modernisation and progress. In this reading -- favoured by tobacco control activists -- development and, by extension, the kinds of "globalised" culture that are presumed to be the hallmark of economic growth become proxies for epidemiological risk. Here, development and culture are constructed as specific and serious threats to public health, with political intervention the favoured solution. Criticism of the simplistic readings of culture that can dominate the politics of NCDs is also a feature of [@bib450] paper in which they explore the recursive relationships between the built environment and the experience of chronic disease in the context of Khayelitsha, one of Cape Town\'s poorest neighbourhoods. Their paper highlights two glaring absences in the problem frames of the global NCD agenda -- mental health and the entanglements of urban environments with upstream determinants of health. The gross inequalities in everyday life within cities not only condition the likelihood of suffering from chronic disease, but also the shape and nature of that suffering. Cultural coping mechanisms, in turn, can be severely compromised by the nature of places and their use. Smit et al. draw attention to the problems of food purchasing and storage, of being physically active and of the depression and stress that emerge from living with the perpetual (fear of) crime and violence. Coping, Smit et al. argue, will only be enhanced through attention to the drivers of risky environments, issues that remain silent in a politics of NCDs that would prefer to blame the failings of culture than acknowledge the complicity of the state in producing risk.
This critique is also a feature of [@bib486] paper in which they argue that the political imaginaries of global health, shaped as they are by inherently neoliberal ideologies, purposefully divert attention from both the social and political economic determinants of NCDs. Instead, they place responsibility for NCDs and their prevention in the hands of individuals, rendering care a matter of successful cultural behavioural interventions. The concern with individual choice, responsibility and empowerment also represents the hope that new, self-governing subjects can be formed that, in turn, can exercise a culture of self-care. Such a culture is essential for the success of most contemporary NCD prevention and treatment strategies, yet so much critical social scientific analysis demonstrates just how problematic these political aspirations are. For example, [@bib482], [@bib492] and [@bib500] papers all engage with the politics of NCDs through the experiences of largely 'improvised' ([@bib77]) treatment options for breast cancer and diabetes available in India, China and Uganda respectively. These papers speak directly to each other in their concern with the new forms of biosociality and therapeutic citizenship that arise in the need to care for those suffering with chronic diseases in places where the medical treatment provided by public hospitals, charities and private clinics is either insufficient or at odds with local cultural models of health and illness. For MacDonald and Whyte, this care takes the form of expert patients and patient groups that help mediate the often inadequate relationship between doctor and patient and provides the empathy and information needed to plug gaps in the existing provision of care. For Bunkenborg, these new forms of sociality and citizenship do not only involve expert patients and patient groups but are also mediated through the commercial world of diabetes treatments and technologies. In his example of China, the doctor-patient relationship is often fraught with mistrust, providing an opportunity for a cacophony of private enterprises to bring the hope of diabetes self-management through a range of products and drugs. In each of these examples, the experience of NCDs, the cultural formations that emerge from them and the demand for care are inextricable from the complex and desperately uneven public-private patchwork of medical services. Inadequate care is rarely a matter of cultural failing, despite the blame tendencies of individualised behavioural framings of NCDs. In understanding the interweaving of politics, care and culture in the problematisation of NCDs in the global South it is hoped that this collection of papers will open up new conversations about these issues and help us think how politics and policies might be reshaped in ways that enhance their ability to alleviate human suffering.
DR thanks George Weisz and Tobias Rees for the insightful discussions on global health and NCDs while a Visiting Scholar at the Department of Social Studies of Medicine at McGill University. DR also gratefully acknowledges the generous financial support from the Wellcome Trust through a Small Medical Humanities Research Grant and a Society & Ethics Research Fellowship. CH thanks the Wellcome Trust for their financial support through a Small Society & Ethics Research Grant.
| {
"pile_set_name": "PubMed Central"
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All relevant data are within the paper and its Supporting Information files.
Introduction {#sec005}
============
Natural and man-made disasters are common worldwide. Various disasters have occurred in South Korea in the twenty-first century, such as typhoons, floods, and subway fires. Among them is the sinking of the Sewol ferry, which occurred on the morning of April 16, 2014. The ferry capsized while carrying 476 people, mostly secondary school students from Danwon High School. In total, 304 passengers and crew members died in the disaster. The Sewol ferry disaster severely shocked Korean society and resulted in widespread social and political reactions in South Korea.
Traumatic symptoms in children and adolescents are expressed in a variety of forms depending on their developmental stage. Children can develop PTSD (Post-Traumatic Stress Disorder) and other mental health problems following traumatic events.\[[@pone.0195235.ref001]\] Moreover, a significant minority of children who are particularly vulnerable have ongoing difficulties.\[[@pone.0195235.ref002]\] Compared with studies of adult samples, studies of youth outcomes after a disaster generally report higher estimates for the prevalence of mental health disorders.\[[@pone.0195235.ref003]\] Therefore, to help children and adolescents, it is very important to evaluate and intervene in situations of psychological trauma.
In South Korea, before April 16, 2014, there were no efforts to prepare the population for coping with disaster. Systematic psychological intervention guides for disaster situations have never been provided.
We searched through guidelines such as the WHO guidelines\[[@pone.0195235.ref004]\], the Mental Health Gap Action Programme (mhGAP) Humanitarian Intervention Guide\[[@pone.0195235.ref005]\] and recommendations by the Inter-Agency Standing Committee (IASC)\[[@pone.0195235.ref006]\]. However, the use of available practical guidelines for disaster and trauma patients might be limited due to cultural differences in medical situations and clinical environments. Therefore, protocols that can more aptly respond to culturally specific situations and issues in South Korea are required.\[[@pone.0195235.ref007]\] The country has suffered from a lack of crisis intervention approaches to follow after disasters. For these reasons, confusion arose when the sinking of MV Sewol occurred on April 16, 2014. Therefore, we seek to study and suggest practical directions for establishing guidelines in South Korea.
In this regard, a Delphi study for disaster care is necessary. The Delphi methodology is a widely used group survey technique typically conducted in three consecutive rounds to evaluate consensus among experts in a field. The quality of the panel of experts and their opinions on the given topic is considered a strength of the Delphi technique.\[[@pone.0195235.ref008]\] The approach has the advantage of obtaining expert opinion with a guarantee of anonymity, thus avoiding potential distortion caused by peer pressure in group situations such as focus group analysis.\[[@pone.0195235.ref009]\] Above all, this technique is most effective when there is a lack of information or only inadequate information on a particular issue.
In this context, it is particularly important to monitor the psychosocial care guidelines for children after a disaster. However, to our knowledge, no researchers have examined expert opinion via a Delphi study in post-disaster situations in South Korea.
This survey details the design of a Delphi study for addressing appropriate psychosocial care guidelines for children and adolescents after a disaster. The agreed-upon measures could constitute a standardized approach to initial clinical evaluation and intervention to help identify individuals in need after a disaster.\[[@pone.0195235.ref010]\] A three-round Delphi study was undertaken to elicit a prioritized list of research topics to guide future research efforts and thus obtain meaningful results.\[[@pone.0195235.ref011]\] Consequently, using the Delphi survey technique, this study aimed to evaluate the usefulness and direction of the development of post-traumatic assessment and intervention based on the opinions of pediatric and disaster- and trauma-related experts.
Methods {#sec006}
=======
The Delphi study consisted of three consultation rounds from January to May 2016. In each Delphi round, we provided the panel with feedback on the results of the previous consultation, and routine communications with panel experts were conducted by e-mail. The study was approved by Eulji University\'s Institutional Review Board (IRB No. EMCS 2015-12-004).
Delphi study {#sec007}
------------
A Delphi study is a structured process that invites experts to complete a series of 'rounds' to gather and refine information related to the study question until an expert consensus is reached.\[[@pone.0195235.ref012]\] A commonly used formal consensus method is the Delphi technique, which involves two or more rounds of postal or online questionnaires.\[[@pone.0195235.ref013]\]
According to previous studies, two or three rounds are frequently used in the Delphi process.\[[@pone.0195235.ref012]\] The survey rounds interactively ask experts to prioritize issues or rate them on implementation-related scales, such as scales measuring feasibility or desirability, enabling controlled feedback on the previous round's group results.\[[@pone.0195235.ref014]\] This group facilitation technique aims to obtain consensus among the opinions of 'experts' through a series of structured questionnaires.\[[@pone.0195235.ref015]\]
Delphi panel {#sec008}
------------
A Delphi study is conducted with a group of individuals considered to have expertise (both professional and experience-based) in the field under investigation.\[[@pone.0195235.ref016]\] The Delphi panel in this study consisted of experts in child and adolescent mental health, professionals providing disaster psychological support, and related practitioners with experience in disasters. Our survey included a range of mental health professionals.\[[@pone.0195235.ref013]\]
The Delphi technique allows for the selection of experts and does not require a representative sample of the population. We note that the literature on Delphi surveys traditionally recommends a panel of 10 to 15 experts, typical of most qualitative research.\[[@pone.0195235.ref017]\] However, a panel size ranging from 20 to 50 has been deemed appropriate.\[[@pone.0195235.ref018]\] Therefore, the present study is informed by recommendations of a sample size from 10 to 50 for qualitative research and Delphi surveys designed to generate hypotheses.\[[@pone.0195235.ref019]\]
The Delphi panel participants were also required to provide basic demographic information and professional characteristics.\[[@pone.0195235.ref020]\] Anonymity was assured for all participants during the study; anonymity prevents the influence of the authority, status, personality, or reputation of group members in the process, thereby preventing biased outcomes.\[[@pone.0195235.ref021]\]
First-round questionnaire {#sec009}
-------------------------
The Round 1 survey consisted of 20 open-ended questions grouped into four themes ([S1 Appendix](#pone.0195235.s001){ref-type="supplementary-material"}). Several open-ended questions were included to ensure that the survey accommodated the opinions of professionals from a multidisciplinary team. After confirming participation, panel participants were e-mailed an invitation to activate the Round 1 questionnaire. We conducted the online interview and received informed consent from all participants on the expert panel before interviewing them. The responses had no word limits, and participants were encouraged to give their opinions freely. Reminders were sent if the survey had not been returned. The survey was open for one month.
Second-round questionnaire {#sec010}
--------------------------
Questions for Rounds 2 were developed based on the participants\' responses in the previous round. Converged answers in Round 1 were classified as evaluation and intervention, and freely presented expert opinions were based on detailed questions. The Round 2 survey consisted of 156 closed-ended questions with responses grouped into 27 themes. The experts received the second-round questionnaire by e-mail and were instructed to rate and score the importance of each indicator on a five-point Likert scale (1 = very unimportant, 3 = neutral and 5 = very important). An item was considered important if ≥80% of the respondents awarded it a score of 4 or 5; otherwise, the item was removed. The experts were encouraged to provide comments freely on each indicator and/or to propose indicators that they considered important. Routine communication with panel experts was conducted by e-mail.
Third-round questionnaire {#sec011}
-------------------------
Round 3 excluded 44 items that did not receive a consensus in Round 2. For 112 items, 80% agreement was reached. In Round 2, the experts freely commented on each indicator that they considered important. Based on these responses, 11 items were modified, and 63 items were added.
Ultimately, 175 items were composed and grouped into 25 themes. In the third round, we asked the panel to rate the importance of each topic on a 5-point Likert scale from 1 (not important) to 5 (very important). The level of consensus was set to 80% of respondents indicating agreement.\[[@pone.0195235.ref009]\] Individual and anonymous opinions were solicited via e-mail.
Data analysis {#sec012}
-------------
Delphi questionnaires were coded individually. Members of the research team alone had access to the codes to facilitate follow-up. Any published data identified individuals, their institution, or organizations.
In Round 1, all topics suggested by the panel experts were categorized using content analysis. We identified words or expressions in conceptual categories to understand and identify the relationships among themes. We performed categorization by removing irrelevant, overlapping and repeated content; looking for common viewpoints; and identifying responses. To analyze the Round 2 and 3 responses, we calculated content validity ratios (CVRs). The minimum CVR was determined by the number of experts participating in each round.
We used the formula CVR = (n~e~ -*N*/2)/ (*N*/2), where n~e~ represents the number of panel experts rating an item as 'essential' (score of 4 or 5) and *N* represents the entire number of panelists.\[[@pone.0195235.ref022]\] The CVR ranges from +1 to −1. A high positive value indicates that the survey experts agreed that a factor or item was essential.\[[@pone.0195235.ref023]\]
Therefore, in Round 2, the CVR values of all items were set to 0.49 for the 16 panels. Additionally, in Round 3, the minimum CVR value was set to 0.38 for the 24 panels.
Results {#sec013}
=======
Demographics of the panel experts {#sec014}
---------------------------------
The demographic characteristics of the experts are described in [Table 1](#pone.0195235.t001){ref-type="table"}.
10.1371/journal.pone.0195235.t001
###### Demographic characteristics of the panel experts.
{#pone.0195235.t001g}
Round 1 (N = 15) Round 2 (N = 16) Round 3 (N = 24)
--------------------------- ------------------ ------------------ ------------------ --------- ---- ---------
Participant response rate 15/18 (83.00) 16/20 (80.00) 24/28 (86.00)
Age, mean (SD) 44.07 (6.84) 43.75 (7.14) 43.83 (8.33)
Gender
Male 5 (33.33) 5 (31.25) 5 (20.83)
Female 10 (66.67) 11 (68.75) 19 (79.17)
Education level
Bachelor\'s degree 2 (13.33) 1 (6.25) 1 (4.16)
Master\'s degree 1 (6.67) 3 (18.75) 4 (16.67)
Doctoral course 2 (13.33) 3 (18.75) 4 (16.67)
Ph.D. 10 (66.67) 9 (56.25) 15 (62.50)
Profession
Psychiatrist 15 (100.00) 10 (62.50) 17 (70.84)
Psychologist 0 (0.00) 5 (31.25) 5 (20.83)
Social worker 0 (0.00) 1 (6.25) 2 (8.33)
SD: standard deviation
In Round 1, 18 experts registered to be members of the Delphi panel, and 15 of them (83%) (10 female, 5 males) returned the Round 1 questionnaire. The mean age of the experts was 44.07 years (standard deviation: 6.84 years). Approximately 10 (66.67%) of the respondents had earned a Ph.D.
In Round 2, 20 participants were included, and 16 (80%) responded; the respondents included psychiatrists (10), psychologists (5), and a social worker (1). The mean age of the experts was 43.75 years (standard deviation: 7.14 years). Approximately 11 (68.75%) of the panel experts were women, and 9 (56.25%) had earned a Ph.D. as their highest level of education.
In Round 3, 28 psychiatric professionals registered to be members of the expert panel, and 24 (86%) returned the questionnaires. The mean age of the experts was 43.83 years (standard deviation: 8.33 years); the experts included psychiatrists (17), psychologists (5), and social workers (2). Most of the experts were females (19), and 15 (62.50%) had earned a Ph.D. Round 3 experts showed an adequate level of agreement on the research topics ([Table 1](#pone.0195235.t001){ref-type="table"}).
Results of first-round Delphi survey {#sec015}
------------------------------------
Qualitative content analysis was used in Round 1. The Round 1 results are described in detail in a previously published paper.\[[@pone.0195235.ref024]\] We found that the following issues have a strong effect on post-disaster interventions: proper timing of the initial interview in the event of a disaster, assessment notification, assessment services for individuals, mandatory enforcement measures, scale screening and treatment intervention elements, symptom degree classification, intervention standardization, program level, care unit environments, and operation plans.
The table in the preliminary research paper that included the Round 1 items and content has been reproduced. We sought permission from previous journals to re-use the table and to add a reference ([Table 2](#pone.0195235.t002){ref-type="table"}).
10.1371/journal.pone.0195235.t002
###### Categories and items of the first round of the Delphi study[\*](#t002fn001){ref-type="table-fn"}.
{#pone.0195235.t002g}
Categories Items
------------------------------------------------------------------------------------------------------- --------------------------------------------------------------------------------
I. Currently used child-adolescent assessment and treatment protocols in disasters Treatment programs that have been proven to be effective in previous disasters
Difficulties when implementing assessment protocols and treatment programs in disasters
Need to promote previous child-adolescent treatment programs
II\. Direction of child-adolescent assessment protocols after disaster Need for child-adolescent psychological assessment intervention after disaster
Adequate means of psychological assessment procedures
Constructing an environment for psychological assessments
Things to consider when using brief scales
Essential factors when selecting assessment scales
III\. Direction of child-adolescent treatment programs after disasters Critical factors in child-adolescent treatment intervention after disasters
Timeframe for treatment program intervention and its evidence
Timeframe for treatment program termination and its evidence
Adequate treatment programs for children and adolescents
Means of operating treatment programs
Need for standardization of the Korean version of foreign treatment programs
IV\. Things to consider in disaster interventions Level and qualifications of treatment professionals
Current level of continuing education system construction for child-adolescent disaster professionals
Ways for disaster professionals to continuously participate in treatment
Effective ways of promoting treatment programs
\*We refer to the table in the previous study.\[[@pone.0195235.ref024]\]
Results of second-round Delphi survey {#sec016}
-------------------------------------
The categories and items on the Delphi panel survey are described in [Table 3](#pone.0195235.t003){ref-type="table"}.
10.1371/journal.pone.0195235.t003
###### Categories and items of the second and third rounds of the Delphi study.
{#pone.0195235.t003g}
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Categories Items Details
------------------------------------------------------------------------------------------ --------------------------------------------------------------------------------- --------------------------------------------------------------------
Concept of child-\ 1\. Concept of trauma in disasters 1\) Unique model for other psychopathologies
Adolescent trauma in disasters
2\) Child-adolescent trauma after disaster
2\. Recovery of trauma in disasters 1\) Return to the daily lives of children and adolescents
2\) Stabilization of developmental tasks (academic function, peer relationships)
Child-adolescent assessment after disasters 1\. Baseline psychological assessments 1\) Importance of assessment
2\) Intake and screening \(1\) Critical factors in screening
\(2\) Children and adolescents
\(2\) Adequate time for screening
\(3\) Things to consider for screening
3\) Developmental recording \(1\) Things to include in the developmental record of children and adolescents
\(2\) Providing assessment service by age
2\. Constructing psychological assessments 1\) Constructing an environment for psychological assessments
2\) Means of operating assessment
3\) Scales recommended for universal screening \(1\) Trauma-related scale
\(2\) Depression/anxiety scale
\(3\) Overall emotion/behavior scale
\(4\) Family-related scale
\(5\) Intelligence test
\(6\) Neuropsychological test
\(7\) Other scales
4\) Things to consider in selecting a scale \(1\) Adequate number of scales
\(2\) Each number of scale items
\(3\) Appropriate age of children and adolescents
5\) Storage and maintenance of scales and analysis report
3\. Assessment professionals 1\) Application plan for disaster assessment professionals
2\) Professionals \(1\) Level of assessment professionals
\(2\) Qualification of assessment professionals
3\) Arrangement of child-adolescent disaster assessment professionals
4\) Education system construction for child-adolescent disaster professionals
4\. Promoting assessments 1\) Participation in assessment \(1\) Ways for system to continuously participate in assessment
\(2\) Awareness of conducting assessment
2\) Effective ways of promoting assessment
3\) Arrange for assessment information system
Child-adolescent treatment programs after disasters 1\. Conducting an intervention 1\) Conducting a treatment program
2\) Essential factors for the treatment program
2\. Traits of participants 1\) Classification of the child-adolescent developmental stage and age
2\) Division of child-adolescent symptoms
3\. Treatment program 1\) Group therapy
2\) Time frame of the treatment program \(1\) Importance of the time frame of treatment
\(2\) Standardization of the Korean version of intervention
3\) Treatment program \(1\) TF-CBT
\(2\) EMDR
\(3\) TRT
\(4\) SSET
\(5\) C-First Aid
\(6\) Play therapy
\(7\) Art therapy
\(8\) Other interventions
4\) Customized programs for symptom levels
5\) Family program \(1\) Family participation program
\(2\) Family camp and crash overnight camp
\(3\) Ways of selecting program participants
6\) Standardization of the Korean version of foreign treatment programs
4\. Facilities in disaster interventions 1\) Providing situations for therapeutic intervention \(1\) Arranging the place for child-adolescent intervention
\(2\) Constructing an environment for intervention
\(3\) Providing treatment program information
2\) Opportunities for the treatment program (time, place)
3\) Keeping materials and artwork in the treatment room
4\) Recognition of differences and complementary cooperation \(1\) Recognition of differences in the related organization
\(2\) Complementary cooperation with organization
5\. Treatment professionals 1\) Methods for practical use of disaster professionals
2\) Professionals \(1\) Level of professionals
\(2\) Qualification of professionals
3\) Arrangement of child-adolescent disaster professionals
4\) Continuing education system construction for child-adolescent disaster professionals
6\. Promoting treatment programs 1\) Participation in treatment \(1\) Continuing participation system for children and adolescents
\(2\) Awareness of participation in a treatment program
\(3\) Education to continuously participate in treatment regularly
2\) Effective ways of promoting a system
3\) Creation of protocol information
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Tables [4](#pone.0195235.t004){ref-type="table"} and [5](#pone.0195235.t005){ref-type="table"} show the evaluation items and intervention items, respectively, for Round 2.
10.1371/journal.pone.0195235.t004
###### Contents of post-disaster evaluation in the Round 2 survey.
{#pone.0195235.t004g}
Variable Item CVR Mean SD
------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------- ------- ------- -------
Concept Unique model for other psychopathologies 0.875 4.440 0.629
Recovery of Trauma Return to daily lives 0.500 4.000 0.730
Attainment of developmental tasks 0.875 4.250 0.775
Screening Importance of screening 0.875 4.310 0.602
Subject of screening Self-report 1.000 4.500 0.516
Family or caregiver report 0.750 4.250 0.856
Acquaintance or friend report[\*](#t004fn001){ref-type="table-fn"} 0.250 3.560 0.814
Teacher report 1.000 4.440 0.512
Contents of screening Checking for coping resources and psychosocial crisis 1.000 4.500 0.516
Time for 20 minutes 0.125 3.560 0.892
Interview with family 0.500 3.880 0.957
Precautions[\*](#t004fn001){ref-type="table-fn"} 0.625 4.130 0.719
Fill out the developmental progress report 0.000 3.310 0.946
Subject of screening for the high-risk group Self-report 1.000 4.560 0.512
Family or caregiver report 1.000 4.690 0.479
Acquaintance or friend report 0.500 4.000 0.730
Teacher report 1.000 4.500 0.516
Evaluation of the high-risk group Required information 1.000 4.560 0.512
Duration of 60 minutes 0.250 3.810 0.911
Interview with family 1.000 4.440 0.512
Precautions[\*](#t004fn001){ref-type="table-fn"} 0.500 4.060 0.772
Scale recommended in screening Trauma-related scale 0.875 4.690 0.602
Grief scale 0.625 4.380 0.957
Depression/anxiety scale 0.875 4.560 0.629
Suicide scale 0.875 4.560 0.629
Drug-related scale 0.500 4.190 0.834
Physical symptom scale 0.750 4.380 0.719
Social resource scale 0.750 4.380 0.885
Family function scale 0.500 4.130 0.957
Adaptation to daily life scale 0.750 4.130 0.957
Additional required evaluation[\*](#t004fn001){ref-type="table-fn"} -0.250 3.380 1.088
Things to consider for screening Fewer than20 questions per scale[\*](#t004fn001){ref-type="table-fn"} 0.500 4.000 1.033
In the individual evaluation, a total of 40--50 questions[\*](#t004fn001){ref-type="table-fn"} 0.125 3.810 1.109
In the group evaluation, a total of 80--100 questions[\*](#t004fn001){ref-type="table-fn"} -0.375 2.810 1.328
Scales and test recommended for the high-risk group Trauma-related scale 1.000 4.750 0.447
Grief scale 0.875 4.560 0.629
Depression/anxiety scale 1.000 4.690 0.479
Suicide scale 1.000 4.690 0.479
Drug-related scale 0.625 4.250 0.931
Physical symptom scale 0.875 4.500 0.632
Social resource scale 1.000 4.750 0.447
Family function scale 0.875 4.630 0.619
Adaptation to daily life scale 0.875 4.630 0.619
Intelligence test -0.125 2.940 1.436
Projection test[\*](#t004fn001){ref-type="table-fn"} -0.500 2.380 1.455
Neuropsychological test -0.500 2.500 1.414
Additional required evaluation[\*](#t004fn001){ref-type="table-fn"} -0.375 3.500 1.155
Things to consider in the high-risk group Less than20 questions per scale[\*](#t004fn001){ref-type="table-fn"} 0.125 3.380 1.455
In the individual evaluation, a total of 40--50 questions[\*](#t004fn001){ref-type="table-fn"} 0.375 3.880 1.204
In the group evaluation, total of 80--100 questions[\*](#t004fn001){ref-type="table-fn"} -0.125 3.250 1.390
Disaster evaluation professionals Importance of disaster evaluation professionals 0.500 4.060 1.063
Professional qualifications and levels 0.875 4.310 0.602
Application plan for disaster assessment professionals 0.625 4.060 1.181
Inclusion in professional education curriculum 0.875 4.560 0.629
Promoting a plan for evaluation processes Importance of promoting evaluation[\*](#t004fn001){ref-type="table-fn"} 1.000 4.440 0.512
Awareness of conducting assessment 1.000 4.560 0.512
Education in school 1.000 4.500 0.516
Campaigns on public TV 0.875 4.500 0.632
Advertisement on the Internet 0.875 4.310 0.602
Advertisement on SNSs 0.625 4.060 0.680
Advertisement in education offices 0.625 4.250 0.775
Advertisement in the community 0.500 4.000 0.894
Prior education 0.875 4.380 0.619
Parents' education 0.750 4.500 0.894
Teacher education 1.000 4.810 0.403
\* Excluded (low CVR) items in Round 3.
10.1371/journal.pone.0195235.t005
###### Contents of post-disaster intervention in the Round 2 survey.
{#pone.0195235.t005g}
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Variable Item CVR Mean SD
----------------------------------------------------------------------------------------------- ----------------------------------------------------------------------------- ------- ------- -------
Conducting treatment programs Importance of intervention 0.750 4.440 0.892
Psychoeducation after disaster 1.000 4.810 0.403
Guideline for coping with the media 1.000 4.810 0.403
Normalization/stabilization education 0.875 4.690 0.602
Practice for physical stabilization 0.875 4.560 0.629
Classification by acute/maintenance intervention 1.000 4.750 0.447
Education for families 1.000 4.810 0.403
Education for teachers 1.000 4.810 0.403
Handling of guilt 0.750 4.690 0.704
Dealing with emotion 0.875 4.630 0.619
Time frame 1--4 sessions 0.500 4.060 0.929
5--8 sessions 0.125 3.880 0.885
9--12 sessions[\*](#t005fn001){ref-type="table-fn"} -0.250 3.250 0.856
Long-term sessions[\*](#t005fn001){ref-type="table-fn"} -0.500 2.750 1.125
Time of intervention Immediately after disaster, interventions as quick and as brief as possible 0.875 4.440 1.031
If there is physical trauma, intervene after pain relief[\*](#t005fn001){ref-type="table-fn"} 0.000 3.310 1.250
Classification of acute/sub-acute/chronic stage 1.000 4.690 0.479
Termination of session If both therapist and client agree[\*](#t005fn001){ref-type="table-fn"} 0.250 3.690 1.014
If the client feels he or she has recovered[\*](#t005fn001){ref-type="table-fn"} 0.125 3.440 1.263
Subject of intervention Categorization by developmental stage/age 1.000 4.810 0.403
Division of child-adolescent symptoms 0.750 4.440 0.727
Combining individual therapy with group therapy 0.250 3.810 1.167
Number of participants in group sessions 2--4[\*](#t005fn001){ref-type="table-fn"} 0.250 3.690 0.946
5--8[\*](#t005fn001){ref-type="table-fn"} 0.250 3.810 0.750
9--12[\*](#t005fn001){ref-type="table-fn"} -0.750 2.750 0.683
13--16[\*](#t005fn001){ref-type="table-fn"} -0.875 2.060 1.063
Whole class[\*](#t005fn001){ref-type="table-fn"} -0.875 2.000 0.894
Time frame For toddlers and preschoolers, 20 minutes with parent participation 0.250 3.630 0.885
In lower grades of elementary school, 30--40 minutes 0.875 4.190 0.544
In upper grade of elementary school, 40 minutes 0.875 4.190 0.544
In middle/high school, 45--50 minutes 0.875 4.130 0.500
Treatment program Importance of intervention guidelines 0.875 4.560 0.629
PFA 0.875 4.560 0.629
TRT 0.625 3.880 0.500
SSET 0.250 3.690 0.602
TF-CBT 0.375 3.810 0.834
EMDR 0.125 3.560 0.727
PE -0.250 3.250 0.856
Trauma-focused play therapy[\*](#t005fn001){ref-type="table-fn"} -0.125 3.250 0.856
Trauma-focused art therapy[\*](#t005fn001){ref-type="table-fn"} -0.375 3.060 0.929
Family program 0.875 4.310 0.602
Additional programs needed[\*](#t005fn001){ref-type="table-fn"} -0.625 3.310 0.704
South Korean version of toddler and preschooler\ Necessity for standardization in the Korean version 0.750 4.250 0.683
therapy
PFA 0.875 4.250 0.775
TRT 0.500 3.880 0.957
SSET[\*](#t005fn001){ref-type="table-fn"} 0.000 3.310 1.302
TF-CBT[\*](#t005fn001){ref-type="table-fn"} 0.000 3.250 1.390
EMDR[\*](#t005fn001){ref-type="table-fn"} -0.250 2.940 1.340
PE[\*](#t005fn001){ref-type="table-fn"} -0.125 3.060 1.289
Trauma-focused play therapy[\*](#t005fn001){ref-type="table-fn"} 0.125 3.630 0.957
Trauma-focused art therapy[\*](#t005fn001){ref-type="table-fn"} -0.125 3.130 1.258
Additional programs needed[\*](#t005fn001){ref-type="table-fn"} -0.625 3.250 0.775
South Korean version of grade-schooler\ Necessity for standardization in the Korean version 0.750 4.380 0.719
therapy
PFA 1.000 4.500 0.516
TRT 0.500 4.060 0.772
SSET[\*](#t005fn001){ref-type="table-fn"} 0.125 3.810 0.834
TF-CBT 0.375 4.000 0.816
EMDR 0.250 3.690 1.014
PE[\*](#t005fn001){ref-type="table-fn"} 0.125 3.630 1.025
Trauma-focused play therapy[\*](#t005fn001){ref-type="table-fn"} 0.000 3.440 1.153
Trauma-focused art therapy[\*](#t005fn001){ref-type="table-fn"} -0.250 3.130 1.204
Additional programs needed[\*](#t005fn001){ref-type="table-fn"} -0.625 3.250 0.775
South Korean version of middle/high school\ Necessity for standardization in the Korean version 0.875 4.440 0.629
therapy
PFA 0.875 4.250 0.577
TRT 0.625 4.060 0.854
SSET 0.625 4.060 0.680
TF-CBT 0.750 4.250 0.683
EMDR 0.500 3.940 0.854
PE[\*](#t005fn001){ref-type="table-fn"} 0.125 3.750 0.775
Trauma-focused play therapy[\*](#t005fn001){ref-type="table-fn"} -0.125 3.130 1.204
Trauma-focused art therapy[\*](#t005fn001){ref-type="table-fn"} 0.125 3.310 1.195
Additional programs needed[\*](#t005fn001){ref-type="table-fn"} -0.625 3.250 0.775
Facilities in disaster interventions Arrange the place for child-adolescent intervention 1.000 4.560 0.512
Providing treatment program information 1.000 4.500 0.516
Treatment program opportunities 0.625 4.250 0.775
Acceptance of in-school counseling as a class[\*](#t005fn001){ref-type="table-fn"} 0.375 4.060 1.289
Arrangement of materials 0.500 3.940 0.998
Complementary cooperation with organization 1.000 4.560 0.512
Disaster intervention professionals Importance of disaster intervention professionals 0.750 4.380 0.885
Professional qualifications and levels 0.875 4.440 0.814
Need for all mental health workers to conduct treatment 0.750 4.310 0.873
Completion of disaster care curriculum 0.875 4.560 0.629
Knowledge of secondary traumatizations 1.000 4.690 0.479
Education system for disaster intervention professionals 1.000 4.690 0.479
Continuous supervision 1.000 4.690 0.479
Promoting treatment programs Continuing the system for child-adolescent participation 0.750 4.380 0.719
Creation of a system for referrals to therapy[\*](#t005fn001){ref-type="table-fn"} -0.250 3.250 1.238
Education of the whole school 1.000 4.630 0.500
Support for medical expenses from the government 0.875 4.560 0.629
Decrease in the stigma of psychiatric treatment 0.500 4.190 0.834
Cooperation with the community 1.000 4.630 0.500
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
\* Excluded (low CVR) items in Round 3.
In the conceptual and semantic domain of trauma in children and adolescents, the CVR was 0.49 or higher, and the content validity was verified for all items. The average value and the CVR were the highest in the 'self-report' and 'teacher-report' assessments. In contrast, the CVR for 'the importance of evaluating an acquaintance (or a friend) of victims from the disaster' was less than 0.49 ([Table 4](#pone.0195235.t004){ref-type="table"}).
The screening questionnaire items 'necessary to meet a family member at the time of screening' and 'caution when interviewing children and adolescents' were validated. The CVR was the highest for 'trauma, depression, anxiety, suicide, physical symptoms, social support, adaptation, and mood response should be included in the screening test'. Nevertheless, the CVR was less than 0.49 for '20 minutes of screening time is needed' and 'children\'s developmental considerations must be considered'. Therefore, the items with low CVRs were excluded in the third round, and supplementary items were developed ([Table 4](#pone.0195235.t004){ref-type="table"}).
In the high-risk group, the CVR was highest for 'child, adolescent, family, teacher evaluation'. However, the CVR for the item 'It takes about one hour to interview the high-risk group' was less than 0.49. Based on an additional comment from the expert panels, it was decided that the third round should include '30 minutes to 1 hour is most appropriate when evaluating a high-risk group'. In addition, many opinions suggested that 'they should evaluate trauma, depression, anxiety, suicide, and social support'. However, the item 'intelligence, projection test, and neuropsychological evaluation are necessary' was excluded from the third round because the CVR was less than 0.49 ([Table 4](#pone.0195235.t004){ref-type="table"}).
In addition, the CVR was lower than 0.49 for 'the number of program sessions is "5 to 8 sessions", "9 to 12 sessions", and "13 sessions or more" is required' if the intervention program is implemented after a disaster. The CVR was also low for 'the treatment was terminated if the child had recovered the level of functioning'. These items should be excluded because of CVR validity; however, we revised those items based on additional comments from the experts, and the revised items were used in the third round ([Table 5](#pone.0195235.t005){ref-type="table"}).
The CVR for the 'need for standardized PFA (psychological first aid) and TRT (teaching recovery techniques)' for the Korean version for infants and children was higher than 0.49. However, the CVRs for 'SSET (support for students exposed to trauma), TF-CBT (trauma-focused cognitive behavior therapy), EMDR (eye movement desensitization and processing), PE (prolonged exposure therapy), trauma-focused play therapy and art therapy' were low. In this case, the opinion of experts on Korean culture was reflected in the third round. However, the need for the Korean version of the PFA, TRT, SSET, TF-CBT, and EMDR was associated with a CVR higher than 0.49 ([Table 5](#pone.0195235.t005){ref-type="table"}).
Results of third-round Delphi survey {#sec017}
------------------------------------
The evaluation items and intervention items for Round 3 are described in detail in Tables [6](#pone.0195235.t006){ref-type="table"} and [7](#pone.0195235.t007){ref-type="table"}, respectively.
10.1371/journal.pone.0195235.t006
###### Contents of post-disaster evaluation in the Round 3 survey.
{#pone.0195235.t006g}
Variable Item CVR Mean SD
------------------------------------------------------------------------------------------------------------------ -------------------------------------------------------------------------------------------------- ------- ------- -------
Concept Unique model for other psychopathologies 0.917 4.375 0.711
Recovery of Trauma Return to daily lives 1.000 4.750 0.442
Attainment of developmental tasks in a long-term stage[\*](#t007fn001){ref-type="table-fn"} 0.833 4.167 0.565
Disappearance of reactions and symptoms of trauma[\*\*](#t006fn002){ref-type="table-fn"} 0.250 3.708 0.624
Stabilization of social functioning[\*\*](#t006fn002){ref-type="table-fn"} 0.583 3.917 0.584
Stabilization of relationships[\*\*](#t006fn002){ref-type="table-fn"} 0.583 3.875 0.637
Stabilization of academic functioning[\*\*](#t006fn002){ref-type="table-fn"} 0.250 3.625 0.770
Significance Guarantee of the usefulness of exceptions in screening[\*](#t007fn001){ref-type="table-fn"} 0.833 4.208 0.588
Subject of screening Self-report 0.917 4.583 0.584
Family or caregiver report 0.750 4.167 0.637
Teacher report 0.750 4.167 0.637
Contents of screening Checking for coping resources and psychosocial crisis 0.917 4.417 0.584
Duration of 10--15 minutes[\*](#t007fn001){ref-type="table-fn"} 0.833 4.375 0.647
Explanation of brief care service[\*\*](#t006fn002){ref-type="table-fn"} 0.917 4.292 0.550
Interview with family 0.000 3.458 0.833
Screening at moderate speed[\*\*](#t006fn002){ref-type="table-fn"} 1.000 4.375 0.495
Importance of attitude of mind[\*\*](#t006fn002){ref-type="table-fn"} 0.917 4.417 0.584
Concern for secondary damage[\*\*](#t006fn002){ref-type="table-fn"} 1.000 4.708 0.464
Importance of safety and mutual trust[\*\*](#t006fn002){ref-type="table-fn"} 1.000 4.833 0.381
For toddlers and preschoolers, fill in developmental progress[\*](#t007fn001){ref-type="table-fn"} 0.417 3.917 0.717
Understanding previous traumatic experience[\*\*](#t006fn002){ref-type="table-fn"} 1.000 4.292 0.464
Checking for separate experiences of parents[\*\*](#t006fn002){ref-type="table-fn"} 0.167 3.583 0.881
Scale recommended in screening Trauma-related scale 1.000 4.708 0.464
Grief scale 0.833 4.458 0.658
Depression/anxiety scale 1.000 4.542 0.509
Suicide scale 0.917 4.542 0.721
Drug-related scale 0.333 3.917 0.974
Addiction scale[\*\*](#t006fn002){ref-type="table-fn"} 0.333 3.875 0.947
Physical symptom scale 0.917 4.417 0.584
Sleep-related scale[\*\*](#t006fn002){ref-type="table-fn"} 1.000 4.500 0.511
Social resource scale 0.667 3.958 0.806
Family function scale 0.417 3.792 1.021
Adaptation to daily life scale 0.500 4.000 0.834
Existing psychological problem scale[\*\*](#t006fn002){ref-type="table-fn"} 0.583 3.917 0.929
Things to consider in screening Minimal screening question[\*\*](#t006fn002){ref-type="table-fn"} 0.833 4.417 0.654
Question of the prediction of a high-risk group[\*\*](#t006fn002){ref-type="table-fn"} 1.000 4.667 0.482
Subject of screening for the high-risk group Self-report 0.917 4.667 0.565
Family or caregiver report 0.917 4.500 0.590
Acquaintance or friend report 0.083 3.625 0.875
Teacher report 0.750 4.292 0.690
Evaluation of the high-risk group Environment of safety and stabilization[\*\*](#t006fn002){ref-type="table-fn"} 1.000 4.750 0.442
Information on medical history and symptoms[\*](#t007fn001){ref-type="table-fn"} 0.833 4.417 0.654
Duration of 30--60 minutes[\*](#t007fn001){ref-type="table-fn"} 0.833 4.375 0.647
Interview with family 0.917 4.417 0.584
Checking for psychological crisis in the family[\*\*](#t006fn002){ref-type="table-fn"} 0.833 4.333 0.637
Scales and tests recommended for the high-risk group Psychiatric interview[\*\*](#t006fn002){ref-type="table-fn"} 0.917 4.542 0.588
Trauma-related scale 1.000 4.667 0.482
Grief scale 0.833 4.417 0.654
Depression/anxiety scale 1.000 4.500 0.511
Suicide scale 1.000 4.667 0.482
Drug-related scale[\*\*](#t006fn002){ref-type="table-fn"} 0.667 4.250 0.847
Addiction scale[\*\*](#t006fn002){ref-type="table-fn"} 0.500 4.042 0.859
Physical symptom scale 0.917 4.417 0.584
Sleep-related scale[\*\*](#t006fn002){ref-type="table-fn"} 1.000 4.583 0.504
Social resource scale 1.000 4.417 0.504
Family function scale 0.833 4.250 0.608
Adaptation to daily life scale 0.833 4.292 0.624
Assessment of school record[\*\*](#t006fn002){ref-type="table-fn"} 0.333 3.792 1.103
Intelligence test 0.417 3.708 1.083
Existing psychological problem scale[\*\*](#t006fn002){ref-type="table-fn"} 0.833 4.333 0.637
Assessment of family's medical history[\*\*](#t006fn002){ref-type="table-fn"} 0.417 3.917 0.830
Neuropsychological test 0.667 4.083 0.881
Assessment of crisis management ability[\*\*](#t006fn002){ref-type="table-fn"} 0.667 4.208 0.721
Things to consider in the high-risk group Importance of personal interviews[\*\*](#t006fn002){ref-type="table-fn"} 1.000 4.583 0.504
Evaluation in a safe place[\*\*](#t006fn002){ref-type="table-fn"} 1.000 4.500 0.511
Disaster evaluation professionals Importance of disaster evaluation professionals 0.917 4.333 0.565
Application plan for disaster assessment professionals 0.750 4.417 0.717
The need for all mental health workers to conduct assessment[\*\*](#t006fn002){ref-type="table-fn"} -0.333 3.167 0.963
Professional qualifications and levels 1.000 4.458 0.509
Training on crisis management in disasters[\*\*](#t006fn002){ref-type="table-fn"} 0.917 4.292 0.550
Upgrading the quality of professionals[\*\*](#t006fn002){ref-type="table-fn"} 1.000 4.583 0.504
Importance of having clinical experience[\*\*](#t006fn002){ref-type="table-fn"} 1.000 4.625 0.495
Education system construction for child-adolescent disaster professionals[\*\*](#t006fn002){ref-type="table-fn"} 0.917 4.417 0.584
Inclusion in professional education curriculum 1.000 4.375 0.495
Promoting a plan for evaluation processes Ways for system of continuous participation in assessment[\*\*](#t006fn002){ref-type="table-fn"} 0.833 4.250 0.608
Arrangement of prior information[\*\*](#t006fn002){ref-type="table-fn"} 1.000 4.458 0.509
Effective ways for early advertisements to the nation[\*\*](#t006fn002){ref-type="table-fn"} 0.667 4.125 0.680
Awareness of conducting assessment 0.917 4.417 0.584
Top-down system from education offices[\*\*](#t006fn002){ref-type="table-fn"} 0.417 3.792 0.833
Setting up guidelines for ethical behavior[\*\*](#t006fn002){ref-type="table-fn"} 0.917 4.417 0.584
Audio-visual education at school[\*](#t007fn001){ref-type="table-fn"} 0.833 4.417 0.565
Campaigns on public TV 0.583 4.208 0.779
Advertisement on the Internet 0.583 4.083 0.717
Advertisement on SNSs 0.417 3.917 0.930
Advertisement from education offices 0.750 4.250 0.794
Advertisement in the community 0.250 3.874 0.900
Prior education 0.667 4.208 0.721
Parents' education 0.750 4.292 0.690
Teacher education 0.750 4.500 0.722
\* Modified items in Round 3.
\*\* Newly added items in Round 3.
10.1371/journal.pone.0195235.t007
###### Contents of post-disaster intervention in the Round 3 survey.
{#pone.0195235.t007g}
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Variable Item CVR Mean SD
----------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------ ------- ------- -------
Conducting treatment programs Importance of intervention 0.833 4.417 0.776
Effective ways to use a precautionary approach[\*\*](#t007fn002){ref-type="table-fn"} 0.833 4.250 0.737
Psychoeducation after disasters 1.000 4.625 0.495
Guideline for coping with the media 0.917 4.500 0.590
Normalization/stabilization education 1.000 4.625 0.495
Practice for physical stabilization 1.000 4.500 0.511
Handling of guilt 0.917 4.417 0.584
Classification by acute/maintenance intervention 0.833 4.458 0.658
Education for families 1.000 4.583 0.504
Education for teachers 1.000 4.583 0.504
Dealing with emotion 1.000 4.542 0.509
Subject of intervention Categorization by developmental stage/age 1.000 4.667 0.482
Division of child-adolescent symptoms 1.000 4.375 0.495
Combining individual therapy with group therapy 0.833 4.125 0.680
Standard of participation and exceptions[\*\*](#t007fn002){ref-type="table-fn"} 1.000 4.500 0.511
Classification of traits in groups[\*\*](#t007fn002){ref-type="table-fn"} 0.833 4.292 0.751
In group therapy, interventions should differ, depending on the trauma type[\*\*](#t007fn002){ref-type="table-fn"} 0.917 4.250 0.532
Conduct disaster intervention on a large scale[\*\*](#t007fn002){ref-type="table-fn"} 0.417 3.833 0.868
In general, psychoeducation and education to the whole class[\*\*](#t007fn002){ref-type="table-fn"} 0.833 4.375 0.647
Time of intervention Immediately after a disaster, interventions as quick and brief as possible 0.500 4.042 0.859
About one week after a disaster, planning psychoeducation[\*\*](#t007fn002){ref-type="table-fn"} 0.583 4.000 0.885
Classification of acute/sub-acute/chronic stage 0.833 4.292 0.624
Immediately after a disaster, stabilization/support-centric acute intervention[\*\*](#t007fn002){ref-type="table-fn"} 1.000 4.458 0.509
One month after a disaster, trauma-focused intervention[\*\*](#t007fn002){ref-type="table-fn"} 0.750 4.125 0.612
Follow-up for the recovery of daily life functioning[\*\*](#t007fn002){ref-type="table-fn"} 1.000 4.375 0.495
End of session Improving post-test scores versus screening[\*\*](#t007fn002){ref-type="table-fn"} 0.167 3.542 1.062
After fixed session ended, refer to follow-up[\*\*](#t007fn002){ref-type="table-fn"} 0.667 4.083 0.654
Time frame In preventing intervention, 1--4 sessions[\*\*](#t007fn002){ref-type="table-fn"} 1.000 4.542 0.509
In therapeutic intervention, 1--4 sessions[\*](#t007fn001){ref-type="table-fn"} 0.167 3.417 1.283
In therapeutic intervention, use 5--8 sessions flexibly[\*](#t007fn001){ref-type="table-fn"} 0.667 4.208 0.833
For toddlers and preschoolers, 30 minutes with parent participation[\*](#t007fn001){ref-type="table-fn"} 0.750 4.167 0.761
In lower grades of elementary school, 30--40 minutes 0.917 4.292 0.550
In upper grades of elementary school, 40 minutes 0.917 4.292 0.550
In middle/high school, 45--50 minutes 0.917 4.292 0.550
Treatment program Importance of intervention guidelines 1.000 4.583 0.504
PFA 1.000 4.542 0.509
TRT 0.833 4.250 0.737
SSET 0.833 4.208 0.721
TF-CBT 0.833 4.333 0.637
EMDR 0.750 4.208 0.658
PE 0.500 3.000 0.834
Family program 0.667 4.042 0.751
Grief program[\*\*](#t007fn002){ref-type="table-fn"} 0.667 4.125 0.680
Personal psychotherapy/medication[\*\*](#t007fn002){ref-type="table-fn"} 1.000 4.500 0.511
South Korean version of toddler and preschooler\ Necessity for standardization in the Korean version 0.917 4.417 0.584
therapy
Verification of the case applied in Korea[\*\*](#t007fn002){ref-type="table-fn"} 0.833 4.208 0.588
PFA 0.917 4.292 0.550
TRT 0.667 4.000 0.722
South Korean version of grade-schooler\ Necessity for standardization in the Korean version 0.917 4.500 0.590
therapy
PFA 0.917 4.417 0.584
TRT 0.833 4.250 0.608
TF-CBT 0.833 4.208 0.588
EMDR 0.417 3.792 0.833
South Korean version of middle/high school\ Necessity for standardization in the Korean version 1.000 4.458 0.509
therapy
PFA 0.917 4.375 0.576
TRT 0.917 4.208 0.509
SSET 0.917 4.250 0.532
TF-CBT 0.833 4.208 0.588
EMDR 0.500 3.875 0.741
Facilities in disaster interventions Arrange a place for child-adolescent intervention 1.000 4.500 0.511
Providing treatment program information 0.917 4.500 0.590
Treatment program opportunities 1.000 4.500 0.511
Keeping materials and artworks in the treatment room[\*\*](#t007fn002){ref-type="table-fn"} 0.917 4.542 0.588
Complementary cooperation with organization 1.000 4.500 0.511
Arrangement of materials 1.000 4.625 0.495
Disaster intervention professionals Importance of disaster intervention professionals 1.000 4.500 0.511
Need for all mental health workers to conduct treatment 0.333 3.792 0.884
Professional qualifications and levels 1.000 4.500 0.511
Arrangement of disaster professionals[\*\*](#t007fn002){ref-type="table-fn"} 1.000 4.500 0.511
Completion of disaster care curriculum 1.000 4.458 0.509
Knowledge of secondary traumatizations 1.000 4.667 0.482
Necessity for peer support groups[\*\*](#t007fn002){ref-type="table-fn"} 0.917 4.583 0.584
Participation of professionals such as psychiatrists and psychologists[\*\*](#t007fn002){ref-type="table-fn"} 1.000 4.625 0.495
Construction of network for in-depth therapy[\*\*](#t007fn002){ref-type="table-fn"} 1.000 4.583 0.504
Development of education system for intervention professionals[\*](#t007fn001){ref-type="table-fn"} 1.000 4.542 0.509
Continuous supervision 0.917 4.375 0.576
Plan for group/online supervision[\*](#t007fn001){ref-type="table-fn"} 0.917 4.208 0.658
Setting up an information network[\*](#t007fn001){ref-type="table-fn"} 1.000 4.417 0.504
Promoting treatment programs Continuing system for child-adolescent participation 0.917 4.583 0584
Creation of a system for referrals to therapy 1.000 4.500 0.511
Education of the whole school 1.000 4.542 0.509
Support for medical expenses from the government 1.000 4.625 0.495
Cooperation with the community 1.000 4.625 0.495
Effective ways to promote the system[\*](#t007fn001){ref-type="table-fn"} 0.833 4.250 0.608
Education to continuously participate in treatment regularly[\*](#t007fn001){ref-type="table-fn"} 1.000 4.417 0.504
Decrease in the stigma of psychiatric treatment 0.833 4.208 0.588
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
\* Modified items in Round 3.
\*\* Newly added items in Round 3.
The CVR for Round 3 was 0.38 or higher, and the content validity was verified for nearly all items. The major items with high CVRs are described as follows.
The CVRs were higher than 0.38 for the following items: 'children and adolescents experiencing trauma should adjust to their current life to recover from trauma', 'stabilize their social and interpersonal functions', and 'fulfill their developmental tasks in the long term' ([Table 6](#pone.0195235.t006){ref-type="table"}).
In particular, in the high-risk group, the average value and the content CVR were the highest for the item 'the child and the family should be evaluated'. The highest CVR was observed for the opinion that a trauma-related scale and scales for depression, anxiety, suicide, sleep, and social resources are needed. The CVR was 0.38 or higher for the items indicating that specialists who perform a psychological assessment in a disaster need 'crisis management training' and the 'ability to cope with various responses of clients' ([Table 6](#pone.0195235.t006){ref-type="table"}).
In terms of the intervention program, the CVR was the highest for 'psychological education for the post-traumatic response, normalization, stabilization, physical stability training, family and teacher education, and emotion education should be included.' With respect to the elements of a therapy program, a high CVR was observed for 'requiring PFA, TRT, SSET, TF-CBT, EMDR, PE, a family participation program, a mourning-themed program, individual psychotherapy and medication'. Opinions suggesting that 'individual psychotherapy and medication are needed' were most frequently observed. In addition, some comments indicated that 'child-parent psychotherapy might be more appropriate than PFA and TRT for toddlers and preschoolers' ([Table 7](#pone.0195235.t007){ref-type="table"}).
With respect to the termination of therapy, CVRs higher than 0.38 were observed for the following items: 'the intervention should be terminated after the prescribed therapy sessions' and 'referrals should be determined thereafter' ([Table 7](#pone.0195235.t007){ref-type="table"}).
A high CVR was found for the item regarding the intervention development strategy: \'establish a therapeutic linkage system based on national support, educate and inform the whole school, support medical expenses (such as with government subsidies), connect with the community, consider the persistence of treatment cases, and reduce the stigma of psychiatry' ([Table 7](#pone.0195235.t007){ref-type="table"}).
Discussion {#sec018}
==========
In South Korea, the dispute over how to evaluate and intervene in the aftermath of the Sewol Ferry Disaster required a consensus regarding the need for disaster planning.\[[@pone.0195235.ref024]\] The Delphi process was a suitable method for surveying experts on this topic.\[[@pone.0195235.ref025]\] Using this method, we propose a multidisciplinary recommendation for treating children exposed to disasters. The results of qualitative and quantitative analyses conducted through the Delphi panel survey demonstrate that psychosocial assessment and intervention are essential to early mental health services following a disaster. We discuss suggestions based on the consensus of the experts involved in the study.
We found that in the event of a disaster, intervention factors such as 'appropriate time for assessment after the disaster', 'prerequisites for screening and in-depth intervention', 'classifying the degree of psychosocial symptoms', and 'social and mental health services' are very important. Recovery from psychological trauma after a disaster means mental stability as well as the recovery of physical health. Screening tests are recommended for all children exposed to disasters, particularly during acute periods of disaster. After the completion of screening tests, assessment should include in-depth interviews and interventions for the high-risk group. First, however, we must distinguish between brief screening and in-depth evaluation. As in our study, many previous studies have suggested mental health assessments and interventions for children.\[[@pone.0195235.ref026]\] These findings are consistent with research findings indicating that screening is appropriate when large numbers of children are exposed to an event or when the level of exposure among a population is unknown.\[[@pone.0195235.ref027]\]
The actual screening assessment performed after a disaster requires the consideration of each stage of the disaster and should consist of appropriate questions.\[[@pone.0195235.ref028]\] In the disaster context, screening tools should reflect the needs of children with mental health problems, including consideration of children's exposure, experience, and subjective reactions to traumatic events and conditions.\[[@pone.0195235.ref029], [@pone.0195235.ref030]\]
Evaluation of children, families, and teachers during the acute phase of a disaster is important. Above all, consensus among experts on the selection of children exposed to a disaster is required. Families and teachers should be evaluated together. The use of multiple informants, such as parents, teachers, and other professionals, as collateral sources of information enables the most comprehensive appraisal of children's reactions and functioning.\[[@pone.0195235.ref028]\] These results are consistent with the opinion that it is important for parents and/or caregivers to participate together in a child\'s treatment session to recover from PTSD symptoms.\[[@pone.0195235.ref026]\] When interviewing a family member, we must check for signs of psychological crisis among family members. This finding is consistent with studies of the family environment, social support, and supportive quality.\[[@pone.0195235.ref031]\] However, it is not necessary to evaluate acquaintances or friends. Furthermore, assessments of grief, depression, anxiety, and suicide risk, as well as trauma-related scales, need to measure PTSD and other psychosocial symptoms. This finding is largely consistent with a previous report that disaster exposure is correlated with PTSD, depression, anxiety, functional impairment, and behavioral problems.\[[@pone.0195235.ref032]\] In addition, trauma assessment of children and adolescents should consider their developmental stage. When treating a child who has experienced trauma, the clinician must understand the child\'s existing psychopathological symptoms and provide appropriate interventions, such as trauma-focused therapy.\[[@pone.0195235.ref026]\] Our results suggest the need to develop a crisis intervention model for children and adolescents.\[[@pone.0195235.ref033]\]
Psychosocial assessments should be conducted in a safe environment and at appropriate durations of 30--60 minutes. Approximately 30 to 60 minutes is needed for screening a high-risk group.
Psychoeducation is also beneficial to children. A post-disaster intervention program should include the following: psychoeducation, guidelines for coping with the media, normalization, stabilization, techniques for handling survivor's guilt and emotion-focused coping strategies. Appropriate access phases can be classified as hyper-acute, acute, sub-acute or chronic stages. Stabilization and psychological support should be provided immediately after a disaster along with intervention to help children adapt to everyday life. This finding is consistent with a report that most interventions are multimodal, incorporating common elements to educate children, normalize their reactions, process their emotions and manage stress, enhance coping and provide social support.\[[@pone.0195235.ref027]\] In addition, the development stage, age, trauma symptoms, and traits of a group should be considered. The number of children participating in a group may vary depending on the type of disaster. In general, psychoeducation can be provided in the class setting at school. For prevention education, holding one to four sessions is recommended, whereas for therapeutic intervention, five to eight sessions are appropriate. If the child is exposed to a national large-scale disaster, intervention to address brief trauma may not be sufficient. Therefore, professional intervention should be provided, particularly for children with symptoms of PTSD.\[[@pone.0195235.ref026]\]
For a preschooler, the appropriate duration of an intervention is 30 minutes with caregiver participation. A proper duration of 30 to 40 minutes is suitable for elementary school students in lower grades. An intermediate duration of 40 minutes is suitable for elementary school students in higher grades. For middle and high school students, intervention programs could last 45 to 50 minutes. The optimal intervention components may not be the same for all children or all situations, which should be examined in future work.\[[@pone.0195235.ref034]\]
We recommend the following available intervention programs: PFA\[[@pone.0195235.ref035]\], TRT\[[@pone.0195235.ref036]\], SSET\[[@pone.0195235.ref037]\], and TF-CBT\[[@pone.0195235.ref038]\]. In South Korea, the South Korean versions of PFA, TRT, and TF-CBT should be standardized for children and adolescents. However, the study findings provided no suggestion related to narrative therapy. Furthermore, an intervention for toddlers and preschoolers should be considered. Multiple evidence-based programs should be considered as well, and an intervention protocol that includes a standardized South Korean version can then be implemented. These results provide a framework for further research. Accordingly, the CIDER (Children In Disaster: Evaluation & Recovery) protocol developed by the authors of this study will be made available. Additionally, we must include not only child-focused therapy but also long-term mental health services. These findings are partially consistent with a prior study.\[[@pone.0195235.ref039]\]
The professionals providing disaster interventions vary with respect to factors such as availability, training, and experience, and the goals and complexity of the intervention differ as well.\[[@pone.0195235.ref027]\] Nevertheless, affected communities do not have enough therapists trained in evidence-based treatments to be able to provide every child with individual therapy.\[[@pone.0195235.ref039]\] It is not necessary for all mental health workers to conduct evaluations and interventions after a disaster. Therefore, disaster experts with experience working in a clinical environment should be called upon; a training and education system for professionals is needed. Such professionals may need additional support and guidance to address their own emotional responses.\[[@pone.0195235.ref027]\] This support can be incorporated into supervision as well as peer support groups. Additionally, the present study shows that good relationships should be cultivated within professional networks of information related to in-depth therapy.
Above all, interventions delivered in groups are particularly well suited for school settings.\[[@pone.0195235.ref027]\] Schools are among the most important links in the chain of public health education for children and adolescents.\[[@pone.0195235.ref040]\] School-based interventions should be developed, regular training in disaster safety measures for school personnel should be mandated, and training programs for children should be established. Moreover, teachers should receive advice on coping with emergencies in either their basic teacher training or in-service training. In summary, schools should identify school crisis emergencies and clearly delineate the roles of children and teachers in coping with disaster. Based on the abovementioned considerations, psychiatric and psychological support should be accessible. Additionally, guiding children to use positive coping strategies and encouraging a warm community atmosphere are recommended.\[[@pone.0195235.ref032]\] Consequently, our confidence in reaching consensus means that we now have a comprehensive framework of competency statements that describe what psychiatric professionals working in the aftermath of a disaster must do. As the National Child Traumatic Stress Network has coordinated collaboration among 10 research development and evaluation sites and 26 community mental health centers across the United States, it is also essential to establish sensible governance between central and local governments, between administrative institutions and institutions that provide services, and between public and civic organizations.\[[@pone.0195235.ref041]\]
This study proposed effective mental health intervention measures and described the implications for developing a post-disaster evaluation treatment protocol. The main strengths of our study include its responses from a panel of defined experts, good response rates and framework of competencies that describe attributes of professionals working within the disaster field. However, some limitations also need to be recognized.
First, the study findings suggest that children in South Korean cultures require disaster-related psychosocial evaluation and interventions, but modifications may be needed to address other cultural issues.
Second, our expert panel was determined by our approach to sampling. E-mails may not have been distributed by some of the professional groups we contacted, and other experts not publishing their work may have been missed. The rich qualitative and quantitative data obtained from this study are very useful for understanding why certain topics are research priorities.\[[@pone.0195235.ref021]\]
Third, the experts who conducted psychological intervention at Danwon High School after the Sewol Ferry Disaster in South Korea were all psychiatrists, except for two psychologists.\[[@pone.0195235.ref042]\] The primary aim was to gather psychiatrists' opinions and experience from the disaster environment. In Round 1, we had limitations in distinguishing between the related areas of expertise in disaster and trauma for the psychological specialists, and these limitations might be reflected in the medical opinions of the panel.
In conclusion, we suggest the need for informed evidence-based assessments, interventions, and treatments for children and adolescents who experience disasters. This survey presents important opinions from trauma care experts and should be utilized by psychiatrists to develop a meaningful protocol for PTSD assessment and treatment. Hence, the results can be applied to existing and future disaster management.
Supporting information {#sec019}
======================
###### The specific 20 questions in Round 1.
(DOCX)
######
Click here for additional data file.
We would like to give our heartfelt thanks to all the panel experts who participated in this study.
[^1]: **Competing Interests:**The authors have declared that no competing interests exist.
| {
"pile_set_name": "PubMed Central"
} |
Cronin EM, Bogun FM, Maury P, et al. 2019 HRS/EHRA/APHRS/LAHRS expert consensus statement on catheter ablation of ventricular arrhythmias: Executive summary. J Arrhythmia. 2020;36:1--58. 10.1002/joa3.12264
\[Correction added on 22 January 2020, after first online publication: typographical errors have been amended on pages 3, 21 and 44.\]
**Document Reviewers:** Samuel J. Asirvatham, MD, FHRS; Eduardo Back Sternick, MD, PhD; Janice Chyou, MD; Sabine Ernst, MD, PhD; Guilherme Fenelon, MD, PhD; Edward P. Gerstenfeld, MD, MS, FACC; Gerhard Hindricks, MD; Koichi Inoue, MD, PhD; Jeffrey J. Kim, MD; Kousik Krishnan, MD, FHRS, FACC; Karl‐Heinz Kuck, MD, FHRS; Martin Ortiz Avalos, MD; Thomas Paul, MD, FACC, FHRS; Mauricio I. Scanavacca, MD, PhD; Roderick Tung, MD, FHRS; Jamie Voss, MBChB; Takumi Yamada, MD; Teiichi Yamane, MD, PhD, FHRS
Developed in partnership with and endorsed by the European Heart Rhythm Association (EHRA), the Asia Pacific Heart Rhythm Society (APHRS), and the Latin American Heart Rhythm Society (LAHRS). Developed in collaboration with and endorsed by the American College of Cardiology (ACC), the American Heart Association (AHA), the Japanese Heart Rhythm Society (JHRS), the Pediatric and Congenital Electrophysiology Society (PACES), and the Sociedade Brasileira de Arritmias Cardíacas (SOBRAC). Endorsed by the Canadian Heart Rhythm Society. For copies of this document, please contact the Elsevier Inc. Reprint Department (<[email protected]>). Permissions: Multiple copies, modification, alteration, enhancement, and/or distribution of this document are not permitted without the express permission of the Heart Rhythm Society. Instructions for obtaining permission are located at <https://www.elsevier.com/about/our-business/policies/copyright/permissions>.
This article has been copublished in ***Heart**Rhythm*, *Europace*, and the *Journal of Arrhythmia*.
© 2019 The Heart Rhythm Society; the European Heart Rhythm Association, a registered branch of the European Society of Cardiology; the Asia Pacific Heart Rhythm Society; and the Latin American Heart Rhythm Society. Published by Elsevier Inc./Oxford University Press/Wiley. This article is published under the Creative Commons CC‐BY license.
AAD
: antiarrhythmic drug
AIV
: anterior interventricular vein
AMC
: aortomitral continuity
ARVC
: arrhythmogenic right ventricular cardiomyopathy
ATP
: antitachycardia pacing
AV
: atrioventricular
BBRVT
: bundle branch reentrant ventricular tachycardia
CHD
: congenital heart disease
CMR
: cardiac magnetic resonance imaging
COR
: class of recommendation
CS
: coronary sinus
DCM
: dilated cardiomyopathy
EAM
: electroanatomical mapping
ECG
: electrocardiogram
GCV
: great cardiac vein
HCM
: hypertrophic cardiomyopathy
HS
: hemodynamic support
ICD
: implantable cardioverter defibrillator
ICE
: intracardiac echocardiography
ICM
: ischemic cardiomyopathy
IHD
: ischemic heart disease
LBB
: left bundle branch
LBBB
: left bundle branch block
LMNA
: lamin A/C
LOE
: level of evidence
LSV
: left sinus of Valsalva
LV
: left ventricle
LVOT
: left ventricular outflow tract
NCSV
: noncoronary sinus of Valsalva
NICM
: nonischemic cardiomyopathy
PES
: programmed electrical stimulation
PVC
: premature ventricular complex
RBB
: right bundle branch
RBBB
: right bundle branch block
RSV
: right sinus of Valsalva
RV
: right ventricle
RVOT
: right ventricular outflow tract
RWI
: relationship with industry and other entities
SHD
: structural heart disease
SV
: sinus of Valsalva
VA
: ventricular arrhythmia
VF
: ventricular fibrillation
VT
: ventricular tachycardia
Table of contents {#joa312264-sec-0001}
=================
1Introduction1.1Document scope and rationale1.2Methods2Background3Clinical evaluation3.1Clinical presentation3.2Diagnostic evaluation3.2.1Resting 12‐lead electrocardiogram3.2.2Assessment of structural heart disease and myocardial ischemia3.2.3Risk stratification in the setting of frequent premature ventricular complexes3.2.4Longitudinal follow‐up in the setting of frequent premature ventricular complexes4Indications for catheter ablation4.1Idiopathic outflow tract ventricular arrhythmia4.2Idiopathic nonoutflow tract ventricular arrhythmia4.3Premature ventricular complexes with or without left ventricular dysfunction4.4Ventricular arrhythmia in ischemic heart disease4.5Nonischemic cardiomyopathy4.6Ventricular arrhythmia involving the His‐Purkinje system, bundle branch reentrant ventricular tachycardia, and fascicular ventricular tachycardia4.7Congenital heart disease4.8Inherited arrhythmia syndromes4.9Ventricular arrhythmia in hypertrophic cardiomyopathy5Procedural planning6Intraprocedural patient care6.1Anesthesia6.2Vascular access6.3Epicardial access6.4Intraprocedural hemodynamic support6.5Intraprocedural anticoagulation7Electrophysiological testing8Mapping and imaging techniques8.1Overview8.2Substrate mapping in sinus rhythm8.3Intraprocedural imaging during catheter ablation of ventricular arrhythmias8.4Electroanatomical mapping systems and robotic navigation9Mapping and ablation9.1Ablation power sources and techniques9.2Idiopathic outflow tract ventricular arrhythmia9.3Idiopathic nonoutflow tract ventricular arrhythmia9.4Bundle branch reentrant ventricular tachycardia and fascicular ventricular tachycardia9.5Postinfarction ventricular tachycardia9.6Dilated cardiomyopathy9.7Ventricular tachycardia ablation in hypertrophic cardiomyopathy9.8Brugada syndrome9.9Polymorphic ventricular tachycardia/ventricular fibrillation triggers9.10Arrhythmogenic right ventricular cardiomyopathy9.11Mapping and ablation in congenital heart disease9.12Sarcoidosis9.13Chagas disease9.14Miscellaneous diseases and clinical scenarios with ventricular tachycardia9.15Surgical therapy9.16Sympathetic modulation9.17Endpoints of catheter ablation of ventricular tachycardia10Postprocedural care10.1Postprocedural care: access, anticoagulation, disposition10.1.1Postprocedural care: access10.1.2Postprocedural care: anticoagulation10.2Incidence and management of complications10.3Hemodynamic deterioration and proarrhythmia10.4Follow‐up of patients post catheter ablation of ventricular tachycardia11Training and institutional requirements and competencies11.1Training requirements and competencies for catheter ablation of ventricular arrhythmias11.2Institutional requirements for catheter ablation of ventricular tachycardia12Future directionsAppendix 1Author disclosure tableAppendix 2Reviewer disclosure table
1. INTRODUCTION {#joa312264-sec-0002}
===============
1.1. Document scope and rationale {#joa312264-sec-0003}
---------------------------------
The field of electrophysiology has undergone rapid progress in the last decade, with advances both in our understanding of the genesis of ventricular arrhythmias (VAs) and in the technology used to treat them. In 2009, a joint task force of the European Heart Rhythm Association (EHRA) and the Heart Rhythm Society (HRS), in collaboration with the American College of Cardiology (ACC) and the American Heart Association (AHA), produced an expert consensus document that outlined the state of the field and defined the indications, techniques, and outcome measures of VA ablation (S1.1.1). In light of advances in the treatment of VAs in the interim, and the growth in the number of VA ablations performed in many countries and regions (S1.1.2, S1.1.3), an updated document is needed. This effort represents a worldwide partnership between transnational cardiac electrophysiology societies, namely, HRS, EHRA, the Asia Pacific Heart Rhythm Society (APHRS), and the Latin American Heart Rhythm Society (LAHRS), and collaboration with ACC, AHA, the Japanese Heart Rhythm Society (JHRS), the Brazilian Society of Cardiac Arrhythmias (Sociedade Brasileira de Arritmias Cardíacas \[SOBRAC\]), and the Pediatric and Congenital Electrophysiology Society (PACES). The consensus statement was also endorsed by the Canadian Heart Rhythm Society (CHRS).
This clinical document is intended to supplement, not replace, the *2017 AHA/ACC/HRS Guideline for Management of Patients with Ventricular Arrhythmias and the Prevention of Sudden Cardiac Death* (S1.1.4) and the *2015 ESC Guidelines for the Management of Patients with Ventricular Arrhythmias and the Prevention of Sudden Cardiac Death* (S1.1.5). The scope of the current document relates to ablation therapy for VAs, from premature ventricular complexes (PVCs) to monomorphic and polymorphic ventricular tachycardia (VT) and triggers of ventricular fibrillation (VF). Due to its narrower scope, the consensus statement delves into greater detail with regard to indications and technical aspects of VA ablation than the above‐mentioned guidelines.
Where possible, the recommendations in this document are evidence based. It is intended to set reasonable standards that can be applicable worldwide, while recognizing the different resources, technological availability, disease prevalence, and health care delivery logistics in various parts of the world. In addition, parts of this document, particularly Section 9, present a practical guide on how to accomplish the procedures described in a manner that reflects the current standard of care, while recognizing that some procedures are better performed, and some disease states better managed, in settings in which there is specific expertise.
References {#joa312264-sec-0004}
----------
S1.1.1. Aliot EM, Stevenson WG, Almendral‐Garrote JM, et al. EHRA/HRS expert consensus on catheter ablation of ventricular arrhythmias: developed in a partnership with the European Heart Rhythm Association (EHRA), a registered branch of the European Society of Cardiology (ESC), and the Heart Rhythm Society (HRS); in collaboration with the American College of Cardiology (ACC) and the American Heart Association (AHA). *Heart Rhythm*. 2009;6:886--933.
S1.1.2. Hosseini SM, Rozen G, Saleh A, et al. Catheter ablation for cardiac arrhythmias: utilization and in‐hospital complications, 2000 to 2013. *JACC Clin Electrophysiol*. 2017;3:1240--8.
S1.1.3. Raatikainen MJP, Arnar DO, Merkely B, Nielsen JC, Hindricks G, Heidbuchel H, Camm J. A decade of information on the use of cardiac implantable electronic devices and interventional electrophysiological procedures in the European Society of Cardiology Countries: 2017 report from the European Heart Rhythm Association. *Europace*. 2017;19(Suppl. 2):ii1--ii90.
S1.1.4. Al‐Khatib SM, Stevenson WG, Ackerman MJ, et al. 2017 AHA/ACC/HRS guideline for management of patients with ventricular arrhythmias and the prevention of sudden cardiac death: a report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines and the Heart Rhythm Society. *Heart Rhythm*. 2018;15:e73--189.
S1.1.5. Priori SG, Blomström‐Lundqvist C, Mazzanti A, et al; Task Force for the Management of Patients with Ventricular Arrhythmias and the Prevention of Sudden Cardiac Death of the European Society of Cardiology (ESC). 2015 ESC guidelines for the management of patients with ventricular arrhythmias and the prevention of sudden cardiac death: the Task Force for the Management of Patients with Ventricular Arrhythmias and the Prevention of Sudden Cardiac Death of the European Society of Cardiology (ESC). *Europace*. 2015;17:1601--87.
1.2. Methods {#joa312264-sec-0005}
------------
The writing group was selected according to each society\'s procedures, including content and methodology experts representing the following organizations: HRS, EHRA, APHRS, LAHRS, ACC, AHA, JHRS, PACES, and SOBRAC. Each partner society nominated a chair and co‐chair, who did not have relevant relationships with industry and other entities (RWIs). In accordance with HRS policies, disclosure of any RWIs was required from the writing committee members (Appendix [1](#joa312264-app-0001){ref-type="app"}) and from all peer reviewers (Appendix [2](#joa312264-app-0002){ref-type="app"}). Of the 38 committee members, 17 (45%) had no relevant RWIs. Recommendations were drafted by the members who did not have relevant RWIs. Members of the writing group conducted comprehensive literature searches of electronic databases, including Medline (via PubMed), Embase, and the Cochrane Library. Evidence tables were constructed to summarize the retrieved studies, with nonrandomized observational designs representing the predominant form of evidence (Appendix [S1](#joa312264-sup-0001){ref-type="supplementary-material"}). Case reports were not used to support recommendations. Supportive text was drafted in the "knowledge byte" format for each recommendation. The writing committee discussed all recommendations and the evidence that informed them before voting. Initial failure to reach consensus was resolved by subsequent discussions, revisions as needed, and re‐voting. Although the consensus threshold was set at 67%, all recommendations were approved by at least 80% of the writing committee members. The mean consensus over all recommendations was 95%. A quorum of two‐thirds of the writing committee was met for all votes (S1.2.1).
Each recommendation in this document was assigned a Class of Recommendation (COR) and a Level of Evidence (LOE) according to the system developed by ACC and AHA (Table [1](#joa312264-tbl-0001){ref-type="table"}) (S1.2.2). The COR denotes the strength of the recommendation based on a careful assessment of the estimated benefits and risks; COR I indicates that the benefit of an intervention far exceeds its risk; COR IIa indicates that the benefit of the intervention moderately exceeds the risk; COR IIb indicates that the benefit may not exceed the risk; and COR III indicates that the benefit is equivalent to or is exceeded by the risk. The LOE reflects the quality of the evidence that supports the recommendation. LOE A is derived from high‐quality randomized controlled trials; LOE B‐R is derived from moderate‐quality randomized controlled trials; LOE B‐NR is derived from well‐designed nonrandomized studies; LOE C‐LD is derived from randomized or nonrandomized studies with limitations of design or execution; and LOE C‐EO indicates that a recommendation was based on expert opinion (S1.2.2).
######
ACC/AHA Recommendation System: Applying Class of Recommendation and Level of Evidence to Clinical Strategies, Interventions, Treatments, and Diagnostic Testing in Patient Care\*
{#nlm-graphic-1}
John Wiley & Sons, Ltd
Unique to this consensus statement is the systematic review commissioned specifically for this document as part of HRS\'s efforts to adopt the rigorous methodology required for guideline development. The systematic review was performed by an experienced evidence‐based practice committee based at the University of Connecticut, which examined the question of VT ablation vs control in patients with VT and ischemic heart disease (IHD) (S1.2.3). The question, in PICOT format, was as follows: In adults with history of sustained VT and IHD, what is the effectiveness and what are the detriments of catheter ablation compared with other interventions? Components of the PICOT were as follows: P = adults with history of sustained VT and IHD; I = catheter ablation; C = control (no therapy or antiarrhythmic drug \[AAD\]); O = outcomes of interest, which included (a) appropriate implantable cardioverter defibrillator (ICD) therapies (ICD shock or antitachycardia pacing \[ATP\]), (b) appropriate ICD shocks, (c) VT storm (defined as three shocks within 24 hours), (d) recurrent VT/VF, (e) cardiac hospitalizations, and (f) all‐cause mortality; and T = no time restrictions.
An industry forum was conducted to achieve a structured dialogue to address technical questions and to gain a better understanding of future directions and challenges. Because of the potential for actual or perceived bias, HRS imposes strict parameters on information sharing to ensure that industry participates only in an advisory capacity and has no role in either the writing of the document or its review.
The draft document underwent review by the HRS Scientific and Clinical Documents Committee and was approved by the writing committee. Recommendations were subject to a period of public comment, and the entire document underwent rigorous peer review by each of the participating societies and revision by the Chairs, before endorsement.
References {#joa312264-sec-0006}
----------
S1.2.1. Indik JH, Patton KK, Beardsall M, et al. HRS clinical document development methodology manual and policies: executive summary. *Heart Rhythm*. 2017;14:e495--500.
S1.2.2. Halperin JL, Levine GN, Al‐Khatib SM, et al. Further evolution of the ACC/AHA clinical practice guideline recommendation classification system: a report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines. *J Am Coll Cardiol*. 2016;67:1572--4.
S1.2.3. Martinez BK, Baker WL, Konopka A, et al. Systematic review and meta‐analysis of catheter ablation of ventricular tachycardia in ischemic heart disease. *Heart Rhythm*. 2019 May 10 \[Epub ahead of print\].
2. BACKGROUND {#joa312264-sec-0007}
=============
This section reviews the history of VT ablation, details the mechanisms of VT, and provides definitions of frequently used terms (Table [2](#joa312264-tbl-0002){ref-type="table"}), including anatomic definitions (Table [3](#joa312264-tbl-0003){ref-type="table"}), as well as illustrating some types of sustained VA (Figure [1](#joa312264-fig-0001){ref-type="fig"}).
######
Definitions
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
**Clinical characteristics**
***Clinical ventricular tachycardia (VT)*** **:** VT that has occurred spontaneously based on analysis of 12‐lead electrocardiogram (ECG) QRS morphology.
***Hemodynamically unstable VT*** **:** causes hemodynamic compromise requiring prompt termination.
***Idiopathic VT*** **:** used to indicate VT that is known to occur in the absence of clinically apparent structural heart disease (SHD).
***Idioventricular rhythm*** **:** three or more consecutive beats at a rate of up to 100 per minute that originate from the ventricles independent of atrial or atrioventricular (AV) nodal conduction. Although various arbitrary rates have been used to distinguish it from VT, the mechanism of ventricular rhythm is more important than the rate. Idioventricular rhythm can be qualified as "accelerated" when the rate exceeds 40 bpm.
***Incessant VT*** **:** continuous sustained VT that recurs promptly despite repeated intervention for termination over several hours.
***Nonclinical VT*** **:** VT induced by programmed electrical stimulation (PES) that has not been documented previously.
***Nonsustained VT*** **:** terminates spontaneously within 30 seconds.
***PVC*** **:** premature ventricular complex; it is an early ventricular depolarization with or without mechanical contraction. We recommend avoiding the use of the terms "ventricular premature depolarization" and "premature ventricular contraction" to standardize the literature and acknowledge that early electrical activity does not necessarily lead to mechanical contraction.
***Presumptive clinical VT*** **:** similar to a spontaneous VT based on rate, limited ECG, or electrogram data available from ICD interrogation, but without the 12‐lead ECG documentation of spontaneous VT.
***PVC burden*** **:** the amount of ventricular extrasystoles, preferably reported as the % of beats of ventricular origin of the total amount of beats over a 24‐hour recording period.
***Repetitive monomorphic VT*** **:** continuously repeating episodes of self‐terminating nonsustained VT.
***Sustained VT*** **:** continuous VT for 30 seconds, or which requires an intervention for termination (such as cardioversion).
***VT*** **:** a tachycardia (rate \>100 bpm) with 3 or more consecutive beats that originates from the ventricles independent of atrial or AV nodal conduction.
***VT storm*** **:** three or more separate episodes of sustained VT within 24 hours, each requiring termination by an intervention.
**VT Morphologies**
***Monomorphic VT*** **:** a similar QRS configuration from beat to beat (Figure [1](#joa312264-fig-0001){ref-type="fig"}A). Some variability in QRS morphology at initiation is not uncommon, followed by stabilization of the QRS morphology.
***Monomorphic VT with indeterminate QRS morphology*** **:** preferred over ***ventricular flutter;*** it is a term that has been applied to rapid VT that has a sinusoidal QRS configuration that prevents identification of the QRS morphology.
***Multiple monomorphic VTs*** **:** more than one morphologically distinct monomorphic VT, occurring as different episodes or induced at different times.
***Pleomorphic VT*** **:** has more than one morphologically distinct QRS complex occurring during the same episode of VT, but the QRS is not continuously changing (Figure [1](#joa312264-fig-0001){ref-type="fig"}B).
***Polymorphic VT*** **:** has a continuously changing QRS configuration from beat to beat, indicating a changing ventricular activation sequence (Figure [1](#joa312264-fig-0001){ref-type="fig"}C).
***Right bundle branch block (RBBB)‐ and left bundle branch block (LBBB)‐like VT configurations*** **:** terms used to describe the dominant deflection in V1, with a dominant R wave described as "RBBB‐like" and a dominant S wave with a negative final component in V1 described as "LBBB‐like" configurations.
***Torsades de pointes*** **:** a form of polymorphic VT with continually varying QRS complexes that appear to spiral around the baseline of the ECG lead in a sinusoidal pattern. It is associated with QT prolongation.
***Unmappable VT*** **:** does not allow interrogation of multiple sites to define the activation sequence or perform entrainment mapping; this could be due to hemodynamic intolerance that necessitates immediate VT termination, spontaneous or pacing‐induced transition to other morphologies of VT, or repeated termination during mapping.
***Ventricular fibrillation (VF):*** a chaotic rhythm defined on the surface ECG by undulations that are irregular in both timing and morphology, without discrete QRS complexes.
**PVC Morphologies**
***Monomorphic PVC*** **:** PVCs felt reasonably to arise from the same focus. Slight changes in QRS morphology due to different exit sites from the same focus can be present.
***Multiple morphologies of PVC*** **:** PVCs originating from several different focal locations.
***Predominant PVC morphology*** **:** the one or more monomorphic PVC morphologies occurring most frequently and serving as the target for ablation.
**Mechanisms**
***Focal VT*** **:** a point source of earliest ventricular activation with a spread of activation away in all directions from that site. The mechanism can be automaticity, triggered activity, or microreentry.
***Scar‐related reentry*** **:** arrhythmias that have characteristics of reentry that originate from an area of myocardial scar identified from electrogram characteristics or myocardial imaging. Large reentry circuits that can be defined over several centimeters are commonly referred to as "macroreentry."
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Abbreviations: AV, atrioventricular; ECG, electrocardiogram; ICD, implantable cardioverter defibrillator; LBBB, left bundle branch block; PES, programmed electrical stimulation; PVC, premature ventricular complex; RBBB, right bundle branch block; SHD, structural heart disease; VT, ventricular tachycardia.
John Wiley & Sons, Ltd
######
Anatomical terminology
Term Definition
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RV inflow The part of the right ventricle (RV) containing the tricuspid valve, chordae, and proximal RV.
RV outflow tract (RVOT) The conus or infundibulum of the RV, derived from the bulbus cordis. It is bounded by the supraventricular crest and the pulmonic valve.
Tricuspid annulus Area immediately adjacent to the tricuspid valve, including septal, free wall, and para‐Hisian regions.
Moderator band A muscular band in the RV, typically located in the mid to apical RV, connecting the interventricular septum to the RV free wall, supporting the anterior papillary muscle. It typically contains a subdivision of the right bundle branch (RBB).
RV papillary muscles Three muscles connecting the RV myocardium to the tricuspid valve via the tricuspid chordae tendineae, usually designated as septal, posterior, and anterior papillary muscles. The septal papillary muscle is closely associated with parts of the RBB.
Supraventricular crest Muscular ridge in the RV between the tricuspid and pulmonic valves, representing the boundary between the conus arteriosus and the rest of the RV. The exact components and terminology are controversial; however, some characterize it as being composed of a parietal band that extends from the anterior RV free wall to meet the septal band, which extends from the septal papillary muscle to meet it.
Pulmonary valves The pulmonic valve includes three cusps and associated sinus, variously named right, left, and anterior; or anterolateral right, anterolateral left, and posterior sinuses. The posterior‐right anterolateral commissure adjoins the aorta (junction of the right and left aortic sinuses). Muscle is present in each of the sinuses, and VA can originate from muscle fibers located within or extending beyond the pulmonary valve apparatus.
Sinuses of Valsalva (SV), aortic cusps, aortic commissures The right (R), left (L), and noncoronary aortic valve cusps are attached to the respective SV. The left sinus of Valsalva (LSV) is posterior and leftward on the aortic root. The noncoronary sinus of Valsalva (NCSV) is typically the most inferior and posterior SV, located posterior and rightward, superior to the His bundle, and anterior and superior to the paraseptal region of the atria near the superior AV junctions, typically adjacent to atrial myocardium. The right sinus of Valsalva (RSV) is the most anterior cusp and may be posterior to the RVOT infundibulum. VAs can also arise from muscle fibers at the commissures (connections) of the cusps, or from myocardium accessible to mapping and ablation from this location, especially from the RSV/LSV junction.
LV outflow tract (LVOT) The aortic vestibule, composed of an infra‐valvular part, bounded by the anterior mitral valve leaflet, but otherwise not clearly distinguishable from the rest of the LV; the aortic valve; and a supra‐valvular part.
LV ostium The opening at the base of the LV to which the mitral and aortic valves attach.
Aortomitral continuity (AMC); aortomitral curtain, or mitral‐aortic intervalvular fibrosa Continuation of the anteromedial aspect of the mitral annulus to the aortic valve; a curtain of fibrous tissue extending from the anterior mitral valve leaflet to the left and noncoronary aortic cusps. The AMC is connected by the left and right fibrous trigones to ventricular myocardium, the right fibrous trigone to the membranous ventricular septum.
Mitral valve annulus Area immediately adjacent to the mitral valve. This can be approached endocardially, or epicardially, either through the coronary venous system or percutaneously.
LV papillary muscles Muscles connecting the mitral valve chordae tendineae to the LV, typically with posteromedial and anterolateral papillary muscles. Papillary muscle anatomy is variable and can have single or multiple heads.
LV false tendon (or LV moderator band) A fibrous or fibromuscular chord‐like band that crosses the LV cavity, attaching to the septum, papillary muscles, trabeculations, or free wall of the LV. They may contain conduction tissue and may impede catheter manipulation in the LV.
Posterior‐superior process The posterior‐superior process of the left ventricle (LV) is the most inferior and posterior aspect of the basal LV, posterior to the plane of the tricuspid valve. VAs originating from the posterior‐superior process of the LV can be accessed from the right atrium, the LV endocardium, and the coronary venous system.
Endocardium Inner lining of the heart.
Purkinje network The specialized conduction system of the ventricles, which includes the His bundle, RBB and left bundle branches (LBB), and the ramifications of these, found in the subendocardium. The Purkinje system can generate focal or reentrant VTs, typically manifesting Purkinje potentials preceding QRS onset.
Interventricular septum Muscular wall between the LV and RV.
Membranous ventricular septum The ventricular septum beneath the RSV and NCSV, through which the penetrating His bundle reaches the ventricular myocardium.
LV summit Triangular region of the most superior part of the LV epicardial surface bounded by the left circumflex coronary artery, the left anterior descending artery, and an approximate line from the first septal coronary artery laterally to the left AV groove. The great cardiac vein (GCV) bisects the triangle. An area superior to the GCV is considered to be inaccessible to catheter ablation due to proximity of the coronary arteries and overlying epicardial fat.
Crux of the heart (crux cordis) Epicardial area formed by the junction of the AV groove and posterior interventricular groove, at the base of the heart, approximately at the junction of the middle cardiac vein and coronary sinus (CS) and near the origin of the posterior descending coronary artery.
Epicardium The outer layer of the heart---the visceral layer of the serous pericardium.
Epicardial fat Adipose tissue variably present over the epicardial surface around coronary arteries, LV apex, RV free wall, left atrial appendage, right atrial appendage, and AV and interventricular grooves.
Pericardial space or cavity The potential space between the parietal and visceral layers of serous pericardium, which normally contains a small amount of serous fluid. This space can be accessed for epicardial procedures.
Parietal pericardium The layer of the serous pericardium that is attached to the inner surface of the fibrous pericardium and is normally apposed to the visceral pericardium, separated by a thin layer of pericardial fluid.
Fibrous pericardium Thick membrane that forms the outer layer of the pericardium.
Subxiphoid area Area inferior to the xiphoid process; typical site for percutaneous epicardial access.
Phrenic nerve The right phrenic nerve lays along the right atrium and does not usually pass over ventricular tissue. The course of the left phrenic nerve on the fibrous pericardium can be quite variable and may run along the lateral margin of the LV near the left obtuse marginal artery and vein; inferior, at the base of the heart; or anterior over the sternocostal surface over the L main stem coronary artery or left anterior descending artery.
Coronary sinus (CS) and branches The CS and its branches comprise the coronary venous system with the ostium of the CS opening into the right atrium. Tributaries of the CS, which runs along the left AV groove, may be used for mapping. These include the anterior interventricular vein (AIV), which arises at the apex and runs along the anterior interventricular septum, connecting to the GCV that continues in the AV groove to the CS; the communicating vein located between aortic and pulmonary annulus; various posterior and lateral marginal branches or perforator veins; and the middle cardiac vein that typically runs along the posterior interventricular septum from the apex to join the CS or empty separately into the right atrium. The junction of the GCV and the CS is at the vein or ligament of Marshall (or persistent left superior vena cava, when present), and the valve of Vieussens (where present).
Anatomical terminology (S2.1--S2.9). See also Figures [3](#joa312264-fig-0003){ref-type="fig"}, [4](#joa312264-fig-0004){ref-type="fig"}, [7](#joa312264-fig-0007){ref-type="fig"}, and [8](#joa312264-fig-0008){ref-type="fig"}.
Abbreviations: AIV, anterior interventricular vein; AMC, aortomitral continuity; AV, atrioventricular; CS, coronary sinus; GCV, great cardiac vein; LBB, left bundle branch; LSV, left sinus of Valsalva; LV, left ventricle; LVOT, left ventricular outflow tract; NCSV, noncoronary sinus of Valsalva; RBB, right bundle branch; RSV, right sinus of Valsalva; RV, right ventricle; RVOT, right ventricular outflow tract; SV, sinus of Valsalva; VA, ventricular arrhythmia; VT, ventricular tachycardia.
John Wiley & Sons, Ltd
{#joa312264-fig-0001}
References {#joa312264-sec-0008}
----------
S2.1. Yamada T, Kay GN. Anatomical consideration in catheter ablation of idiopathic ventricular arrhythmias. *Arrhythm Electrophysiol Rev*. 2016;5:203--9.
S2.2. Della Bella P, Maccabelli G, Carbucicchio C. Anatomical assessment for catheter ablation of ventricular tachycardia. In: Auricchio A, editor. Cardiac imaging in electrophysiology. London: Springer‐Verlag, 2012. p. 95--104.
S2.3. Enriquez A, Malavassi F, Saenz LC, Supple G, Santangeli P, Marchlinski FE, Garcia FC. How to map and ablate left ventricular summit arrhythmias. *Heart Rhythm*. 2017;14:141--8.
S2.4. Saremi F, Muresian H, Sanchez‐Quintana D. Coronary veins: comprehensive CT‐anatomic classification and review of variants and clinical implications. *Radiographics*. 2012;32:E1--32.
S2.5. Ho SY. Anatomic insights for catheter ablation of ventricular tachycardia. *Heart Rhythm*. 2009;6(Suppl. 8):S77--80.
S2.6. Ho SY, Nihoyannopoulos P. Anatomy, echocardiography, and normal right ventricular dimensions. *Heart*. 2006;92(Suppl. 1):i2--i13.
S2.7. Sánchez‐Quintana D, Ho SY, Climent V, Murillo M, Cabrera JA. Anatomic evaluation of the left phrenic nerve relevant to epicardial and endocardial catheter ablation: implications for phrenic nerve injury. *Heart Rhythm*. 2009;6:764--8.
S2.8. Yamada T, Litovsky SH, Kay GN. The left ventricular ostium: an anatomic concept relevant to idiopathic ventricular arrhythmias. *Circ Arrhythm Electrophysiol*. 2008;1:396--404.
S2.9. McAlpine WA. Heart and coronary arteries: an anatomical atlas for clinical diagnosis, radiological investigation, and surgical treatment. New York: Springer‐Verlag; 1975.
3. CLINICAL EVALUATION {#joa312264-sec-0009}
======================
This section discusses clinical presentations of patients with VAs and their workup as it pertains to documentation of arrhythmias and appropriate testing to assess for the presence of SHD.
3.1. Clinical presentation {#joa312264-sec-0010}
--------------------------
Recommendation for clinical evaluation of patients with VAs
CORLOERecommendationIC‐EO1. A careful clinical evaluation including history, physical examination, review of available cardiac rhythm data, prior imaging, and relevant laboratory workup should be performed in patients presenting with VAs.
3.2. Diagnostic evaluation {#joa312264-sec-0011}
--------------------------
### 3.2.1. Resting 12‐lead electrocardiogram {#joa312264-sec-0012}
Recommendations for resting 12‐lead ECG
CORLOERecommendationsReferencesIB‐NR1. In patients with wide complex tachycardia, a 12‐lead ECG during tachycardia should be obtained whenever possible.S3.2.1.1--S3.2.1.15IB‐NR2. In patients with suspected or documented VA, a 12‐lead ECG should be obtained in sinus rhythm to look for evidence of underlying heart disease.S3.2.1.16
### References {#joa312264-sec-0013}
S3.2.1.1. Brugada P, Brugada J, Mont L, Smeets J, Andries EW. A new approach to the differential diagnosis of a regular tachycardia with a wide QRS complex. *Circulation*. 1991;83:1649--59.
S3.2.1.2. Wellens HJ, Bar FW, Lie KI. The value of the electrocardiogram in the differential diagnosis of a tachycardia with a widened QRS complex. *Am J Med*. 1978;64:27--33.
S3.2.1.3. Vereckei A, Duray G, Szenasi G, Altemose GT, Miller JM. New algorithm using only lead aVR for differential diagnosis of wide QRS complex tachycardia. *Heart Rhythm*. 2008;5:89--98.
S3.2.1.4. Ohe T, Shimomura K, Aihara N, et al. Idiopathic sustained left ventricular tachycardia: clinical and electrophysiologic characteristics. *Circulation*. 1988;77:560--8.
S3.2.1.5. Dixit S, Gerstenfeld EP, Callans DJ, Marchlinski FE. Electrocardiographic patterns of superior right ventricular outflow tract tachycardias: distinguishing septal and free‐wall sites of origin. *J Cardiovasc Electrophysiol*. 2003;14:1--7.
S3.2.1.6. Callans DJ, Menz V, Schwartzman D, Gottlieb CD, Marchlinski FE. Repetitive monomorphic tachycardia from the left ventricular outflow tract: electrocardiographic patterns consistent with a left ventricular site of origin. *J Am Coll Cardiol*. 1997;29:1023--7.
S3.2.1.7. Kanagaratnam L, Tomassoni G, Schweikert R, et al. Ventricular tachycardias arising from the aortic sinus of Valsalva: an under‐recognized variant of left outflow tract ventricular tachycardia. *J Am Coll Cardiol*. 2001;37:1408--14.
S3.2.1.8. Crawford T, Mueller G, Good E, et al. Ventricular arrhythmias originating from papillary muscles in the right ventricle. *Heart Rhythm*. 2010;7:725--30.
S3.2.1.9. Yamada T, McElderry HT, Okada T, et al. Idiopathic focal ventricular arrhythmias originating from the anterior papillary muscle in the left ventricle. *J Cardiovasc Electrophysiol*. 2009;20:866--72.
S3.2.1.10. Li S, Wang Z, Shan Z, et al. Surface electrocardiography characteristics and radiofrequency catheter ablation of idiopathic ventricular arrhythmias originating from the left infero‐septal papillary muscles: differences from those originating from the left posterior fascicle. *Europace*. 2018;20:1028--34.
S3.2.1.11. Berruezo A, Mont L, Nava S, Chueca E, Bartholomay E, Brugada J. Electrocardiographic recognition of the epicardial origin of ventricular tachycardias. *Circulation*. 2004;109:1842--7.
S3.2.1.12. Daniels DV, Lu YY, Morton JB, et al. Idiopathic epicardial left ventricular tachycardia originating remote from the sinus of Valsalva: electrophysiological characteristics, catheter ablation, and identification from the 12‐lead electrocardiogram. *Circulation*. 2006;113:1659--66.
S3.2.1.13. Bazan V, Gerstenfeld EP, Garcia FC, et al. Site‐specific twelve‐lead ECG features to identify an epicardial origin for left ventricular tachycardia in the absence of myocardial infarction. *Heart Rhythm*. 2007;4:1403--10.
S3.2.1.14. Valles E, Bazan V, Marchlinski FE. ECG Criteria to identify epicardial ventricular tachycardia in nonischemic cardiomyopathy. *Circ Arrhythm Electrophysiol*. 2010;3:63--71.
S3.2.1.15. Bazan V, Bala R, Garcia FC, et al. Twelve‐lead ECG features to identify ventricular tachycardia arising from the epicardial right ventricle. *Heart Rhythm*. 2006;3:1132--9.
S3.2.1.16. Perez‐Rodon J, Martinez‐Alday J, Baron‐Esquivias G, et al. Prognostic value of the electrocardiogram in patients with syncope: data from the group for syncope study in the emergency room (GESINUR). *Heart Rhythm*. 2014;11:2035--44.
### 3.2.2. Assessment of Structural Heart Disease and Myocardial Ischemia {#joa312264-sec-0014}
Recommendations for assessment of SHD and myocardial ischemia
CORLOERecommendationsReferencesIB‐NR1. In patients with known or suspected VA, echocardiography is recommended for evaluation of cardiac structure and function.S3.2.2.1, S3.2.2.2IIaB‐NR2. In patients presenting with VA who are suspected of having SHD, even after normal echocardiographic evaluation, advanced cardiac imaging can be useful to detect and characterize underlying SHD.S3.2.2.3--S3.2.2.7IIaC‐EO3. In patients with VA in whom myocardial ischemia is suspected, stress testing and/or coronary angiography and subsequent revascularization can be beneficial before catheter ablation to avoid significant ischemia during induced VT.III: No BenefitB‐NR4. In patients presenting with monomorphic VT, revascularization alone is not effective to prevent VT recurrence.S3.2.2.8--S3.2.2.10
### References {#joa312264-sec-0015}
S3.2.2.1. Solomon SD, Zelenkofske S, McMurray JJ, et al. Sudden death in patients with myocardial infarction and left ventricular dysfunction, heart failure, or both. *N Engl J Med*. 2005;352:2581--8.
S3.2.2.2. Gula LJ, Klein GJ, Hellkamp AS, et al. Ejection fraction assessment and survival: an analysis of the Sudden Cardiac Death in Heart Failure Trial (SCD‐HeFT). *Am Heart J*. 2008;156:1196--200.
S3.2.2.3. Yoon Y, Ktagawa K, Kato S, et al. Prognostic value of unrecognised myocardial infarction detected by late gadolinium‐enhanced MRI in diabetic patients with normal global and regional left ventricular systolic function. *Eur Radiol*. 2013;23:2101--8.
S3.2.2.4. Olivotto I, Maron M, Autore C, et al. Assessment and significance of left ventricular mass by cardiovascular magnetic resonance in hypertrophic cardiomyopathy. *J Am Coll Cardiol*. 2008;52:559--66.
S3.2.2.5. Desjardins B, Yokokawa M, Good E, et al. Characteristics of intramural scar in patients with nonischemic cardiomyopathy and relation to intramural ventricular arrhythmias. *Circ Arrhythm Electrophysiol*. 2013;6:891--7.
S3.2.2.6. Dweck M, Abgral R, Trivieri M, et al. Hybrid magnetic resonance imaging and positron emission tomography with fluorodeoxyglucose to diagnose active cardiac sarcoidosis. *JACC Cardiovasc Imaging*. 2018;11:94--107.
S3.2.2.7. Piers SR, Tao Q, van Huls van Taxis CF, Schalij MJ, van der Geest RJ, Zeppenfeld K. Contrast‐enhanced MRI‐derived scar patterns and associated ventricular tachycardias in nonischemic cardiomyopathy: implications for the ablation strategy. *Circ Arrhythm Electrophysiol*. 2013;6:875--83.
S3.2.2.8. Brugada J, Aguinaga L, Mont L, Betriu A, Mulet J, Sanz G. Coronary artery revascularization in patients with sustained ventricular arrhythmias in the chronic phase of a myocardial infarction: effects on the electrophysiologic substrate and outcome. *J Am Coll Cardiol*. 2001;37:529--33.
S3.2.2.9. Nageh M, Kim J, Chen L, Yao JF. Implantable defibrillators for secondary prevention of sudden cardiac death in cardiac surgery patients with perioperative ventricular arrhythmias. *J Am Heart Assoc*. 2014;3:e000686.
S3.2.2.10. Elsokkari I, Parkash R, Gray C, et al. Effect of coronary revascularization on long‐term clinical outcomes in patients with ischemic cardiomyopathy and recurrent ventricular arrhythmia. *Pacing Clin Electrophysiol*. 2018;41:775--9.
### 3.2.3. Risk stratification in the setting of frequent premature ventricular complexes {#joa312264-sec-0016}
Recommendations for cardiac magnetic resonance imaging (CMR) in patients with frequent PVCs and for PES in patients with SHD and frequent PVCs
CORLOERecommendationsReferencesIIaB‐NR1. CMR can be useful for risk stratification for sudden cardiac death in patients with frequent PVCs.S3.2.3.1, S3.2.3.2IIaC‐LD2. PES can be useful for risk stratification for sudden cardiac death in patients with SHD undergoing ablation of frequent PVCs.S3.2.3.2
### References {#joa312264-sec-0017}
S3.2.3.1. Aquaro GD, Pingitore A, Strata E, Di Bella G, Molinaro S, Lombardi M. Cardiac magnetic resonance predicts outcome in patients with premature ventricular complexes of left bundle branch block morphology. *J Am Coll Cardiol*. 2010;56:1235--43.
S3.2.3.2. Yokokawa M, Siontis KC, Kim HM, et al. Value of cardiac magnetic resonance imaging and programmed ventricular stimulation in patients with frequent premature ventricular complexes undergoing radiofrequency ablation. *Heart Rhythm*. 2017;14:1695--1701.
### 3.2.4. Longitudinal follow‐up in the setting of frequent premature ventricular complexes {#joa312264-sec-0018}
Recommendation for longitudinal follow‐up of patients with frequent PVCs
CORLOERecommendationReferenceIIaB‐NR1. Periodic monitoring of PVC burden and LV function and dimensions can be useful in patients with frequent, asymptomatic PVCs and normal LV function and dimensions.S3.2.4.1
### Reference {#joa312264-sec-0019}
S3.2.4.1. Niwano SY, Wakisaka H, Niwano H, et al. Prognostic significance of frequent premature ventricular contractions originating from the ventricular outflow tract in patients with normal left ventricular function. *Heart*. 2009;95:1230--7.
4. INDICATIONS FOR CATHETER ABLATION {#joa312264-sec-0020}
====================================
Following are the consensus recommendations for catheter ablation of VAs organized by underlying diagnosis and substrate. These recommendations are each assigned a COR and an LOE according to the current recommendation classification system (S4.1). In drafting each of these recommendations, the writing committee took into account the published literature in the specific area, including the methodological quality and size of each study, as well as the collective clinical experience of the writing group when published data were not available. Implicit in each recommendation are several points: (a) the procedure is being performed by an electrophysiologist with appropriate training and experience in the procedure and in a facility with appropriate resources; (b) patient and procedural complexity vary widely, and some patients or situations merit a more experienced operator or a center with more capabilities than others, even within the same recommendation (eg, when an epicardial procedure is indicated and the operator or institution has limited experience with this procedure, it might be preferable to refer the patient to an operator or institution with adequate experience in performing epicardial procedures); (c) the patient is an appropriate candidate for the procedure, as outlined in Section 5, recognizing that the level of patient suitability for a procedure will vary widely with the clinical scenario; and (d) the patient\'s (or designee\'s) informed consent, values, and overall clinical trajectory are fundamental to a decision to proceed (or not) with any procedure. Therefore, in some clinical scenarios, initiation or continuation of medical therapy instead of an ablation procedure may be the most appropriate option, even when a class 1 recommendation for ablation is present. There may also be scenarios not explicitly covered in this document, and on which little or no published literature is available, in which the physician and patient must rely solely on their own judgment.
Figure [2](#joa312264-fig-0002){ref-type="fig"} provides an overview of care for the patient with congenital heart disease (CHD) and VA.
{#joa312264-fig-0002}
Reference {#joa312264-sec-0021}
---------
S4.1. Halperin JL, Levine GN, Al‐Khatib SM, et al. Further evolution of the ACC/AHA clinical practice guideline recommendation classification system: a report of the American College of Cardiology Foundation/American Heart Association Task Force on Clinical Practice Guidelines. *Circulation*. 2006;133:1426--8.
4.1. Idiopathic outflow tract ventricular arrhythmia {#joa312264-sec-0022}
----------------------------------------------------
Recommendations for catheter ablation of idiopathic outflow tract VA
CORLOERecommendationsReferencesIB‐R1. In patients with frequent and symptomatic PVCs originating from the RVOT in an otherwise normal heart, catheter ablation is recommended in preference to metoprolol or propafenone.S4.1.1IB‐NR2. In patients with symptomatic VAs from the RVOT in an otherwise normal heart for whom antiarrhythmic medications are ineffective, not tolerated, or not the patient\'s preference, catheter ablation is useful.S4.1.2--S4.1.12IB‐NR3. In patients with symptomatic idiopathic sustained monomorphic VT, catheter ablation is useful.S4.1.13--S4.1.17IIaB‐NR4. In patients with symptomatic VAs from the endocardial LVOT, including the SV, in an otherwise normal heart for whom antiarrhythmic medications are ineffective, not tolerated, or not the patient\'s preference, catheter ablation can be useful.S4.1.18--S4.1.27IIaB‐NR5. In patients with symptomatic VAs from the epicardial outflow tract or LV summit in an otherwise normal heart for whom antiarrhythmic medications are ineffective, not tolerated, or not the patient\'s preference, catheter ablation can be useful.S4.1.28--S4.1.32
References {#joa312264-sec-0023}
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S4.1.1. Ling Z, Liu Z, Su L, et al. Radiofrequency ablation versus antiarrhythmic medication for treatment of ventricular premature beats from the right ventricular outflow tract: prospective randomized study. *Circ Arrhythm Electrophysiol*. 2014;7:237--43.
S4.1.2. Zhang F, Yang B, Chen H, Ju W, Kojodjojo P, Cao K, Chen M. Magnetic versus manual catheter navigation for mapping and ablation of right ventricular outflow tract ventricular arrhythmias: a randomized controlled study. *Heart Rhythm*. 2013;10:1178--83.
S4.1.3. Krittayaphong R, Sriratanasathavorn C, Dumavibhat C, et al. Electrocardiographic predictors of long term outcomes after radiofrequency ablation in patients with right‐ventricular outflow tract tachycardia. *Europace*. 2006;8:601--6.
S4.1.4. Vestal M, Wen MS, Yeh SJ, Wang CC, Lin FC, Wu D. Electrocardiographic predictors of failure and recurrence in patients with idiopathic right ventricular outflow tract tachycardia and ectopy who underwent radiofrequency catheter ablation. *J Electrocardiol*. 2003;36:327--32.
S4.1.5. Miyazawa K, Ueda M, Kondo Y, Hayashi T, Nakano M, Ishimura M, Nakano M, Kobayashi Y. Rapid mapping and differentiation in ventricular outflow tract arrhythmia using non‐contact mapping. *J Interv Card Electrophysiol*. 2017;49:41--9.
S4.1.6. Akdeniz C, Gul EE, Celik N, Karacan M, Tuzcu V. Catheter ablation of idiopathic right ventricular arrhythmias in children with limited fluoroscopy. *J Interv Card Electrophysiol*. 2016;46:355--60.
S4.1.7. Morady F, Kadish AH, DiCarlo L, Kou WH, Winston S, deBuitlier M, Calkins H, Rosenheck S, Sousa J. Long‐term results of catheter ablation of idiopathic right ventricular tachycardia. *Circulation*. 1990;82:2093--9.
S4.1.8. Liao Z, Zhan X, Wu S, et al. Idiopathic ventricular arrhythmias originating from the pulmonary sinus cusp: prevalence, electrocardiographic/electrophysiological characteristics, and catheter ablation. *J Am Coll Cardiol*. 2015;66:2633--44.
S4.1.9. Bogun F, Crawford T, Reich S, Koelling TM, Armstrong W, Good E, Jongnarangsin K, Marine JE, Chugh A, Pelosi F, Oral H, Morady F. Radiofrequency ablation of frequent, idiopathic premature ventricular complexes: comparison with a control group without intervention. *Heart Rhythm*. 2007;4:863--7.
S4.1.10. Chen H, Shehata M, Swerdlow C, et al. Intramural outflow tract ventricular tachycardia: anatomy, mapping, and ablation. *Circ Arrhythm Electrophysiol*. 2014;7:978--81.
S4.1.11. Teh AW, Reddy VY, Koruth JS, et al. Bipolar radiofrequency catheter ablation for refractory ventricular outflow tract arrhythmias. *J Cardiovasc Electrophysiol*. 2014;25:1093--9.
S4.1.12. Lamba J, Redfearn DP, Michael KA, Simpson CS, Abdollah H, Baranchuk A. Radiofrequency catheter ablation for the treatment of idiopathic premature ventricular contractions originating from the right ventricular outflow tract: a systematic review and meta‐analysis. *Pacing Clin Electrophysiol*. 2014;37:73--8.
S4.1.13. Calkins H, Kalbfleisch J, El‐Atassi R, Langberg J, Morady F. Relation between efficacy of radiofrequency catheter ablation and site of origin of idiopathic ventricular tachycardia. *Am J Cardiol*. 1993;71:827--33.
S4.1.14. Rodriguez LM, Smeets JL, Timmermans C, Wellens HJ. Predictors for successful ablation of right‐ and left‐sided idiopathic ventricular tachycardia. *Am J Cardiol*. 1997;79:309--14.
S4.1.15. Coggins DL, Lee RJ, Sweeney J, et al. Radiofrequency catheter ablation as a cure for idiopathic tachycardia of both left and right ventricular origin. *J Am Coll Cardiol*. 1994;23:1333--41.
S4.1.16. Wen MS, Yeh SJ, Wang CC, Lin FC, Chen IC, Wu D. Radiofrequency ablation therapy in idiopathic left ventricular tachycardia with no obvious structural heart disease. *Circulation*. 1994;89:1690--6.
S4.1.17. Movsowitz C, Schwartzman D, Callans DJ, et al. Idiopathic right ventricular outflow tract tachycardia: narrowing the anatomic location for successful ablation. *Am Heart J*. 1996;131:930--6.
S4.1.18. Frey B, Kreiner G, Fritsch S, Veit F, Gossinger HD. Successful treatment of idiopathic left ventricular outflow tract tachycardia by catheter ablation or minimally invasive surgical cryoablation. *Pacing Clin Electrophysiol*. 2000;23:870--6.
S4.1.19. Krebs ME, Krause PC, Engelstein ED, Zipes DP, Miles WM. Ventricular tachycardias mimicking those arising from the right ventricular outflow tract. *J Cardiovasc Electrophysiol*. 2000;11:45--51.
S4.1.20. Kumagai K, Fukuda K, Wakayama Y, et al. Electrocardiographic characteristics of the variants of idiopathic left ventricular outflow tract ventricular tachyarrhythmias. *J Cardiovasc Electrophysiol*. 2008;19:495--501.
S4.1.21. Latchamsetty R, Yokokawa M, Morady F, et al. Multicenter outcomes for catheter ablation of idiopathic premature ventricular complexes. *JACC Clin Electrophysiol*. 2015;1:116--23.
S4.1.22. Kamakura S, Shimizu W, Matsuo K, et al. Localization of optimal ablation site of idiopathic ventricular tachycardia from right and left ventricular outflow tract by body surface ECG. *Circulation*. 1998;98:1525--33.
S4.1.23. Yamada T, Yoshida N, Murakami Y, et al. Electrocardiographic characteristics of ventricular arrhythmias originating from the junction of the left and right coronary sinuses of Valsalva in the aorta: the activation pattern as a rationale for the electrocardiographic characteristics. *Heart Rhythm*. 2008;5:184--92.
S4.1.24. Tada H, Naito S, Ito S, et al. Significance of two potentials for predicting successful catheter ablation from the left sinus of Valsalva for left ventricular epicardial tachycardia. *Pacing Clin Electrophysiol*. 2004;27:1053--9.
S4.1.25. Ouyang F, Fotuhi P, Ho SY, et al. Repetitive monomorphic ventricular tachycardia originating from the aortic sinus cusp: electrocardiographic characterization for guiding catheter ablation. *J Am Coll Cardiol*. 2002;39:500--8.
S4.1.26. Callans DJ, Menz V, Schwartzman D, Gottlieb CD, Marchlinski FE. Repetitive monomorphic tachycardia from the left ventricular outflow tract: electrocardiographic patterns consistent with a left ventricular site of origin. *J Am Coll Cardiol*. 1997;29:1023--7.
S4.1.27. Bala R, Garcia FC, Hutchinson MD, et al. Electrocardiographic and electrophysiologic features of ventricular arrhythmias originating from the right/left coronary cusp commissure. *Heart Rhythm*. 2010;7:312--22.
S4.1.28. Tada H, Nogami A, Naito S, et al. Left ventricular epicardial outflow tract tachycardia: a new distinct subgroup of outflow tract tachycardia. *Jpn Circ J*. 2001;65:723--30.
S4.1.29. Baman TS, Ilg KJ, Gupta SK, et al. Mapping and ablation of epicardial idiopathic ventricular arrhythmias from within the coronary venous system. *Circ Arrhythm Electrophysiol*. 2010;3:274--9.
S4.1.30. Carrigan T, Patel S, Yokokawa M, Schmidlin E, Swanson S, Morady F, Bogun F. Anatomic relationships between the coronary venous system, surrounding structures, and the site of origin of epicardial ventricular arrhythmias. *J Cardiovasc Electrophysiol*. 2014;25:1336--42.
S4.1.31. Santangeli P, Marchlinski FE, Zado ES, et al. Percutaneous epicardial ablation of ventricular arrhythmias arising from the left ventricular summit: outcomes and electrocardiogram correlates of success. *Circ Arrhythm Electrophysiol*. 2015;8:337--43.
S4.1.32. Nagashima K, Choi EK, Lin KY, et al. Ventricular arrhythmias near the distal great cardiac vein: challenging arrhythmia for ablation. *Circ Arrhythm Electrophysiol*. 2014;7:906--12.
4.2. Idiopathic nonoutflow tract ventricular arrhythmia {#joa312264-sec-0024}
-------------------------------------------------------
Recommendations for catheter ablation of nonoutflow tract VAs in the absence of SHD
CORLOERecommendationsReferencesIB‐NR1. In patients with symptomatic VAs from the RV at sites other than the outflow tracts (tricuspid annulus, moderator band, parietal band, or papillary muscles) in an otherwise normal heart for whom antiarrhythmic medications are ineffective, not tolerated, or not the patient\'s preference, catheter ablation is useful.S4.2.1--S4.2.14IB‐NR2. In patients with symptomatic VAs from the LV at sites other than the outflow tracts (mitral annulus, papillary muscles, or AMC) in an otherwise normal heart for whom antiarrhythmic medications are ineffective, not tolerated, or not the patient\'s preference, catheter ablation is useful.S4.2.15--S4.2.31IIaB‐NR3. In patients with symptomatic VAs from the epicardial coronary venous system in an otherwise normal heart for whom antiarrhythmic medications are ineffective, not tolerated, or not the patient\'s preference, catheter ablation can be useful.S4.2.32--S4.2.43IIaB‐NR4. In patients with symptomatic VAs from para‐Hisian sites in an otherwise normal heart for whom antiarrhythmic medications are ineffective, not tolerated, or not the patient\'s preference, catheter ablation can be useful.S4.2.7, S4.2.13, S4.2.14, S4.2.44--S4.2.49IIaC‐LD5. In patients with symptomatic VAs from the posterior‐superior process of the LV in an otherwise normal heart for whom antiarrhythmic medications are ineffective, not tolerated, or not the patient\'s preference, catheter ablation from the LV endocardium, right atrium, or CS, can be useful.S4.2.50--S4.2.52
References {#joa312264-sec-0025}
----------
S4.2.1. Van Herendael H, Garcia F, Lin D, et al. Idiopathic right ventricular arrhythmias not arising from the outflow tract: prevalence, electrocardiographic characteristics, and outcome of catheter ablation. *Heart Rhythm*. 2011;8:511--8.
S4.2.2. Sadek MM, Benhayon D, Sureddi R, et al. Idiopathic ventricular arrhythmias originating from the moderator band: electrocardiographic characteristics and treatment by catheter ablation. *Heart Rhythm*. 2015;12:67--75.
S4.2.3. Crawford T, Mueller G, Good E, et al. Ventricular arrhythmias originating from papillary muscles in the right ventricle. *Heart Rhythm*. 2010;7:725--30.
S4.2.4. Tada H, Tadokoro K, Miyaji K, et al. Idiopathic ventricular arrhythmias arising from the pulmonary artery: prevalence, characteristics, and topography of the arrhythmia origin. *Heart Rhythm*. 2008;5:419--26.
S4.2.5. Tada H, Tadokoro K, Ito S, et al. Idiopathic ventricular arrhythmias originating from the tricuspid annulus: prevalence, electrocardiographic characteristics, and results of radiofrequency catheter ablation. *Heart Rhythm*. 2007;4:7--16.
S4.2.6. Santoro F, DiBiase L, Hranitzky P, et al. Ventricular tachycardia originating from the septal papillary muscle of the right ventricle: electrocardiographic and electrophysiological characteristics. *J Cardiovasc Electrophysiol*. 2015;26:145--50.
S4.2.7. Sasaki K, Sasaki S, Kimura M, et al. Catheter ablation of ventricular arrhythmias arising from the basal septum of the right ventricle: characteristics and significance of junctional rhythm appearing during ablation. *J Interv Card Electrophysiol*. 2016;45:159--67.
S4.2.8. Yue‐Chun L, Wen‐Wu Z, Na‐Dan Z, et al. Idiopathic premature ventricular contractions and ventricular tachycardias originating from the vicinity of tricuspid annulus: results of radiofrequency catheter ablation in thirty‐five patients. *BMC Cardiovasc Disord*. 2012;10:12--32.
S4.2.9. Yamada T, Yoshida N, Itoh T, Litovsky SH, Doppalapudi H, McElderry HT, Kay GN. Idiopathic ventricular arrhythmias originating from the parietal band: electrocardiographic and electrophysiological characteristics and outcome of catheter ablation. *Circ Arrhythm Electrophysiol*. 2017;10:e005099.
S4.2.10. Ceresnak SR, Pass RH, Krumerman AK, Kim SG, Nappo L, Fisher JD. Characteristics of ventricular tachycardia arising from the inflow region of the right ventricle. *J Electrocardiol*. 2012;45:385--90.
S4.2.11. Yamada T, Yoshida N, Litovsky SH, Itoh T, Doppalapudi H, Kay GN. Idiopathic ventricular arrhythmias originating from the infundibular muscles: prevalence, electrocardiographic and electrophysiological characteristics, and outcome of catheter ablation. *Circ Arrhythm Electrophysiol*. 2018;11:e005749.
S4.2.12. Li T, Zhan XZ, Xue YM, et al. Combined approach improves the outcomes of catheter ablation of idiopathic ventricular arrhythmias originating from the vicinity of tricuspid annulus. *Pacing Clin Electrophysiol*. 2014;37:624--9.
S4.2.13. Lian‐Pin W, Yue‐Chun L, Jing‐Lin Z, et al. Catheter ablation of idiopathic premature ventricular contractions and ventricular tachycardias originating from right ventricular septum. *PLoS ONE*. 2013;8:e67038.
S4.2.14. Enriquez A, Pathak RK, Santangeli P, et al. Inferior lead discordance in ventricular arrhythmias: a specific marker for certain arrhythmia locations. *J Cardiovasc Electrophysiol*. 2017;28:1179--86.
S4.2.15. Doppalapudi H, Yamada T, McElderry HT, Plumb VJ, Epstein AE, Kay GN. Ventricular tachycardia originating from the posterior papillary muscle in the left ventricle: a distinct clinical syndrome. *Circ Arrhythm Electrophysiol*. 2008;1:23--9.
S4.2.16. Yamada T, McElderry HT, Okada T, et al. Idiopathic focal ventricular arrhythmias originating from the anterior papillary muscle in the left ventricle. *J Cardiovasc Electrophysiol*. 2009;20:866--72.
S4.2.17. Yamada T, Doppalapudi H, McElderry HT, et al. Idiopathic ventricular arrhythmias originating from the papillary muscles in the left ventricle: prevalence, electrocardiographic and electrophysiological characteristics, and results of the radiofrequency catheter ablation. *J Cardiovasc Electrophysiol*. 2010;21:62--9.
S4.2.18. Yamada T, Doppalapudi H, McElderry HT, et al. Electrocardiographic and electrophysiological characteristics in idiopathic ventricular arrhythmias originating from the papillary muscles in the left ventricle: relevance for catheter ablation. *Circ Arrhythm Electrophysiol*. 2010;3:324--31.
S4.2.19. Bassil G, Liu CF, Markowitz SM, et al. Comparison of robotic magnetic navigation‐guided and manual catheter ablation of ventricular arrhythmias arising from the papillary muscles. *Europace*. 2018;20(Suppl. 2):ii5--10.
S4.2.20. Ban JE, Lee HS, Lee DI, et al. Electrophysiological characteristics related to outcome after catheter ablation of idiopathic ventricular arrhythmia originating from the papillary muscle in the left ventricle. *Korean Circ J*. 2013;43:811--8.
S4.2.21. Yokokawa M, Good E, Desjardins B, et al. Predictors of successful catheter ablation of ventricular arrhythmias arising from the papillary muscles. *Heart Rhythm*. 2010;7:1654--9.
S4.2.22. Rivera S, Ricapito Mde L, Tomas L, et al. Results of cryoenergy and radiofrequency‐based catheter ablation for treating ventricular arrhythmias arising from the papillary muscles of the left ventricle, guided by intracardiac echocardiography and image integration. *Circ Arrhythm Electrophysiol*. 2016;9:e003874.
S4.2.23. Al\'Aref SJ, Ip JE, Markowitz SM, et al. Differentiation of papillary muscle from fascicular and mitral annular ventricular arrhythmias in patients with and without structural heart disease. *Circ Arrhythm Electrophysiol*. 2015;8:616--24.
S4.2.24. Wasmer K, Köbe J, Dechering DG, et al. Ventricular arrhythmias from the mitral annulus: patient characteristics, electrophysiological findings, ablation, and prognosis. *Heart Rhythm*. 2013;10:783--8.
S4.2.25. Kumagai K, Yamauchi Y, Takahashi A, et al. Idiopathic left ventricular tachycardia originating from the mitral annulus. *J Cardiovasc Electrophysiol*. 2005;16:1029--36.
S4.2.26. Tada H, Ito S, Naito S, et al. Idiopathic ventricular arrhythmia arising from the mitral annulus: a distinct subgroup of idiopathic ventricular arrhythmias. *J Am Coll Cardiol*. 2005;45:877--86.
S4.2.27. Yue‐Chun L, Cheng Z, Jun H, Jun‐Hua C, Jing‐Lin Z, Jia‐Feng L. Catheter ablation of idiopathic premature ventricular contractions and ventricular tachycardias originating from the vicinity of endocardial and epicardial mitral annulus. *PLoS ONE*. 2013;8:e80777.
S4.2.28. Yamada T, Doppalapudi H, McElderry HT, Kay GN. Idiopathic mitral annular PVCs with multiple breakouts and preferential conduction unmasked by radiofrequency catheter ablation. *Pacing Clin Electrophysiol*. 2012;35:e112--5.
S4.2.29. Yamada T, Litovsky SH, Kay GN. The left ventricular ostium: an anatomic concept relevant to idiopathic ventricular arrhythmias. *Circ Arrhythmia Electrophysiol*. 2008;1:396--404.
S4.2.30. Chen J, Hoff PI, Rossvoll O, et al. Ventricular arrhythmias originating from the aortomitral continuity: an uncommon variant of left ventricular outflow tract tachycardia. *Europace*. 2012;14:388--95.
S4.2.31. Hai JJ, Chahal AA, Friedman PA, et al. Electrophysiologic characteristics of ventricular arrhythmias arising from the aortic mitral continuity‐potential role of the conduction system. *J Cardiovasc Electrophysiol*. 2015;26:158--63.
S4.2.32. Yamada T, McElderry HT, Doppalapudi H., et al. Idiopathic ventricular arrhythmias originating from the left ventricular summit: anatomic concepts relevant to ablation. *Circ Arrhythm Electrophysiol*. 2010;3:616--23.
S4.2.33. Baman TS, Ilg KJ, Gupta SK, et al. Mapping and ablation of epicardial idiopathic ventricular arrhythmias from within the coronary venous system. *Circ Arrhythm Electrophysiol*. 2010;3:274--9.
S4.2.34. Mountantonakis SE, Frankel DS, Tschabrunn CM, et al. Ventricular arrhythmias from the coronary venous system: prevalence, mapping, and ablation. *Heart Rhythm*. 2015;12:1145--53.
S4.2.35. Meininger GR, Berger RD. Idiopathic ventricular tachycardia originating in the great cardiac vein. *Heart Rhythm*. 2006;3:464--6.
S4.2.36. Yamada T, Doppalapudi H, Litovsky SH, McElderry HT, Kay GN. Challenging radiofrequency catheter ablation of idiopathic ventricular arrhythmias originating from the left ventricular summit near the left main coronary artery. *Circ Arrhythm Electrophysiol*. 2016;9:e004202.
S4.2.37. Yokokawa M, Latchamsetty R, Good E, et al. Ablation of epicardial ventricular arrhythmias from nonepicardial sites. *Heart Rhythm*. 2011;8:1525--9.
S4.2.38. Jauregui Abularach ME, Campos B, Park KM, et al. Ablation of ventricular arrhythmias arising near the anterior epicardial veins from the left sinus of Valsalva region: ECG features, anatomic distance, and outcome. *Heart Rhythm*. 2012;9:865--73.
S4.2.39. Yokokawa M, Good E, Chugh A, et al. Intramural idiopathic ventricular arrhythmias originating in the intraventricular septum: mapping and ablation. *Circ Arrhythm Electrophysiol*. 2012;5:258--63.
S4.2.40. Doppalapudi H, Yamada T, Ramaswamy K, Ahn J, Kay GN. Idiopathic focal epicardial ventricular tachycardia originating from the crux of the heart. *Heart Rhythm*. 2009;6:44--50.
S4.2.41. Kawamura M, Gerstenfeld EP, Vedantham V, et al. Idiopathic ventricular arrhythmia originating from the cardiac crux or inferior septum. *Circ Arrhythm Electrophysiol*. 2014;7:1152--8.
S4.2.42. Larroussi L, Badhwar N. Ventricular tachycardia arising from cardiac crux: electrocardiogram recognition and site of ablation. *Card Electrophysiol Clin*. 2016;8:109--13.
S4.2.43. Yui Y, Sekiguchi Y, Nogami A, et al. Electrophysiological characteristics and radiofrequency catheter ablation treatment of idiopathic ventricular arrhythmias successfully ablated from the ostium of the coronary sinus. *Circ J*. 2017;81:1807--15.
S4.2.44. Komatsu Y, Otomo K, Taniguchi H, et al. Catheter ablation of ventricular arrhythmias arising from the right ventricular septum close to the His bundle: features of the local electrogram at the optimal ablation site. *J Cardiovasc Electrophysiol*. 2011;22:878--85.
S4.2.45. Yamada T, Plumb VJ, McElderry HT, Doppalapudi H, Epstein AE, Kay GN. Focal ventricular arrhythmias originating from the left ventricle adjacent to the membranous septum. *Europace*. 2010;12:1467--74.
S4.2.46. Wei HQ, Guo XG, Liu X, et al. Safety and efficacy of catheter ablation of ventricular arrhythmias with para‐Hisian origin via a systematic direct approach from the aortic sinus cusp. *Heart Rhythm*. 2018;15:1626--33.
S4.2.47. Yamauchi Y, Aonuma K, Takahashi A, et al. Electrocardiographic characteristics of repetitive monomorphic right ventricular tachycardia originating near the His‐bundle. *J Cardiovasc Electrophysiol*. 2005;16:1041--8.
S4.2.48. Komatsu Y, Taniguchi H, Miyazaki S, et al. Two distinct electrocardiographic forms of idiopathic ventricular arrhythmia originating in the vicinity of the His bundle. *Europace*. 2012;14:1778--85.
S4.2.49. Enriquez A, Tapias C, Rodriguez D, et al. How to map and ablate parahisian ventricular arrhythmias. *Heart Rhythm*. 2018;15:1268--74.
S4.2.50. Santangeli P, Hutchinson MD, Supple GE, Callans DJ, Marchlinski FE, Garcia FC. Right atrial approach for ablation of ventricular arrhythmias arising from the left posterior‐superior process of the left ventricle. *Circ Arrhythm Electrophysiol*. 2016;9:e004048.
S4.2.51. Li A, Zuberi Z, Bradfield JS, et al. Endocardial ablation of ventricular ectopic beats arising from the basal inferoseptal process of the left ventricle. *Heart Rhythm*. 2018;15:1356--62.
S4.2.52. Tavares L, Dave A, Valderrábano M. Successful ablation of premature ventricular contractions originating from the inferoseptal process of the left ventricle using a coronary sinus approach. *HeartRhythm Case Rep*. 2018;4:371--4.
4.3. Premature ventricular complexes with or without left ventricular dysfunction {#joa312264-sec-0026}
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Recommendations for catheter ablation of PVCs in patients with or without LV dysfunction
CORLOERecommendationsReferencesIB‐NR1. In patients with cardiomyopathy suspected to be caused by frequent and predominately monomorphic PVCs and for whom AADs are ineffective, not tolerated, or not preferred for long‐term therapy, catheter ablation is recommended.S4.3.1--S4.3.10IIaB‐NR2. In patients with SHD in whom frequent PVCs are suspected to be contributing to a cardiomyopathy and for whom AADs are ineffective, not tolerated, or not preferred for long‐term therapy, catheter ablation can be useful.S4.3.3, S4.3.11, S4.3.12IIaB‐NR3. In patients with focally triggered VF refractory to AADs and triggered by a similar PVC, catheter ablation can be useful.S4.3.13--S4.3.17IIaC‐LD4. In nonresponders to cardiac resynchronization therapy with very frequent unifocal PVCs limiting optimal biventricular pacing despite pharmacological therapy, catheter ablation can be useful.S4.3.18
References {#joa312264-sec-0027}
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S4.3.1. Latchamsetty RY, Morady M, Kim F, et al. Multicenter outcomes for catheter ablation of idiopathic premature ventricular complexes. *JACC Clinical Electrophysiol*. 2015;1:116--23.
S4.3.2. Singh SN, Fletcher RD, Fisher SG, et al. Amiodarone in patients with congestive heart failure and asymptomatic ventricular arrhythmia: survival trial of antiarrhythmic therapy in congestive heart failure. *N Engl J Med*. 1995;333:77--82.
S4.3.3. Mountantonakis SE, Frankel DS, Gerstenfeld EP, et al. Reversal of outflow tract ventricular premature depolarization‐induced cardiomyopathy with ablation: effect of residual arrhythmia burden and preexisting cardiomyopathy on outcome. *Heart Rhythm*. 2011;8:1608--14.
S4.3.4. Zang M, Zhang T, Mao J, Zhou S, He B. Beneficial effects of catheter ablation of frequent premature ventricular complexes on left ventricular function. *Heart*. 2014;100:787--93.
S4.3.5. Lee A, Denman R, Haqqani HM. Ventricular ectopy in the context of left ventricular systolic dysfunction: risk factors and outcomes following catheter ablation. *Heart Lung Circ*. 2019;28:379--88.
S4.3.6. Bogun F, Crawford T, Reich S, et al. Radiofrequency ablation of frequent, idiopathic premature ventricular complexes: comparison with a control group without intervention. *Heart Rhythm*. 2007;4:863--7.
S4.3.7. Takemoto M, Yoshimura H, Ohba Y, et al. Radiofrequency catheter ablation of premature ventricular complexes from right ventricular outflow tract improves left ventricular dilation and clinical status in patients without structural heart disease. *J Am Coll Cardiol*. 2005;45:1259--65.
S4.3.8. Baman TS, Lange DC, Ilg KJ, et al. Relationship between burden of premature ventricular complexes and left ventricular function. *Heart Rhythm*. 2010;7:865--9.
S4.3.9. Yarlagadda RK, Iwai S, Stein KM, et al. Reversal of cardiomyopathy in patients with repetitive monomorphic ventricular ectopy originating from the right ventricular outflow tract. *Circulation*. 2005;112:1092--7.
S4.3.10. Wijnmaalen AP, Delgado V, Schalij MJ, et al. Beneficial effects of catheter ablation on left ventricular and right ventricular function in patients with frequent premature ventricular contractions and preserved ejection fraction. *Heart*. 2010;96:1275--80.
S4.3.11. Sarrazin JF, Labounty T, Kuhne M, et al. Impact of radiofrequency ablation of frequent post‐infarction premature ventricular complexes on left ventricular ejection fraction. *Heart Rhythm*. 2009;6:1543--9.
S4.3.12. El Kadri M, Yokokawa M, Labounty T, et al. Effect of ablation of frequent premature ventricular complexes on left ventricular function in patients with nonischemic cardiomyopathy. *Heart Rhythm*. 2015;12:706--13.
S4.3.13. Haïssaguerre M, Shoda M, Jaïs P, et al. Mapping and ablation of idiopathic ventricular fibrillation. *Circulation*. 2002;106:962--7.
S4.3.14. Knecht S, Sacher F, Wright M, et al. Long‐term follow‐up of idiopathic ventricular fibrillation ablation: a multicenter study. *J Am Coll Cardiol*. 2009;54:522--8.
S4.3.15. Peichl P, Cihák R, Kozeluhová M, Wichterle D, Vancura V, Kautzner J. Catheter ablation of arrhythmic storm triggered by monomorphic ectopic beats in patients with coronary artery disease. *J Interv Card Electrophysiol*. 2010;27:51--9.
S4.3.16. Haïssaguerre M, Extramiana F, Hocini M, et al. Mapping and ablation of ventricular fibrillation associated with long‐QT and Brugada syndromes. *Circulation*. 2003;108:925--8.
S4.3.17. Sadek MM, Benhayon D, Sureddi R, et al. Idiopathic ventricular arrhythmias originating from the moderator band: electrocardiographic characteristics and treatment by catheter ablation. *Heart Rhythm*. 2015;12:67--75.
S4.3.18. Lakkireddy D, Di Biase L, Ryschon K, et al. Radiofrequency ablation of premature ventricular ectopy improves the efficacy of cardiac resynchronization therapy in nonresponders. *J Am Coll Cardiol*. 2012;60:1531--9.
4.4. Ventricular arrhythmia in ischemic heart disease {#joa312264-sec-0028}
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Recommendations for catheter ablation of VAs in patients with IHD
CORLOERecommendationsReferencesIB‐R1. In patients with IHD who experience recurrent monomorphic VT despite chronic amiodarone therapy, catheter ablation is recommended in preference to escalating AAD therapy.S4.4.1IB‐NR2. In patients with IHD and recurrent symptomatic monomorphic VT despite AAD therapy, or when AAD therapy is contraindicated or not tolerated, catheter ablation is recommended to reduce recurrent VT.S4.4.2--S4.4.4IB‐NR3. In patients with IHD and VT storm refractory to AAD therapy, catheter ablation is recommended.S4.4.5--S4.4.9IIaC‐EO4. In patients with IHD and recurrent monomorphic VT, in whom AADs are not desired, catheter ablation can be useful.IIbA5. In patients with IHD and an ICD who experience a first episode of monomorphic VT, catheter ablation may be considered to reduce the risk of recurrent VT or ICD therapies.S4.4.10--S4.4.14IIbC‐LD6. In patients with prior myocardial infarction and recurrent episodes of symptomatic sustained VT for whom prior endocardial catheter ablation has not been successful and who have ECG, endocardial mapping, or imaging evidence of a subepicardial VT substrate, epicardial ablation may be considered.S4.4.15--S4.4.19
References {#joa312264-sec-0029}
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S4.4.1. Sapp JL, Wells GA, Parkash R, et al. Ventricular tachycardia ablation versus escalation of antiarrhythmic drugs. *N Engl J Med*. 2016;375:111--21.
S4.4.2. Stevenson WG, Wilber DJ, Natale A, et al; Multicenter Thermocool VT Ablation Trial Investigators. Irrigated radiofrequency catheter ablation guided by electroanatomic mapping for recurrent ventricular tachycardia after myocardial infarction: the multicenter thermocool ventricular tachycardia ablation trial. *Circulation*. 2008;118:2773--82.
S4.4.3. Tanner H, Hindricks G, Volkmer M, et al. Catheter ablation of recurrent scar‐related ventricular tachycardia using electroanatomical mapping and irrigated ablation technology: results of the prospective multicenter Euro‐VT‐study. *J Cardiovasc Electrophysiol*. 2010;21:47--53.
S4.4.4. Marchlinski FE, Haffajee CI, Beshai JF, et al. Long‐term success of irrigated radiofrequency catheter ablation of sustained ventricular tachycardia: post‐approval THERMOCOOL VT trial. *J Am Coll Cardiol*. 2016;67:674--83.
S4.4.5. Carbucicchio C, Santamaria M, Trevisi N, et al. Catheter ablation for the treatment of electrical storm in patients with implantable cardioverter‐defibrillators: short‐ and long‐term outcomes in a prospective single‐center study. *Circulation*. 2008;117:462--9.
S4.4.6. Deneke T, Shin D, Lawo T, et al. Catheter ablation of electrical storm in a collaborative hospital network. *Am J Cardiol*. 2011;108:233--9.
S4.4.7. Muser D, Liang JJ, Pathak RK, et al. Long‐term outcomes of catheter ablation of electrical storm in nonischemic dilated cardiomyopathy compared with ischemic cardiomyopathy. *JACC Clin Electrophysiol*. 2017;3:767--78.
S4.4.8. Kumar S, Fujii A, Kapur S, et al. Beyond the storm: comparison of clinical factors, arrhythmogenic substrate, and catheter ablation outcomes in structural heart disease patients with versus those without a history of ventricular tachycardia storm. *J Cardiovasc Electrophysiol*. 2017;28:56--67.
S4.4.9. Nayyar S, Ganesan AN, Brooks AG, Sullivan T, Roberts‐Thomson KC, Sanders P. Venturing into ventricular arrhythmia storm: a systematic review and meta‐analysis. *Eur Heart J*. 2013;34:560--9.
S4.4.10. Reddy VY, Reynolds MR, Neuzil P, et al. Prophylactic catheter ablation for the prevention of defibrillator therapy. *N Engl J Med*. 2007;357:2657--65.
S4.4.11. Kuck KH, Schaumann A, Eckhardt L, et al; for the VTACH Study Group. Catheter ablation of stable ventricular tachycardia before defibrillator implantation in patients with coronary heart disease (VTACH): a multicentre randomised controlled trial. *Lancet*. 2010;375:31--40.
S4.4.12. Al‐Khatib SM, Daubert JP, Anstrom KJ, et al. Catheter ablation for ventricular tachycardia in patients with an implantable cardioverter defibrillator (CALYPSO) pilot trial. *J Cardiovasc Electrophysiol*. 2015;26:151--7.
S4.4.13. Kuck KH, Tilz RR, Deneke T, et al; SMS Investigators. Impact of substrate modification by catheter ablation on implantable cardioverter--defibrillator interventions in patients with unstable ventricular arrhythmias and coronary artery disease: results from the multicenter randomized controlled SMS (Substrate Modification Study). *Circ Arrhythm Electrophysiol*. 2017;10:e004422.
S4.4.14. Martinez BK, Baker WL, Konopka A, et al. Systematic review and meta‐analysis of catheter ablation of ventricular tachycardia in ischemic heart disease. *Heart Rhythm*. 2019 May 10 \[Epub ahead of print\].
S4.4.15. Littmann L, Svenson RH, Gallagher JJ, et al. Functional role of the epicardium in postinfarction ventricular tachycardia: observations derived from computerized epicardial activation mapping, entrainment, and epicardial laser photoablation. *Circulation*. 1991;83:1577--91.
S4.4.16. Sosa E, Scanavacca M, d\'Avila A, Oliveira F, Ramires JA. Nonsurgical transthoracic epicardial catheter ablation to treat recurrent ventricular tachycardia occurring late after myocardial infarction. *J Am Coll Cardiol*. 2000;35:1442--9.
S4.4.17. Schmidt B, Chun KR, Baensch D, Antz M, Koektuerk B, Tilz RR, Metzner A, Ouyang F, Kuck KH. Catheter ablation for ventricular tachycardia after failed endocardial ablation: epicardial substrate or inappropriate endocardial ablation? *Heart Rhythm*. 2010;7:1746--52.
S4.4.18. Di Biase L, Santangeli P, Burkhardt DJ, et al. Endo‐epicardial homogenization of the scar versus limited endocardial substrate ablation for the treatment of electrical storms in patients with ischemic cardiomyopathy. *J Am Coll Cardiol*. 2012;60:132--41.
S4.4.19. Izquierdo M, Sánchez‐Gómez JM, Ferrero de Loma‐Osorio A, Martínez A, Bellver A, Peláez A, Núñez J, Núñez C, Chorro J, Ruiz‐Granell R. Endo‐epicardial versus only‐endocardial ablation as a first line strategy for the treatment of ventricular tachycardia in patients with ischemic heart disease. *Circ Arrhythm Electrophysiol*. 2015;8:882--9.
4.5. Nonischemic cardiomyopathy {#joa312264-sec-0030}
-------------------------------
Recommendations for catheter ablation of VT in nonischemic cardiomyopathy (NICM)
CORLOERecommendationsReferencesIB‐NR1. In patients with NICM and recurrent sustained monomorphic VT for whom antiarrhythmic medications are ineffective, contraindicated, or not tolerated, catheter ablation is useful for reducing recurrent VT and ICD shocks.S4.5.1--S4.5.6IB‐NR2. In patients with NICM and electrical storm refractory to AAD therapy, catheter ablation is useful for reducing recurrent VT and ICD shocks.S4.5.7--S4.5.9IIaB‐NR3. In patients with NICM, epicardial catheter ablation of VT can be useful after failure of endocardial ablation or as the initial ablation approach when there is a suspicion of an epicardial substrate or circuit.S4.5.4, S4.5.10--S4.5.13IIaB‐NR4. In patients with cardiac sarcoidosis and recurrent VT despite medical therapy, catheter ablation can be useful to reduce the risk of VT recurrence and ICD shocks.S4.5.14--S4.5.18IIaC‐EO5. In patients with NICM and recurrent sustained monomorphic VT for whom antiarrhythmic medications are not desired, catheter ablation can be useful for reducing recurrent VT and ICD shocks.IIbB‐NR6. In patients with NICM related to lamin A/C (*LMNA*) mutations and recurrent VT, catheter ablation may be considered as a palliative strategy for short‐term arrhythmia control.S4.5.19
References {#joa312264-sec-0031}
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S4.5.1. Muser D, Santangeli P, Castro SA, et al. Long‐term outcome after catheter ablation of ventricular tachycardia in patients with nonischemic dilated cardiomyopathy. *Circ Arrhythm Electrophysiol*. 2016;9:e004328.
S4.5.2. Proietti R, Essebag V, Beardsall J, et al. Substrate‐guided ablation of haemodynamically tolerated and untolerated ventricular tachycardia in patients with structural heart disease: effect of cardiomyopathy type and acute success on long‐term outcome. *Europace*. 2015;17:461--7.
S4.5.3. Dinov B, Arya A, Bertagnolli L, et al. Early referral for ablation of scar‐related ventricular tachycardia is associated with improved acute and long‐term outcomes: results from the Heart Center of Leipzig ventricular tachycardia registry. *Circ Arrhythm Electrophysiol*. 2014;7:1144--51.
S4.5.4. Dinov B, Fiedler L, Schönbauer R, et al. Outcomes in catheter ablation of ventricular tachycardia in dilated nonischemic cardiomyopathy compared with ischemic cardiomyopathy: results from the Prospective Heart Centre of Leipzig VT (HELP‐VT) study. *Circulation*. 2014;129:728--36.
S4.5.5. Tokuda M, Tedrow UB, Kojodjojo P, et al. Catheter ablation of ventricular tachycardia in nonischemic heart disease. *Circ Arrhythm Electrophysiol*. 2012;5:992--1000.
S4.5.6. Tung R, Vaseghi M, Frankel DS, et al. Freedom from recurrent ventricular tachycardia after catheter ablation is associated with improved survival in patients with structural heart disease: an International VT Ablation Center Collaborative Group study. *Heart Rhythm*. 2015;12:1997--2007.
S4.5.7. Muser D, Liang JJ, Pathak RK, et al. Long‐term outcomes of catheter ablation of electrical storm in nonischemic dilated cardiomyopathy compared with ischemic cardiomyopathy. *JACC Clin Electrophysiol*. 2017;3:767--78.
S4.5.8. Arya A, Bode K, Piorkowski C, et al. Catheter ablation of electrical storm due to monomorphic ventricular tachycardia in patients with nonischemic cardiomyopathy: acute results and its effect on long‐term survival. *Pacing Clin Electrophysiol*. 2010;33:1504--9.
S4.5.9. Carbucicchio C, Santamaria M, Trevisi N, et al. Catheter ablation for the treatment of electrical storm in patients with implantable cardioverter‐defibrillators: short‐ and long‐term outcomes in a prospective single‐center study. *Circulation*. 2008;117:462--9.
S4.5.10. Hu J, Zeng S, Zhou Q, et al. Can ventricular tachycardia non‐inducibility after ablation predict reduced ventricular tachycardia recurrence and mortality in patients with non‐ischemic cardiomyopathy? A meta‐analysis of twenty‐four observational studies. *Int J Cardiol*. 2016;222:689--95.
S4.5.11. Della Bella P, Brugada J, Zeppenfeld K, et al. Epicardial ablation for ventricular tachycardia: a European multicenter study. *Circ Arrhythm Electrophysiol*. 2011;4:653--9.
S4.5.12. Sacher F, Roberts‐Thomson K, Maury P, et al. Epicardial ventricular tachycardia ablation a multicenter safety study. *J Am Coll Cardiol*. 2010;55:2366--72.
S4.5.13. Cano O, Hutchinson M, Lin D, et al. Electroanatomic substrate and ablation outcome for suspected epicardial ventricular tachycardia in left ventricular nonischemic cardiomyopathy. *J Am Coll Cardiol*. 2009;54:799--808.
S4.5.14. Jefic D, Joel B, Good E, et al. Role of radiofrequency catheter ablation of ventricular tachycardia in cardiac sarcoidosis: report from a multicenter registry. *Heart Rhythm*. 2009;6:189--95.
S4.5.15. Naruse Y, Sekiguchi Y, Nogami A, et al. Systematic treatment approach to ventricular tachycardia in cardiac sarcoidosis. *Circ Arrhythm Electrophysiol*. 2014;7:407--13.
S4.5.16. Kumar S, Barbhaiya C, Nagashima K, et al. Ventricular tachycardia in cardiac sarcoidosis: characterization of ventricular substrate and outcomes of catheter ablation. *Circ Arrhythm Electrophysiol*. 2015;8:87--93.
S4.5.17. Muser D, Santangeli P, Pathak RK, et al. Long‐term outcomes of catheter ablation of ventricular tachycardia in patients with cardiac sarcoidosis. *Circ Arrhythm Electrophysiol*. 2016;9:e004333.
S4.5.18. Papageorgiou N, Providência R, Bronis K, et al. Catheter ablation for ventricular tachycardia in patients with cardiac sarcoidosis: a systematic review. *Europace*. 2018;20:682--91.
S4.5.19. Kumar S, Androulakis AF, Sellal JM, et al. Multicenter experience with catheter ablation for ventricular tachycardia in lamin A/C cardiomyopathy. *Circ Arrhythm Electrophysiol*. 2016;9:e004357.
4.6. Ventricular arrhythmia involving the His‐Purkinje system, bundle branch reentrant ventricular tachycardia, and fascicular ventricular tachycardia {#joa312264-sec-0032}
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Recommendations for catheter ablation of bundle branch reentrant VT and for catheter ablation of fascicular VT
CORLOERecommendationsReferencesIB‐NR1. In patients with bundle branch reentrant VT, catheter ablation is useful for reducing the risk of recurrent VT.S4.6.1--S4.6.9IB‐NR2. In patients with idiopathic left fascicular reentrant VT for whom medications are ineffective, not tolerated, or not the patient\'s preference, catheter ablation is useful.S4.6.10--S4.6.22IB‐NR3. In larger pediatric patients (≥15 kg) with idiopathic left fascicular reentrant VT in whom medical treatment is ineffective or not tolerated, catheter ablation is useful.S4.6.23--S4.6.26IB‐NR4. In patients with focal fascicular VT with or without SHD, catheter ablation is useful.S4.6.11, S4.6.27--S4.6.29IB‐NR5. In patients with postinfarction reentrant Purkinje fiber‐mediated VT, catheter ablation is useful.S4.6.30--S4.6.32
References {#joa312264-sec-0033}
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S4.6.1. Cohen TJ, Chien WW, Lurie KG, et al. Radiofrequency catheter ablation for treatment of bundle branch reentrant ventricular tachycardia: results and long‐term follow‐up. *J Am Coll Cardiol*. 1991;18:1767--73.
S4.6.2. Blank Z, Dhala A, Deshpande S, Sra J, Jazayeri M, Akhtar M. Bundle branch reentrant ventricular tachycardia: Cumulative experience in 48 patients. *J Cardiovasc Electrophysiol*. 1993;4:253--62.
S4.6.3. Mehdirad AA, Keim S, Rist K, Tchou P. Long‐term clinical outcome of right bundle branch radiofrequency catheter ablation for treatment of bundle branch reentrant ventricular tachycardia. *Pacing Clin Electrophysiol*. 1995;18(12 Pt 1):2135--43.
S4.6.4. Pathak RK, Fahed J, Santangeli P, et al. Long‐term outcome of catheter ablation for treatment of bundle branch re‐entrant tachycardia. *JACC Clin Electrophysiol*. 2018;4:331--8.
S4.6.5. Narasimhan C, Jazayeri MR, Sra J, et al. Ventricular tachycardia in valvular heart disease: facilitation of bundle‐branch reentry by valve surgery. *Circulation*. 1997;96:4307--13.
S4.6.6. Li YG, Grönefeld G, Israel C, Bogun F, Hohnloser SH. Bundle branch reentrant tachycardia in patients with apparent normal His‐Purkinje conduction: the role of functional conduction impairment. *J Cardiovasc Electrophysiol*. 2002;13:1233--9.
S4.6.7. Schmidt B, Tang M, Chun KR, et al. Left bundle branch‐Purkinje system in patients with bundle branch reentrant tachycardia: lessons from catheter ablation and electroanatomic mapping. *Heart Rhythm*. 2009;6:51--8.
S4.6.8. Blanck Z, Jazayeri M, Dhala A, Deshpande S, Sra J, Akhtar M. Bundle branch reentry: a mechanism of ventricular tachycardia in the absence of myocardial or valvular dysfunction. *J Am Coll Cardiol*. 1993;22:1718--22.
S4.6.9. Chen H, Shi L, Yang B, et al. Electrophysiological characteristics of bundle branch reentry ventricular tachycardia in patients without structural heart disease. *Circ Arrhythm Electrophysiol*. 2018;11:e006049.
S4.6.10. Nogami A, Naito S, Tada H, et al. Demonstration of diastolic and presystolic Purkinje potentials as critical potentials in a macroreentry circuit of verapamil‐sensitive idiopathic left ventricular tachycardia. *J Am Coll Cardiol*. 2000;36:811--23.
S4.6.11. Ouyang F, Cappato R, Ernst S, et al. Electroanatomic substrate of idiopathic left ventricular tachycardia: unidirectional block and macroreentry within the Purkinje network. *Circulation*. 2002;105:462--9.
S4.6.12. Liu Y, Fang Z, Yang B, et al. Catheter ablation of fascicular ventricular tachycardia: long‐term clinical outcomes and mechanisms of recurrence. *Circ Arrhythm Electrophysiol*. 2015;8:1443--51.
S4.6.13. Nakagawa H, Beckman KJ, McClelland JH, et al. Radiofrequency catheter ablation of idiopathic left ventricular tachycardia guided by a Purkinje potential. *Circulation*. 1993;88:2607--17.
S4.6.14. Chen M, Yang B, Zou J, et al. Non‐contact mapping and linear ablation of the left posterior fascicle during sinus rhythm in the treatment of idiopathic left ventricular tachycardia. *Europace*. 2005;7:138--44.
S4.6.15. Kottkamp H, Chen X, Hindricks G, et al. Idiopathic left ventricular tachycardia: new insights into electrophysiological characteristics and radiofrequency catheter ablation. *Pacing Clin Electrophysiol*. 1995;18:1285--97.
S4.6.16. Lin D, Hsia HH, Gerstenfeld EP, et al. Idiopathic fascicular left ventricular tachycardia: linear ablation lesion strategy for noninducible or nonsustained tachycardia. *Heart Rhythm*. 2005;2:934--9.
S4.6.17. Tada H, Nogami A, Naito S, et al. Retrograde Purkinje potential activation during sinus rhythm following catheter ablation of idiopathic left ventricular tachycardia. *J Cardiovasc Electrophysiol*. 1998;9:1218--24.
S4.6.18. Tsuchiya T, Okumura K, Honda T, et al. Significance of late diastolic potential preceding Purkinje potential in verapamil‐sensitive idiopathic left ventricular tachycardia. *Circulation*. 1999;99:2408--13.
S4.6.19. Wen MS, Yeh SJ, Wang CC, Lin FC, Wu D. Successful radiofrequency ablation of idiopathic left ventricular tachycardia at a site away from the tachycardia exit. *J Am Coll Cardiol*. 1997;30:1024--31.
S4.6.20. Arya A, Haghjoo M, Emkanjoo Z, et al. Comparison of presystolic Purkinje and late diastolic potentials for selection of ablation site in idiopathic verapamil sensitive left ventricular tachycardia. *J Interv Card Electrophysiol*. 2004;11:135--41.
S4.6. 21. Liu Q, Shehata M, Jiang R, et al. Macroreentrant loop in ventricular tachycardia from the left posterior fascicle: new implications for mapping and ablation. *Circ Arrhythm Electrophysiol*. 2016;9:e004272.
S4.6.22. Guo XG, Liu X, Zhou GB, et al. Clinical, electrocardiographic, and electrophysiological characteristics of left upper septal fascicular ventricular tachycardia. *Europace*. 2018;20:673--81.
S4.6.23. Collins KK, Schaffer MS, Liberman L, et al. Fascicular and nonfascicular left ventricular tachycardias in the young: an international multicenter study. *J Cardiovasc Electrophysiol*. 2013;24:640--8.
S4.6.24. Suzuki T, Nakamura Y, Yoshida S, et al. Radiofrequency catheter ablation of idiopathic left anterior fascicular ventricular tachycardia in children. *Heart Rhythm*. 2014;11:1948--56.
S4.6.25. Fishberger SB, Olen MM, Rollinson NL, Rossi AF. Creation of partial fascicular block: an approach to ablation of idiopathic left ventricular tachycardia in the pediatric population. *Pacing Clin Electrophysiol*. 2015;38:209--15.
S4.6.26. Saul JP, Kanter RJ, Abrams D, et al. PACES/HRS expert consensus statement on the use of catheter ablation in children and patients with congenital heart disease: developed in partnership with the Pediatric and Congenital Electrophysiology Society (PACES) and the Heart Rhythm Society (HRS). Endorsed by the governing bodies of PACES, HRS, the American Academy of Pediatrics (AAP), the American Heart Association (AHA), and the Association for European Pediatric and Congenital Cardiology (AEPC). *Heart Rhythm*. 2016;13:e251--89.
S4.6.27. Talib AK, Nogami A, Morishima I, et al. Non‐reentrant fascicular tachycardia: clinical and electrophysiological characteristics of a distinct type of idiopathic ventricular tachycardia. *Circ Arrhythm Electrophysiol*. 2016;9:e004177.
S4.6.28. Lopera G, Stevenson WG, Soejima K, et al. Identification and ablation of three types of ventricular tachycardia involving the His‐Purkinje system in patients with heart disease. *J Cardiovasc Electrophysiol*. 2004;15:52--8.
S4.6.29. Gonzalez RP, Scheinman MM, Lesh MD, Helmy I, Torres V, Van Hare GF. Clinical and electrophysiologic spectrum of fascicular tachycardias. *Am Heart J*. 1994;128:147--56.
S4.6.30. Nogami A. Purkinje‐related arrhythmias. Part I: monomorphic ventricular tachycardias. *Pacing Clin Electrophysiol*. 2011;34:624--50.
S4.6.31. Hayashi M, Kobayashi Y, Iwasaki YK, et al. Novel mechanism of postinfarction ventricular tachycardia originating in surviving left posterior Purkinje fibers. *Heart Rhythm*. 2006;3:908--18.
S4.6.32. Bogun F, Good E, Reich S, et al. Role of Purkinje fibers in post‐infarction ventricular tachycardia. *J Am Coll Cardiol*. 2006;48:2500--7.
4.7. Congenital heart disease {#joa312264-sec-0034}
-----------------------------
Recommendations for catheter ablation of VA in patients with CHD
CORLOERecommendationsReferencesIB‐NR1. In patients with CHD presenting with sustained VAs, evaluation for potential residual anatomical or coronary abnormalities should be performed.S4.7.1--S4.7.6IB‐NR2. In patients with CHD presenting with sustained VT in the presence of important hemodynamic lesions, treatment of hemodynamic abnormalities as feasible should be performed in conjunction with consideration for ablation.S4.7.2, S4.7.7--S4.7.16IB‐NR3. In patients with repaired tetralogy of Fallot and sustained monomorphic VT or recurrent appropriate ICD therapy for VAs, catheter ablation is effective.S4.7.17--S4.7.24IIaB‐NR4. In select patients with CHD and clinical episodes of sustained VT who are undergoing surgical repair of residual hemodynamic abnormalities, surgical ablation of VT guided by preoperative or intraoperative electroanatomical mapping (EAM) can be beneficial.S4.7.2, S4.7.8, S4.7.9, S4.7.11, S4.7.25
References {#joa312264-sec-0035}
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S4.7.1. Gatzoulis MA, Till JA, Somerville J, Redington AN. Mechanoelectrical interaction in tetralogy of Fallot. QRS prolongation relates to right ventricular size and predicts malignant ventricular arrhythmias and sudden death. *Circulation*. 1995;92:231--7.
S4.7.2. Harrison DA, Harris L, Siu SC, et al. Sustained ventricular tachycardia in adult patients late after repair of tetralogy of Fallot. *J Am Coll Cardiol*. 1997;30:1368--73.
S4.7.3. Knauth AL, Gauvreau K, Powell AJ, et al. Ventricular size and function assessed by cardiac MRI predict major adverse clinical outcomes late after tetralogy of Fallot repair. *Heart*. 2008;94:211--6.
S4.7.4. Diller GP, Kempny A, Liodakis E, et al. Left ventricular longitudinal function predicts life‐threatening ventricular arrhythmia and death in adults with repaired tetralogy of Fallot. *Circulation*. 2012;125:2440--6.
S4.7.5. Koyak Z, Harris L, de Groot JR, et al. Sudden cardiac death in adult congenital heart disease. *Circulation*. 2012;126:1944--54.
S4.7.6. Koyak Z, de Groot JR, Bouma BJ, et al. Sudden cardiac death in adult congenital heart disease: can the unpredictable be foreseen? *Europace*. 2017;19:401--6.
S4.7.7. Deal BJ, Scagliotti D, Miller SM, Gallastegui JL, Hariman RJ, Levitsky S. Electrophysiologic drug testing in symptomatic ventricular arrhythmias after repair of tetralogy of Fallot. *Am J Cardiol*. 1987;59:1380--5.
S4.7.8. Oechslin EN, Harrison DA, Harris L, et al. Reoperation in adults with repair of tetralogy of Fallot: indications and outcomes. *J Thorac Cardiovasc Surg*. 1999;118:245--51.
S4.7.9. Therrien J, Siu SC, Harris L, et al. Impact of pulmonary valve replacement on arrhythmia propensity late after repair of tetralogy of Fallot. *Circulation*. 2001;103:2489--94.
S4.7.10. Therrien J, Provost Y, Merchant N, Williams W, Colman J, Webb G. Optimal timing for pulmonary valve replacement in adults after tetralogy of Fallot repair. *Am J Cardiol*. 2005;95:779--82.
S4.7.11. Mavroudis C, Deal BJ, Backer CL, Tsao S. Arrhythmia surgery in patients with and without congenital heart disease. *Ann Thorac Surg*. 2008;86:857--68.
S4.7.12. Adamson L, Vohra HA, Haw MP. Does pulmonary valve replacement post repair of tetralogy of Fallot improve right ventricular function? *Interact Cardiovasc Thorac Surg*. 2009;9:520--7.
S4.7.13. Miyazaki A, Sakaguchi H, Ohuchi H, et al. Efficacy of hemodynamic‐based management of tachyarrhythmia after repair of tetralogy of Fallot. *Circ J*. 2012;76:2855--62.
S4.7.14. Khairy P, Van Hare GF, Balaji S, et al. PACES/HRS expert consensus statement on the recognition and management of arrhythmias in adult congenital heart disease: developed in partnership between the Pediatric and Congenital Electrophysiology Society (PACES) and the Heart Rhythm Society (HRS). Endorsed by the governing bodies of PACES, HRS, the American College of Cardiology (ACC), the American Heart Association (AHA), the European Heart Rhythm Association (EHRA), the Canadian Heart Rhythm Society (CHRS), and the International Society for Adult Congenital Heart Disease (ISACHD). *Heart Rhythm*. 2014;11:e102--65.
S4.7.15. Lin YS, Liu PH, Wu LS, Chen YM, Chang CJ, Chu PH. Major adverse cardiovascular events in adult congenital heart disease: a population‐based follow‐up study from Taiwan. *BMC Cardiovasc Disord*. 2014;14:38.
S4.7.16. Sabate Rotes A, Connolly HM, Warnes CA, et al. Ventricular arrhythmia risk stratification in patients with tetralogy of Fallot at the time of pulmonary valve replacement. *Circ Arrhythm Electrophysiol*. 2015;8:110--6.
S4.7.17. Gonska BD, Cao K, Raab J, Eigster G, Kreuzer H. Radiofrequency catheter ablation of right ventricular tachycardia late after repair of congenital heart defects. *Circulation*. 1996;94:1902--8.
S4.7.18. Morwood JG, Triedman JK, Berul CI, et al. Radiofrequency catheter ablation of ventricular tachycardia in children and young adults with congenital heart disease. *Heart Rhythm*. 2004;1:301--8.
S4.7.19. Zeppenfeld K, Schalij MJ, Bartelings MM, et al. Catheter ablation of ventricular tachycardia after repair of congenital heart disease: electroanatomic identification of the critical right ventricular isthmus. *Circulation*. 2007;116:2241--52.
S4.7.20. Kriebel T, Saul JP, Schneider H, Sigler M, Paul T. Noncontact mapping and radiofrequency catheter ablation of fast and hemodynamically unstable ventricular tachycardia after surgical repair of tetralogy of Fallot. *J Am Coll Cardiol*. 2007;50:2162--8.
S4.7.21. Kapel GF, Reichlin T, Wijnmaalen AP, et al. Left‐sided ablation of ventricular tachycardia in adults with repaired tetralogy of Fallot: a case series. *Circ Arrhythm Electrophysiol*. 2014;7:889--97.
S4.7.22. Kapel GF, Reichlin T, Wijnmaalen AP, et al. Re‐entry using anatomically determined isthmuses: a curable ventricular tachycardia in repaired congenital heart disease. *Circ Arrhythm Electrophysiol*. 2015;8:102--9.
S4.7.23. van Zyl M, Kapa S, Padmanabhan D, et al. Mechanism and outcomes of catheter ablation for ventricular tachycardia in adults with repaired congenital heart disease. *Heart Rhythm*. 2016;13:1449--54.
S4.7.24. Kapel GF, Sacher F, Dekkers OM, et al. Arrhythmogenic anatomical isthmuses identified by electroanatomical mapping are the substrate for ventricular tachycardia in repaired tetralogy of Fallot. *Eur Heart J*. 2017;38:268--76.
S4.7.25. Sandhu A, Ruckdeschel E, Sauer WH, et al. Perioperative electrophysiology study in patients with tetralogy of Fallot undergoing pulmonary valve replacement will identify those at high risk of subsequent ventricular tachycardia. *Heart Rhythm*. 2018;15:679--85.
S4.7.26. Tracy CM, Epstein AE, Darbar D, et al. 2012 ACCF/AHA/HRS focused update of the 2008 guidelines for device‐based therapy of cardiac rhythm abnormalities: a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines. *Heart Rhythm*. 2012;9:1737--53.
4.8. Inherited arrhythmia syndromes {#joa312264-sec-0036}
-----------------------------------
Recommendations for catheter ablation of VA in inherited primary arrhythmia disorders
CORLOERecommendationsReferencesIB‐NR1. In patients with arrhythmogenic right ventricular cardiomyopathy (ARVC) who experience recurrent sustained VT or frequent appropriate ICD interventions for VT in whom AAD therapy is ineffective or not tolerated, catheter ablation, at a center with specific expertise, is recommended.S4.8.1--S4.8.11IB‐NR2. In patients with ARVC who have failed one or more attempts of endocardial VT catheter ablation, an epicardial approach for VT ablation is recommended.S4.8.3--S4.8.7, S4.8.12, S4.8.13IIaB‐NR3. In patients with ARVC who experience recurrent sustained VT or frequent appropriate ICD interventions for VT in whom AAD therapy is not desired or preferred, catheter ablation, at a center with specific expertise, is reasonable.S4.8.1, S4.8.3--S4.8.6, S4.8.8IIaB‐NR4. In patients with Brugada syndrome who experience recurrent sustained VAs or frequent appropriate ICD interventions, catheter ablation can be useful.S4.8.14--S4.8.17IIaC‐LD5. In patients with ARVC, a first‐line combined endocardial/epicardial approach for VT ablation is reasonable.S4.8.1, S4.8.6, S4.8.12, S4.8.18
References {#joa312264-sec-0037}
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S4.8.1. Berruezo A, Acosta J, Fernández‐Armenta J, et al. Safety, long‐term outcomes and predictors of recurrence after first‐line combined endoepicardial ventricular tachycardia substrate ablation in arrhythmogenic cardiomyopathy. Impact of arrhythmic substrate distribution pattern. A prospective multicentre study. *Europace*. 2017;19:607--16.
S4.8.2. Jiang H, Zhang X, Yang Q, et al. Catheter ablation for ventricular tachycardia in patients with arrhythmogenic right ventricular dysplasia/cardiomyopathy: a systematic review and meta‐analysis. *Acta Cardiol*. 2016;71:639--49.
S4.8.3. Philips B, te Riele AS, Sawant A, et al. Outcomes and ventricular tachycardia recurrence characteristics after epicardial ablation of ventricular tachycardia in arrhythmogenic right ventricular dysplasia/cardiomyopathy. *Heart Rhythm*. 2015;12:716--25.
S4.8.4. Santangeli P, Zado ES, Supple GE, et al. Long‐term outcome with catheter ablation of ventricular tachycardia in patients with arrhythmogenic right ventricular cardiomyopathy. *Circ Arrhythm Electrophysiol*. 2015;8:1413--21.
S4.8.5. Philips B, Madhavan S, James C, et al. Outcomes of catheter ablation of ventricular tachycardia in arrhythmogenic right ventricular dysplasia/cardiomyopathy. *Circ Arrhythm Electrophysiol*. 2012;5:499--505.
S4.8.6. Bai R, Di Biase L, Shivkumar K, et al. Ablation of ventricular arrhythmias in arrhythmogenic right ventricular dysplasia/cardiomyopathy: arrhythmia‐free survival after endo‐epicardial substrate based mapping and ablation. *Circ Arrhythm Electrophysiol*. 2011;4:478--85.
S4.8.7. Garcia FC, Bazan V, Zado ES, Ren JF, Marchlinski FE. Epicardial substrate and outcome with epicardial ablation of ventricular tachycardia in arrhythmogenic right ventricular cardiomyopathy/dysplasia. *Circulation*. 2009;120:366--75.
S4.8.8. Dalal D, Jain R, Tandri H, et al. Long‐term efficacy of catheter ablation of ventricular tachycardia in patients with arrhythmogenic right ventricular dysplasia/cardiomyopathy. *J Am Coll Cardiol*. 2007;50:432--40.
S4.8.9. Verma A, Kilicaslan F, Schweikert RA, et al. Short‐ and long‐term success of substrate‐based mapping and ablation of ventricular tachycardia in arrhythmogenic right ventricular dysplasia. *Circulation*. 2005;111:3209--16.
S4.8.10. Marchlinski FE, Zado E, Dixit S, et al. Electroanatomic substrate and outcome of catheter ablative therapy for ventricular tachycardia in setting of right ventricular cardiomyopathy. *Circulation*. 2004;110:2293--8.
S4.8.11. Nogami A, Sugiyasu A, Tada H, et al. Changes in the isolated delayed component as an endpoint of catheter ablation in arrhythmogenic right ventricular cardiomyopathy: predictor for long‐term success. *J Cardiovasc Electrophysiol*. 2008;19:681--8.
S4.8.12. Müssigbrodt A, Efimova E, Knopp H, et al. Should all patients with arrhythmogenic right ventricular dysplasia/cardiomyopathy undergo epicardial catheter ablation? *J Interv Card Electrophysiol*. 2017;48:193--9.
S4.8.13. Pokushalov E, Romanov A, Turov A, Artyomenko S, Shirokova N, Karaskov A. Percutaneous epicardial ablation of ventricular tachycardia after failure of endocardial approach in the pediatric population with arrhythmogenic right ventricular dysplasia. *Heart Rhythm*. 2010;7:1406--10.
S4.8.14. Pappone C, Brugada J, Vicedomini G, et al. Electrical substrate elimination in 135 consecutive patients with Brugada syndrome. *Circ Arrhythm Electrophysiol*. 2017;10:e005053.
S4.8.15. Brugada J, Pappone C, Berruezo A, et al. Brugada syndrome phenotype elimination by epicardial substrate ablation. *Circ Arrhythm Electrophysiol*. 2015;8:1373--81.
S4.8.16. Zhang P, Tung R, Zhang Z, et al. Characterization of the epicardial substrate for catheter ablation of Brugada syndrome. *Heart Rhythm*. 2016;13:2151--8.
S4.8.17. Nademanee K, Veerakul G, Chandanamattha P, et al. Prevention of ventricular fibrillation episodes in Brugada syndrome by catheter ablation over the anterior right ventricular outflow tract epicardium. *Circulation*. 2011;123:1270--9.
S4.8.18. Berruezo A, Fernández‐Armenta J, Mont L, et al. Combined endocardial and epicardial catheter ablation in arrhythmogenic right ventricular dysplasia incorporating scar dechanneling technique. *Circ Arrhythm Electrophysiol*. 2012;5:111--21.
4.9. Ventricular arrhythmia in hypertrophic cardiomyopathy {#joa312264-sec-0038}
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Recommendation for VA ablation in hypertrophic cardiomyopathy (HCM)
CORLOERecommendationReferencesIIaB‐NR1. In patients with HCM and recurrent monomorphic VT in whom AAD therapy is ineffective or not tolerated, catheter ablation can be useful.S4.9.1--S4.9.5
References {#joa312264-sec-0039}
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S4.9.1. Dukkipati SR, d\'Avila A, Soejima K, et al. Long‐term outcomes of combined epicardial and endocardial ablation of monomorphic ventricular tachycardia related to hypertrophic cardiomyopathy. *Circ Arrhythm Electrophysiol*. 2011;4:185--94.
S4.9.2. Santangeli P, Di Biase L, Lakkireddy D, et al. Radiofrequency catheter ablation of ventricular arrhythmias in patients with hypertrophic cardiomyopathy: safety and feasibility. *Heart Rhythm*. 2010;7:1036--42.
S4.9.3. Ueda A, Fukamizu S, Soejima K, et al. Clinical and electrophysiological characteristics in patients with sustained monomorphic reentrant ventricular tachycardia associated with dilated‐phase hypertrophic cardiomyopathy. *Europace*. 2012;14:734--40.
S4.9.4. Inada K, Seiler J, Roberts‐Thomson KC, et al. Substrate characterization and catheter ablation for monomorphic ventricular tachycardia in patients with apical hypertrophic cardiomyopathy. *J Cardiovasc Electrophysiol*. 2011;22:41--8.
S4.9.5. Igarashi M, Nogami A, Kurosaki K, et al. Radiofrequency catheter ablation of ventricular tachycardia in patients with hypertrophic cardiomyopathy and apical aneurysm. *JACC Clin Electrophysiol*. 2018;4:339--50.
5. PROCEDURAL PLANNING {#joa312264-sec-0040}
======================
This section includes preprocedural risk assessment (Table [4](#joa312264-tbl-0004){ref-type="table"}), preprocedural patient preparation, and preprocedural arrhythmia documentation with a focus on the regionalizing information of the ECG regarding the origin of VAs (Figures [3](#joa312264-fig-0003){ref-type="fig"} and [4](#joa312264-fig-0004){ref-type="fig"}). Furthermore, the capabilities of multimodality imaging in localizing the arrhythmogenic substrate are discussed in detail. Topics including the required equipment, personnel, and facility are detailed in this section.
######
The PAAINESD Score, developed to predict the risk of periprocedural hemodynamic decompensation
Variable Points
------------------------------ --------
**P**ulmonary disease (COPD) 5
**A**ge \>60 3
General **a**nesthesia 4
**I**schemic cardiomyopathy 6
**N**YHA class III/IV 6
**E**F \<25% 3
VT **s**torm 5
**D**iabetes mellitus 3
The PAAINESD Score, developed to predict the risk of periprocedural hemodynamic decompensation, has values that range from 0 to 35 points (or 0 to 31 \[PAINESD\] when the modifiable intraprocedural variable "general anesthesia" is excluded) (Santangeli et al. *Circ Arrhythm Electrophysiol*. 2015;8:68--75).
Abbreviations: COPD, chronic obstructive pulmonary disease; EF, ejection fraction; NYHA, New York Heart Association; VT, ventricular tachycardia.
John Wiley & Sons, Ltd
![Examples of 12‐lead ECGs of premature ventricular complexes from different LV sites, as corroborated by successful focal ablation. (A) shows 12‐lead ECG patterns of common ventricular arrhythmia origins in patients without SHD \[1‐9\] from the left ventricle. All leads are displayed at the same amplification and sweep speed. These locations are illustrated in (B) based on 3D reconstruction of a cardiac computed tomography using the MUSIC software that was developed at the University of Bordeaux. The reconstruction shows an anterolateral view of the left ventricle, aorta, and left atrium. Also shown are the coronary arteries (red), the coronary venous system (blue), and the phrenic nerve (green). Abbreviations: AIV, anterior interventricular vein; AL PAP, anterolateral papillary muscle; AMC, aortomitral continuity; ECG, electrocardiogram; GCV, great cardiac vein; ant. MA, anterior mitral valve annulus; PM PAP, posteromedial papillary muscle; R/L, right‐left; SHD, structural heart disease; SoV, sinus of Valsalva](JOA3-36-1-g003){#joa312264-fig-0003}
![Examples of 12‐lead ECGs of premature ventricular complexes from different right ventricular sites, as corroborated by successful focal ablation. All leads are displayed at the same amplification and sweep speed. (A) shows the 12‐lead ECG pattern of common origins of right ventricular arrhythmias in patients without SHD \[1‐6\]. The locations are detailed in a 3D reconstruction of the computed tomography using the MUSIC software that was developed at the University of Bordeaux. The reconstruction shown in (B) illustrates the septal view of the right ventricle. Indicated are the pulmonary artery, the tricuspid valve annulus, and the right ventricular apex. Abbreviations: ECG, electrocardiogram; PA, pulmonary artery; RVOT, right ventricular outflow tract; SHD, structural heart disease; TVA, tricuspid valve annulus](JOA3-36-1-g004){#joa312264-fig-0004}
Recommendations for preprocedural imaging for VA catheter ablation
CORLOERecommendationsReferencesIB‐NR1. In patients with LV dysfunction undergoing catheter ablation of VA, preprocedural or intraprocedural imaging is recommended to rule out cardiac thrombi.S5.1--S5.6IIaB‐NR2. In patients with NICM or ischemic cardiomyopathy (ICM) undergoing catheter ablation of VT, preprocedural CMR can be useful to reduce VT recurrence.S5.7--S5.9IIaB‐NR3. In patients with NICM or ICM undergoing catheter ablation of VA, preprocedural imaging can be useful for procedural planning.S5.10--S5.26IIaC‐EO4. In patients with NICM, CMR can be useful prior to ICD implantation to allow imaging without device‐related artifact for diagnostic purposes and identification of potential arrhythmogenic substrate.IIbC‐EO5. In patients with ICM, CMR may be considered prior to ICD implantation to allow imaging without device‐related artifact for identification of the potential arrhythmogenic substrate.
References {#joa312264-sec-0041}
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S5.1. Visser CA, Kan G, David GK, Lie KI, Durrer D. Two dimensional echocardiography in the diagnosis of left ventricular thrombus: a prospective study of 67 patients with anatomic validation. *Chest*. 1983;83:228--32.
S5.2. Ezekowitz MD, Wilson DA, Smith EO, et al. Comparison of Indium‐111 platelet scintigraphy and two‐dimensional echocardiography in the diagnosis of left ventricular thrombi. *N Engl J Med*. 1982;306:1509--13.
S5.3. Stratton JR, Lighty Jr. GW, Pearlman AS, Ritchie JL. Detection of left ventricular thrombus by two‐dimensional echocardiography: sensitivity, specificity, and causes of uncertainty. *Circulation*. 1982;66:156--66.
S5.4. Thanigaraj S, Schechtman KB, Perez JE. Improved echocardiographic delineation of left ventricular thrombus with the use of intravenous second‐generation contrast image enhancement. *J Am Soc Echocardiogr*. 1999;12:1022--6.
S5.5. Weinsaft JW, Kim HW, Shah DJ, et al. Detection of left ventricular thrombus by delayed‐enhancement cardiovascular magnetic resonance prevalence and markers in patients with systolic dysfunction. *J Am Coll Cardiol*. 2008;52:148--57.
S5.6. Weinsaft JW, Kim RJ, Ross M, et al. Contrast‐enhanced anatomic imaging as compared to contrast‐enhanced tissue characterization for detection of left ventricular thrombus. *JACC Cardiovasc Imaging*. 2009;2:969--79.
S5.7. Siontis KC, Kim HM, Sharaf Dabbagh G, et al. Association of preprocedural cardiac magnetic resonance imaging with outcomes of ventricular tachycardia ablation in patients with idiopathic dilated cardiomyopathy. *Heart Rhythm*. 2017;14:1487--93.
S5.8. Zghaib T, Ipek EG, Hansford R, et al. Standard ablation versus magnetic resonance imaging‐guided ablation in the treatment of ventricular tachycardia. *Circ Arrhythm Electrophysiol*. 2018;11:e005973.
S5.9. Andreu D, Penela D, Acosta J, et al. Cardiac magnetic resonance‐aided scar dechanneling: Influence on acute and long‐term outcomes. *Heart Rhythm*. 2017;14:1121--8.
S5.10. Codreanu A, Odille F, Aliot E, et al. Electroanatomic characterization of post‐infarct scars comparison with 3‐dimensional myocardial scar reconstruction based on magnetic resonance imaging. *J Am Coll Cardiol*. 2008;52:839--42.
S5.11. Desjardins B, Crawford T, Good E, et al. Infarct architecture and characteristics on delayed enhanced magnetic resonance imaging and electroanatomic mapping in patients with postinfarction ventricular arrhythmia. *Heart Rhythm*. 2009;6:644--51.
S5.12. Bogun FM, Desjardins B, Good E, et al. Delayed‐enhanced magnetic resonance imaging in nonischemic cardiomyopathy: utility for identifying the ventricular arrhythmia substrate. *J Am Coll Cardiol*. 2009;53:1138--45.
S5.13. Dickfeld T, Tian J, Ahmad G, et al. MRI‐guided ventricular tachycardia ablation: integration of late gadolinium‐enhanced 3D scar in patients with implantable cardioverter‐defibrillators. *Circ Arrhythm Electrophysiol*. 2011;4:172--84.
S5.14. Fernandez‐Armenta J, Berruezo A, Andreu D, et al. Three‐dimensional architecture of scar and conducting channels based on high resolution ce‐CMR: insights for ventricular tachycardia ablation. *Circ Arrhythm Electrophysiol*. 2013;6:528--37.
S5.15. Gupta S, Desjardins B, Baman T, et al. Delayed‐enhanced MR scar imaging and intraprocedural registration into an electroanatomical mapping system in post‐infarction patients. *JACC Cardiovasc Imaging*. 2012;5:207--10.
S5.16. Marra MP, Leoni L, Bauce B, et al. Imaging study of ventricular scar in arrhythmogenic right ventricular cardiomyopathy: comparison of 3D standard electroanatomical voltage mapping and contrast‐enhanced cardiac magnetic resonance. *Circ Arrhythm Electrophysiol*. 2012;5:91--100.
S5.17. Nakahara S, Vaseghi M, Ramirez RJ, et al. Characterization of myocardial scars: electrophysiological imaging correlates in a porcine infarct model. *Heart Rhythm*. 2011;8:1060--7.
S5.18. Ghannam M, Cochet H, Jais P, et al. Correlation between computer tomography‐derived scar topography and critical ablation sites in postinfarction ventricular tachycardia. *J Cardiovasc Electrophysiol*. 2018;29:438--45.
S5.19. Esposito A, Palmisano A, Antunes S, et al. Cardiac CT with delayed enhancement in the characterization of ventricular tachycardia structural substrate: relationship between CT‐segmented scar and electro‐anatomic mapping. *JACC Cardiovasc Imaging*. 2016;9:822--32.
S5.20. Tian J, Jeudy J, Smith MF, et al. Three‐dimensional contrast‐enhanced multidetector CT for anatomic, dynamic, and perfusion characterization of abnormal myocardium to guide ventricular tachycardia ablations. *Circ Arrhythm Electrophysiol*. 2010;3:496--504.
S5.21. Yamashita S, Sacher F, Mahida S, et al. Image integration to guide catheter ablation in scar‐related ventricular tachycardia. *J Cardiovasc Electrophysiol*. 2016;27:699--708.
S5.22. Komatsu Y, Cochet H, Jadidi A, et al. Regional myocardial wall thinning at multidetector computed tomography correlates to arrhythmogenic substrate in postinfarction ventricular tachycardia: assessment of structural and electrical substrate. *Circ Arrhythm Electrophysiol*. 2013;6:342--50.
S5.23. Dickfeld T, Lei P, Dilsizian V, et al. Integration of three‐dimensional scar maps for ventricular tachycardia ablation with positron emission tomography‐computed tomography. *JACC Cardiovasc Imaging*. 2008;1:73--82.
S5.24. Tian J, Smith MF, Chinnadurai P, et al. Clinical application of PET/CT fusion imaging for three‐dimensional myocardial scar and left ventricular anatomy during ventricular tachycardia ablation. *J Cardiovasc Electrophysiol*. 2009;20:567--604.
S5.25. Andreu D, Ortiz‐Perez JT, Boussy T, et al. Usefulness of contrast‐enhanced cardiac magnetic resonance in identifying the ventricular arrhythmia substrate and the approach needed for ablation. *Eur Heart J*. 2014;35:1316--26.
S5.26. Soto‐Iglesias D, Acosta J, Penela D, et al. Image‐based criteria to identify the presence of epicardial arrhythmogenic substrate in patients with transmural myocardial infarction. *Heart Rhythm*. 2018;15:814--21.
6. INTRAPROCEDURAL PATIENT CARE {#joa312264-sec-0042}
===============================
Important aspects regarding intraprocedural sedation and its potential problems are highlighted in this section. Furthermore, vascular access, epicardial access with its many potential complications are discussed in detail, as well as anticoagulation and the indications for the use of hemodynamic support (HS) during VT ablation procedures.
6.1. Anesthesia {#joa312264-sec-0043}
---------------
Recommendations for anesthesia during catheter ablation of VA
CORLOERecommendationsReferencesIC‐EO1. Provision of variable depth of sedation, analgesia, and anesthesia during mapping and ablation of VA is recommended.IC‐EO2. In patients undergoing VA ablation, careful preprocedural assessment is indicated to define the ideal strategy for sedation and analgesia.IIaC‐LD3. It is reasonable to avoid general anesthesia and deeper levels of sedation in patients with idiopathic VA, particularly if the arrhythmia is suspected to be catecholamine‐sensitive or was not inducible at a prior procedure.S6.1.1IIbB‐NR4. Moderate to deep sedation under close hemodynamic and respiratory monitoring might be considered for VA ablation in stable patients with idiopathic or scar‐related VAs expected to have a longer procedure or undergo a painful technique, such as epicardial access.S6.1.1--S6.1.3
References {#joa312264-sec-0044}
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S6.1.1. Wutzler A, Mueller A, Loehr L, et al. Minimal and deep sedation during ablation of ventricular tachycardia. *Int J Cardiol*. 2014;172:161--4.
S6.1.2. Servatius H, Höfeler T, Hoffmann BA, et al. Propofol sedation administered by cardiologists for patients undergoing catheter ablation for ventricular tachycardia. *Europace*. 2016;18:1245--51.
S6.1.3. Nazer B, Woods C, Dewland T, Moyers B, Badhwar N, Gerstenfeld EP. Importance of ventricular tachycardia induction and mapping for patients referred for epicardial ablation. *Pacing Clin Electrophysiol*. 2015;38:1333--42.
6.2. Vascular access {#joa312264-sec-0045}
--------------------
Recommendation for vascular access during catheter ablation of VA
CORLOERecommendationReferencesIB‐NR1. Ultrasound‐guided femoral arterial and venous access is recommended to reduce the incidence of vascular access complications during VA ablation.S6.2.1--S6.2.5
References {#joa312264-sec-0046}
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S6.2.1. Sharma PS, Padala SK, Gunda S, Koneru JN, Ellenbogen KA. Vascular complications during catheter ablation of cardiac arrhythmias: a comparison between vascular ultrasound guided access and conventional vascular access. *J Cardiovasc Electrophysiol*. 2016;27:1160--6.
S6.2.2. Tanaka‐Esposito CC, Chung MK, Abraham JM, Cantillon DJ, Abi‐Saleh B, Tchou PJ. Real‐time ultrasound guidance reduces total and major vascular complications in patients undergoing pulmonary vein antral isolation on therapeutic warfarin. *J Interv Card Electrophysiol*. 2013;37:163--8.
S6.2.3. Yamagata K, Wichterle D, Roubíček T, et al. Ultrasound‐guided versus conventional femoral venipuncture for catheter ablation of atrial fibrillation: a multicentre randomized efficacy and safety trial (ULTRA‐FAST trial). *Europace*. 2018;20:1107--14.
S6.2.4. Sobolev M, Shiloh AL, Di Biase L, Slovut DP. Ultrasound‐guided cannulation of the femoral vein in electrophysiological procedures: a systematic review and meta‐analysis. *Europace*. 2017;19:850--5.
S6.2.5. Seto AH, Abu‐Fadel MS, Sparling JM, et al. Real‐time ultrasound guidance facilitates femoral arterial access and reduces vascular complications: FAUST (Femoral Arterial Access With Ultrasound Trial). *JACC Cardiovasc Interv*. 2010;3:751--8.
6.3. Epicardial access {#joa312264-sec-0047}
----------------------
Recommendations for epicardial access for catheter ablation
CORLOERecommendationsIC‐EO1. In patients undergoing epicardial VT ablation, imaging of the epicardial coronary arteries by coronary arteriography or coronary computed tomography angiogram prior to ablation is recommended to reduce the risk of arterial injury.IC‐EO2. In patients undergoing epicardial VT ablation via a percutaneous approach, provision for immediate echocardiography, blood transfusion, and onsite cardiothoracic surgical backup is recommended.IC‐EO3. In patients with prior cardiac surgery or pericardial adhesions for whom epicardial VT ablation via a percutaneous approach is considered, careful assessment of the risk/benefit ratio and alternative therapies such as surgical dissection are recommended.IC‐EO4. In patients undergoing epicardial VT ablation, pacing with high stimulus intensity from the ablation electrode to rule out diaphragmatic stimulation is recommended to avoid phrenic nerve injury.
6.4. Intraprocedural hemodynamic support {#joa312264-sec-0048}
----------------------------------------
Recommendations for catheter ablation of VA with mechanical HS
CORLOERecommendationsReferencesIC‐EO1. In select patients at risk of requiring HS, a decision to proceed with catheter ablation of VA should be made in collaboration with specialists in advanced heart failure management.IIaB‐NR2. In select patients, HS with a percutaneous ventricular assist device and extracorporeal membrane oxygenation during VT ablation can be useful to avoid acute hemodynamic deterioration.S6.4.1--S6.4.7IIbB‐NR3. Mechanical HS may be considered in select cases to allow mapping and ablation of unstable VTs.S6.4.1--S6.4.6
References {#joa312264-sec-0049}
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S6.4.1. Miller MA, Dukkipati SR, Mittnacht AJ, et al. Activation and entrainment mapping of hemodynamically unstable ventricular tachycardia using a percutaneous left ventricular assist device. *J Am Coll Cardiol*. 2011;58:1363--71.
S6.4.2. Reddy YM, Chinitz L, Mansour M, et al. Percutaneous left ventricular assist devices in ventricular tachycardia ablation: multicenter experience. *Circ Arrhythm Electrophysiol*. 2014;7:244--50.
S6.4.3. Baratto F, Pappalardo F, Oloriz T, et al. Extracorporeal membrane oxygenation for hemodynamic support of ventricular tachycardia ablation. *Circ Arrhythm Electrophysiol*. 2016;9:e004492.
S6.4.4. Kusa S, Miller MA, Whang W, et al. Outcomes of ventricular tachycardia ablation using percutaneous left ventricular assist devices. *Circ Arrhythm Electrophysiol*. 2017;10:e004717.
S6.4.5. Mathuria N, Wu G, Rojas‐Delgado F, et al. Outcomes of pre‐emptive and rescue use of percutaneous left ventricular assist device in patients with structural heart disease undergoing catheter ablation of ventricular tachycardia. *J Interv Card Electrophysiol*. 2017;48:27--34.
S6.4.6. Turagam MK, Vuddanda V, Atkins D, et al. Hemodynamic support in ventricular tachycardia ablation: an International VT Ablation Center Collaborative Group Study. *JACC Clin Electrophysiol*. 2017;3:1534--43.
S6.4.7. Enriquez A, Liang J, Gentile J, et al. Outcomes of rescue cardiopulmonary support for periprocedural acute hemodynamic decompensation in patients undergoing catheter ablation of electrical storm. *Heart Rhythm*. 2018;15:75--80.
6.5. Intraprocedural anticoagulation {#joa312264-sec-0050}
------------------------------------
Recommendations for intraprocedural anticoagulation
CORLOERecommendationsReferencesIB‐NR1. In patients undergoing endocardial LV catheter mapping and/or ablation, intraprocedural systemic anticoagulation with intravenous heparin is recommended.S6.5.1--S6.5.6IC‐EO2. In patients undergoing RV endocardial mapping and/or ablation who are considered high risk for thromboembolism, intraprocedural systemic anticoagulation with intravenous heparin is recommended.IIaC‐LD3. In patients undergoing epicardial access after systemic heparinization, reversal of heparin with protamine is reasonable.S6.5.7, S6.5.8
References {#joa312264-sec-0051}
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S6.5.1. Reddy VY, Reynolds MR, Neuzil P, et al. Prophylactic catheter ablation for the prevention of defibrillator therapy. *N Engl J Med*. 2007;357:2657--65.
S6.5.2. Kuck KH, Schaumann A, Eckardt L, et al; VTACH Study Group. Catheter ablation of stable ventricular tachycardia before defibrillator implantation in patients with coronary heart disease (VTACH): a multicentre randomised controlled trial. *Lancet*. 2010;375:31--40.
S6.5.3. Al‐Khatib SM, Daubert JP, Anstrom KJ, et al. Catheter ablation for ventricular tachycardia in patients with an implantable cardioverter defibrillator (CALYPSO) pilot trial. *J Cardiovasc Electrophysiol*. 2015;26:151--7.
S6.5.4. Sapp JL, Wells GA, Parkash R, et al. Ventricular tachycardia ablation versus escalation of antiarrhythmic drugs. *N Engl J Med*. 2016;375:111--21.
S6.5.5. Kuck KH, Tilz RR, Deneke T, et al; SMS Investigators. Impact of substrate modification by catheter ablation on implantable cardioverter‐defibrillator interventions in patients with unstable ventricular arrhythmias and coronary artery disease: results from the multicenter randomized controlled SMS (Substrate Modification Study). *Circ Arrhythm Electrophysiol*. 2017;10:e004422.
S6.5.6. Calkins H, Epstein A, Packer D, et al. Catheter ablation of ventricular tachycardia in patients with structural heart disease using cooled radiofrequency energy: results of a prospective multicenter study. Cooled RF Multi Center Investigators Group. *J Am Coll Cardiol*. 2000;35:1905--14.
S6.5.7. Siontis KC, Jamé S, Sharaf Dabbagh G, et al. Thromboembolic prophylaxis protocol with warfarin after radiofrequency catheter ablation of infarct‐related ventricular tachycardia. *J Cardiovasc Electrophysiol*. 2018;29:584--90.
S6.5.8. Tanner H, Hindricks G, Volkmer M, et al. Catheter ablation of recurrent scar‐related ventricular tachycardia using electroanatomical mapping and irrigated ablation technology: results of the prospective multicenter Euro‐VT‐study. *J Cardiovasc Electrophysiol*. 2010;21:47--53.
7. ELECTROPHYSIOLOGICAL TESTING {#joa312264-sec-0052}
===============================
The benefits and limitations of PES are detailed in this section.
8. MAPPING AND IMAGING TECHNIQUES {#joa312264-sec-0053}
=================================
8.1. Overview {#joa312264-sec-0054}
-------------
Activation mapping with multipolar catheters, entrainment mapping (Figures [5](#joa312264-fig-0005){ref-type="fig"} and [6](#joa312264-fig-0006){ref-type="fig"}), and pace mapping are the main techniques used to map VAs. This section reviews these techniques including the technique of substrate mapping aiming to identify the arrhythmogenic substrate in sinus rhythm. Furthermore, intraprocedural imaging as it pertains to procedural safety and to identification of the arrhythmogenic substrate is reviewed in this section.
{#joa312264-fig-0005}
{#joa312264-fig-0006}
8.2. Substrate mapping in sinus rhythm {#joa312264-sec-0055}
--------------------------------------
Recommendations for substrate mapping in sinus rhythm
CORLOERecommendationsReferencesIB‐NR1. In patients with scar‐related VT, substrate‐guided ablation is useful for prevention of arrhythmia recurrences.S8.2.1--S8.2.11IIaB‐NR2. High‐density multielectrode mapping to obtain a more comprehensive characterization of the arrhythmogenic tissue during catheter ablation of scar‐related VT can be useful.S8.2.12--S8.2.14IIaB‐NR3. In patients with no or minimal endocardial bipolar electrogram abnormalities, reduced unipolar voltage can be useful for detection of epicardial or intramural scar.S8.2.15--S8.2.19
References {#joa312264-sec-0056}
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S8.2.1. Reddy VY, Reynolds MR, Neuzil P, et al. Prophylactic catheter ablation for the prevention of defibrillator therapy. *N Engl J Med*. 2007;357:2657--65.
S8.2.2. Di Biase L, Burkhardt JD, Lakkireddy D, et al. Ablation of stable VTs versus substrate ablation in ischemic cardiomyopathy: the VISTA randomized multicenter trial. *J Am Coll Cardiol*. 2015;66:2872--82.
S8.2.3. Calkins H, Epstein A, Packer D, et al. Catheter ablation of ventricular tachycardia in patients with structural heart disease using cooled radiofrequency energy: results of a prospective multicenter study. Cooled RF Multi Center Investigators Group. *J Am Coll Cardiol*. 2000;35:1905--14.
S8.2.4. Stevenson WG, Wilber DJ, Natale A, et al. Irrigated radiofrequency catheter ablation guided by electroanatomic mapping for recurrent ventricular tachycardia after myocardial infarction: the multicenter thermocool ventricular tachycardia ablation trial. *Circulation*. 2008;118:2773--82.
S8.2.5. Sapp JL, Wells GA, Parkash R, et al. Ventricular tachycardia ablation versus escalation of antiarrhythmic drugs. *N Engl J Med*. 2016;375:111--21.
S8.2.6. Kuck K‐H, Tilz RR, Deneke T, et al. Impact of substrate modification by catheter ablation on implantable cardioverter‐defibrillator interventions in patients with unstable ventricular arrhythmias and coronary artery disease: results from the multicenter randomized controlled SMS (Substrate Modification Study). *Circ Arrhythm Electrophysiol*. 2017;10:e004422.
S8.2.7. Volkmer M, Ouyang F, Deger F, et al. Substrate mapping vs. tachycardia mapping using CARTO in patients with coronary artery disease and ventricular tachycardia: impact on outcome of catheter ablation. *Europace*. 2006;8:968--76.
S8.2.8. Makimoto H, Nakajima I, Miyamoto K, et al. Clinical impact of mapping strategies for treatment of ventricular tachycardias in patients with structural heart disease. *Pacing Clin Electrophysiol*. 2015;38:630--40.
S8.2.9. Carbucicchio C, Ahmad Raja N, Di Biase L, et al. High‐density substrate‐guided ventricular tachycardia ablation: role of activation mapping in an attempt to improve procedural effectiveness. *Heart Rhythm*. 2013;10:1850--8.
S8.2.10. Briceño DF, Romero J, Villablanca PA, et al. Long‐term outcomes of different ablation strategies for ventricular tachycardia in patients with structural heart disease: systematic review and meta‐analysis. *Europace*. 2018;20:104--15.
S8.2.11. Kumar S, Baldinger SH, Romero J, et al. Substrate‐based ablation versus ablation guided by activation and entrainment mapping for ventricular tachycardia: a systematic review and meta‐analysis. *J Cardiovasc Electrophysiol*. 2016;27:1437--47.
S8.2.12. Acosta J, Penela D, Andreu D, et al. Multielectrode vs. point‐by‐point mapping for ventricular tachycardia substrate ablation: a randomized study. *Europace*. 2018;20:512--19.
S8.2.13. Berte B, Relan J, Sacher F, et al. Impact of electrode type on mapping of scar‐related VT. *J Cardiovasc Electrophysiol*. 2015;26:1213--23.
S8.2.14. Yamashita S, Cochet H, Sacher F, et al. Impact of new technologies and approaches for post‐myocardial infarction ventricular tachycardia ablation during long‐term follow‐up. *Circ Arrhythm Electrophysiol*. 2016;9:e003901.
S8.2.15. Hutchinson MD, Gerstenfeld EP, Desjardins B, et al. Endocardial unipolar voltage mapping to detect epicardial ventricular tachycardia substrate in patients with nonischemic left ventricular cardiomyopathy. *Circ Arrhythmia Electrophysiol*. 2011;4:49--55.
S8.2.16. Polin GM, Haqqani H, Tzou W, et al. Endocardial unipolar voltage mapping to identify epicardial substrate in arrhythmogenic right ventricular cardiomyopathy/dysplasia. *Heart Rhythm*. 2011;8:76--83.
S8.2.17. Chopra N, Tokuda M, Ng J, et al. Relation of the unipolar low‐voltage penumbra surrounding the endocardial low‐voltage scar to ventricular tachycardia circuit sites and ablation outcomes in ischemic cardiomyopathy. *J Cardiovasc Electrophysiol*. 2014;25:602--8.
S8.2.18. Soto‐Becerra R, Bazan V, Bautista W, et al. Ventricular tachycardia in the setting of Chagasic cardiomyopathy. *Circ Arrhythm Electrophysiol*. 2017;10:e004950.
S8.2.19. Desjardins B, Yokokawa M, Good E, et al. Characteristics of intramural scar in patients with nonischemic cardiomyopathy and relation to intramural ventricular arrhythmias. *Circ Arrhythm Electrophysiol*. 2013;6:891--7.
8.3. Intraprocedural imaging during catheter ablation of ventricular arrhythmias {#joa312264-sec-0057}
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Recommendations for intraprocedural imaging during catheter ablation of VAs
CORLOERecommendationsReferencesIB‐NR1. Coronary angiography or intracardiac echocardiography (ICE) is recommended to localize the ostia of the coronary arteries prior to ablation within the SV.S8.3.1--S8.3.4IB‐NR2. Coronary angiography is recommended to identify the course of the coronary arteries when ablation is performed in the coronary venous system or in the epicardium.S8.3.5--S8.3.8IB‐NR3. ICE is beneficial to identify and target the papillary muscles with ablation and to assess for catheter stability.S8.3.9--S8.3.20IB‐NR4. ICE or transthoracic echocardiography is useful to assess for pericardial effusion in case of hemodynamic deterioration of the patient.S8.3.21--S8.3.23IC‐LD5. ICE is useful for early recognition of complications, including pericardial effusion.S8.3.21--S8.3.23IIbB‐NR6. ICE may be useful as an adjuvant technique to identify wall segments with wall thinning, wall motion abnormalities, and segments with increased echogenicity, and also to identify intracardiac thrombi.S8.3.24, S8.3.25
References {#joa312264-sec-0058}
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S8.3.1. Ouyang F, Fotuhi P, Ho SY, et al. Repetitive monomorphic ventricular tachycardia originating from the aortic sinus cusp: electrocardiographic characterization for guiding catheter ablation. *J Am Coll Cardiol*. 2002;39:500--8.
S8.3.2. Hoffmayer KS, Dewland TA, Hsia HH, et al. Safety of radiofrequency catheter ablation without coronary angiography in aortic cusp ventricular arrhythmias. *Heart Rhythm*. 2014;11:1117--21.
S8.3.3. Hachiya H, Aonuma K, Yamauchi Y, Igawa M, Nogami A, Iesaka Y. How to diagnose, locate, and ablate coronary cusp ventricular tachycardia. *J Cardiovasc Electrophysiol*. 2002;13:551--6.
S8.3.4. Yamada T, McElderry HT, Doppalapudi H, et al. Idiopathic ventricular arrhythmias originating from the aortic root prevalence, electrocardiographic and electrophysiologic characteristics, and results of radiofrequency catheter ablation. *J Am Coll Cardiol*. 2008;52:139--47.
S8.3.5. Baman TS, Ilg KJ, Gupta SK, et al. Mapping and ablation of epicardial idiopathic ventricular arrhythmias from within the coronary venous system. *Circ Arrhythm Electrophysiol*. 2010;3:274--9.
S8.3.6. Yokokawa M, Latchamsetty R, Good E, et al. Ablation of epicardial ventricular arrhythmias from nonepicardial sites. *Heart Rhythm*. 2011;8:1525--9.
S8.3.7. D\'Avila A, Gutierrez P, Scanavacca M, et al. Effects of radiofrequency pulses delivered in the vicinity of the coronary arteries: implications for nonsurgical transthoracic epicardial catheter ablation to treat ventricular tachycardia. *Pacing Clin Electrophysiol*. 2002;25:1488--95.
S8.3.8. Sacher F, Roberts‐Thomson K, Maury P, et al. Epicardial ventricular tachycardia ablation a multicenter safety study. *J Am Coll Cardiol*. 2010;55:2366--72.
S8.3.9. Good E, Desjardins B, Jongnarangsin K, et al. Ventricular arrhythmias originating from a papillary muscle in patients without prior infarction: a comparison with fascicular arrhythmias. *Heart Rhythm*. 2008;5:1530--7.
S8.3.10. Yokokawa M, Good E, Desjardins B, et al. Predictors of successful catheter ablation of ventricular arrhythmias arising from the papillary muscles. *Heart Rhythm*. 2010;7:1654--9.
S8.3.11. Yamada T, McElderry HT, Okada T, et al. Idiopathic focal ventricular arrhythmias originating from the anterior papillary muscle in the left ventricle. *J Cardiovasc Electrophysiol*. 2009;20:866--72.
S8.3.12. Yamada T, Doppalapudi H, McElderry HT, et al. Electrocardiographic and electrophysiological characteristics in idiopathic ventricular arrhythmias originating from the papillary muscles in the left ventricle: relevance for catheter ablation. *Circ Arrhythm Electrophysiol*. 2010;3:324--31.
S8.3.13. Yamada T, Doppalapudi H, McElderry HT, et al. Idiopathic ventricular arrhythmias originating from the papillary muscles in the left ventricle: prevalence, electrocardiographic and electrophysiological characteristics, and results of the radiofrequency catheter ablation. *J Cardiovasc Electrophysiol*. 2010;21:62--9.
S8.3.14. Bassil G, Liu CF, Markowitz SM, et al. Comparison of robotic magnetic navigation‐guided and manual catheter ablation of ventricular arrhythmias arising from the papillary muscles. *Europace*. 2018;20:ii5--10.
S8.3.15. Ban JE, Lee HS, Lee DI, et al. Electrophysiological characteristics related to outcome after catheter ablation of idiopathic ventricular arrhythmia originating from the papillary muscle in the left ventricle. *Korean Circ J*. 2013;43:811--8.
S8.3.16. Crawford T, Mueller G, Good E, et al. Ventricular arrhythmias originating from papillary muscles in the right ventricle. *Heart Rhythm*. 2010;7:725--30.
S8.3.17. Rivera S, Ricapito Mde L, Tomas L, et al. Results of cryoenergy and radiofrequency‐based catheter ablation for treating ventricular arrhythmias arising from the papillary muscles of the left ventricle, guided by intracardiac echocardiography and image integration. *Circ Arrhythm Electrophysiol*. 2016;9:e003874.
S8.3.18. Proietti R, Rivera S, Dussault C, et al. Intracardiac echo‐facilitated 3D electroanatomical mapping of ventricular arrhythmias from the papillary muscles: assessing the 'fourth dimension' during ablation. *Europace*. 2017;19:21--8.
S8.3.19. Peichl P, Baran J, Wichterle D, et al. The tip of the muscle is a dominant location of ventricular ectopy originating from papillary muscles in the left ventricle. *J Cardiovasc Electrophysiol*. 2018;29:64--70.
S8.3.20. Lee A, Hamilton‐Craig C, Denman R, Haqqani HM. Catheter ablation of papillary muscle arrhythmias: implications of mitral valve prolapse and systolic dysfunction. *Pacing Clin Electrophysiol*. 2018;41:750--8.
S8.3.21. Ren JF, Marchlinski FE. Early detection of iatrogenic pericardial effusion: importance of intracardiac echocardiography. *JACC Cardiovasc Interv*. 2010;3:127.
S8.3.22. Filgueiras‐Rama D, de Torres‐Alba F, Castrejón‐Castrejón S, et al. Utility of intracardiac echocardiography for catheter ablation of complex cardiac arrhythmias in a medium‐volume training center. *Echocardiography*. 2015;32:660--70.
S8.3.23. Weintraub AR, Schwartz SL, Smith J, Hsu TL, Pandian NG. Intracardiac two‐dimensional echocardiography in patients with pericardial effusion and cardiac tamponade. *J Am Soc Echocardiogr*. 1991;4:571--6.
S8.3.24. Bala R, Ren JF, Hutchinson MD, et al. Assessing epicardial substrate using intracardiac echocardiography during VT ablation. *Circ Arrhythm Electrophysiol*. 2011;4:667--73.
S8.3.25. Peichl P, Wichterle D, Cihak R, Aldhoon B, Kautzner J. Catheter ablation of ventricular tachycardia in the presence of an old endocavitary thrombus guided by intracardiac echocardiography. *Pacing Clin Electrophysiol*. 2016;39:581--7.
8.4. Electroanatomical mapping systems and robotic navigation {#joa312264-sec-0059}
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Recommendations for the use of EAM systems and remote navigation in ablation procedures for VAs
CORLOERecommendationsReferencesIB‐NR1. In patients with VA due to SHD undergoing an ablation procedure, EAM is useful.S8.4.1--S8.4.9IIaB‐NR2. In patients with idiopathic VA undergoing an ablation procedure, EAM can be useful.S8.4.4, S8.4.6IIaB‐NR3. In patients undergoing an ablation procedure for VA, magnetic catheter navigation can be useful to reduce fluoroscopy use.S8.4.10--S8.4.14
References {#joa312264-sec-0060}
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S8.4.1. Khaykin Y, Skanes A, Whaley B, et al. Real‐time integration of 2D intracardiac echocardiography and 3D electroanatomical mapping to guide ventricular tachycardia ablation. *Heart Rhythm*. 2008;5:1396--402.
S8.4.2. Sporton SC, Earley MJ, Nathan AW, Schilling RJ. Electroanatomic versus fluoroscopic mapping for catheter ablation procedures: a prospective randomized study. *J Cardiovasc Electrophysiol*. 2004;15:310--5.
S8.4.3. Earley MJ, Showkathali R, Alzetani M, et al. Radiofrequency ablation of arrhythmias guided by non‐fluoroscopic catheter location: a prospective randomized trial. *Eur Heart J*. 2006;27:1223--9.
S8.4.4. Marchlinski FE, Callans DJ, Gottlieb CD, Zado E. Linear ablation lesions for control of unmappable ventricular tachycardia in patients with ischemic and nonischemic cardiomyopathy. *Circulation*. 2000;101:1288--96.
S8.4.5. Khongphatthanayothin A, Kosar E, Nademanee K. Nonfluoroscopic three‐dimensional mapping for arrhythmia ablation: tool or toy? *J Cardiovasc Electrophysiol*. 2000;11:239--43.
S8.4.6. Stevenson WG, Wilber DJ, Natale A, et al. Irrigated radiofrequency catheter ablation guided by electroanatomic mapping for recurrent ventricular tachycardia after myocardial infarction: the multicenter thermocool ventricular tachycardia ablation trial. *Circulation*. 2008;118:2773--82.
S8.4.7. Reithmann C, Hahnefeld A, Remp T, et al. Electroanatomic mapping of endocardial right ventricular activation as a guide for catheter ablation in patients with arrhythmogenic right ventricular dysplasia. *Pacing Clin Electrophysiol*. 2003;26:1308--16.
S8.4.8. Zeppenfeld K, Schalij MJ, Bartelings MM, et al. Catheter ablation of ventricular tachycardia after repair of congenital heart disease. *Circulation*. 2007;116:2241--52.
S8.4.9. Codreanu A, Odille F, Aliot E, et al. Electroanatomic characterization of post‐infarct scars comparison with 3‐dimensional myocardial scar reconstruction based on magnetic resonance imaging. *J Am Coll Cardiol*. 2008;52:839--42.
S8.4.10. Zhang F, Yang B, Chen H, et al. Magnetic versus manual catheter navigation for mapping and ablation of right ventricular outflow tract ventricular arrhythmias: a randomized controlled study. *Heart Rhythm*. 2013;10:1178--83.
S8.4.11. Dinov B, Schoenbauer R, Wojdyla‐Horodynska A, et al. Long‐term efficacy of single procedure remote magnetic catheter navigation for ablation of ischemic ventricular tachycardia: a retrospective study. *J Cardiovasc Electrophysiol*. 2012;23:499--505.
S8.4.12. Szili‐Torok T, Schwagten B, Akca F, et al. Catheter ablation of ventricular tachycardias using remote magnetic navigation: a consecutive case‐control study. *J Cardiovasc Electrophysiol*. 2012;23:948--54.
S8.4.13. Akca F, Theuns DA, Abkenari LD, de Groot NM, Jordaens L, Szili‐Torok T. Outcomes of repeat catheter ablation using magnetic navigation or conventional ablation. *Europace*. 2013;15:1426--31.
S8.4.14. Bauernfeind T, Akca F, Schwagten B, et al. The magnetic navigation system allows safety and high efficacy for ablation of arrhythmias. *Europace*. 2011;13:1015--21.
9. MAPPING AND ABLATION {#joa312264-sec-0061}
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This section is designed as a "how‐to" section that details the procedural steps of VT ablation in different patient populations ranging from ablation of PVCs in patients without heart disease to ablation of VT/VF in patients with different types of SHD (Figures [7](#joa312264-fig-0007){ref-type="fig"}, [8](#joa312264-fig-0008){ref-type="fig"}, [9](#joa312264-fig-0009){ref-type="fig"}, [10](#joa312264-fig-0010){ref-type="fig"}, [11](#joa312264-fig-0011){ref-type="fig"}, [12](#joa312264-fig-0012){ref-type="fig"} and Tables [5](#joa312264-tbl-0005){ref-type="table"}, [6](#joa312264-tbl-0006){ref-type="table"}, [7](#joa312264-tbl-0007){ref-type="table"}, [8](#joa312264-tbl-0008){ref-type="table"}). Bullet points summarize the key points in this section.
![Anatomical boundaries of the LV summit, with the inaccessible \[1\] and accessible \[2\] parts. Shown are the left anterior descending artery (LAD), the circumflex artery (Cx), the great cardiac vein (GCV), the anterior interventricular vein (AIV), and the first and second diagonal branch of the LAD (D1, D2)](JOA3-36-1-g007){#joa312264-fig-0007}
{#joa312264-fig-0008}
{#joa312264-fig-0009}
{#joa312264-fig-0010}
{#joa312264-fig-0011}
{#joa312264-fig-0012}
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Types of bundle branch reentrant tachycardia
Type A Type B (Interfascicular tachycardia) Type C
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ECG morphology LBBB pattern RBBB pattern RBBB pattern
Anterograde limb RBB LAF or LPF LBB
Retrograde limb LBB LPF or LAF RBB
Abbreviations: LAF, left anterior fascicle; LBB, left bundle branch; LBBB, left bundle branch block; LPF, left posterior fascicle; RBB, right bundle branch; RBBB, right bundle branch block.
John Wiley & Sons, Ltd
######
Fascicular ventricular tachycardias
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**I. Verapamil‐sensitive fascicular reentrant VT**
**1. Left posterior type**
i\. Left posterior septal fascicular reentrant VT
ii\. Left posterior papillary muscle fascicular reentrant VT
**2. Left anterior type**
i\. Left anterior septal fascicular reentrant VT
ii\. Left anterior papillary muscle fascicular reentrant VT
**3. Upper septal type**
**II. Nonreentrant fascicular VT**
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Abbreviation: VT, ventricular tachycardia.
John Wiley & Sons, Ltd
######
Select recent radiofrequency catheter ablation studies in patients post myocardial infarction with a focus on substrate‐based ablation strategies
Study N EF (%) Prior CABG (%) Inclusion Access mapping catheter Mapping strategy Ablation strategy Procedural endpoint RF time procedural duration complications VT recurrence and burden (follow‐up)
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Jais et al. (2012) (S9.5.1)Two centers observational 70 35 ± 10 NR 1\. Sustained VT resistant to AAD therapy and requiring external cardioversion or ICD therapies2) SHD with ischemic or nonischemic dilated cardiomyopathyExclusions:1. VA attributable to an acute or reversible cause2. Repetitive PVCs or nonsustained VT without sustained VT Retrograde in 61 pts (87%)Transseptal in 32 pts (46%); epicardial access in 21 pts (31%)Dual access encouraged3.5‐mm external irrigated ablation catheter; multielectrode mapping catheter in 50% endocardial procedures and in all epicardial procedures 1\. PES and activation mapping of induced stable VTs2. Substrate mapping for LAVAs---sharp high‐frequency electrograms often of low amplitude, occurring during or after the far‐field ventricular electrogram, sometimes fractionated or multicomponent, poorly coupled to the rest of the myocardium 1\. Ablation of LAVA in SR2. Ablation of tolerated VTs guided by entrainment and activation mapping3. Remapping (in stable patients) with further ablation if residual LAVA or persistent inducibility 1\. Complete LAVA elimination---achieved in 47 of 67 pts with LAVA (70.1%)2. Noninducibility---achieved in 70%, similar if LAVA eliminated or not RF time 23 ± 11 minProcedure time 148 ± 73 minComplications 6 pts (8.6%): tamponade or bleeding managed conservatively (3), RV perforation requiring surgical repair (1); 3 pts died within 24 h due to low‐flow state (2) plus arrhythmia recurrence (1), PEA (1) Combined endpoint of VT recurrence or death occurred in 39 pts (55.7%); 45% of pts with LAVA elimination and 80% of those withoutVT recurrence in 32 (46%); 32% of pts with LAVA elimination and 75% of those without7 cardiac deaths (10%) over 22 months of median follow‐up
Di Biase et al. (2015) (S9.5.2)VISTA trialMulticenter RCT 118 Group 133 ± 14Group 232 ± 10 34% 1\. Post‐MI2. Recurrent stable AAD refractory VT (symptomatic or requiring ICD therapy)Exclusion: syncope, cardiac arrest, prior failed ablation, renal failure, end‐stage heart failure EndocardialEpicardial when clinical VTs were inducible after endocardial ablation + no CABGGroup 1: 11.7%Group 2: 10.3%3.5‐mm tip 1\. Substrate mapping (BV ≤1.5 mV) + Group 12. PES and activation mapping/pace mapping for clinical and stable nonclinical VT (unstable VT not targeted) Group 1: Clinical VT ablation, linear lesion to transect VT isthmusGroup 2: Extensive substrate ablation targeting any abnormal potential (=fractionated and/or LP) Group 1:Noninducibility of clinical VT---achieved in 100%Group 2:1. Elimination of abnormal potentials2. No capture from within the scar (20 mA)3) Noninducibility of clinical VT---achieved in 100% Group 1:RF time 35 ± 27 minProcedural time 4.6 ± 1.6 hGroup 2:RF time 68 ± 27 min (*P* \< .001)Procedural time24.2 ± 1.3 h (*P* = .13)Complications 5% VT recurrence at 12 monthsGroup 1: 48.3%Group 2: 15.5%*P* \< .001Mortality at 12 monthsGroup 1: 15%Group 2: 8.6%*P* = .21
Tilz et al. (2014) (S9.5.3)Single center observational 1212/117 pts with post‐MI VT 32 ± 13 --- 1\. Presence of a circumscribed dense scar (BV \<1.5 mV, area \<100 cm2)2. Recurrent unmappable VT3. Post‐MIExclusion: patchy scar/multiple scars Endocardial3.5‐mm tip 1\. PES2. Substrate mapping: area of BV \<1.5 mV + double, fractionated or LP3. PES after ablation Circumferential linear lesion along BZ (BV \<1.5 mV) to isolate substrate 1\. Lack of abnormal EGMs within area2. No capture within area---achieved in 50%3. Max. 40 RF lesionNoninducibility of any VT (no predefined endpoint) ---observed in 92% RF time 53 ± 15 minProcedure time 195 ± 64 minNo complication VT recurrence 33%Median follow‐up 497 days
Tzou et al. (2015) (S9.5.4)Two centers observational 44Post‐MI 3244/566 pts with SHD 31 ± 13 --- 1\. SHD2. AAD refractory VT3. Intention to achieve core isolation EndocardialEpicardial post‐MI 6%3.5‐mm tipSelected patients: multi‐electrode catheters for exit block evaluation 1\. BV mapping2. PES3. Activation mapping4. Substrate mappingDense scar BV \<0.5 mV; BZ BV 0.5--1.5 mV/voltage channels/ fractionated/LP; pace‐match, S‐QRS \>40 ms5. PES after core isolation 1\. Circumferential linear lesion to isolate core (=confluent area of BV \<0.5 mV area and regions with BV \<1 mV harbouring VT‐related sites2. Targeting fractionated and LP within core3. Targeting VT‐related sites outside core (2 and 3 in 59%) 1\. No capture of the ventricle during pacing inside core2. Dissociation of isolated potentials --- core isolation achieved in 70% post‐MI3. Noninducibility ---achieved in 84% RF lesions111 ± 91Procedure time326 ± 121 minComplications 2.2%No death VT recurrence 14%Follow‐up 17.5 ± 9 months
Silberbauer et al. (2014) (S9.5.5)One center observational 160 28 ± 9.5 inducible after RFCA34 ± 9.2 endpoint reached 22.5% 1\. Post‐MI2) AAD refractory VT3) First VT ablation at the center EndocardialCombined endoepicardial (20%)--- Clinical findings--- Prior ablation--- Research protocol3.5‐mm tip/4‐mm tip 1\. Substrate mapping: BV \<1.5 mV + LP (=continuous, fragmented bridging to components after QRS offset/inscribing after QRS, no voltage cutoff) + early potentials (EP = fragmented \<1.5 mV)Pace‐match2) PES3) Activation mapping4) PES after substrate ablation 1\. Ablation mappable VT2. Ablation of all LPLP present at baselineEndocardium 100/160 ptsEpicardium 19/32 pts 1\. Abolition of all LP --- achieved at endocardium in 79 pts (49%), at epicardium 12/32 pts (37%)2. Noninducibility of any VT --- achieved in 88% RF time endocardial median ≈25 min epicardial ≈6 minProcedure timeMedian 210--270 minComplications3.1%In‐hospital mortality2.5% VT recurrence 32% after median 82 (16‐192) daysVT recurrence according to endpoint 1+2 achieved (16.4%)Endpoint 2 achieved (46%)No endpoint achieved (47.4%)
Wolf et al. (2018) (S9.5.6)One center observational 159 34 ± 11 25% 1\. Post‐MI2. First VT ablation3. Recurrent, AAD refractory episodes VT EndocardialCombined endoepicardial 27%--- Epicardial access was encouraged--- Epicardial ablation 27/46 pts3.5‐mm tip (70 pts)Multielectrode catheters (89 pts) 1\. PES2. Activation mapping3. Substrate mapping: BV mapping (\<1.5 mV) + LAVA (=sharp high‐frequency EGMs, possibly of low amplitude, distinct from the far‐field EGM occurring anytime during or after the far‐field EGM4. PES 1\. Ablation of mappable VT2. Ablation of LAVA (until local no capture)LAVA present at baselineEndocardium 141/157 ptsEpicardium 36/46 pts 1\. Abolition of LAVA --- achieved in 93/146 pts (64%)2. Noninducibility---achieved in 94/110 tested pts RF time 36 ± 20 minProcedure time 250 ± 78 minComplications 7.5% (4 surgical interventions)Procedure‐related mortality 1.3% VT‐free survival 55% during 47 months (33--82)Outcome according to endpoints:LAVA abolished vs not abolished 63% vs 44%VT‐free survival at 1 year 73%
Berruezo et al. (2015) (S9.5.7)One center observational 101Post‐MI 75 36 ± 13 --- 1\. Scar‐related VT EndocardialCombined endoepicardial (27/101 pts, among post‐MI not provided)--- Endo no substrate/suggestive epi--- CE‐MRI--- VT ECG3.5‐mm tip 1\. Substrate mapping: BV (\<1.5 mV) + EGMs with delayed components: identification of entrance (shortest delay) of conducting channels2. PES3. Activation mapping + pace‐match 1\. Scar dechanneling targeting entrance2. Short linear lesions (eg, between scar and mitral annulus)3. Ablation of VT‐related sites --- performed in 45% 1\. Scar dechanneling--- Achieved in 85 pts (84.2%)--- Noninducible after 1.55 pts (54.5%)2. Noninducibility ---achieved in 78% RF time24 ± 10 min only scar dechanneling (31 ± 18 min + additional RFCA)Procedure time227 ± 69 minComplications 6.9%No death VT recurrence 27% after a median follow‐up of 21 months (11--29)1‐year VT‐free survival according to endpoint: scar dechanneling complete vs incomplete (≈82% vs ≈65%)
Porta‐Sánchez et al. (2018) (S9.5.8)Multicenter observational 20 33 ± 11 --- 1\. Post‐MI2. Recurrent VT Endocardial3.5‐mm tip 4 ptsMultielectrode catheters 16 pts 1\. Substrate mapping: annotation of LP (=fractionated/isolated after QRS offset) and assessment if LP showed additional delay of \>10 ms after RV extrastimuli (S1 600 ms, S2 VERP + 20 ms) defined as DEEP2. PES3. Additional mapping 1\. Targeting areas with DEEP2. Ablation of VT‐related sites discretion of operator 1\. Noninducibility--- achieved in 80% after DEEP ablation--- Remains 80% after additional ablation in those inducible RF time 30.6 ± 21.4 minProcedure time and complications not reported VT recurrence 25% at 6‐month follow‐up
de Riva et al. (2018) (S9.5.9)One center observational 60 33 ± 12 30% 1\. Post‐MI2) Sustained VT EndocardialEpicardial 10%--- Endocardial failure--- Epicardial substrate suspected3.5‐mm tip catheter 1\. PES2. Substrate mapping: systematic assessment of presumed infarct area independent of BV during SR and RV extrastimuliPacing (S1 500 ms, S2 VRP + 50 ms): EDP (evoked delayed potentials) = low voltage (\<1.5 mV) EGM with conduction delay \>10 ms or block in response to S23. Activation and pace mapping 1\. Targeting EDPs only2. Ablation of VT‐related sites based on activation/pace mapping 1\. Elimination of EDPs --- achieved in all2. Noninducibility of targeted VT (fast VT with VTCL≈VERP not targeted)--- Achieved in 67% after EDP ablation--- Achieved in 90% after additional ablation RF time15 min (10--21)Procedure time173 min (150--205)Complications3.3%One procedure‐related death VT recurrence 22% at median follow‐up of 16 months (8--23)Subgroup of patients with EDPs in normal‐voltage areas at baseline (hidden substrate) compared to historical matched group without EDP mappingVT‐free survival at 1 year 89% vs 73%
Included studies: post myocardial infarction (or data for patients post myocardial infarction provided).
Abbreviations: AAD, antiarrhythmic drug; BV, bipolar voltage; BZ, border zone; CABG, coronary artery bypass grafting; CE‐MRI, contrast‐enhanced magnetic resonance imaging; DC, delayed component; DEEP, decremental evoked potential; ECG, electrocardiogram; EDP, evoked delayed potential; EF, ejection fraction; EGM, electrogram; ICD, implantable cardioverter defibrillator; LAVA, local abnormal ventricular activity; MI, myocardial infarction; PEA, pulseless electrical activity; PES, programmed electrical stimulation; pts, patients; PVC, premature ventricular complex; RCT, randomized controlled trial; RF, radiofrequency; RFCA, radiofrequency catheter ablation; RV, right ventricle; SHD, structural heart disease; SR, sinus rhythm; VT, ventricular tachycardia.
John Wiley & Sons, Ltd
######
Catheter ablation of ventricular arrhythmias in cardiac sarcoidosis
Study N LVEF, % Concurrent immunosuppressive therapy, n (%) VTs induced, mean ± SD Mapping, Endo n/Epi n Ablation, Endo n/Epi n Patients undergoing repeated procedures, n (%) VT Recurrence, n (%) VT Burden decrease, n (%) Major complications Follow‐up, months
---------------------------- ---- --------- --------------------------------------------- ------------------------ ----------------------- ------------------------ ------------------------------------------------ ---------------------- --------------------------- --------------------- -------------------
Koplan et al. (S9.12.5) 8 35 ± 15 5 (63) 4 ± 2 6/2 8/2 1 (13) 6 (75) 4 (44) NR 6
Jefic et al. (S9.12.2) 9 42 ± 14 8 (89) 5 ± 7 8/1 NR 3 (33) 4 (44) 9 (100) NR 20
Naruse et al. (S9.12.3) 14 40 ± 12 12 (86) 3 ± 1 14/0 14/0 4 (29) 6 (43) NR NR 33
Dechering et al. (S9.12.1) 8 36 ± 19 NR 4 ± 2 NR NR NR 1 (13) 7 (88) NR 6
Kumar et al. (S9.12.6) 21 36 ± 14 12 (57) Median 3 (range 1--8) 21/8 21/5 11 (52) 15 (71) 16 (76) 4.7% 24
Muser et al. (S9.12.4) 31 42 ± 15 22 (71) Median 3 (range 1--5) 31/11 31/8 9 (29) 16 (52) 28 (90) 4.5% 30
Abbreviations: LVEF, left ventricular ejection fraction; N, number; NR, not reported; VT, ventricular tachycardia.
John Wiley & Sons, Ltd
9.1. Ablation power sources and techniques {#joa312264-sec-0062}
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Key PointsAn impedance drop ≥10 ohms or a contact force ≥10 g is commonly used as a target for radiofrequency energy delivery.The use of half normal saline generates larger ablation lesions but can result in steam pops.Simultaneous bipolar or unipolar ablation can result in larger ablation lesions.Cryoablation can be beneficial for achieving more stable contact on the papillary muscles.Ethanol ablation can generate lesions in areas where the arrhythmogenic substrate cannot be otherwise reached, provided that suitable target vessels are present.Stereotactic radiotherapy is an emerging alternative to ablation, requiring identification of a region of interest that can be targeted prior to the radiation treatment.
9.2. Idiopathic outflow tract ventricular arrhythmia {#joa312264-sec-0063}
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Key PointsThe RVOT, pulmonary arteries, SVs, LV epicardium and endocardium contain most of the outflow tract arrhythmias.Activation mapping and pace mapping can be used to guide ablation in the RVOT.Imaging of coronary artery ostia is essential before ablation in the aortic SVs.The LV summit is a challenging site of origin, often requiring mapping and/or ablation from the RVOT, LVOT, SVs, coronary venous system, and sometimes the epicardial space.Deep intraseptal VA origins can be challenging to reach.
9.3. Idiopathic nonoutflow tract ventricular arrhythmia {#joa312264-sec-0064}
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Key PointsVAs originating from the papillary muscles can be challenging due to multiple morphologies of the VA and the difficulty in achieving and maintaining sufficient contact during ablation.VAs originate in LV papillary muscles more often than in RV papillary muscles; they more often originate from the posteromedial than the anterolateral papillary muscle and occur more often at the tip than at the base.Pace mapping is less accurate than in other focal VAs.ICE is particularly useful for assessing contact and stability.Cryoablation can also aid in catheter stability during lesion delivery.
9.4. Bundle branch reentrant ventricular tachycardia and fascicular ventricular tachycardia {#joa312264-sec-0065}
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Key PointsBundle branch reentry can occur in a variety of patients in whom the conduction system can be affected, including patients with dilated cardiomyopathy (DCM), valvular heart disease, myocardial infarction, myotonic dystrophy, Brugada syndrome, and ARVC, among others.Ablation of either the right or left bundle branch eliminates bundle branch reentrant ventricular tachycardia (BBRVT) but does not eliminate other arrhythmic substrates.A correct diagnosis of BBRVT is crucial and should employ established criteria prior to ablation of either of the bundle branches.Ablation of the AV node does not cure BBRVT.Ablation of either bundle branch does not cure interfascicular VT.For posterior fascicular VTs, the P1 potential is targeted during VT; if P1 cannot be identified or VT is not tolerated, an anatomical approach can be used.Purkinje fibers can extend to the papillary muscles, and these can be part of the VT circuit.For anterior fascicular VTs, the P1 potential is targeted with ablation.Focal nonreentrant fascicular VT is infrequent and can occur in patients with IHD; however, it cannot be induced with programmed stimulation, and the target is the earliest Purkinje potential during VT.
9.5. Postinfarction ventricular tachycardia {#joa312264-sec-0066}
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Key PointsIn cases of multiple inducible VTs, the clinical VT should be preferentially targeted.Elimination of all inducible VTs reduces VT recurrence and is associated with prolonged arrhythmia‐free survival.For tolerated VTs, entrainment mapping allows for focal ablation of the critical isthmus.For nontolerated VTs, various ablation strategies have been described, including targeting abnormal potentials, matching pace mapping sites, areas of slow conduction, linear lesions, and scar homogenization.Imaging can be beneficial in identifying the arrhythmogenic substrate.Epicardial ablation is infrequently required, but epicardial substrate is an important reason for VT recurrence after VT ablation in patients with prior infarcts.
References {#joa312264-sec-0067}
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S9.5.1. Jais P, Maury P, Khairy P, et al. Elimination of local abnormal ventricular activities: a new end point for substrate modification in patients with scar‐related ventricular tachycardia. *Circulation*. 2012;125:2184--96.
S9.5.2. Di Biase L, Burkhardt JD, Lakkireddy D, et al. Ablation of stable VTs versus substrate ablation in ischemic cardiomyopathy: the VISTA randomized multicenter trial. *J Am Coll Cardiol*. 2015;66:2872--82.
S9.5.3. Tilz RR, Makimoto H, Lin T, et al. Electrical isolation of a substrate after myocardial infarction: a novel ablation strategy for unmappable ventricular tachycardias--feasibility and clinical outcome. *Europace*. 2014;16:1040--52.
S9.5.4. Tzou WS, Frankel DS, Hegeman T, et al. Core isolation of critical arrhythmia elements for treatment of multiple scar‐based ventricular tachycardias. *Circ Arrhythm Electrophysiol*. 2015;8:353--61.
S9.5.5. Silberbauer J, Oloriz T, Maccabelli G, et al. Noninducibility and late potential abolition: a novel combined prognostic procedural end point for catheter ablation of postinfarction ventricular tachycardia. *Circ Arrhythm Electrophysiol*. 2014;7:424--35.
S9.5.6. Wolf M, Sacher F, Cochet H, et al. Long‐term outcome of substrate modification in ablation of post‐myocardial infarction ventricular tachycardia. *Circ Arrhythm Electrophysiol*. 2018;11:e005635.
S9.5.7. Berruezo A, Fernandez‐Armenta J, Andreu D, et al. Scar dechanneling: new method for scar‐related left ventricular tachycardia substrate ablation. *Circ Arrhythm Electrophysiol*. 2015;8:326--36.
S9.5.8. Porta‐Sanchez A, Jackson N, Lukac P, et al. Multicenter study of ischemic ventricular tachycardia ablation with decrement‐evoked potential (DEEP) mapping with extra stimulus. *JACC Clin Electrophysiol*. 2018;4:307--15.
S9.5.9. de Riva M, Naruse Y, Ebert M, et al. Targeting the hidden substrate unmasked by right ventricular extrastimulation improves ventricular tachycardia ablation outcome after myocardial infarction. *JACC Clin Electrophysiol*. 2018;4:316--27.
9.6. Dilated cardiomyopathy {#joa312264-sec-0068}
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Key PointsIdentifying the location and extent of scarring on CMR is beneficial in procedural planning and has improved the outcomes of ablation in patients with DCM.The ablation strategy is similar to postinfarction VT.An intramural substrate is more frequently encountered in DCM than in postinfarction patients and requires a different ablation strategy than for patients with either epicardial or endocardial scarring.Epicardial ablation is beneficial if the scar is located in the epicardium of the LV free wall.For intramural circuits involving the septum, epicardial ablation is not beneficial.In the absence of CMR, unipolar voltage mapping has been described as a method to indicate a deeper‐seated scar.
9.7. Ventricular tachycardia ablation in hypertrophic cardiomyopathy {#joa312264-sec-0069}
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Key PointsPolymorphic VT and VF are the most common VAs in HCM; monomorphic VT is less common.The arrhythmogenic substrate in HCM often involves the septum but can extend to the epicardium, often necessitating combined endocardial and epicardial ablation procedures to eliminate the VT.VT associated with apical aneurysms is often ablated endocardially.
9.8. Brugada syndrome {#joa312264-sec-0070}
---------------------
Key PointsPVC‐triggered VF or polymorphic VT are the most prevalent VAs that motivate device therapy in patients with Brugada syndrome.Monomorphic VT is less frequent but can be caused by BBRVT in patients with Brugada syndrome.The arrhythmogenic substrate is located in the RV epicardium and can be demonstrated by sodium channel blockers.Ablation targets include fractionated prolonged electrograms on the epicardial aspect of the RV.
9.9. Polymorphic ventricular tachycardia/ventricular fibrillation triggers {#joa312264-sec-0071}
--------------------------------------------------------------------------
Key PointsRecurrent PVC‐induced VF is most often triggered by PVCs originating from Purkinje fibers, located in the RVOT, the moderator band, or the LV.Patients with a single triggering PVC are better ablation candidates; however, there are often multiple triggers.Patients with healed myocardial infarction often require extensive ablation of the Purkinje fiber system within or at the scar border.Ischemia should be ruled out as a trigger for VF prior to ablation.
9.10. Arrhythmogenic right ventricular cardiomyopathy {#joa312264-sec-0072}
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Key PointsThe arrhythmogenic substrate in ARVC is located in the epicardium and can involve the endocardium in advanced stages.The most commonly affected areas are the subtricuspid and RV outflow regions.LV involvement is not uncommon.Endocardial‐epicardial ablation is often required and results in higher acute success and lower recurrence rates compared with endocardial ablation alone.Conventional mapping and ablation techniques, including entrainment mapping of tolerated VT, pace mapping, and substrate ablation, are used.
9.11. Mapping and ablation in congenital heart disease {#joa312264-sec-0073}
------------------------------------------------------
Key PointsPatients with a VT substrate after congenital heart defect surgery include those with repaired tetralogy of Fallot, repaired ventricular septal defect, and repaired d‐transposition of the great arteries (D‐TGA), as well as Ebstein\'s anomaly among other disease processes.VT isthmuses are often located between anatomical barriers and surgical incisions or patch material.An anatomical isthmus can be identified and targeted during sinus rhythm.For tolerated VTs, entrainment mapping is the method of choice for identifying critical components of the reentry circuit.
9.12. Sarcoidosis {#joa312264-sec-0074}
-----------------
Key PointsThe arrhythmogenic substrate in cardiac sarcoidosis is often intramurally located but can include the endocardium and epicardium.A CMR is beneficial in planning an ablation procedure in cardiac sarcoidosis.The arrhythmogenic substrate can be complex and can include areas of active inflammation and chronic scarring.The VT recurrence rate after ablation is high.
References {#joa312264-sec-0075}
----------
S9.12.1. Dechering DG, Kochhaüser S, Wasmer K, et al. Electrophysiological characteristics of ventricular tachyarrhythmias in cardiac sarcoidosis versus arrhythmogenic right ventricular cardiomyopathy. *Heart Rhythm*. 2013;10:158--64.
S9.12.2. Jefic D, Joel B, Good E, et al. Role of radiofrequency catheter ablation of ventricular tachycardia in cardiac sarcoidosis: report from a multicenter registry. *Heart Rhythm*. 2009;6:189--95.
S9.12.3. Naruse Y, Sekiguchi Y, Nogami A, et al. Systematic treatment approach to ventricular tachycardia in cardiac sarcoidosis. *Circ Arrhythm Electrophysiol*. 2014;7:407--13.
S9.12.4. Muser D, Santangeli P, Pathak RK, et al. Long‐term outcomes of catheter ablation of ventricular tachycardia in patients with cardiac sarcoidosis. *Circ Arrhythm Electrophysiol*. 2016;9:e004333.
S9.12.5. Koplan BA, Soejima K, Baughman KL, Epstein LM, Stevenson WG. Refractory ventricular tachycardia secondary to cardiac sarcoid: electrophysiologic characteristics, mapping, and ablation. *Heart Rhythm*. 2006;3:924--9.
S9.12.6. Kumar S, Barbhaiya C, Nagashima K, et al. Ventricular tachycardia in cardiac sarcoidosis: characterization of ventricular substrate and outcomes of catheter ablation. *Circ Arrhythm Electrophysiol*. 2015;8:87--93.
9.13. Chagas disease {#joa312264-sec-0076}
--------------------
Key PointsThe pathogenesis of Chagas disease is poorly understood but often results in an inferolateral LV aneurysm.The arrhythmogenic substrate is located intramurally and on the epicardial surface, often necessitating an epicardial ablation procedure.
9.14. Miscellaneous diseases and clinical scenarios with ventricular tachycardia {#joa312264-sec-0077}
--------------------------------------------------------------------------------
Key PointsLamin cardiomyopathy often has a poor prognosis, progressing to end‐stage heart failure.VT ablation is challenging due to intramural substrates.VT recurrence rate is high after ablations.VT in patients with noncompaction tends to originate from regions of noncompacted myocardium where scar can be identified in the midapical LV.VT ablation in patients with LV assist device can be challenging due to the limitation of preprocedural imaging, and the electromagnetic noise generated by the LV assist device.
9.15. Surgical therapy {#joa312264-sec-0078}
----------------------
Key PointsSurgery‐facilitated access to the epicardium via a limited subxiphoid incision can be helpful in the case of adhesions.Cryoablation via thoracotomy is possible for posterolateral substrates and via sternotomy for anterior substrates.
9.16. Sympathetic modulation {#joa312264-sec-0079}
----------------------------
Key PointsSympathetic modulation targeting the stellate ganglia by video‐assisted thoracoscopy may be considered for failed VT ablation procedures or VF storms.A temporary effect can be obtained with the percutaneous injection or infusion of local anesthetics.
9.17. Endpoints of catheter ablation of ventricular tachycardia {#joa312264-sec-0080}
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Key PointsNoninducibility of VT by PES after ablation is a reasonable endpoint and predictor for VT recurrence after VT ablation in patients with SHD.Due to the limitations of programmed stimulation, endpoints other than noninducibility have been described, including elimination of excitability, elimination of late potentials or local abnormal ventricular activity, dechanneling, substrate homogenization, core isolation, image‐guided ablation, and anatomically fixed substrate ablation.
10. POSTPROCEDURAL CARE {#joa312264-sec-0081}
=======================
Access‐related issues, anticoagulation (Table [9](#joa312264-tbl-0009){ref-type="table"}), and complications (Table [10](#joa312264-tbl-0010){ref-type="table"}), as well as the management thereof, are reviewed in this section. Furthermore, assessment of outcomes and determinants of outcomes are detailed (Figure [13](#joa312264-fig-0013){ref-type="fig"}).
######
Postprocedural care in prospective studies of ventricular tachycardia catheter ablation
Study Postprocedure NIPS AAD type AAD duration Follow‐up ICD programming Anticoagulation postablation Bleeding and thromboembolic events (ablation arm)
------------------------------- -------------------- ---------------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------ ----------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ ---------------------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------
Calkins 2000 (S10.1.2.5) No Patients were continued on the type of antiarrhythmic therapy they had received before ablation. At least the first 3 months after hospital discharge Evaluation at 1, 3, 6, 9, 12, and 24 months after ablation Not specified Not specified Four of 146 (2.7%) stroke or TIA, 4 (2.7%) episodes of pericardial tamponade
SMASH‐VT 2007 (S10.1.2.6) No No patient received an AAD (other than beta blockers) before the primary endpoint was reached. N/A Followed in the ICD clinic at 3, 6, 9, 12, 18, and 24 months; echocardiography at 3 and 12 months Not specified Oral anticoagulation 4--6 weeks, aspirin if fewer than 5 ablation lesions One pericardial effusion without tamponade, managed conservatively; 1 deep venous thrombosis
Stevenson 2008 (S10.1.2.1) No The previously ineffective AAD was continued for the first 6 months, after which time drug therapy was left to the discretion of the investigator. Six months, after which time drug therapy was left to the discretion of the investigator Echocardiogram and neurologist examination before and after ablation; office visit at 2 and 6 months, with ICD interrogation where applicable Not specified Three months with either 325 mg/day aspirin or warfarin if ablation had been performed over an area over 3 cm in length Vascular access complications in 4.7%; no thromboembolic complications
Euro‐VT 2010 (S10.1.2.7) No Drug management during follow‐up was at the discretion of the investigator. Drug management during follow‐up was at the discretion of the investigator. At 2, 6, and 12 months, with ICD interrogation where applicable Investigators were encouraged to program ICD detection for slow VT for at least 20 beats or 10 seconds to allow nonsustained VT to terminate before therapy is triggered. Not specified No major bleeding or thromboembolic complications
VTACH 2010 (S10.1.2.8) No Discouraged Discouraged Every 3 months from ICD implantation until completion of the study VF zone with a cutoff rate of 200--220 bpm and a VT zone with a cutoff CL of 60 ms above the slowest documented VT and ATP followed by shock Not specified One transient ischemic ST‐segment elevation; 1 TIA
CALYPSO 2015 (S10.1.2.9) No Discouraged Discouraged At 3 and 6 months Investigators were required to ensure that VT detection in the ICD is programmed at least 10 beats below the rate of the slowest documented VT. At the discretion of the treating physician, anticoagulation recommended with aspirin or warfarin for 6‐12 weeks
Marchlinski 2016 (S10.1.2.10) Not required Not dictated by the study protocol Not dictated by the study protocol At 6 months and at 1, 2, and 3 years Not dictated by the study protocol Per clinical conditions and physician preference Cardiac perforation (n = 1), pericardial effusion (n = 3)
VANISH 2016 (S10.1.2.11) No Continued preprocedure antiarrhythmic medications Not specified A 3‐month office visit, echo, ICD check; a 6‐month office visit, ICD check; every 6 months thereafter, an office visit, ICD check VT detection at 150 bpm or with a 10--20 bpm margin if the patient was known to have a slower VT. ATP was recommended in all zones. The protocol was modified to recommend prolonged arrhythmia detection duration for all patients. Intravenous heparin (without bolus) 6 hours after sheath removal, then warfarin if substrate‐mapping approach used or if more than 10 minutes of RF time Major bleeding in 3 patients; vascular injury in 3 patients; cardiac perforation in 2 patients
SMS 2017 (S10.1.2.12) No At the discretion of the investigator At the discretion of the investigator At 3, 6, 9, and 12 months, and at 3‐ or 6‐month intervals until completion of the study or until 33‐month follow‐up was reached VF zone at 200--220 bpm, detection 18 of 24 beats, shock only; VT zone detection at least 16 consecutive beats, ATP, and shocks. Where VT rates were exclusively \>220 bpm, VT zone at 160--180 bpm was recommended; where VT rates were \<220 bpm, VT zone with a CL 60 ms above the slowest VT was recommended Aspirin (250 mg/day) or warfarin as necessitated by the underlying heart disease Two tamponades requiring pericardiocentesis
Abbreviations: AAD, antiarrhythmic dug; ATP, antitachycardia pacing; CL, cycle length; ICD, implantable cardioverter defibrillator; NIPS, noninvasive programmed stimulation; RF, radiofrequency; TIA, transient ischemic attack; VF, ventricular fibrillation; VT, ventricular tachycardia.
John Wiley & Sons, Ltd
######
Major complications of ventricular arrhythmia ablation in patients with structural heart disease
Complication Incidence Mechanisms Presentation Prevention Treatment Ref.
----------------------------------------------------------------------------- ------------------------------------ ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- -------------------------------------------------------------------------------------------------- --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ------------------
In‐hospital mortality 0%‐3% VT recurrence, heart failure, complications of catheter ablation Not applicable Correct electrolyte disturbances and optimize medical status before ablation --- S10.2.1--S10.2.5
Long‐term mortality 3%‐35% (12‐39 months of follow‐up) VT recurrence and progression of heart failure Cardiac nonarrhythmic death (heart failure) and VT recurrence Identification of patients with indication for heart transplantation --- S10.2.2--S10.2.5
Neurological complication (stroke, TIA, cerebral hemorrhage) 0%‐2.7% Emboli from left ventricle, aortic valve, or aorta; cerebral bleeding Focal or global neurological deficits Careful anticoagulation control; ICE can help detection of thrombus formation, and of aortic valve calcification; TEE to assess aortic arch Thrombolytic therapy S10.2.1--S10.2.5
Pericardial complications: cardiac tamponade, hemopericardium, pericarditis 0%‐2.7% Catheter manipulation, RF delivery, epicardial perforation Abrupt or gradual fall in blood pressure; arterial line is recommended in ablation of complex VT Contact force can be useful, careful in RF delivery in perivenous foci and RVOT Pericardiocentesis; if necessary, surgical drainage, reversal heparin; steroids and colchicine in pericarditis S10.2.1--S10.2.5
AV block 0%‐1.4% Energy delivery near the conduction system Fall in blood pressure and ECG changes Careful monitoring when ablation is performed near the conduction system; consider cryoablation Pacemaker; upgrade to a biventricular pacing device might be necessary S10.2.1--S10.2.4
Coronary artery damage/MI 0.4%‐1.9% Ablation near coronary artery, unintended coronary damage during catheter manipulation in the aortic root or crossing the aortic valve Acute coronary syndrome; confirmation with coronary catheterization Limit power near coronary arteries and avoid energy delivery \<5 mm from coronary vessel; ICE is useful to visualize the coronary ostium Percutaneous coronary intervention S10.2.1--S10.2.5
Heart failure/pulmonary edema 0%‐3% External irrigation, sympathetic response due to ablation, and VT induction Heart failure symptoms Urinary catheter and careful attention to fluid balance and diuresis, optimize clinical status before ablation, reduce irrigation volume if possible (decrease flow rates or use closed irrigation catheters) New/increased diuretics S10.2.2--S10.2.5
Valvular injury 0%‐0.7% Catheter manipulation, especially retrograde crossing the aortic valve and entrapment in the mitral valve; energy delivery to subvalvular structures, including papillary muscle Acute cardiovascular collapse, new murmurs, progressive heart failure symptoms Careful catheter manipulation; ICE can be useful for identification of precise location of energy delivery Echocardiography is essential in the diagnosis; medical therapy, including vasodilators and dobutamine before surgery; IABP is useful in acute mitral regurgitation and is contraindicated in aortic regurgitation S10.2.2--S10.2.5
Acute periprocedural hemodynamic decompensation, cardiogenic shock 0%‐11% Fluid overloading, general anesthesia, sustained VT Sustained hypotension despite optimized therapy Close monitoring of fluid infusion and hemodynamic status‐Optimize medical status before ablation‐pLVAD‐Substrate mapping preferred, avoid VT induction in higher‐risk patients Mechanical HS S10.2.2--S10.2.6
Vascular injury: hematomas, pseudoaneurysm, AV fistulae 0%‐6.9% Access to femoral arterial and catheter manipulation Groin hematomas, groin pain, fall in hemoglobin Ultrasound‐guided access Ultrasound‐guided compression, thrombin injection, and surgical closure S10.2.1--S10.2.5
Overall major complications with SHD 3.8%‐11.24% S10.2.1--S10.2.5
Overall all complications 7%‐14.7% S10.2.3,S10.2.7,S10.2.8
Abbreviations: AV, atrioventricular; ECG, electrocardiogram; HS, hemodynamic support; IABP, intra‐aortic balloon pump; ICE, intracardiac echocardiography; MI, myocardial infarction; pLVAD, percutaneous left ventricular assist device; RF, radiofrequency; RVOT, right ventricular outflow tract; SHD, structural heart disease; TEE, transesophageal echocardiography; TIA, transient ischemic attack; VT, ventricular tachycardia.
John Wiley & Sons, Ltd
{#joa312264-fig-0013}
10.1. Postprocedural care: access, anticoagulation, disposition {#joa312264-sec-0082}
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### 10.1.1. Postprocedural care: access {#joa312264-sec-0083}
Recommendations for management of venous access sites after catheter ablation of VA
CORLOERecommendationsReferencesIA1. Manual compression is effective in achieving hemostasis after venous access for VT ablation.S10.1.1.1--S10.1.1.3IIaB‐R2. Venous access closure using temporary purse‐string or figure‐of‐8 suture techniques can be useful in achieving faster hemostasis and earlier ambulation and reducing pain or discomfort associated with hemostasis compared to manual compression.S10.1.1.1, S10.1.1.2
Recommendation for management of arterial access sites after catheter ablation of VA
CORLOERecommendationReferencesIA1. Achieving arterial access site hemostasis using either manual compression or a vascular closure device is recommended.S10.1.1.4, S10.1.1.5
Recommendations for management of epicardial access sites after catheter ablation of VA
CORLOERecommendationsReferencesIC‐EO1. If pericardial bleeding or cardiac tamponade has occurred during epicardial VT ablation, a pericardial drain should be left in place until bleeding has resolved.IIaB‐NR2. The instillation of intrapericardial corticosteroids can be effective in reducing pericarditic chest pain after epicardial VT mapping or ablation.S10.1.1.6, S10.1.1.7IIaB‐NR3. To reduce pericardial pain after epicardial VT ablation, unless pericardial bleeding or cardiac tamponade has occurred, it is reasonable to remove all pericardial access sheaths at the end of the procedure.S10.1.1.6, S10.1.1.7IIbC‐EO4. Leaving a pericardial drain in place might be reasonable in patients at high risk for late bleeding or cardiac tamponade after epicardial VT ablation.
### References {#joa312264-sec-0084}
S10.1.1.1. Jackson N, McGee M, Ahmed W, et al. Groin haemostasis with a purse string suture for patients following catheter ablation procedures (GITAR study). *Heart Lung Circ*. 2018 March 20 <https://doi.org/10.1016/j.hlc.2018.03.011>. \[Epub ahead of print\].
S10.1.1.2. Pracon R, Bangalore S, Henzel J, et al. A randomized comparison of modified subcutaneous "Z"‐stitch versus manual compression to achieve hemostasis after large caliber femoral venous sheath removal. *Catheter Cardiovasc Interv*. 2018;91:105--112.
S10.1.1.3. Ben‐Dor I, Craig P, Torguson R, et al. MynxGrip vascular closure device versus manual compression for hemostasis of percutaneous transfemoral venous access closure: results from a prospective multicenter randomized study. *Cardiovasc Revasc Med*. 2018;19:418--22.
S10.1.1.4. Robertson L, Andras A, Colgan F, Jackson R. Vascular closure devices for femoral arterial puncture site haemostasis. *Cochrane Database Syst Rev*. 2016;3:CD009541.
S10.1.1.5. Jiang J, Zou J, Ma H, et al. Network meta‐analysis of randomized trials on the safety of vascular closure devices for femoral arterial puncture site haemostasis. *Sci Rep*. 2015;5:13761.
S10.1.1.6. Della Bella P, Brugada J, Zeppenfeld K, et al. Epicardial ablation for ventricular tachycardia: a European multicenter study. *Circ Arrhythm Electrophysiol*. 2011;4:653--59.
S10.1.1.7. Dyrda K, Piers SR, van Huls van Taxis CF, Schalij MJ, Zeppenfeld K. Influence of steroid therapy on the incidence of pericarditis and atrial fibrillation after percutaneous epicardial mapping and ablation for ventricular tachycardia. *Circ Arrhythm Electrophysiol*. 2014;7:671--6.
### 10.1.2. Postprocedural care: anticoagulation {#joa312264-sec-0085}
Recommendations for anticoagulation after VA ablation procedures
CORLOERecommendationsReferencesIIaC‐LD1. After less extensive endocardial VT ablation, treatment with an antiplatelet agent for a limited period of time is reasonable.S10.1.2.1, S10.1.2.2IIaC‐LD2. Heparin reversal with protamine for sheath removal after ablation is reasonable.S10.1.2.3, S10.1.2.4IIbC‐LD3. After extensive endocardial VT ablation, treatment with an oral anticoagulant for a limited period of time might be reasonable.S10.1.2.1, S10.1.2.2IIbC‐EO4. The use of heparin bridging after endocardial VT ablation may be considered but can be associated with an increased risk of periprocedural bleeding.
### References {#joa312264-sec-0086}
S10.1.2.1. Stevenson WG, Wilber DJ, Natale A, et al. Irrigated radiofrequency catheter ablation guided by electroanatomic mapping for recurrent ventricular tachycardia after myocardial infarction: the multicenter Thermocool ventricular tachycardia ablation trial. *Circulation*. 2008;118:2773--82.
S10.1.2.2. Siontis KC, Jame S, Sharaf Dabbagh G, et al. Thromboembolic prophylaxis protocol with warfarin after radiofrequency catheter ablation of infarct‐related ventricular tachycardia. *J Cardiovasc Electrophysiol*. 2018;29:584--90.
S10.1.2.3. Patel AA, Clyne CA, Henyan NN, et al. The use of protamine after radiofrequency catheter ablation: a pilot study. *J Interv Card Electrophysiol*. 2007;18:155--8.
S10.1.2.4. Ghannam M, Chugh A, Dillon P, et al. Protamine to expedite vascular hemostasis after catheter ablation of atrial fibrillation: a randomized controlled trial. *Heart Rhythm*. 2018;15:1642--7.
S10.1.2.5. Calkins H, Epstein A, Packer D, et al; Cooled RF Multi Center Investigators Group. Catheter ablation of ventricular tachycardia in patients with structural heart disease using cooled radiofrequency energy: results of a prospective multicenter study. *J Am Coll Cardiol*. 2000;35:1905--14.
S10.1.2.6. Reddy VY, Reynolds MR, Neuzil P, et al. Prophylactic catheter ablation for the prevention of defibrillator therapy. *N Engl J Med*. 2007;357:2657--65.
S10.1.2.7. Tanner H, Hindricks G, Volkmer M, et al. Catheter ablation of recurrent scar‐related ventricular tachycardia using electroanatomical mapping and irrigated ablation technology: results of the prospective multicenter Euro‐VT‐study. *J Cardiovasc Electrophysiol*. 2010;21:47--53.
S10.1.2.8. Kuck KH, Schaumann A, Eckardt L, et al. Catheter ablation of stable ventricular tachycardia before defibrillator implantation in patients with coronary heart disease (VTACH): a multicentre randomised controlled trial. *Lancet*. 2010;375:31--40.
S10.1.2.9. Al‐Khatib SM, Daubert JP, Anstrom KJ, et al. Catheter ablation for ventricular tachycardia in patients with an implantable cardioverter defibrillator (CALYPSO) pilot trial. *J Cardiovasc Electrophysiol*. 2015;26:151--7.
S10.1.2.10. Marchlinski FE, Haffajee CI, Beshai JF, et al. Long‐term success of irrigated radiofrequency catheter ablation of sustained ventricular tachycardia: post‐approval THERMOCOOL VT trial. *J Am Coll Cardiol*. 2016;67:674--83.
S10.1.2.11. Sapp JL, Wells GA, Parkash R, et al. Ventricular tachycardia ablation versus escalation of antiarrhythmic drugs. *N Engl J Med*. 2016;375:111--21.
S10.1.2.12. Kuck KH, Tilz RR, Deneke T, et al; SMS Investigators. Impact of substrate modification by catheter ablation on implantable cardioverter‐defibrillator interventions in patients with unstable ventricular arrhythmias and coronary artery disease: results from the multicenter randomized controlled SMS (Substrate Modification Study). *Circ Arrhythm Electrophysiol*. 2017;10:e004422.
10.2. Incidence and management of complications {#joa312264-sec-0087}
-----------------------------------------------
### References {#joa312264-sec-0088}
S10.2.1. Palaniswamy C, Kolte D, Harikrishnan P, et al. Catheter ablation of postinfarction ventricular tachycardia: ten‐year trends in utilization, in‐hospital complications, and in‐hospital mortality in the United States. *Heart Rhythm*. 2014;11:2056--63.
S10.2.2. Stevenson WG, Wilber DJ, Natale A, et al. Irrigated radiofrequency catheter ablation guided by electroanatomic mapping for recurrent ventricular tachycardia after myocardial infarction: the multicenter thermocool ventricular tachycardia ablation trial. *Circulation*. 2008;118:2773--82.
S10.2.3. Calkins H, Epstein A, Packer D, et al. Catheter ablation of ventricular tachycardia in patients with structural heart disease using cooled radiofrequency energy: results of a prospective multicenter study. Cooled RF Multi Center Investigators Group. *J Am Coll Cardiol*. 2000;35:1905--14.
S10.2.4. Reddy VY, Reynolds MR, Neuzil P, et al. Prophylactic catheter ablation for the prevention of defibrillator therapy. *N Engl J Med*. 2007;357:2657--65.
S10.2.5. Kuck KH, Schaumann A, Eckardt L, et al; VTACH Study Group. Catheter ablation of stable ventricular tachycardia before defibrillator implantation in patients with coronary heart disease (VTACH): a multicentre randomised controlled trial. *Lancet*. 2010;375:31--40.
S10.2.6. Santangeli P, Muser D, Zado ES, et al. Acute hemodynamic decompensation during catheter ablation of scar‐related ventricular tachycardia: incidence, predictors, and impact on mortality. *Circ Arrhythm Electrophysiol*. 2015;8:68--75.
S10.2.7. Katz DF, Turakhia MP, Sauer WH, et al. Safety of ventricular tachycardia ablation in clinical practice: findings from 9699 hospital discharge records. *Circ Arrhythmia Electrophysiol*. 2015;8:362--70.
S10.2.8. Santangeli P, Frankel DS, Tung R, et al. Early mortality after catheter ablation of ventricular tachycardia in patients with structural heart disease. *J Am Coll Cardiol*. 2017;69:2105--15.
10.3. Hemodynamic deterioration and proarrhythmia {#joa312264-sec-0089}
-------------------------------------------------
Recommendation for echocardiography after VA ablation
CORLOERecommendationReferenceIC‐LD1. Echocardiography should be performed in case of hemodynamic deterioration post‐VT ablation to assess for pericardial effusion and cardiac tamponade.S10.3.1
Reference {#joa312264-sec-0090}
---------
S10.3.1. Cheitlin MD, Alpert JS, Armstrong WF, et al. ACC/AHA guidelines for the clinical application of echocardiography: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Committee on Clinical Application of Echocardiography). *Circulation*. 1997;95:1686--744.
10.4. Follow‐up of patients post catheter ablation of ventricular tachycardia {#joa312264-sec-0091}
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Recommendation for noninvasive programmed stimulation after catheter ablation of VT
CORLOERecommendationReferencesIIaB‐NR1. Noninvasive programmed stimulation can be useful in the several days following VT catheter ablation to inform further management, including ICD programming, predicting the risk of VT recurrence, and/or considering a repeat VT catheter ablation.S10.4.1, S10.4.2
References {#joa312264-sec-0092}
----------
S10.4.1. Frankel DS, Mountantonakis SE, Zado ES, et al. Noninvasive programmed ventricular stimulation early after ventricular tachycardia ablation to predict risk of late recurrence. *J Am Coll Cardiol*. 2012;59:1529--35.
S10.4.2. Oloriz T, Baratto F, Trevisi N, et al. Defining the outcome of ventricular tachycardia ablation: timing and value of programmed ventricular stimulation. *Circ Arrhythm Electrophysiol*. 2018;11:e005602.
11. TRAINING AND INSTITUTIONAL REQUIREMENTS AND COMPETENCIES {#joa312264-sec-0093}
============================================================
This section contains the general training and institutional requirements with an emphasis on lifelong learning, professionalism, and acquisition and maintenance of knowledge and skills. In addition, institutional requirements for specific procedures are reviewed.
11.1. Training requirements and competencies for catheter ablation of ventricular arrhythmias {#joa312264-sec-0094}
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Recommendation for training requirements and competencies for catheter ablation of VA
CORLOERecommendationIC‐EO1. For clinical cardiac electrophysiologists who perform catheter ablation for VAs, appropriate advanced training and continued lifelong learning is recommended.
11.2. Institutional requirements for catheter ablation of ventricular tachycardia {#joa312264-sec-0095}
---------------------------------------------------------------------------------
Recommendations for institutional requirements for catheter ablation of VT
CORLOERecommendationsIC‐EO1. Patients with certain underlying medical conditions and comorbidities undergoing complex VA ablations who are deemed to have increased procedural risk should undergo procedures in a hospital‐based electrophysiology laboratory.IC‐EO2. Onsite interventional cardiology expertise is recommended for electrophysiology procedures requiring coronary imaging to delineate coronary anatomy for epicardial ablation, aortography to delineate coronary ostia for SV VT ablation, and need for placement of HS devices.IC‐EO3. Onsite cardiothoracic surgical backup is recommended for electrophysiology procedures requiring pericardial access due to the potential need for emergent sternotomy and cardiopulmonary bypass.IC‐EO4. Availability of anesthesia personnel is recommended for all patients undergoing catheter ablation of VAs.
12. FUTURE DIRECTIONS {#joa312264-sec-0096}
=====================
This section summarizes ongoing trials and the need for prospective evaluation of different clinical problems. It further reviews recent advances and limitations of various mapping techniques and addresses unanswered questions requiring future investigations.
Supporting information
======================
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Click here for additional data file.
{#joa312264-sec-0097}
Writing group memberEmploymentHonoraria/Speaking/ConsultingSpeakers\' bureauResearch[a](#joa312264-note-1016){ref-type="fn"}Fellowship support[a](#joa312264-note-1016){ref-type="fn"}Ownership/Partnership/Principal/Majority stockholderStock or stock optionsIntellectual property/RoyaltiesOtherEdmond M. Cronin, MB, BCh, BAO, FHRS, CCDS, CEPS‐A (Chair)Hartford Hospital, Hartford, ConnecticutNoneNoneNoneNoneNoneNoneNoneNoneFrank M. Bogun, MD (Vice‐Chair)University of Michigan, Ann Arbor, MichiganNoneNoneNoneNoneNoneNoneNoneNonePhilippe Maury, MD (EHRA Chair)University Hospital Rangueil, Toulouse, FranceNoneNoneNoneNoneNoneNoneNoneNonePetr Peichl, MD, PhD (EHRA Vice‐Chair)Institute for Clinical and Experimental Medicine, Prague, Czech Republic1: Abbott; 1: Biosense Webster; 1: BIOTRONIKNoneNoneNoneNoneNoneNoneNoneMinglong Chen, MD, PhD, FHRS (APHRS Chair)Jiangsu Province Hospital, The First Affiliated Hospital of Nanjing Medical University, Nanjing, ChinaNoneNoneNoneNoneNoneNoneNone0: APHRS Board MemberNarayanan Namboodiri, MBBS, MD (APHRS Vice‐Chair)Sree Chitra Institute for Medical Sciences and Technology, Thiruvananthapuram, IndiaNoneNoneNoneNoneNoneNoneNoneNoneLuis Aguinaga, MD, PhD, FESC, FACC (LAHRS Chair)Centro Privado de Cardiología, Tucuman, ArgentinaNoneNoneNoneNoneNoneNoneNoneNoneLuiz Roberto Leite, MD, PhD, FHRS (LAHRS Vice‐Chair)Instituto Brasília de Arritmia, Brasília, BrazilNone1: Boehringer IngelheimNoneNoneNoneNoneNoneNoneSana M. Al‐Khatib, MD, MHS, FHRS, CCDSDuke University Medical Center, Durham, North CarolinaNoneNoneNoneNoneNoneNoneNoneNoneElad Anter, MDBeth Israel Deaconess Medical Center, Boston, Massachusetts2: Itamar Medical; 3: Boston Scientific; 4: Biosense WebsterNoneNoneNoneNoneNoneNoneNoneAntonio Berruezo, MD, PhDHeart Institute, Teknon Medical Center, Barcelona, Spain1: Biosense Webster1: Biosense Webster2: Biosense WebsterNoneNoneNoneNoneNoneDavid J. Callans, MD, FHRS, CCDSUniversity of Pennsylvania, Philadelphia, Pennsylvania1: Abbott Laboratories; 1: BIOTRONIK; 1: Medtronic; 1: Wolters Kluwer; 2: Boston ScientificNone4: NIH2: BIOTRONIK; 2: Boston Scientific; 4: Abbott; 4: Biosense Webster; 4: MedtronicNoneNoneNone1: Acutus Medical; 1: AtriCure; 1: Impulse Dynamics; 1: Thermedical; 3: BayerMina K. Chung, MD, FHRSCleveland Clinic, Cleveland, Ohio2: ABIMNone5: AHA; 5: NIHNoneNoneNone1: Elsevier; 1: Up to Date0: ACC (EP Section Leadership Council member); 0: AHA (Chair, ECG & Arrhythmias Committee; Member, Clinical Cardiology Leadership Committee; Member, Committee on Scientific Sessions Programming); 0: Amarin (Data monitoring committee member); 0: BIOTRONIK; 2: AHA (Associate Editor, *Circulation Arrhythmia & Electrophysiology*)Phillip Cuculich, MDWashington University School of Medicine, St. Louis, Missouri2: MedtronicNoneNoneNoneNoneNoneNoneNoneAndre d\'Avila, MD, PhDHospital Cardiologico SOS Cardio, Florianopolis, BrazilNoneNoneNoneNoneNoneNoneNoneNoneBarbara J. Deal, MD, FACCNorthwestern University Feinberg School of Medicine, Chicago, IllinoisNoneNoneNoneNoneNoneNoneNoneNonePaolo Della Bella, MDOspedale San Raffaele, Milan, Italy2: Biosense Webster; 2: Boston Scientific; 3: AbbottNoneNone2: Medtronic; 3: Abbott Vascular; 3: Biosense Webster; 3: BIOTRONIK; 4: Boston ScientificNoneNoneNoneNoneThomas Deneke, MD, PhD, FHRSHerz‐ und Gefäß‐Klinik, Bad Neustadt, GermanyNoneNoneNoneNoneNoneNoneNoneNoneTimm‐Michael Dickfeld, MD, PhD, FACC, FHRSUniversity of Maryland, Baltimore, Maryland1: Abbott Laboratories; 1: Biosense Webster; 1: Impulse Dynamics; 1: PhilipsNone1: GE HealthcareNoneNoneNoneNoneNoneClaudio Hadid, MDHospital General de Agudos Cosme Argerich, Buenos Aires, ArgentinaNoneNoneNoneNoneNoneNoneNoneNoneHaris M. Haqqani, MBBS, PhD, FHRSUniversity of Queensland, The Prince Charles Hospital, Chermside, Australia0: Abbott Laboratories; 0: Boston Scientific; 0: MedtronicNone3: Biosense WebsterNoneNoneNoneNoneNoneG. Neal Kay, MD, CCDSUniversity of Alabama at Birmingham, Birmingham, AlabamaNoneNoneNoneNoneNoneNoneNoneNoneRakesh Latchamsetty, MD, FHRSUniversity of Michigan, Ann Arbor, MichiganNone1: BIOTRONIKNoneNoneNoneNoneNoneNoneFrancis Marchlinski, MD, FHRSUniversity of Pennsylvania, Philadelphia, Pennsylvania1: Abbott; 1: Biosense Webster; 1: Boston Scientific; 1: Medtronic; 2: BIOTRONIKNone1: Abbott; 4: Biosense Webster2: BIOTRONIK; 2: Boston Scientific; 4: Abbott; 4: Biosense Webster; 4: MedtronicNoneNoneNoneNoneJohn M. Miller, MD, FHRSIndiana University School of Medicine, Krannert Institute of Cardiology, Indianapolis, Indiana1: Abbott Laboratories; 1: Biosense Webster; 1: Boston Scientific; 1: BIOTRONIK; 1: MedtronicNoneNone3: Biosense Webster; 3: Boston Scientific; 3: MedtronicNoneNone1: ElsevierNoneAkihiko Nogami, MD, PhDUniversity of Tsukuba, Ibaraki, Japan1: Abbott LaboratoriesNone4: MedtronicNoneNoneNoneNoneNoneAkash R. Patel, MD, FHRS, CEPS‐PUniversity of California San Francisco Benioff Children\'s Hospital, San Francisco, CaliforniaNoneNoneNoneNoneNoneNoneNoneNoneRajeev Kumar Pathak, MBBS, PhD, FHRSAustralian National University, Canberra Hospital, Canberra, Australia1: BIOTRONIK1: MedtronicNoneNoneNoneNoneNoneNoneLuis C. Saenz Morales, MDCardioInfantil Foundation, Cardiac Institute, Bogota, ColumbiaNoneNoneNoneNoneNoneNoneNoneNonePasquale Santangeli, MD, PhDUniversity of Pennsylvania, Philadelphia, Pennsylvania1: Abiome; 1: Baylis Medical; 1: Biosense Webster; 1: Medtronic; 1: Stereotaxis; 2: Abbott LaboratoriesNoneNoneNoneNoneNoneNoneNoneJohn L. Sapp, Jr., MD, FHRSQueen Elizabeth II Health Sciences Centre, Halifax, Canada1: Abbott Vascular; 1: Biosense Webster; 1: MedtronicNone4: Abbott Vascular; 5: Biosense WebsterNoneNoneNone1: Biosense WebsterNoneAndrea Sarkozy, MD, PhD, FEHRAUniversity Hospital Antwerp, University of Antwerp, Antwerp, Belgium1: Biosense Webster; 1: BIOTRONIKNoneNoneNoneNoneNoneNone0: EHRA Board memberKyoko Soejima, MDKyorin University School of Medicine, Tokyo, Japan2: Abbott Laboratories; 2: Boehringer Ingelheim3: MedtronicNoneNoneNoneNoneNoneNoneWilliam G. Stevenson, MD, FHRSVanderbilt University Heart and Vascular Center, Nashville, Tennessee1: Abbott; 1: BIOTRONIK; 1: Boston Scientific; 1: MedtronicNoneNoneNoneNoneNone0: Biosense Webster; 0: Brigham and Women\'s HospitalNoneUsha B. Tedrow, MD, MS, FHRSBrigham and Women\'s Hospital, Boston, Massachusetts1: Abbott; 1: Biosense Webster; 1: MedtronicNoneNoneNoneNoneNoneNoneNoneWendy S. Tzou, MD, FHRSUniversity of Colorado Denver, Aurora, Colorado1: Abbott; 1: Biosense Webster; 1: BIOTRONIK; 1: Boston Scientific; 1: MedtronicNoneNoneNoneNoneNoneNoneNoneNiraj Varma, MD, PhDCleveland Clinic, Cleveland, Ohio1: BIOTRONIK; 1: MedtronicNone3: AbbottNoneNoneNoneNoneNoneKatja Zeppenfeld, MD, PhD, FESC, FEHRALeiden University Medical Center, Leiden, the Netherlands1: AbbottNone5: Biosense Webster3: Biosense WebsterNoneNoneNoneNoneNotes[^10][^11][^12]
{#joa312264-sec-0098}
Peer reviewerEmploymentHonoraria/Speaking/ConsultingSpeakers\' bureauResearch[a](#joa312264-note-1018){ref-type="fn"}Fellowship support[a](#joa312264-note-1018){ref-type="fn"}Ownership/Partnership/Principal/Majority stockholderStock or stock optionsIntellectual property/RoyaltiesOtherSamuel J. Asirvatham, MD, FHRSMayo Clinic College of Medicine, Rochester, Minnesota1: Abbott; 1: BIOTRONIK; 1: Boston Scientific; 1: MedtronicNoneNoneNoneNoneNone1: AliveCorNoneEduardo Back Sternick, MD, PhDFaculdade Ciências Médicas de Minas, Gerais, BrazilNoneNoneNoneNoneNoneNoneNoneNoneJanice Chyou, MDNorthport VA Medical Center, Northport, New York; Agile Health, New York, New YorkNoneNoneNoneNoneNoneNoneNoneNoneSabine Ernst, MD, PhDRoyal Brompton and Harefield Hospitals, London, England2: Biosense Webster; 2: StereotaxisNone3: Other; 4: Baylis; 4: Spectrum DynamicsNoneNoneNoneNoneNoneGuilherme Fenelon, MD, PhDHospital Israelita Albert Einstein, Sao Paulo, Sao Paulo, Brazil1: LibbsNoneNoneNoneNoneNoneNoneNoneEdward P. Gerstenfeld, MD, MS, FACCUniversity of California, San Francisco, San Francisco, California1: Abbott Vascular; 1: Biosense Webster; 1: Boston Scientific; 1: MedtronicNone4: Abbott Vascular; 4: Biosense WebsterNoneNoneNoneNoneNoneGerhard Hindricks, MDHeart Center Leipzig at the University of Leipzig, Leipzig, GermanyNoneNone4: Abbott VascularNoneNoneNoneNoneNoneKoichi Inoue, MD, PhDSakurabashi‐Watanabe Hospital, Osaka, Japan2: Biosense Webster; 2: Medtronic JapanNoneNoneNoneNoneNoneNoneNoneJeffrey J. Kim, MDBaylor College of Medicine, Texas Children\'s Hospital, Houston, TexasNoneNoneNone3: MedtronicNoneNoneNoneNoneKousik Krishnan, MD, FHRS, FACCRush University Medical Center, Chicago, Illinois1: ZOLLNoneNoneNoneNoneNoneNoneNoneKarl‐Heinz Kuck, MD, FHRSAsklepios Klinik St. Georg, Hamburg, Germany1: Abbott Vascular; 1: Biosense Webster; 1: Boston Scientific; 1: Edwards Lifesciences; 1: MedtronicNoneNoneNoneNoneNoneNoneNoneMartin Ortiz Avalos, MDHospital San Angel Inn Universidad, Mexico City, MexicoNoneNoneNoneNoneNoneNoneNoneNoneThomas Paul, MD, FACC, FHRSGeorg August University Medical Center, Gottingen, GermanyNoneNoneNoneNoneNoneNoneNoneNoneMauricio I. Scanavacca, MD, PhDInstituto Do Coracao, Sao Paulo, BrazilNoneNoneNoneNoneNoneNoneNoneNoneRoderick Tung, MD, FHRSThe University of Chicago Medicine, Center for Arrhythmia Care, Heart and Vascular Center, Chicago, Illinois2: AbbottNone2: Abbott3: Abbott; 3: Medtronic; 3: Boston ScientificNoneNoneNoneNoneJamie Voss, MBChBMiddlemore Hospital, Auckland, New ZealandNoneNoneNoneNoneNoneNoneNoneNoneTakumi Yamada, MDUniversity of Alabama at Birmingham, Birmingham, Alabama1: Nihon Kohden; 2: Abbott; 2: Japan LifelineNoneNoneNoneNo; neNoneNoneNoneTeiichi Yamane, MD, PhD, FHRSJikei University School of Medicine, Tokyo, Japan1: Boehringer Ingelheim; 1: Bristol‐Myers Squibb; 2: Abbott Laboratories; 2: Medtronic JapanNoneNoneNoneNoneNoneNoneNone[^13][^14]
[^1]: Representative of the European Heart Rhythm Association (EHRA)
[^2]: Representative of the American College of Cardiology (ACC)
[^3]: Representative of the Sociedade Brasileira de Arritmias Cardíacas (SOBRAC)
[^4]: Representative of the American Heart Association (AHA)
[^5]: Representative of the Latin American Heart Rhythm Society (LAHRS)
[^6]: Representative of the Asia Pacific Heart Rhythm Society (APHRS)
[^7]: Representative of the Japanese Heart Rhythm Society (JHRS)
[^8]: Representative of the Pediatric and Congenital Electrophysiology Society (PACES)
[^9]: Representative of the Heart Rhythm Society (HRS)
[^10]: Number value: **0** = \$0; **1** = ≤\$10 000; **2** = \>\$10 000 to ≤\$25 000; **3** = \>\$25 000 to ≤\$50 000; **4** = \>\$50 000 to ≤\$100 000; **5** = \>\$100 000.
[^11]: Abbreviations: ABIM, American Board of Internal Medicine; ACC, American College of Cardiology; AHA, American Heart Association; APHRS, Asia Pacific Heart Rhythm Society; EHRA, European Heart Rhythm Association; LAHRS, Latin American Heart Rhythm Society; NIH, National Institutes of Health.
[^12]: Research and fellowship support are classed as programmatic support. Sources of programmatic support are disclosed but are not regarded as a relevant relationship with industry for writing group members or reviewers.
[^13]: Number value: 0 = \$0; 1 = ≤\$10 000; 2 = \>\$10 000 to ≤\$25 000; 3 = \>\$25 000 to ≤\$50 000; 4 = \>\$50 000 to ≤\$100 000; 5 = \>\$100 000.
[^14]: Research and fellowship support are classed as programmatic support. Sources of programmatic support are disclosed but are not regarded as a relevant relationship with industry for writing group members or reviewers.
| {
"pile_set_name": "PubMed Central"
} |
Background {#Sec1}
==========
In 2015, 303,000 maternal deaths were reported globally, of which 99% occurred in developing countries. About 60% of this global smaternal mortality burden is shared by only ten countries including India, Nigeria, Afghanistan, Ethiopia, Democratic Republic of the Congo, Tanzania, Kenya, Uganda, Bangladesh, and Pakistan \[[@CR1]\]. A lack of access and availability of emergency obstetric care accounts for a large majority of maternal mortality in these countries \[[@CR2]\]. Although maternal mortality ratio in Pakistan declined from 521 in 1990 to 178 in 2015, the country still faces a high maternal mortality ratio compared to the regional countries. In bordering India, maternal mortality ratio declined from 556 to 174, in Bangladesh from 569 to 176, in Afghanistan from 1340 to 396, between 1990 and 2015 \[[@CR3]\]. The Pakistan Demographic and Health Survey in 2012--2013 reported a maternal mortality ratio of 276 per 100, 000 and a neonatal mortality rate of 55 per 1000 \[[@CR4]\]. In the province of Sindh, the maternal mortality ratio reported was 314, which was much higher than the national average, indicating wide disparities within country with regard to access to maternal and child healthcare services \[[@CR5]\]. The country also faltered in its achievement of Millennium Development Goal of reducing maternal mortality ratio by three quarters, between 1990 and 2015 \[[@CR6]\]. Such a failure is reflected by the fact that maternal mortality burden has increased from previous estimates for some parts of Pakistan. For instance, the maternal mortality ratio in Thatta district, in Southern Sindh province, increased more than 50% from 219 in 2010 to 333 per 100,000 live births in 2013 \[[@CR7]\].
Pakistan's maternal and child health burden continues to remain high and coverage of maternal survival interventions is still low despite the investments in recent years, by both public sector and non-governmental organizations. The country's total maternal, newborn and child health expenditure increased by 67% between 2001 and 2010 \[[@CR8]\]. Maternal and newborn deaths in Pakistan could be prevented by improving the access to and availability of basic emergency obstetric and newborn care (BEmONC) and comprehensive emergency obstetric and newborn care (CEmONC) \[[@CR9]\].
BEmONC and CEmONC services are offered at various tiers of public healthcare system. BEmONC services including the normal vaginal deliveries, administer oxytocin, newborns resuscitation services are expected to be provided at primary healthcare facilities. CEmONC services were offered for breech presentation, prolonged labor and caesarean section, blood transfusion and care of sick newborns is provided at secondary and tertiary care hospitals. In Sindh, the health system consists of basic health units (BHUs), rural health centers (RHCs), *Taluka* (subdistrict) headquarter hospitals (THQs), mother and child health (MCH) centers and district headquarters hospitals (DHQs) or civil hospitals. Despite of health facility availability in almost all administrative areas in the province, the availability of and access to quality maternal and newborn care services has been poor \[[@CR10], [@CR11]\]. The present study was sanctioned considering the need for an evaluation of the essential services and resources related to maternal and newborn care provision in the province of Sindh. The study was aimed to encompass the level of infrastructure, equipment and commodities required to deliver emergency obstetric and newborn care services in the province.
Methods {#Sec2}
=======
This cross-sectional study was conducted in twelve of the 29 districts of Sindh Province (Fig. [1](#Fig1){ref-type="fig"}). The province had a total population of 47.9 million in 2017 \[[@CR12]\]. These districts had poor maternal and child health indicators according to a ranking based on the multiple indicator cluster survey conducted in Sindh in 2014. This Survey was designed to provide estimates for more than 100 indicators about the women and children's health and social status \[[@CR13]\]. The main maternal and child healthcare indicators in the survey were: antenatal and postnatal care, contraception use rate, breastfeeding rate, vaccination coverage, institutional births with skilled providers and proportion of low birth weight babies. We adopted universal sampling technique by interviewing all hospital administrators from 25 RHC, 28 THQ, and 10 DHQ hospitals (Table [1](#Tab1){ref-type="table"}). Questionnaire from World Health Organization (WHO) monitoring emergency obstetric care tool in developing countries was adapted and used after pretesting in adjacent district of Sindh and the availability of EmONC signal functions was assessed by adopting the direct observations WHO recommended check list during this study as described in Table [2](#Tab2){ref-type="table"} \[[@CR14], [@CR15]\]. The tool comprised of items on demographic information, availability of equipment and instruments necessary for EmONC. It also contained a checklist to assess EmONC and other maternal child healthcare services like; availability of newborn ward, kangaroo mother care, skilled birth attendance, caesarean section, facility environment and assessment of signal functions of BEmONC and CEmONC. A team of data collectors was trained and data collection quality was ensured by the principal investigator. The data were entered in Microsoft Excel and imported into Statistical Package for Social Sciences version 20 for analysis. Fig. 1Geographical distribution of twelve districts surveyed in Sindh province (Developed by using WHO health mapper freely available; <https://health-mapper.informer.com/4.3/>) Table 1Sociodemographic characteristics of the health facilities assessed in Sindh province (*n* = 63)Selected DistrictsPopulationNumber of district headquarters hospitalsNumber of Taluka headquarters hospitalsNumber of Rural Health CentersTotal number of health facilities visitedArea (Km^2^)Catchment populationTotalSelectedTotalSelectedTotalSelectedDadu25511,089,1691143515Ghotki19,8081,649,6611153315Jacobabad7705979,8171132336Kamber Shahdadkot20271,612,3731174427Kashmore27711,006,2971--31423Larkana25771,231,4811--43525Mithi/ Tharparkar55991,341,0421162214Nausheroferoz65061,647,23911521225Shikarpur19061,524,3911142947Sujawal8699781,9671142225Thatta80341,550,2661131646UmerKot55031,073,1461153615Total12105328612563 Table 2Reported emergency obstetric and neonatal care signal functions in the health facilities of Sindh ProvinceBEmONC Signal functionsAll health facilities (*n* = 63)RHCs (*n* = 25)THQs (*n* = 28)DHQs (*n* = 10)n(%)95%CIn(%)95%CIn(%)95%CIn(%)95%CI1. Parenteral antibiotics58(92)92(88.0--96.0)23(92)92(75.0--97.0)25(89)89(72.0--96.0)10(100)100(72.0--100.0)2. Parenteral Oxytocin57(90)90(80.0--95.0)22(88)88(70.0--95.0)25(89)89(72.0--96.0)10(100)100(72.0--100.0)3. Parenteral anticonvulsants54(83)83(75.0--92.0)21(84)84(65.0--93.0)25(89)89(72.0--96.0)8(80)80(49.0--94.0)4. Manual removal of placenta58(92)92(88.0--96.0)23(92)92(75.0--97.0)26(93)92(77.0--98.0)9(90)90(59.0--98.0)5. Removal of retained products55(87)87(76.0--93.0)20(80)80(60.0--91.0)25(89)89(72.0--96.0)10(100)100(72.0--100.0)6. Assisted vaginal delivery52(83)82(71.0--89.0)19(76)76(56.0--88.0)23(82)82(64.0--92.0)10 (100)100 (72.0--100.0)7. Neonatal resuscitation51(81)80(69.0--88.0)20(80)80(60.0--91.0)22(79)78(60.0--89.0)9(90)90 (59.0--98.0)CEmONC Services 1. Caesarean birth15(24)23(14.0--35.0)0 (0)0(0.0--13.0)9(54)32(17.0--50.0)6(60)60(31.0--83.0) 2. Blood Transfusion36(57)57(44.0--68.0)11(44)44(26.0--62.0)16(57)57(39.0--73.0)9(90)90(59.0--98.0)
Ethical approval was obtained from the Institutional Review Board of Health Services Academy, Islamabad; administrative permission from health department of Sindh province was also taken for the study.
Results {#Sec3}
=======
EmONC service availability in the health facilities {#Sec4}
---------------------------------------------------
From the 63 health facilities surveyed, 25 (40%) were RHCs; and 28 (44%) and 10 (16%) were THQ and DHQ hospitals, respectively. Most of the health facilities 58(92%) surveyed had parenteral antibiotics available, 57(90%) of them had uterotonic drugs available for the management of the third stage of labor and postpartum hemorrhage prevention. Fifty two (83%) facilities had trained staff available to manage eclampsia and pre-eclampsia by anticonvulsant drug administration and 58(92%) had trained staff available to perform manual removal of placenta. Most 55(87%) health facility staff reported that they could perform for dilatation and curettage and vacuum extraction of retained products of placenta and 52(83%) facilities reported presence of services for assisted vaginal delivery. Newborn care services were available in 51(81%) of the health facilities. The availability of the BEmONC signal functions was better in DHQ hospitals compared to RHCs and THQs. However, with regard to CEmONC services, only 15(24%) of the health facilities reported offering caesarean section services and blood transfusion service was available only in 36(57%) of the health facilities (Table [2](#Tab2){ref-type="table"}). These services have been verified through observation of the previous hospital record.
Maternal and child healthcare service-related structures and services {#Sec5}
---------------------------------------------------------------------
Almost all facilities had antenatal care clinics except three health centers, including RHC New Jatoi, Buxapur and THQ hospital Kashmore. A postnatal ward was available at all DHQ and 18 THQ hospitals. A newborn ward was available in nine DHQ hospitals and about half of THQ hospitals. Labor rooms were available in all surveyed DHQ and THQ hospitals and RHCs. Equipment for assisted birth (vacuum extraction) was present in all DHQ and 26 THQ hospitals, and in 23 RHCs. Services for the active management of third stage of labor were available in most (84%) health facilities, and services for the management of pre-eclampsia and eclampsia were available in 81% of the health facilities and newborn resuscitation service was present in 85% of the health facilities. Post abortion care services, like manual vacuum aspiration and use of misoprostol were available in 81% of the health facilities. Protocol for assisted vaginal delivery was present in 84% facilities. However, a proper referral mechanism did not exist between these facilities (Table [3](#Tab3){ref-type="table"}). Table 3Reported availability of maternal and child health related services in surveyed health facilities of Sindh ProvinceIndicatorsAll health facilities (*n* = 63)RHCs (*n* = 25)THQs (*n* = 28)DHQs (*n* = 10)*n*Percent (95% CI)*n*Percent (95% CI)*n*Percent (95% CI)*n*Percent (95% CI)Adequate number of beds6095(86.0--98.0)2496(80.0--99.0)2692(77.0--98.0)10100(72.0--100.0)Newborn ward3757(46.0--70.0)1352 (33.0--69.0)1553(35.0--70.0)990(59.0--98.0)Kangaroo mother care area /room2945(34.0--58.0)1144(26.0--62.0)1242(26.0--60.0)660(31.0--83.0)Postnatal ward471(60.0--82.0)1872(52.0--85.0)1864(45.0--79.0)10100(72.0--100.0)Labor room63100(94.0--100.0)25100(86.0--100.0)28100(87.0--100.0)1086(72.0--90.0)Assisted birth (vacuum extraction)5993(84.0--97.0)2392(75.0--97.0)2689(77.0--98.0)10100(72.0--100.0)Skilled birth attendants (nurse, midwife, Lady health visitor and/or a medical doctor)6195(89.0--99.0)2496(80.0--99.0)2796(82.0--99.0)10100(72.0--100.0)Stock out of EmONC specific medicines is available as per Government supply4773(62.0--83.0)1768 (48.0--82.0)2175(56.0--87.0)990(59.0--98.0)Facilities ready to provide EmONC services as per WHO criteria4671(60.0--82.0)1664(44.0--79.0)2280(60.0--89.0)880(49.0--94.0)Facilities with free caesarean-section2539(28.0--52.0)1664(44.0--79.0)2278(60.0--89.0)880(49.0--94.0)
Assessment of guidelines and policies for maternal and child healthcare services {#Sec6}
--------------------------------------------------------------------------------
Guidelines and polices related to maternal and child healthcare provision, case management and quality control mechanism were available in most of the surveyed health facilities; however, these guidelines were more likely to be available within DHQ hospitals compared to lower level health facilities. Less than half of the healthcare staff including doctors and paramedics had received any refresher training on EmONC service provision in the past 6 months. Most health facilities responded that they were gathering and communicating health data regularly (Table [4](#Tab4){ref-type="table"}). Table 4Reported availability of guidelines and policies supporting maternal and child health Services in surveyed health facilities of Sindh provinceEmONC service monitoring indicatorsAll health facilities (*n* = 63)RHCs (*n* = 25)THQs (*n* = 28)DHQs (*n* = 10)*n*Percent (95% CI)*n*Percent (95% CI)*n*Percent (95% CI)*n*Percent (95% CI)Availability of MNCH policy and guideline documents4977(66.0--86.0)1872(52.0--85.0)2175(56.0--87.0)10100(72.0--100.0)EmONC quality check-up team nominated at the hospital4469(57.0--79.0)180.72(52.0--85.0)1760(42.0--76.0)990(59.0--98.0)Regular data reporting63100(94.0--100.0)25100(86.0--100.0)28100(87.0--100.0)10100(72.0--100.0)Facilities supervised in last 3 months6095(86.0--98.0)2392(75.0--97.0)2796(82.0--99.0)10100(72.0--100.0)Any training in EmONC in 6 months3149(37.0--61.0)1040(23.0--59.0)1553(35.0--70.0)660(31.0--83.0)Availability of Soap/running water or alcohol rub in Labor Room5688(78.0--94.0)2080(60.0--91.0)2692(77.0--98.0)10100(72.0--100.0)Any referral mechanism for patients during emergency5688(78.0--94.0)2184(65.0--93.0)2589(72.0--96.0)10100(72.0--100.0)
Discussion {#Sec7}
==========
In the present study, availability of BEmONC and CEmNOC signal functions was assessed at the three levels of health care system in twelve districts of Sindh province where the status of maternal and child health, and other social indicators has been historically low. Even though most of the health facilities we surveyed reported high availability of the BEmONC services, the availability of CEmONC sginal functions of cesarean section and blood transfusion was substandard. Almost half of these health facilities lacked necessary guidelines and policies to manage maternal and child health related cases and about half of their staff lacked a recent training on maternal and child health related topic. Our findings are consistent with a previous multi-country survey which reported high availability of BEmONC services \[[@CR16]\]. Studies have consistently showed that a low EmONC coverage in developing countries is linked with poor maternal and child health indicators \[[@CR17]--[@CR19]\]. The reason for this is that very few health facilities provide accessible caesarean section and blood transfusion services in many developing countries \[[@CR15]\]. Our results are also consistent with other similar studies which show a reasonably acceptable availability of basic services; yet more advanced maternal and child health services have been lacking; thereby limiting communities' access to these services and resulting in poor outcomes around birth \[[@CR20]--[@CR22]\]. Most of the maternal and newborn deaths in Pakistan occur at birth and improving access to EmONC services can tremendously improve birth outcomes \[[@CR9]\].
Availability of CEmONC signal functions in secondary and tertiary level health facilities is necessary to ensure women and children have access to essential emergency care at birth. Not only the THQ hospitals in our study had low availability of caesarean section service; alarmingly, even most of the DHQ hospitals lacked it. Considering that these districts are geographically large, unavailability of an affordable caesarean section service within their main hospitals may result in access related challenges to the communities. These communities may not afford expenses related to caesarean birth in private hospitals and possibly experience hardships while travelling to the districts where such care may be available. This usually leads to families spending out of pocket leading to catastrophic expenditures. Nevertheless, private hospitals in some districts offer an alternative to absent caesarean section service in public sector hospitals \[[@CR11]\].
As with caesarean section, blood transfusion availability as part of the national blood transfusion system, even in the hospitals offering CEmONC services, is still a challenge in countries like Pakistan. Previous research has also identified similar challenges to the provision of blood transfusion in other parts of Pakistan \[[@CR21]\], as well as in other countries including Malawi and India which also have high maternal and child mortality \[[@CR22]\]. Most patients in Pakistan access blood transfusion service through private blood transfusion centers by paying out of pocket; these facilities often require patients to arrange a blood donor \[[@CR23]\]. Ensuring the availability of blood transfusion facilities could prevent a significant proportion of maternal deaths This is crucial considering that post-partum hemorrhage causes one-fourth maternal mortality burden in the developing countries \[[@CR24]\].
Inadequately trained staff with less exposure to refresher trainings was another aspect of poor readiness of the health facilities to provide EmONC services. Availability of skilled and trained human resources for health could also be another challenge for the high maternal mortality ratio in surveyed districts, because employees are often poorly satisfied with their jobs and their work environment is usually suboptimal \[[@CR25], [@CR26]\]. Adequate training of health workers is another area of concern where the trained staff would be able to perform their responsibilities more efficiently which can impact better maternal and newborn health outcomes \[[@CR27]\].
Strengths and limitations {#Sec8}
-------------------------
Direct access to the health facilities, use of a standard validated checklist and observations of the key structures and functions in the facilities was a strength of this study. However, since our data collectors relied upon health facility staff and managers for reporting, the actual availability of the services could be lower than is presented. Also, observations of the health facilities were conducted, yet they did not focus on the real time utilization of services by the patients. Although, it was found that the equipment and infrastructure for emergency obstetric care were available in the 12 surveyed districts of Sindh, and some of the health facilities also provided CEmONC services, the quality and patient satisfaction with the range of services was not assessed as it was not one of the aims of the present study.
Conclusion {#Sec9}
==========
Although BEmONC service coverage was high in most of the health facilities in the province; the CEmONC service availability was dismal. Ensuring the availability of cesarean section and blood transfusion facilities in most strategically located secondary and tertiary care health facilities will improve the communities' access to these two essential services and may lead to better maternal and newborn survival. Further research is recommended to directly determine the utilization and quality of EmONC services in the health facilities.
BEmONC
: Basic Emergency Obstetric and Newborn care
CEmONC
: Comprehensive Emergency Obstetric and Newborn care
EmONC
: Emergency Obstetric and Newborn Care
**Publisher's Note**
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
We acknowledge the support provided by Rachadapisek Sompote Fund for Postdoctoral Fellowship, Chulalongkorn University Thailand.
RK conceptualized this study, JA drafted the manuscript and helped in the data analysis, FA did data analysis and RS revised manuscript critically and added her intellectual content and finalized the manuscript. All authors have read and approved the final manuscript.
Authors did not receive any funding for this research.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Written consent was obtained from participants of the study and Institutional Review Committee of the Health Services Academy, Islamabad Pakistan has approved the ethical clearance prior to start the data collection (7--82/2017-IERB).
Not applicable.
Author Ramesh Kumar is an Editorial Board Member for this journal. All other authors declare that they have no competing interests.
| {
"pile_set_name": "PubMed Central"
} |
Introduction
============
Sexual violence against children is a major public health problem in its own right, and it is directly and indirectly relevant to the HIV epidemic. The physical trauma of sexual violence can increase the risk of HIV transmission directly[@b1; @b2; @b3] and the psychological damage related to abuse can result in increased risk-taking behaviours, re-victimisation[@b4; @b5; @b6; @b7] and perpetration of sexual violence.[@b8] [@b9] Even for those not directly involved, having a friend or neighbour who suffers sexual abuse builds an environment where sexual violence is seen as an everyday occurrence.[@b10] Gender violence in general (including forms other than sexual violence) is an important factor increasing the risk of HIV infection among young women in southern Africa.[@b11]
Sexual abuse of children is believed to be common in East and southern Africa but there are few quantitative studies, mostly in South Africa.[@b12] [@b13] Different methods of data collection and differing definitions of what is included within the term sexual violence can produce very different estimates of occurrence. A national South African study using a self-administered questionnaire reported 10% of school-going youth (both females and males) suffered forced or coerced sex each year, with around 35% affected by the age of 18 years.[@b14] A smaller study with face-to-face questioning, also in South Africa, reported only 1.6% of girls experienced forced sex before the age of 15 years.[@b15] A study among young women in Swaziland found that 35% of girls reported being 'touched sexually or forced to have sex' by the age of 18 years.[@b16]
Despite its importance as a public health problem and human rights violation, and its clear relevance to the HIV epidemic in southern Africa, there is little empirical data available on sexual violence against children in this region, especially data allowing comparisons between countries and over time. Internationally comparable surveys such as the Demographic and Health Survey do not collect information about sexual violence in this age group. Using data from nationally representative samples in eight southern African countries in 2003 and these same countries plus another two in 2007, we examined the frequency, changes over time and risk factors for experience of forced or coerced sex among school-going youth aged 11--16 years, as well as the frequency and risk factors of perpetration of forced sex in 2007. The surveys used the same instruments, training and data collection methods in the same settings on both occasions.
Methods
=======
Sample
------
In 2002/2003, we drew a stratified (urban/rural) random sample of census enumeration areas (EAs) in Botswana, Lesotho, Malawi, Mozambique, Namibia, Swaziland, Zambia and Zimbabwe, covering 25--30 EAs in each country. The schools\' sample reported here comprised the schools serving these EAs that included grades 6--9 (students aged approximately 11--17 years). In 2007, we added South Africa and Tanzania to cover a total of 259 EAs and 445 schools across the 10 countries. Within each school, the field teams randomly selected one class per grade for the survey, in grades covering students aged 11 years and above. In a few schools, at the request of the head teacher, they covered all the two to three classes per grade.
Data collection
---------------
For both surveys, we standardised training in one country and then repeated this in each country, training 25--30 field workers in each country over 1 week. The training specifically covered methods of ensuring privacy of responses in a crowded classroom, asking about sensitive issues and how to handle students who might become upset or who might have questions or seek advice because of their participation in the survey. Field coordinators approached the head teacher of each sample school and explained the survey aims. They only rarely needed to share the actual questionnaire and then only shared it with the head teacher. Head teachers of many schools chose to send information to parents about the survey (without sending details of the contents); they did not seek opt-in consent from parents for their children to participate. In each classroom, with the teacher absent, the facilitator first explained that participation was entirely voluntary and that students could leave out any questions they did not want to answer or leave the questionnaire blank. He or she then advised students to use open exercise books to ensure privacy of their responses and read each question in turn, in the language of choice of the class, encouraging participants to wait until they *heard* each question before writing their answer on the scannable form. Most answers required respondents to fill in one or more bubbles for response options. In each class, at least one assistant (two or more in particularly big classes) checked that the privacy arrangements were working and alerted the facilitator if any students were having difficulty with the process. The whole session, including the explanations and instructions and collection of completed response forms, took \<1 hour.
The questionnaire, translated into 27 languages, asked the respondent 'has anyone ever forced or persuaded you to have sex when you did not want to?' We counted as suffering sexual violence those who responded positively to this closed direct question. The questionnaire used exactly the same words in 2003 and 2007. The questionnaire also asked if the respondent had ever perpetrated sexual violence ('forced sex with someone without their consent').
The questionnaire also documented age and sex of the respondent, whether they drank alcohol, the degree of crowding in their homes and whether there was enough food in their house in the last week (as an indicator of serious poverty).
We derived several school-level variables from 2007 data, potentially related to the risk of experience of sexual violence. Based on the youth questionnaire responses, we categorised schools as having above or below the mean (for each country) proportion of students having experienced forced or coerced sex and having perpetrated forced sex and drinking alcohol. We also documented community-level variables: whether the community was urban or rural, whether it had tar road access and whether it had any active government HIV prevention programmes. Other community-level variables came from a household survey of adults which took place in the sample EAs served by the schools in late 2002 and 2007. Trained interviewers administered a questionnaire to adults aged 16--59 years present in households, covering 24 069 respondents across the 10 countries in 2007. Other publications describe the household survey in more detail.[@b17] [@b18] We categorised communities as having above or below the 2007 country mean in access by good tar road; active government HIV prevention programme; proportion of adults saying that "women sometimes deserve to be beaten"; proportion of adults saying it is "okay for an older man to have sex with teenagers"; proportion of adults saying "men have the right to sex with their girlfriends if they buy them gifts" and proportion of adults reporting intimate partner violence in the last year. We also coded school-level variables as above or below the national average: proportion of students reporting experience of sexual violence; proportion of students knowing of three child rights; proportion of students agreeing that boys and girls are equal; proportion of students reporting perpetration of sexual violence and proportion of students reporting drinking alcohol.
Analysis
--------
Operators scanned self-administered questionnaires using Remark[@b19] and analysis relied on CIETmap open-source software.[@b20] The analysis excluded students who did not answer the questions about sexual violence and those who did not give their age or who reported their age as '17 years or older'. We weighted individual country frequency estimates to account for any rural/urban disproportion in the sample compared with the population. In addition, we weighted regional frequency estimates in proportion to population of the countries; some countries were over-sampled and others under-sampled in relation to their population. To compare reported experience of forced or coerced sex between 2003 and 2007, we restricted the comparison to schools included in both surveys in eight countries, examined male and female changes separately and standardised on the age distribution of the Botswana male sample in 2007. Risk analysis of factors related to experience of forced or coerced sex reported in 2007 began by examining bivariate associations using the Mantel--Haenszel procedure.[@b21] We adjusted these bivariate estimates of association by country and for clustering (at school level) using a method described by Lamothe[@b22] [@b23] based on a variance estimator to weight the Mantel--Haenszel OR for cluster-correlated data. We report the OR and cluster-adjusted CIs. Multivariate analysis of factors significant in bivariate analysis began with a saturated model, with backwards deletion excluding the weakest association, until only significant associations remained. For the multivariate analysis, we used a generalised linear mixed model (GLMM) to examine personal variables like age and sex, together with household variables like crowding and food sufficiency, community-level variables like high or low prevalence of intimate partner violence and negative attitudes about gender and gender violence and school-level variables like high or low proportion of children who said they were victims or perpetrators of forced sex or drank alcohol. For GLMM, we used the R package lme4,[@b24] achieving a fit of fixed and random effects (country) by the Laplace approximation.[@b25]
Ethical aspects
---------------
The accredited international ethical review board of CIET international approved the project in addition to an ethical review board in each country: the Health Research and Development Committee, Ministry of Health in Botswana; Research and Ethics Committee, Ministry of Health and Social Welfare in Lesotho; the National Health Sciences Research Committee, Ministry of Health in Malawi; the Comité Nacional de Bioética para a Saude, Ministerio da Saude in Mozambique; the Research Management Committee, Ministry of Health and Social Services in Namibia; the CIET Trust Research Ethics Committee in South Africa; the Scientific and Ethics Committee, Ministry of Health and Social Welfare in Swaziland; the Institutional Review Board, Ifakara Health Research and Development Centre in Tanzania; the Permanent Secretary, Ministry of Health, Zambia and the Medical Research Council of Zimbabwe. In each country, we also received written authority from the Ministry of Education to interview children in school. School head teachers gave consent to survey students in their school and contacted parents to inform them about the survey in general terms and give them the option to opt-out their child.
Results
=======
From 60 646 facilitated self-administered questionnaires in schools in the 10 countries in 2007, we obtained 59 986 usable records (1.1% did not complete or spoiled their questionnaires). We excluded 10 631 respondents (17.7%) who did not report their age or reported their age as '17 years or older' (9623). Of 49 355 respondents aged 11--16 years, 48 586 (98.4%) answered the question about forced or coerced sex. In 2003, 28 896 students aged 11--16 years in eight of the countries completed the questionnaire and 27 772 (96.1%) of them answered the question about forced or coerced sex.
In 2007, some 27.5% (based on 13 216/47 102) of respondents lived in houses with more than three people per room and 13.6% (based on 8498/48 614) reported they had insufficient food in their households in the week before the survey. Some 29.2% (based on 14 178/49 355) correctly recognised three child rights (to go to school, to be safe and not to be abused). Based on direct observation and key informant responses, 46.4% (based on 17 202/42 028 for whom this information was available) lived in a community that could be accessed by good tar road and 67.7% (based on 30 953/44 661) lived near an active government HIV prevention programme.
Experience of forced sex
------------------------
Weighting for country size and urban/rural proportions in each country, in 2007, 19.6% (based on 4432/25 840) of female youth and 21.1% (based on 4080/21 613) of male youth aged 11--16 years reported they had experienced forced or coerced sex. [Figure 1](#fig1){ref-type="fig"} shows the age- and sex-specific rates, each point representing the population weighted average across 10 countries, for ages 11--16 years and for male and female respondents. Up until the age of 14 years, male students reported higher rates than female students. [Table 1](#tbl1){ref-type="table"} shows the weighted percentages of male and female students aged 16 years who had ever experienced forced or coerced sex in each of the 10 countries. Across the 10 countries, 25.4% of male students and 28.8% of female students had experienced forced or coerced sex by the time they were 16. There was considerable variation between countries. Among males, the rates ranged from 11.9% in Botswana to 37.8% in Malawi, and in females, they ranged from 15.0% in Botswana to 43.2% in Tanzania.
{#fig1}
######
Reported experience of forced or coerced sex among male and female students aged 16 years in 2007, by country
Country Fraction (weighted %) who ever experienced forced or coerced sex
------------------------ ------------------------------------------------------------------ ------------------
Botswana 48/408 (11.5) 44/299 (14.7)
Lesotho 127/587 (22.6) 174/790 (21.5)
Malawi 165/436 (37.8) 87/237 (36.9)
Mozambique 124/472 (27.0) 65/290 (21.5)
Namibia 80/334 (24.4) 93/330 (29.4)
South Africa 142/767 (17.7) 176/955 (18.3)
Swaziland 79/562 (13.9) 84/496 (17.2)
Tanzania 119/356 (32.0) 196/460 (42.6)
Zambia 152/572 (26.6) 159/491 (33.4)
Zimbabwe 45/219 (20.2) 23/140 (15.7)
All countries combined 1093/4762 (25.4) 1118/4554 (28.8)
[Table 2](#tbl2){ref-type="table"} shows country-specific rates of the experience of forced or coerced sex in 2003 and 2007, separately for male and female respondents, age standardised on the age distribution in the Botswana 2007 male sample. The comparison is limited to those schools covered in the survey in eight countries in both 2003 and 2007. Among male respondents, age-standardised rates decreased significantly in two countries, increased significantly in two and did not change significantly in the other four. Among female respondents, in all but one country, there was a lower rate in 2007 than in 2003, but none of the changes on their own were significant.
######
Age-standardised comparison between 2003 and 2007: experience of forced or coerced sex among school-going youth aged 11--16 years, in schools which conducted both surveys
Country Male Age-standardised contrast 2007/2003, RR (95% CI) Female Age-standardised contrast 2007/2003, RR (95% CI)
------------ ---------- -------------------------------------------------- ---------- -------------------------------------------------- ------------------------- ---------- ------- ---------- ------- ---------------------
Botswana 163/1006 0.164 295/2812 0.0995 **0.61 (0.49 to 0.75)** 142/1215 0.113 348/3342 0.099 0.88 (0.74 to 1.05)
Lesotho 352/1917 0.170 416/2071 0.211 **1.24 (1.04 to 1.49)** 451/2805 0.156 499/3269 0.137 0.88 (0.75 to 1.02)
Malawi 521/1829 0.258 517/1904 0.246 0.95 (0.83 to 1.10) 479/1822 0.249 465/1698 0.255 1.02 (0.89 to 1.17)
Mozambique 233/947 0.268 479/2043 0.264 0.98 (0.69 to 1.39) 153/698 0.180 291/1562 0.179 0.99 (0.77 to 1.28)
Namibia 325/1313 0.234 332/1355 0.238 1.02 (0.84 to 1.23) 397/1736 0.219 394/1698 0.209 0.95 (0.84 to 1.12)
Swaziland 259/2024 0.122 249/2389 0.091 **0.75 (0.62 to 0.90)** 360/2792 0.121 358/2948 0.116 0.96 (0.83 to 1.13)
Zambia 501/1807 0.241 418/1699 0.236 0.98 (0.79 to 1.21) 526/1837 0.249 495/2012 0.221 0.89 (0.75 to 1.03)
Zimbabwe 202/1247 0.109 511/2772 0.183 **1.68 (1.41 to 1.94)** 326/1577 0.192 527/3461 0.154 0.80 (0.53 to 1.20)
Values in bold indicate a 2003--2007 difference significant at the 5% level.
Direct age standardisation on Botswana 2007 male population.
[Table 3](#tbl3){ref-type="table"} shows the bivariate associations, adjusted for country and clustering, between personal and cluster-level variables and experience of forced or coerced sex, among male and female students. The patterns were similar among the males and females: older youth were more likely to have experienced forced or coerced sex, as were those who did not have enough food in the house in the last week. Students attending schools where experience and perpetration of forced sex was more common and where more students used alcohol were more likely to report experiencing forced or coerced sex.
######
Risk factors for lifetime experience of sexual violence in school-going male and female youth aged 11--16 years in 2007
Characteristics Categories Lifetime experience of sexual violence[\*](#table-fn1){ref-type="table-fn"}
-------------------------------------------------------------------------------------------------------- --------------- ----------------------------------------------------------------------------- ------------------------- ------------- -------------------------
Individual and household variables
Age group 11--13 years 1137/6591 **1.13 (1.03 to 1.23)** 1114/9076 **1.71 (1.52 to 1.92)**
14--16 years 2943/15 022 3318/16 764
Area of residence Urban 1685/10 007 1.10 (0.98 to 1.25) 1840/12 237 1.09 (0.96 to 1.25)
Rural 2395/11 606 2592/13 603
Crowding in the house 1--3 per room 2710/14 840 0.96 (0.89 to 1.04) 3055/17 814 0.94 (0.86 to 1.02)
4--10 per room 1154/5767 1206/7006
Enough food in the house in the last week Yes 3199/17 621 **1.33 (1.20 to 1.46)** 3409/21 041 **1.51 (1.35 to 1.69)**
No 824/3724 952/4457
Community-level variables
Access by good tar road Yes 1319/7405 0.93 (0.80 to 1.09) 1496/9190 1.03 (0.88 to 1.20)
No 2260/11 190 2309/12 673
Active government HIV prevention programme Yes 2390/13 124 1.01 (0.86 to 1.17) 2524/15 361 1.01 (0.85 to 1.19)
No 1185/5551 1196/6538
Proportion of adults saying that "women sometimes deserve to be beaten" Below average 1979/10 366 0.94 (0.83 to 1.07) 2139/12 442 0.91 (0.80 to 1.04)
Above average 1884/10 104 2028/12 142
Proportion of adults saying it is "okay for an older man to have sex with teenagers" Below average 2582/13 517 0.98 (1.86 to 1.12) 2865/16 462 0.91 (0.80 to 1.03)
Above average 1281/6953 1302/8122
Proportion of adults saying "men have the right to sex with their girlfriends if they buy them gifts" Below average 2213/11 879 1.06 (0.94 to 1.20) 2515/14 535 0.98 (0.85 to 1.13)
Above average 1650/8591 1652/10 049
Proportion of adults reporting intimate partner violence in last year Below average 1654/8619 0.94 (0.83 to 1.07) 1785/10 435 1.01 (0.88 to 1.15)
Above average 2209/11 851 2385/14 149
School-level variables
Proportion of students reporting experience of sexual violence Below average 1540/10 909 **1.94 (1.73 to 2.17)** 1819/13 561 **1.84 (1.61 to 2.11)**
Above average 2540/10 704 2613/12 279
Proportion of students knowing of three child rights Above average 1678/9588 1.11 (0.98 to 1.25) 2095/12 263 0.99 (0.87 to 1.13)
Below average 2402/12 025 2337/13 577
Proportion of students agreeing that boys and girls are equal Above average 1851/10 491 1.13 (1.00 to 1.27) 2255/13 342 1.01 (0.90 to 1.14)
Below average 2229/11 122 2177/12 498
Proportion of students reporting perpetration of sexual violence Below average 1939/12 293 **1.60 (1.42 to 1.81)** 2298/15 320 **1.48 (1.29 to 1.70)**
Above average 2141/9320 2134/10 520
Proportion of students reporting drinking alcohol Below average 2182/12 700 **1.34 (1.19 to 1.52)** 2370/14 831 **1.31 (1.15 to 1.49)**
Above average 1898/8913 2062/11 009
Values in bold indicate associations significant at the 5% level.
Defined as those who responded positively to the question: "Has anyone ever forced or persuaded you to have sex when you did not want to?"
OR and 95% CI from bivariate analysis of group with characteristic compared with counterfactual group (eg, age 14--16 years compared with age 11--13 years), stratified by country and adjusted for clustering.
Multilevel analysis (GLMM) treated country as a random effect ([table 4](#tbl4){ref-type="table"}). The final GLMM model for female youth included one personal factor (age over 13 years), a household factor (insufficient food in the last week), three school group factors (a higher proportion who experienced forced or coerced sex, a higher proportion who perpetrated forced sex and a lower proportion who knew about child rights) and two community factors (where more adults said a man could expect sex if he gave a gift to a woman and where more adults reported intimate partner violence in the last year). Among male youth, there was one household factor (insufficient food in the last week), three school group factors (a higher proportion who experienced forced or coerced sex, a higher proportion who perpetrated forced sex and a higher proportion who reported use of alcohol) and one community factor (where more adults said a man could expect sex if he gave a gift to a woman).
######
GLMM of factors associated with forced or coerced sex in male and female youth aged 11--16 years
Variables in final GLMM models Adjusted OR (95% CI)
-------------------------------------------------------------------------------------------------------------------- ---------------------- ---------------------
Age over 13 years 1.49 (1.38 to 1.61)
Insufficient food in the last week 1.22 (1.12 to 1.34) 1.40 (1.29 to 1.53)
Attending a school where there was a higher proportion of students who said they had suffered sexual violence 1.79 (1.65 to 1.95) 1.76 (1.62 to 1.90)
Attending a school where a lower proportion of students knew about child rights 1.15 (1.08 to 1.25)
Attending a school where there was a higher proportion of students who said they had perpetrated sexual violence 1.22 (1.12 to 1.33) 1.18 (1.09 to 1.28)
Attending a school where alcohol use was more common among students 1.11 (1.03 to 1.20)
Living in a community where a higher proportion of adults said a man could expect sex if he gave a gift to a woman 1.16 (1.08 to 1.26) 1.16 (1.07 to 1.24)
Living in a community where a higher proportion of adults reported intimate partner violence in the last year 1.09 (1.01 to 1.17)
Country was treated as a random effect in the models. The initial saturated models for males and females included all the variables in [table 2](#tbl2){ref-type="table"}.
GLMM, generalised linear mixed model.
While the school group variables in [table 4](#tbl4){ref-type="table"} each had an effect in their own right, there was also evidence that the factors combined to increase the risk of sexual violence. Of female youth at schools where fewer people reported being a victim and fewer claimed to be perpetrators, 13.2% (1460/11 030) had suffered sexual violence; of those at schools where fewer than average were victims and more were perpetrators, 14.2% (359/2531) suffered sexual violence; of those with more victims and fewer perpetrators, 19.5% (838/4290) suffered sexual violence and of those at schools with more victims and more perpetrators, 22.2% (1775/7989) suffered sexual violence.
Perpetration of forced sex
--------------------------
In 2007, weighted for country population and urban/rural proportions in each country, 4.7% of female students (based on 1157/25 902) and 11.7% of male students (based on 2413/21 701) said they had perpetrated forced sex. The final GLMM model of risk factors for female perpetration of forced sex ([table 5](#tbl5){ref-type="table"}) included one personal factor (experienced forced or coerced sex), four school group factors (a higher proportion of students who experienced sexual violence, a higher proportion who perpetrated sexual violence, a lower proportion who knew about child rights and a higher proportion who used alcohol) and three community factors (where more adults said it is okay for older men to have sex with teenagers, where more adults reported intimate partner violence in the last year and communities that could not be accessed by tar road). The final GLMM model of risk factors for male perpetration of forced sex ([table 5](#tbl5){ref-type="table"}) included one personal factor (experienced forced or coerced sex), a household factor (insufficient food in the last week), three school group factors (a higher proportion of students who experienced sexual violence, a higher proportion who perpetrated sexual violence and a higher proportion who used alcohol) and one community factor (communities that could not be accessed by tar road).
######
GLMM of factors associated with being a perpetrator of forced sex, among male and female youth aged 11--16 years
Variables in final GLMM models Adjusted OR (95% CI)
------------------------------------------------------------------------------------------------------------------------------ ---------------------- ---------------------
Experienced forced or coerced sex 4.37 (3.96 to 4.82) 5.34 (4.66 to 6.13)
Insufficient food in the last week in household 1.30 (1.16 to 1.45)
Attending a school where there was a higher proportion of students who said they had suffered sexual violence 1.51 (1.28 to 1.78)
Attending a school where a lower proportion of students knew about child rights 1.29 (1.16 to 1.43) 1.35 (1.16 to 1.57)
Attending a school where a higher proportion of students said they had perpetrated forced sex 2.23 (2.01 to 2.49) 2.13 (1.81 to 2.51)
Attending a school where a higher proportion of students drank alcohol 1.25 (1.13 to 1.38) 1.17 (1.01 to 1.36)
Living in a community that is not accessible by tar road 1.33 (1.20 to 1.48) 1.51 (1.30 to 1.75)
Living in a community where a higher proportion of adults said it is acceptable for an older man to have sex with a teenager 1.17 (1.01 to 1.35)
Living in a community where a higher proportion of adults reported intimate partner violence in the last year 1.23 (1.07 to 1.42)
Country was treated as a random effect in the models.
GLMM, generalised linear mixed model.
Discussion
==========
Sexual violence was very common among school children in the 10 countries of southern Africa, affecting one in every five children aged 11--16 years of both sexes. The occurrence increased with age, so that by 16 years, a quarter of male students and a higher proportion of female students said they had experienced forced or coerced sex.
There was little evidence of a reduction in rates of forced sex between 2003 and 2007; the small reduction among female students was not significant in any country and the pattern among male students was inconsistent, with decreases in some countries but increases in others.
Risk factors for female and male students were similar, including personal factors (age), household-level factors (insufficient food as an indicator of poverty), school-level factors (attending schools where sexual violence and alcohol use was more common) and community-level factors (communities with more support for transactional sex and more intimate partner violence).
One in every 10 male students and one in 20 female students in 2007 said they had perpetrated forced sex. Victimisation was a strong risk factor for perpetration in both male and female students, and again, there were school- and community-level risk factors.
The 2007 study applied the same instrument in 10 countries within a period of 4 months, generating comparable data about coerced sex among youth of these 10 countries. This is the first school-based study of sexual violence using the same instrument at the same time across multiple countries in southern Africa. The anonymous self-administered questionnaire under carefully arranged conditions may have increased disclosure compared with face-to-face interviews, and this might help to explain the higher rates of forced sex than a study in South Africa that used face-to-face interviews.[@b15] Our measure of sexual violence was limited specifically to coerced physical sex. This leads to lower estimates of sexual violence than studies that include unwanted touching and verbal abuse as well as forced sex.[@b16]
The school-based surveys probably underestimated the rates of forced and coerced sex among all children since we excluded children not in school, who may have a higher risk of experiencing sexual violence or who may have left school because they experienced sexual violence. We have no details about enrolment and attendance other than on the day of the survey. The school-based surveys did not contact young women who were unable to attend school due to pregnancy, a possible result of sexual abuse. The percentage of female students illustrates their dropout with age: 60%, 59%, 57%, 55%, 53% and 49% with increasing age from 11 through to 16 years. If girls who experience forced or coerced sex leave school as a result, this could explain the apparently small gender gap or, in many country- and age-specific groups, more frequent reports of sexual violence among male than female respondents.
As with any self-reported experience, some students declined to answer questions and some may have given false answers. We recognise reasons not to report but we have no basis to expect respondents to fabricate a history of coerced sex; we expect this bias underestimated true rates. It is possible that under-reporting of forced or coerced sex was more marked among the female students. If so, this could explain our finding of a similar reported rate of forced sex between the sexes, even if the female youth actually experienced more forced sex.
There is a prevailing belief that child sexual abuse affects predominantly girls. Studies in Europe, the USA and Australia have generally reported higher rates of experience of sexual violence among female than male youth,[@b26; @b27; @b28; @b29; @b30; @b31] although a recent study from Ireland reported male rates of experience of sexual abuse in childhood not much lower than female rates.[@b32] Few studies from Africa report both male and female rates of experience of child sexual abuse. Collings[@b33] reported that 29% of a small sample of male university students in South Africa had experienced contact or non-contact sexual abuse as children and later reported 35% of female students in the same university had experienced contact forms of sexual abuse as children.[@b34] Two studies from a province of South Africa found similar rates of experience of childhood sexual violence among male and female youth[@b35] [@b36] and a large study of school-going youth in South Africa found similar rates of experience of forced or coerced sex among males and females.[@b14] [@b37] The problem is much less studied among male youth, especially in Africa, with many enquiries limited to female youth.
By definition, sex with children is abuse whether or not the child 'consents'. The age of consent is complicated, with differing ages in different forms of legislation. The age of consent in the countries included in our study is generally 16 years and 18 years in Tanzania. Thus, nearly all coerced sex reported in our study was child sexual abuse as a matter of definition. The questionnaire asked those who reported forced or coerced sex how old they were when it first occurred. Of the 1,118 sixteen-year-old females who reported forced or coerced sex, 498 said this first occurred when they were aged 16 years or did not give an age when it occurred, similarly among the 1093 sixteen-year-old males reporting forced or coerced sex, 377 said it first occurred when they were aged 16 years or did not specify the age of first occurrence. A sensitivity analysis excluded these 875 youth; we could detect no shift in the pattern of risk factors.
The risk factors we included in the survey and analysis were based on evidence from other studies,[@b12] [@b26] [@b27] [@b33] [@b34] [@b38; @b39; @b40] and a belief that since sexual violence is a clustered phenomenon, factors at school and community level may be important. We recognise other risk factors for forced sex among children that this study did not measure.[@b12] [@b33] [@b34]
Our findings on perpetration of forced sex are consistent with those reported elsewhere.[@b12] Male students were more likely to admit to forcing sex on someone else, but some female students also admitted to it. And being a victim of forced or coerced sex was a strong risk factor for being a perpetrator. In this cross-sectional study, we cannot say which came first, but the finding is compatible with the finding that many child perpetrators of rape have themselves been victims of sexual abuse.[@b41]
School-based group variables were strong risk factors for experience of forced or coerced sex and indeed perpetration of forced sex, illustrating the social nature of sexual violence. In this cross-sectional study, we cannot draw conclusions about which came first: personal experiences leading to school characteristics or the other way around. It seems plausible that some schools foster a culture of sexual violence, while others foster a culture of protection. If true, this could be key for school-based strategies to reduction of sexual violence among the students. Raghavan and colleagues[@b42] showed that witnessing community violence influenced social support networks, and these in turn influenced gender violence. The counterpoint is that *not* witnessing community violence might also influence gender violence but in a positive way.
Sexual abuse in childhood is profoundly linked to the risk of HIV, largely through high-risk behaviours among survivors[@b10]; the high rates of forced and coerced sex we found among school students are a cause for serious concern. Increasing resources and developing approaches for reducing sexual abuse of children in southern Africa, including randomised controlled trials of school-based interventions, should be a public health priority.
Supplementary Material
======================
###### Supporting Statement
###### Supporting Statement
###### Supporting Statement
###### Reviewer comments
These findings emanate from further analysis of the "Soul City regional programme audience reception and impact evaluation", for which CIET were commissioned to undertake surveys in eight countries in 2002--2003 and 2007. The Tanzania survey was part of the African Development of AIDS Prevention Trials capacities (ADAPT) project, funded by the Global Health Research Initiative through Canada\'s International Development Research Centre IDRC grant 104051-005. CIET Trust funded the South African survey. We thank the Ministries of Education that authorised the survey in the 10 countries, CIET field teams in each country and the 60 000 school-going youth who contributed to the survey.
**To cite:** Andersson N, Paredes-Solís S, Milne D, *et al*. Prevalence and risk factors for forced or coerced sex among school-going youth: national cross-sectional studies in 10 southern African countries in 2003 and 2007. *BMJ Open* 2012;**2**:e000754. doi:[10.1136/bmjopen-2011-000754](http://dx.doi.org/10.1136/bmjopen-2011-000754)
**Contributors:** NA designed the studies, provided oversight and training for fieldwork, conducted the analysis and wrote the present manuscript. SPS, DM, KO, NM and DL conducted the fieldwork and contributed to the writing. AC provided training and field supervision for the 2007 study and assisted with writing of the current manuscript. NA and AC are guarantors.
**Funding:** The Soul City commissioned surveys were funded by the European Union. The Tanzania survey was funded by the Global Health Research Initiative through the International Development Research Centre.
**Competing interests:** None.
**Ethics approval:** Health Research and Development Committee, Ministry of Health in Botswana; Research and Ethics Committee, Ministry of Health and Social Welfare in Lesotho; the National Health Sciences Research Committee, Ministry of Health in Malawi; the Comité Nacional de Bioética para a Saude, Ministerio da Saude in Mozambique; the Research Management Committee, Ministry of Health and Social Services in Namibia; the CIET Trust Research Ethics Committee in South Africa; the Scientific and Ethics Committee, Ministry of Health and Social Welfare in Swaziland; the Institutional Review Board, Ifakara Health Research and Development Centre in Tanzania; the Permanent Secretary, Ministry of Health, Zambia and the Medical Research Council of Zimbabwe. In each country, we also received written authority from the Ministry of Education to interview children in school.
**Provenance and peer review:** Not commissioned; externally peer reviewed.
**Data sharing statement:** Data from this study are not in the public domain.
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Main text {#Sec1}
=========
Central giant cell granuloma (CGCG) is defined by the World Health Organization as an intraosseous lesion consisting of cellular fibrous tissue that contains multiple foci of haemorrhage, aggregations of multiple nucleated giant cells, and occasionally trabeculae of woven bone \[[@CR1]\].
It is uncommon (7% of all benign jaw lesions), and the biologic behaviour ranges from quiescent to aggressive, with pain, root resorption and a tendency to recurrence after excision \[[@CR1]\]. In the great part of cases, CGCG lesion is unilateral. Sometime the lesion is located in a mandibular angle. And very few rare cases are reported in literature of bilateral CGCG located at the two angles of the mandible \[[@CR2]--[@CR4]\].
A case of bilateral CGCG of the mandibular angle has been reported in a 12 years old female, and classified as idiopathic, as none of the family members of the young girl presented with a similar lesion \[[@CR2]\]. Another sporadic case has been reported in an 18 years old girl, associated with neurofibromatosis type 1 \[[@CR3]\]. Finally, another case of bilateral CGCG of the mandibular angle was reported in a 8 years old female with Noonan's syndrome \[[@CR4]\].
In this cases series, we describe the first report in literature of a repetitive bilateral CGCG of the two mandibular angles, in three females from the same family. These rare presentations of CGCG may be defined as hereditary bilateral CGCG of the mandibular angles or also cherubism-like lesions.
In 1990, a 24-year-old young athlete was exposed to clinical observation at the maxillofacial surgery of the University of L'Aquila, central Italy, for the appearance of two osteolytic lesions at branches and mandibular angles (Fig. [1](#Fig1){ref-type="fig"}).Fig. 1The family
These lesions appeared symmetrical to radiological examinations (Fig. [2a, b](#Fig2){ref-type="fig"}).Fig. 2Mother: diagnosis at age 23. Mandibular x-Ray tomography: the right ramus (**a**) and the left ramus (**b**) show bilateral and symmetric radiolucenct areas. **c** Histopathological pattern suggests central giant cell granuloma. **d** Panoramic radiography 23 years after surgery; note the complete restoring of the mandibular bone structure
The patient underwent surgical intervention and histological examination (Fig. [2c](#Fig2){ref-type="fig"}) revealed a case of GCGC. The patient was then subjected to regular follow-up over the years.
We currently have an x-ray performed after 23 years from surgery, which confirms the absence of relapses and a good mandibular bone restructuring.
After getting married in 1995 she had three children: a son in 1996 and two daughters, respectively, in 1999 and 2006.
The mother, due to her previous pathological lesion, had made radiological controls in childhood to the male child, with negative results.
On the contrary, at the age of 9 years, two symmetrical bilateral osteolytic lesions of the jaw were observed in the first female daughter, in the same sites as the mother (Figs. [3](#Fig3){ref-type="fig"} and [4](#Fig4){ref-type="fig"}).Fig. 3TA, female, diagnosis at age 9 *(4-gen-2008) -* **a** Panoramic radiography shows on both side of the mandible two symmetric large multilocular radiolucent lesions involving the angle and the ramus regions (white arrows). In the lower dental arch, there are only the first molar at right side (**b**) and the first and second molars at left side (**c**) CTCB study of the mandible, respectively, of the right and the left site, shows the extension of the lesions. Note their critical relationship with the mandibular canal and its neurovascular structures, in particular the inferior alveolar nervesFig. 4TA, female, diagnosis at age 9 -- **a**-**c** and **d**-**f**: respectively the lesion of the right mandibular side and the left mandibular side. Note for each one the intraoperative aspect, and e.e. 10× and e.e. 20× histopatological speciments that show a moderately cellular and partially collagenized stroma, characterized by melted cells with dense nuclei and giant cells osteoclast like
Subsequently, Cone beam CT scans showed the same lesions to the second female daughter, but earlier, at age of 6 years \[[@CR5]\].
All the lesions were surgically removed (Figs. [4a](#Fig4){ref-type="fig"}, [5](#Fig5){ref-type="fig"}, [6](#Fig6){ref-type="fig"} and [7a, b](#Fig7){ref-type="fig"}, and [f](#Fig7){ref-type="fig"}) and the histopathologic diagnosis was always identical (Fig. [8](#Fig8){ref-type="fig"}): giant cell central granulomas, with patterns that showed an absolute correspondence between them and with the mother (compare Figs. [2c](#Fig2){ref-type="fig"},[3](#Fig3){ref-type="fig"}, [4b, c](#Fig4){ref-type="fig"}, [5](#Fig5){ref-type="fig"}, [6](#Fig6){ref-type="fig"} and [7c, d, g, h](#Fig7){ref-type="fig"}).Fig. 5TA, female, diagnosis at age 9 -- Follow-up at 5 years *(16-avr-2013)*. Panoramic radiography shows the good aspect of the bone mandibular structures and the absence of relapseFig. 6TC, female, diagnosis at age 6 (29-mar-2012) - **a** Panoramic radiography shows on both side of the mandible two symmetric large multilocular radiolucent lesions involving the angle and the ramus regions and the second molars. (white arrows). In the lower dental arch there are the first and the second molars (**b**, **c**) CTCB study of the mandible, respectively, of the right and the left site, shows the extension of the lesions. Note their critical relationship with the mandibular canal and its neurovascular structures, in particular the inferior alveolar nervesFig. 7TA, female, diagnosis at age 9 -- **a**-**d** and **e-h**: respectively the lesion of the right mandibular side and the left mandibular side. Note for each one the intraoperative aspect, the excised tissue, e.e. 10× and e.e. 20× histopatological speciments that show a moderately cellular and partially collagenized stroma, characterized by melted cells with dense nuclei and giant cells osteoclast likeFig. 8Absolute correspondence of the histological aspect of the lesions in the two sisters: the histological framework consisted of a moderately cellular and partially collagenized stroma, characterized by melted cells with dense nuclei and giant cells osteoclast like
After the surgery, radiological follow-up examinations showed no relapses and good restructuring of the mandibular bone structure (Figs. [2c](#Fig2){ref-type="fig"},[3](#Fig3){ref-type="fig"}, [4](#Fig4){ref-type="fig"}, [5](#Fig5){ref-type="fig"}, [6](#Fig6){ref-type="fig"}, [7](#Fig7){ref-type="fig"} and [9](#Fig9){ref-type="fig"}). The father was free from this disease. Periodical yearly follow-up was suggested for the two sisters until the end of puberty.Fig. 9TA, female, diagnosis at age 9. Follow-up at 8 months years *(16-oct-2013)*. Panoramic radiography shows the good aspect of the bone mandibular structures and the absence of relapse
To the best of the authors' knowledge, this is the first report of three cases of bilateral CGCG of the mandibular angles in three females from the same family. Considering the repetition of the lesion in subjects belonging to the same family, considering the particular location of the lesions (the mandibular angles in all three subjects), this situation may be attributed to the presence of a grade I (low level) Cherubism, or to the occurrence of cherubism-like lesions, as the cases did not show the other peculiar characteristics of Cherubism \[[@CR6]--[@CR8]\]. Table [1](#Tab1){ref-type="table"} shows the summary of the differences between Cherubism and idiopathic CGCG lesions that was followed in order to classify the lesion of the present cases. CGCG lesions may be associated with other disorders like Neurofibromatosis type 1 \[[@CR3]\], gingival fibromatosis as well as Noonan's syndrome \[[@CR4]\], all of them are Rasopathies. Noonan syndrome is an autosomal dominantly inherited syndrome with variable expressivity. And multiple CGCG lesions in Noonan's syndrome may be aggressive and cause complications. For these reasons, the diagnosis of Noonan's syndrome was firstly taken in consideration. But the physical examination of these subjects contributed to discard the diagnosis of Noonan's syndrome, that is characterized by short stature and atypical face like a broad or webbed neck, low set and posteriorly angulated ears, ptosis, hypertelorism, and downward-slanting eyes \[[@CR9]\].Table 1Comparison of the characteristics of Cherubism and idiopathic CGCGsCherubismIdiopathic CGCGAetiologyCaused by a gain-of-function mutation in the gene coding a c-Abltyrosine kinase-binding protein (SH3BP2) located on the short arm of chromosome 4The true aetiology is unknown and still controversial. It was thought that it is a reparative component. However, the evidence is not available to classify the lesions as reparative. The CGCG is thought by many to be reactive, but it is classified as a benign, non-neoplastic lesion.Gender distributionMore diffused in males (or equally diffused between males and females)More diffused in femalesAge distributionMore prevalently diagnosed in childrenCGCGs mainly affect patients between 10 and 30 yearsFacial aspectSwelling of bilateral mandibular angle region, typical of Cherubism (accompanied by hypertelorism)NormalOther signsA marked cervical lymphadenopathy is common.NoneDefinition (concept)Cherubism is an autosomal dominantly inherited condition, with variable expressivity, that is characterized by multi-quadrant radiolucent lesions of the jaws and a progressive and clinically, symmetrical enlargement of the mandible and/or the maxilla.Central giant cell granuloma (CGCG) is defined by the World Health Organization as an intraosseous lesion. The biologic behaviour ranges from quiescent to aggressive, with pain, root resorption and a tendency to recurrence after puberty.Mandibular LesionsSymmetrical mandibular lesionsLesions are typically found unilaterally in the frontal region of the mandible. Sometime the lesion is located in a mandibular angle.Family occurrenceThere is usually a familial history of similarly affected family members.Sometime they show an autosomal inheritance. In these cases, when bilateral, they are defined cherubism-like lesions.Histological aspect of lesionsThe lesions appear microscopically generally indistinguishable from CGCG, except occasionally, when a fairly characteristic condensation of perivascular collagen is evidentCellular fibrous tissue that contains multiple foci of haemorrhage, aggregations of multiple nucleated giant cells, and occasionally trabeculae of woven bone.Images and Rx aspectsMulti-quadrant radiolucent lesions of the jaws\
At the Rx can be observed a marked displacement or agenesia of second and third molars as well as premature exfoliation of primary teeth.Osteolytic lesions of the jawDifferential diagnosisNeurofibromatosis type 1, gingival fibromatosis as well as Noonan's syndrome, all of them are RasopathiesNeurofibromatosis type 1, gingival fibromatosis as well as Noonan's syndrome, all of them are RasopathiesTreatmentsTreatment of lesions consists of local curettage, jaw contouring, intralesional steroid injections, and systemic calcitonin administration as wellCommonly treated by surgical curettage.Long-term clinical managementLong-term follow-upLong-term follow-upPrognosisThe regression of the lesions is often seen following pubertyThese lesions tend to increase before the puberty (perhaps due to ovarian hormones) and to stabilize after puberty.
Cherubism is an autosomal dominantly inherited condition, with variable expressivity, that is characterized by multi-quadrant radiolucent lesions of the jaws and a progressive and clinically, symmetrical enlargement of the mandible and/or the maxilla \[[@CR10]--[@CR12]\]. There is usually a familial history of similarly affected family members and the regression of the lesions is often seen following puberty \[[@CR8]\]. In the present family the mother was 24-year-old at the time of the first diagnosis, consequently she could probably be considered as a missed diagnosis until that age.
From a cellular point of view, the cherubism-like lesions appear microscopically generally indistinguishable from CGCG, except occasionally, when a fairly characteristic condensation of perivascular collagen is evident \[[@CR10]\]. Consequently, the clinical aspects provide helpful clues to distinguish cherubism from CGCGs. CGCGs mainly affect patients between 10 and 30 years (while cherubism is more prevalent in children) and are typically found unilaterally in the frontal region of the mandible, whereas symmetrical lesions are found in cherubism \[[@CR13]\]. The present cases show cherubism-like lesions.
Cherubism originates from genetic alteration in the SH3BP2 gene, and currently, it is believed to be caused by a gain-of-function mutation in the gene coding a c-Abltyrosine kinase-binding protein (SH3BP2) located on the short arm of chromosome 4 \[[@CR14]\]. Only a sporadic case of CGCG with mutation of this gene was previously published \[[@CR11], [@CR15]\]. While another study conducted on a group of patients with an aggressive CGCG did not show any mutations, indicating that Cherubism is indeed a distinct entity from CGCG \[[@CR16]\]. In the present cases, the patients do not present the typical swelling of bilateral mandibular angle region, typical of Cherubism (accompanied by hypertelorism[1](#Fn1){ref-type="fn"}). But the repetition of the same cherubin-like lesions in three female subjects belonging to the same family, is suggestive for this diagnosis. Unfortunately, the family refused to perform genetic analysis to investigate the mutation in the SH3BP2 gene.
Dental findings in Cherubism include marked displacement of developing or agenesia of second and third molars as well as premature exfoliation of primary teeth \[[@CR17]\]. In addition, in Cherubism a marked cervical lymphadenopathy is common.
In the present cases, there were not all common clinical aspects of Cherubism and only females were characterized by lesions. While Cherubism, in the scientific literature, is reported to be more common in males or equally distributed between males and females \[[@CR17]\]. For the CGCG lesions, instead, the predominant distribution among females, respect to males, is certain \[[@CR2]\], correlated to the hormonal influence due to ovarian hormones, oestrogen and progesterone, which are supposed to be responsible for the development of CGCG as for other pathologies \[[@CR18]--[@CR21]\].
For example, some cases of central giant cell lesion in pregnant patients have showed a proliferation, and also in subjects during a hormonal therapy \[[@CR18]\]. But an immunostaining research, aimed to the detection of of estrogen and progesterone receptor proteins in 10 CGCG lesions, failed to evidence estrogen receptor protein, except for an occasional mononuclear cell stained weakly positive for estrogen receptor protein \[[@CR18]\]. In other cases, estrogen receptor positivity was found in stromal cells. In ten of these, osteoclast-type giant cells also exhibited estrogen receptor immunostaining \[[@CR22]\]. Due to the different results in literature, the direct influence of the ovarian hormones, estrogen and progesterone, in the development and growth of these lesions is still to be considered only a hypothesis.
For the present three cases, therefore, the hypothesis may be a hereditary form of bilateral CGCG of the mandibular angles - lesions that could be defined as cherubism-like lesions - or a rare manifestation of grade I Cherubism. CGCGs of the jaws are commonly treated by surgical curettage. And the management generally involves long-term follow-up, with the assumption that these lesions will stabilize during puberty. Thus, a yearly follow-up was suggested to the patients until the end of puberty.
Conclusion {#Sec2}
==========
Three females from the same family presented identical bilateral CGCG of the mandibular angles. In literature there are few reports about multiple CGCG, but this case clearly report the autosomal inheritance of this pathology, and even with a repetitive cherubism-like location of the lesion at the mandibular angles. Thus, should be important to perform the genetic analysis in order to investigate the presence of the related gene mutations.
CGCG
: Central Giant Cells Granuloma
Clinically, cherubism most commonly manifests as a progressive and symmetrical enlargement of the mandible and/or the maxilla, and is first noted between between 2 and 7 years of age, after which, lesions proliferate and increase in size until puberty. The lesions subsequently begin to regress, fill with bone and remodel until age 30, when they are frequently not detectable.
Mandibular swelling produces plump cheeks and maxillary enlargement causes retraction of the lower eyelids and elevation of the pupils upward, resulting in an "angel-like" appearance reminiscent of the cherubs depicted in Renaissance art.
The authors acknowledge dr Maria Tecco for her support in the editorial managing.
Funding {#FPar1}
=======
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Availability of data and materials {#FPar2}
==================================
The data that support the findings of this study are available from the archive of the University of L'Aquila, but restrictions apply to the availability of these data, which were used under permission and consent for the current study, and so are not publicly available. Data are however available from the authors upon reasonable request and with permission of the patients and the Ethic Committee of the University of L'Aquila.
ST and AN wrote the manuscript, analysed and interpreted the patient data regarding the disease and the treatment. PL and GC performed the histological examination and was a major contributor in writing the manuscript. SC revised the entire manuscript. TC and RG treated the patients. All authors read and approved the final manuscript.
Ethics approval and consent to participate {#FPar3}
==========================================
Ethics approval was obtained by the Ethic Committee of the University of L'Aquila, Italy. The consent to the treatment was obtained by the patients before the beginning of the therapy.
The partecipants have signed consent to the surgical intervention, the processing of personal data and the use of clinical material for scientific purposes (C.F.Uni.L'AquilaHosp.S.S.1995 and C.F.Uni.L'AquilaHosp.S.S.2012).
Consent for publication {#FPar4}
=======================
The consent to publish the present data was obtained from the subjects, also for the children. The partecipants have signed consent to the surgical intervention, the processing of personal data and the use of clinical material for scientific purposes (C.F.Uni.L'AquilaHosp.S.S.1995 and C.F.Uni.L'AquilaHosp.S.S.2012).
Competing interests {#FPar5}
===================
The authors declare that they have no competing interests.
Publisher's Note {#FPar6}
================
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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1. Introduction {#sec1-cancers-11-00163}
===============
Animal models have been extensively used in cancer research to investigate biology and genetics of human malignancies, including breast cancer, as well as efficacy and toxicity of chemical and biological agents \[[@B1-cancers-11-00163]\]. After the recent advent of immunotherapy, much attention has been focused on animal models outbred and with preserved immune system \[[@B2-cancers-11-00163]\]. Since dogs develop spontaneously mammary tumours, exhibiting a number of clinical and molecular similarities with human breast cancer \[[@B3-cancers-11-00163]\], canine mammary tumour could be an excellent model to study human disease.
Mammary gland carcinomas are a very common malignancy in adult bitches.
Reported annual incidence rates range between 145 and 250 per 100,000 dogs-year at risk amongst female dogs \[[@B4-cancers-11-00163],[@B5-cancers-11-00163],[@B6-cancers-11-00163],[@B7-cancers-11-00163]\]. In general, more than 40% of tumours in female dogs are mammary tumours (MT) \[[@B6-cancers-11-00163],[@B8-cancers-11-00163],[@B9-cancers-11-00163]\] and approximately 30--50% of canine MT are malignant \[[@B8-cancers-11-00163],[@B10-cancers-11-00163]\].
Regarding neutering status, it has been reported that ovariohysterectomy before the first or second oestrus cycle significantly reduces the relative risk of developing MT, while ovariohysterectomy later in life had no significant effect \[[@B8-cancers-11-00163],[@B10-cancers-11-00163],[@B11-cancers-11-00163]\]. More recently, the authors of a systematic review concluded that due to the limited evidence available and the risk of bias in the published results the evidences that neutering reduces the risk of MT and that age at neutering has an effect, are weak \[[@B12-cancers-11-00163]\].
Approximately 50% of canine mammary gland tumours are malignant, and 50% of these tend to infiltrate the surrounding tissues and metastasize to regional lymph nodes and lungs \[[@B13-cancers-11-00163],[@B14-cancers-11-00163]\].
While surgery represents the main therapy with curative intent for WHO stage I--III canine mammary carcinoma, the metastatic spread to regional lymph nodes and distant sites accounts for the vast majority of cancer-related deaths \[[@B15-cancers-11-00163],[@B16-cancers-11-00163]\]. The metastatic potential of canine mammary carcinoma is currently based on clinical stage and histopathological features of the surgical specimen, such as histotype, grade and status of the margins \[[@B17-cancers-11-00163],[@B18-cancers-11-00163]\]. However, no reliable markers exist to predict the early development of metastases before they occur \[[@B19-cancers-11-00163],[@B20-cancers-11-00163]\].
Circulating tumour cells (CTCs) are rare and defined as heterogeneous cells that shed from the primary tumour and circulate in the peripheral blood (PB) of human patients with cancer. Since sites of secondary disease are consistent with blood vessels' anatomy, hematogenous dissemination of tumour cells has for a long time supposed to have been a key step of metastatic dissemination \[[@B21-cancers-11-00163],[@B22-cancers-11-00163]\]
Furthermore, in the last fifteen years, direct and indirect evidences contrasted the view that hematogenous dissemination of tumour cells to secondary sites is a late and minor event respect to the lymphatic dissemination, challenging the traditional model of metastasis. In fact, since bone marrow can be invaded by tumour cells only through the blood flow, it is noteworthy that at diagnosis Braun and colleagues detected micro-metastases in the bone marrow of 30.6% of human breast cancers, regardless of disease stage \[[@B23-cancers-11-00163]\]; these tumour cells, indicated as disseminated tumour cells (DTCs), were associated with poor outcome stage \[[@B23-cancers-11-00163]\].
Moreover, karyotypic abnormalities of micro-metastasis in bone marrow, both in breast cancer patients and in animal models, indicate that tumour cells disseminate already during the pre-invasive stage of disease \[[@B24-cancers-11-00163]\], when the primary tumour is still undetectable. According to a general consensus, bone marrow can hence act as a reservoir of tumour cells, namely DTCs, which can then re-enter the circulation and spread to distant organs, finally giving overt metastasis \[[@B25-cancers-11-00163]\].
By injecting intravenous C57BL/6J mice with radiolabelled melanoma cells cultured in vitro, Fidler and colleagues \[[@B26-cancers-11-00163]\] extrapolated that less than 0.01% of the injected cells displayed competence to grow metastasis, since very few surviving tumour cells were needed to establish a metastasis. Several years later, the author itself provided consistent evidence in animal model that a single tumour cell may be enough to develop metastasis in vivo \[[@B27-cancers-11-00163]\]. Indeed, although rare in human malignancies, CTCs have been associated with progression-free survival and treatment efficacy in different solid tumours, including breast cancer \[[@B28-cancers-11-00163]\].
Moreover, CTCs have been proposed as a potential biological marker for early detection, prognosis and treatment selection and as a promising tool for monitoring disease progression, since a high number of CTCs measured at any time during treatment has been associated, in several studies, with a shorter time to progression \[[@B29-cancers-11-00163],[@B30-cancers-11-00163]\]. Conversely, a decreasing CTC number during treatment has been associated with therapeutic success \[[@B30-cancers-11-00163],[@B31-cancers-11-00163]\].
Over the past two decades, many systems for CTC/DTC detection have been developed. In 2004, CTC enumeration using the CellSearch (CS) system was shown to be significantly associated with outcome in women with metastatic breast cancer \[[@B29-cancers-11-00163]\], leading the Food and Drug Administration to approve this technique as a method to monitor breast cancer treatment and to indicate its effectiveness. In particular, a risk threshold could be established, since human patients with ≥5 CTCs per 7.5 mL PB had a shorter progression-free survival and overall survival (OS) \[[@B29-cancers-11-00163]\]. Notably, a pooled analysis of individual data obtained from 1944 metastatic breast cancer patients from 17 European centres confirmed that CTC enumeration at baseline is a strong independent prognostic marker that adds value to the existing clinical prognostic variables \[[@B30-cancers-11-00163],[@B31-cancers-11-00163]\].
In veterinary medicine, only few studies have aimed, so far, to identify CTCs in the canine species \[[@B32-cancers-11-00163]\]. Specifically in mammary carcinoma, CTCs have been detected by means of a nucleic acid-based method based on reverse transcriptase polymerase chain reaction (RT-PCR) assays \[[@B33-cancers-11-00163],[@B34-cancers-11-00163]\]. However, with this method, no association was found between CTCs and outcome in a clinical setting.
On these bases, we designed a prospective, single-centre pilot study, whose primary objective was: to enumerate CTCs and DTCs in dogs with metastatic mammary carcinoma (MMC), using CS, before medical treatment and at the first follow-up visit. The secondary end-points were: (1) to correlate CTC and DTC enumeration with survival time (ST); (2) to establish a cut-off value of prognostic significance.
2. Results {#sec2-cancers-11-00163}
==========
2.1. Clinical Characteristics {#sec2dot1-cancers-11-00163}
-----------------------------
Thirty-two female dogs were enrolled: 21 (65.6%) purebred and 11 (34.4%) crossbreed dogs. Among pure bred dogs, Bichon frisè (*n* = 2, 10%), Poodles (*n* = 2, 10%) and Golden retrievers (*n* = 2, 10%) were the most common. Twenty-nine (90.6%) dogs were spayed and 3 (9.4%) were intact. Median age was 11 years (range, 5--15 years) and median weight was 19.6 kg (range, 3.2--44.4 kg).
Overall, 25 (78.1%) dogs had undergone previous surgery and 7 (21.9%) dogs were considered non-surgical candidates.
At presentation, all dogs had measurable disease. Twenty-three (71.9%) had unilateral mammary cancer, whereas 9 (28.1%) had bilateral involvement.
Regarding clinical stage, 1 (3.1%) dog had stage III disease, 9 (28.1%) had stage IV disease (nodal metastasis) and 22 (68.8%) had stage V disease. The dog with stage III disease had histologically proven neoplastic emboli. Among dogs with stage V disease, 10 (45.5%) had cutaneous metastasis, 5 (22.7%) had cutaneous and pulmonary metastasis, 2 (9.1%) had cutaneous and splenic metastasis, 1 (4.5%) had cutaneous and liver metastasis, 1 (4.5%) had lung metastasis, 1 (4.5%) had cutaneous, lung and spleen metastasis, 1 (4.5%) had cutaneous, lung, spleen and liver metastasis and 1 (4.5%) had bone metastasis.
2.2. Histopathology {#sec2dot2-cancers-11-00163}
-------------------
Overall, 26 out of 32 paraffin blocks were retrieved and underwent histological and immune histochemical investigations, whereas the remaining 6 samples (referred as 5 simple carcinomas and 1 anaplastic carcinoma with lymphatic invasion) were unavailable for revision. Based on the WHO classification, 3 (11.5%) samples were classified as complex carcinoma (1 grade I and 2 grade III), 14 (53.8%) as simple carcinoma (7 grade II and 7 grade III), 5 (19.2%) as anaplastic carcinoma (grade III) and4 (15.4%) as solid carcinoma (grade III).
Eleven out of 26 tumours (42.3%) were further classified into the clinical-pathologic entity of inflammatory mammary carcinoma, based on the presence of neoplastic emboli within the lymphatic vessels of the dermis ([Figure 1](#cancers-11-00163-f001){ref-type="fig"}A).
By immunohistochemistry, 6 (23.1%) samples were positive for ERα, 6 (23.1%) were positive for HER-2, 1 (3.8%) was positive for PR and 1 (3.8%) sample showed co-expression of ER/PR, whereas 12 out of 26 (46.2%) samples were negative for all three antibodies tested.
2.3. Treatment and Response {#sec2dot3-cancers-11-00163}
---------------------------
Thirty out of 32 dogs (93.8%) received medical treatment, consisting of a tyrosine kinase inhibitor (*n* = 18) or systemic cytotoxic chemotherapy (*n* = 12). Two (6.2%) dogs died shortly after the pre-treatment evaluation and received no treatment.
Among the 30 dogs that were evaluable for treatment response, 5 (16.7%) achieved complete response (CR), defined as disappearance of all lesions for \>4 weeks; 6 (20%) achieved partial response (PR), defined as a decrease of at least 30% in the diameter of a lesion for \>4 weeks; 2 (6.7%) were stable (\<30% reduction or 20% increase in the diameter of a lesion); and 17 (56.7%) progressed, (appearance of a new metastatic lesion or at least a 20% increase of the diameter of a lesion).
At the end of the study, 25 dogs had died (21 due to their cancer and 4 for unrelated causes), 5 were still alive and 2 dogs were lost at follow-up. The median follow-up time was 167 days (25th percentile: 97 days, 75th percentile: 315 days) while the median ST was 189 days (25th percentile: 104 days, 75th percentile: 454 days). The 4 dogs that had died for unrelated causes were censored.
2.4. CTC and DTC Prevalence in MMC {#sec2dot4-cancers-11-00163}
----------------------------------
Overall, 32 and 19 dogs had PB and BM drawn at baseline (T1) for CTC and DTC enumeration, respectively. At the first follow-up (T2), 15 and 6 dogs underwent CTC and DTC enumeration, respectively. The number of evaluable dogs at the following time point decreased because of death, occurring in the meantime.
Considering the feasibility of CS assay in the veterinary setting, at T1 informative results were obtained in 27 out of 32 (84.4%) PB samples and in 13 out of 19 (68.4%) BM samples, respectively. At T2, informative results were obtained in 15 out of 15 (100%) PB samples and in 3 out of 6 (50%) BM samples. The main reasons for unsuccessful tests were partially clotted samples or slides not fully satisfying internal quality controls (at least two DAPI+ events for doing autofocus, in at least 6 out of 9 selected microscopic fields), so that the automated scanner of the slide failed.
At T1, different levels of tumour cells in PB and BM were documented. Indeed, 12 out of 27 (44.4%) dogs had at least 1 CTC/7.5 mL PB and 11 out of 14 (78.6%) dogs had at least 1 DTC/1 mL BM, respectively ([Figure 1](#cancers-11-00163-f001){ref-type="fig"}B). Conversely, we did not find any CTCs in PB of healthy, negative control dogs (*n* = 5).
The expression of M30, a marker of early apoptosis in epithelial cells, was high both in PB and in BM samples, with a median value of 75% and 90% in CTCs and DTCs, respectively and high inter-samples variability (range 0 to 100%, in both districts).
2.5. Prognostic Significance of CTC/DTC Enumeration {#sec2dot5-cancers-11-00163}
---------------------------------------------------
[Table 1](#cancers-11-00163-t001){ref-type="table"} summarizes the distribution of clinical-pathological characteristics of the cohort, according to the presence of CTCs. For all but one variables (the expression of progesterone receptor), we had more than 1 case, hence we could perform statistics, by appropriate tests for a pilot study (Chi-square test with Yates correction or Fisher's exact test).
Overall, CTCs were not associated with clinical-pathological characteristics ([Table 1](#cancers-11-00163-t001){ref-type="table"}), including the presence of single versus multiple sites of metastasis. Notably, prior surgery and neutering status did not affect CTC prevalence at baseline.
Inflammatory carcinoma is the only remarkable exception, since it was significantly associated with the occurrence of CTCs ([Table 1](#cancers-11-00163-t001){ref-type="table"}, *p* = 0.035, Fisher's exact test).
At T2, only 4 out of 15 (26.7%) evaluable PB samples were CTC-positive, meanwhile 2 out of 3 (66.7%) evaluable BM samples resulted DTC-positive. By comparing T1 and T2 results, 6 out of 8 (75%) samples that were CTC-negative at T1 remained negative at T2, while 5 out of 6 (83%) positive samples at T1 turned out to be negative at T2. The dogs of this last group were too few to do further speculations, however, we recorded that 4 out of these 5 dogs died because of mammary cancer after 50, 72, 189 and 294 days, respectively, whilst the fifth dog was alive at data analysis closure after 523 days.
Concerning changes in DTC levels, in paired T1--T2 samples we recorded not evaluable, 8 and 41 cells at T1 versus 2, 0 and 8 DTCs at T2, in cases \#21, \#22 and \#24 respectively.
Moreover, in both PB and BM, the levels of tumour cells decreased at T2, with a median number of 1 CTC and 2 DTCs, respectively.
By stratifying dogs according to CTCs measured at T1 (*n* = 27), a significant association with ST was documented; indeed, the median ST was 339 days in CTC-negative dogs (*n* = 15) versus 105 days in dogs with at least 1 CTC (*n* = 12) (*p*-value *=* 0.0361, Wilcoxon test; [Figure 2](#cancers-11-00163-f002){ref-type="fig"}A). Considering a cut-off of CTCs ≥ 2, a stronger association with ST was observed (305 vs.105 days for CTC numbers \< 2 vs. ≥ 2, respectively) (*p*-value = 0.0113, Wilcoxon test; [Figure 2](#cancers-11-00163-f002){ref-type="fig"}B). Conversely, the DTC count at T1 did not show any significant association with ST (*p*-value = 0.8299, Wilcoxon test), even though dogs with at least 1 DTC (*n* = 11) showed shorter median ST (189 days) than DTC-negative dogs (*n* = 3; 454 days).
Finally, the presence of at least 1 cell, regardless of the site (PB or BM) showed a weak association with ST (*p*-value = 0.0989, Wilcoxon test; [Figure 2](#cancers-11-00163-f002){ref-type="fig"}C).
3. Discussion {#sec3-cancers-11-00163}
=============
In dogs, MMC represents a therapeutic challenge, mainly due to the very limited treatment options. Once distant metastases have occurred, the disease remains largely incurable, with a median ST that rarely exceeds a couple of months. While this holds true for the majority of dogs, the risk of outcome is not identical among given individuals. Acknowledging the paucity of effective treatment options for dogs with MMC, the identification of prognostic markers is clearly crucial to identify those dogs that may harbour a better prognosis and benefit from treatment, thereby guiding the clinical decision-making process.
Herein, we document for the first time the feasibility to quantify CTCs and DTCs in canine MMC with the automated CS platform. Sampling of PB and BM was uneventful and less invasive than multiple biopsies of metastatic sites and the use of an automated platform permitted the serial monitoring of the enrolled dogs.
Secondly, the levels of CTCs/DTCs and the prevalence of positive dogs closely resemble the results obtained by CS in human metastatic breast cancer (MBC) at diagnosis \[[@B29-cancers-11-00163]\], further extending the similarities between humans and dogs regarding mammary tumour biology \[[@B3-cancers-11-00163]\]. Indeed, our results demonstrate that MMC is a systemic disease that involves BM in the great majority of cases, whilst the presence of CTCs is associated with a worse outcome. Therefore, CTC enumeration in canine MMC may yield prognostic information once the tumour is first diagnosed, since the provisional cut-off of at least 2 CTCs was significantly associated to a shorter median ST (105 days). By contrast, dogs with less than 2 CTCs had a median ST of 305 days, supporting the role of CTC assay to identify those subsets of dogs that would benefit from medical treatment.
In our study, CTC enumeration resulted to be an independent prognostic factor, not related with other pathological and clinical data, such as tumour type, histological grading and clinical staging. Although this finding might be considered with caution, because of the small sample size of our pilot study, and it warrants further investigations, it is noteworthy that it is similar to what it has been observed in more than two thousand human MBC \[[@B29-cancers-11-00163],[@B31-cancers-11-00163]\].
Interestingly, one third of MMCs included in this pilot study were inflammatory carcinomas and 70% of them were CTC-positive, a condition that closely resembles what has been observed in humans, in whom inflammatory breast cancer is a rare disease, burdened by early metastatic dissemination, high CTC levels and poor prognosis \[[@B35-cancers-11-00163]\].
Moreover, when we analysed the expression of M30, a marker of epithelial apoptosis, we found a high level of apoptosis of tumour cells both in PB and in BM, similarly to the findings previously reported in newly diagnosed, treatment-naive metastatic solid tumours \[[@B36-cancers-11-00163]\]. The expression of apoptosis markers in tumour cells might seem to be counterintuitive, especially when analysing metastatic disease; however, a higher histological grade and an increased proliferation are often associated with tumour necrosis and apoptosis, which we may regard as adverse prognostic features \[[@B37-cancers-11-00163]\]. In our canine cohort, this finding is consistent with the advanced stage of the MMCs included.
In human MBC, CTC enumeration over time is important in treatment monitoring \[[@B30-cancers-11-00163]\]. We can also confirm in our canine cohort that the CTC/DTC assay is dynamic enough to document decreasing levels of CTCs and DTCs at the first follow-up visit. Herein, we analysed all cases cumulatively, regardless of the type of treatment, since the primary objective of the study was to test the feasibility of the automated test and the sample size was accordingly small. The determination of the better therapeutic choice was beyond the purpose of this pilot study, an objective that warrants evaluation in larger cohorts, in ad hoc planned protocols \[[@B38-cancers-11-00163]\]. Nevertheless, based on our findings, CTC assay looks like a promising marker of treatment efficacy also in dog.
The main limit of our study is the low number of samples that prevented complete statistical analysis. In addition, the number of controls, close to 15% of the full cohort entered in the study, is low because of ethical reasons. However, the findings herein reported have to be considered a first milestone for further investigations.
At this regard, the heterogeneity of the population enrolled in this pilot study (as for breed, treatments and neutered status) mimics that of human population and it might be a plus, in view of potential application in translational research on human cancer.
In addition, since sampling the iliac crest allows, as opposed to other sites (including the humerus or ribs), a better and safer animal restraint, we could examine PB and BM in parallel, even if in few cases only. This is of utmost importance for translational studies, since bone marrow screening for occult metastatic tumour cells has not been included, in human cancer, as standard clinical routine in the majority of the European Member States \[[@B39-cancers-11-00163]\], because of the procedure's invasiveness \[[@B40-cancers-11-00163]\].
In conclusion, our study documents that CTCs and DTCs are an actionable prognostic biomarker of canine MMC, as the minimal invasiveness of sampling procedures and the use of an automated platform enable high robustness and reproducibility of findings. Taken together, these two aspects will lead to the design of prospective studies to evaluate treatment efficacy in veterinary oncology.
4. Methods {#sec4-cancers-11-00163}
==========
4.1. Animals and Samples {#sec4dot1-cancers-11-00163}
------------------------
The study protocol was approved by Committee of the "Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe)" (ethic code: \# 18913-P) responsible for animal protection and welfare and a mandatory written consent from all dog's owners was obtained. All the experiments were performed in accordance with the relevant guidelines and regulations, namely the Italian D.L. no. 26, 4 March 2014 that implements the European Directive 2010/63/UE regarding the protection of animals used for experimental and other scientific purposes and the Good Clinical Practice.
Overall, between December 2014 and January 2018, 32 client-owned, treatment-naïve dogs with a clinical and histopathological diagnosis of measurable MMC were eligible for recruitment.
We further analysed 5 PB samples from healthy dogs (from December 2014 and January 2019), which served as negative controls for CTC detection. Dogs used as negative controls were healthy female dogs, belonging to the blood donor register of the Istituto Zooprofilattico Sperimentale delle Venezie, that underwent periodically medical and laboratory screening, to exclude pathological status. The complete signalament of controls was: 1 Golden retriever, neutered female, 8 years old; 1 Newfoundland, intact female, 3 years old; 1 Labrador neutered female, 5 years old; 1 mixed breed, neutered female, 7 years old; 1 American Staffordshire terrier, 2 years, entire female. We did not sample the BM for ethical reasons.
Study inclusion criteria were as follows: 1.At least one unidimensional lesion measurable by ultrasound or computed tomography (CT), according to the canine Response Evaluation Criteria In Solid Tumours version 1.0 (cRECIST v1.0) \[[@B41-cancers-11-00163]\];2.Clinically detectable metastatic disease or the presence of lymphatic emboli, confirmed by histopathology;3.Prior surgery without systemic chemotherapy permitted.
A further optional inclusion criterion was:4.Availability of tumour tissue for histopathological evaluation and immune-histochemical staining.
Exclusion criteria were previous systemic chemotherapy or molecular target therapy.
At enrolment, all dogs underwent a complete routine staging work-up, consisting of tumour biopsy, CT or abdominal ultrasound and thoracic radiographs.
Additionally, a clinician was asked to collect into CellSave tubes (Menarini-Silicon Biosystems, Castel Maggiore, BO, Italy) both PB (7.5 mL) and BM (2 mL) of dogs from the iliac crest. In dogs, the iliac crest is commonly used for BM aspiration due to the greater feasibility compared to other sites. The dogs were not anesthetized for the procedure and sampling the iliac crest allowed as opposed to other sites (including the humerus or ribs) for a better and safer animal restraint.
Subsequently, we maintained samples at room temperature during the transport to the CTC-lab of IOV-IRCCS, where we performed baseline CTC/DTC enumeration within 96 h from the blood draw, according to manufacturer′s instructions.
The type of treatment was at the investigator's personal discretion and included traditional cytotoxic chemotherapy or target therapy and surgery when indicated.
Reassessment of disease status was conducted at the first follow-up (T2; 6--8 weeks, depending on treatment type and schedule) with clinical examination and imaging using cRECIST criteria \[[@B41-cancers-11-00163]\], without knowledge of baseline CTC/DTC results.
Briefly, disappearance of all lesions for \>4 weeks was defined as complete response (CR); a decrease of at least 30% in the diameter of a lesion for \>4 weeks was defined as partial response (PR); the appearance of new MCTs or at least a 20% increase of the diameter of a lesion, was defined as progressive disease (PD). Less than 30% reduction or 20% increase in the diameter of a lesion, was defined as stable disease (SD).
4.2. Histopathological Analysis {#sec4dot2-cancers-11-00163}
-------------------------------
Surgical or bioptic paraffin-embedded samples of the cases included were retrieved by different histopathological services and further sections were stained with haematoxylin and eosin (HE) for histological evaluation. Tumours were classified according to the WHO international classification of mammary tumours of domestic animals \[[@B17-cancers-11-00163],[@B42-cancers-11-00163]\] and grading was applied according with Peña and colleagues \[[@B18-cancers-11-00163]\] with the agreement of two pathologists (CZ, MV).
In addition, 3-μm sections from each sample were stained by the BenchMark ULTRA automated immunostainer (Ventana Medical Systems, Tucson, AZ, USA) using 3 different antibodies (already validated in dogs by Jaillardon and coll. \[[@B43-cancers-11-00163]\]):
\(a\) The anti-progesterone receptor monoclonal rabbit pre-diluted antibody (clone 1E2, Roche Diagnostics) for 24 min at 37 °C;
\(b\) The Pathway anti/HER-2/neu (HER-2) monoclonal rabbit pre-diluted antibody (clone 4B5, Roche Diagnostics) for 32 min at 36 °C;
\(c\) The anti-oestrogen-α receptor monoclonal mouse antibody (Clone C311, Santa Cruz Biotechnology, Dallas, TX, USA) applied at 1:50 dilution for 44 min at room temperature.
For all the three antibodies, dewaxing was performed by the instrument at 72 °C for 8 min. Antigen retrieval was performed using a commercial pre-diluted solution (pH 8.4) (ULTRA cell conditioning solution (CC1), Ventana Medical System, Tucson, AZ, USA), at 95 °C for 64 min for PR and for 52 min for HER-2. No antigen retrieval was applied for ERα antibody.
An indirect biotin-free system (UltraView universal DAB detection kit (052 697 806 001 code), Ventana Medical Systems, Tucson, AZ, USA) was used as detection system. Haematoxylin was used as contrast stain.
The immunohistochemical protocols for all antibodies were developed at the Laboratory of Histopathology of the Istituto Zooprofilattico Sperimentale delle Venezie using positive controls in each run, consisting of a canine mammary gland for PR and ERα antibodies and a control slide pathway for HER2 provided by the Company. Each run also included negative controls obtained by omitting the primary antibody during the labelling steps.
4.3. CTCs/DTCs Enumeration {#sec4dot3-cancers-11-00163}
--------------------------
The presence of CTCs and DTCs in canine PB or BM, respectively, was assessed by CellSearch^™^ System (Menarini-Silicon Biosystems, Castel Maggiore (BO), Italy), according to the manufacturer′s instructions, with minimal modification \[[@B28-cancers-11-00163],[@B29-cancers-11-00163]\].
Since it has been previously reported the cross-species reactivity man/dog of monoclonal antibodies (mAbs), in particular for highly conserved proteins as cytokeratins and EpCAM \[[@B44-cancers-11-00163],[@B45-cancers-11-00163]\], we used the standard CTC kit (Menarini), developed by the manufacturers for human samples and based on immune-magnetic enrichment and fluorescent labelling. The CellSearch platform is an automated, closed system, whose reagents are provided in a ready-to-use package that includes the following mouse mAbs: ferrofluid-conjugated anti-EpCAM, PE-conjugated anti-cytokeratins (8, 18 and 19) and APC-conjugated anti-CD45; DAPI is also included as nucleic acid dye.
Secondly, we examined a median volume of 7.5 mL PB and 1 mL BM for CTC and DTC samples, respectively; volume variations depended on the dogs' weight, as previously reported for mouse model \[[@B46-cancers-11-00163]\]. Finally, we adjusted the red blood cell level by diluting canine blood draws with run's buffer, according to the maximum permitted by the CellSearch platform.
The classification of cells, instead, fully satisfied the standard CellSearch criteria. Briefly, we classified an event as a CTC, when its morphological features were consistent with those of a tumour cell and the phenotype EpCAM+, CK+, DAPI+ and CD45- was exhibited.
We also quantified apoptotic CTCs by integrating the standard assay with an anti-M30 mAb (ALX-804-590, Alexis Biochemicals, San Diego, CA, USA); M30 is a neoepitope disclosed by caspase cleavage of cytokeratin 18 (CK18) in early apoptosis of epithelial cells \[[@B36-cancers-11-00163]\]. Quantitative results were expressed as the total number of cells and of M30-positive cells per 7.5 mL PB or 1 mL BM, for CTCs and DTCs, respectively.
4.4. Statistical Analysis {#sec4dot4-cancers-11-00163}
-------------------------
The association with CTC level was assessed using Chi-square test with Yates' correction or Fisher′ exact test for clinical-pathological characteristics and Student *t*-test for age distribution.
The Kaplan-Meier method was used to draw the cumulative survival curves, applied to time between the date of diagnosis and the date of death for mammary tumour; the dogs alive at the end of follow-up period or those dead for other causes were censored using the time between the date of diagnosis and their most recent follow-up evaluations.
The proportional-hazards assumption was evaluated by means of a log-log plot. Then, the Wilcoxon test was adopted \[[@B47-cancers-11-00163]\] to compare survival curves between MMC groups, defined as: (a) CTC ≥ 1 and CTC \< 1 cell; (b) CTC ≥ 2 and CTC \< 2 cells; (c) DTC ≥ 1 and DTC \< 1 cell; and (d) dogs with CTC or DTC ≥ 1 versus dogs with a cell number \< 1 cells for both CTC and DTC.
All statistical analyses were carried out using STATA version 12.1.
5. Conclusions {#sec5-cancers-11-00163}
==============
Finally and importantly, our findings have a translational interest, since canine invasive mammary tumours share many features with human breast cancer and develop spontaneously in dogs with an intact immune system, without genetic or chemical manipulation \[[@B3-cancers-11-00163]\]. The value of this opportunity has been increasingly recognized in different fields of cancer research, such as tumour biology and cancer progression, identification of cancer-associated genes and the development of novel cancer therapeutics. To similar purposes, CTC derived mouse models (CDXs) have been proposed by several authors in malignancies of lung \[[@B48-cancers-11-00163],[@B49-cancers-11-00163]\], liver \[[@B50-cancers-11-00163]\], breast \[[@B46-cancers-11-00163],[@B51-cancers-11-00163]\] and prostate \[[@B46-cancers-11-00163]\], among the others. However, as the patient-derived mouse models (PDXs) \[[@B52-cancers-11-00163]\], CDXs have low success rate, require long time to be established and are unsuitable to investigate tumour/microenvironment and tumour/immune system interplay.
Herein, we show that the model of canine MMC, monitored by dog CTCs, addresses these challenges, in particular for clinical trials purposes, since the shorter lifespan of dogs with respect to humans represents an excellent resource for testing new therapeutics in a pre-clinical setting. Our study reinforces the validity of dogs as spontaneous models for cancer, highlighting further similarities in tumour spread and progression between dogs and humans.
The authors thank Christina Drace (IOV-IRCCS, Padua, Italy) for English editing.
M.V., R.Z., E.R. and L.M. designed research; L.M. and P.L. enrolled and treated affected dogs; M.V., E.M. and C.Z. performed histopathological analyses; A.F., E.R., R.V. and R.Z. performed CTC/DTC tests; R.Z. and K.C. performed statistical analyses; L.M., V.M. and R.Z. wrote the paper. All authors reviewed the manuscript.
This work was supported by a grant from the Italian Ministry of Health, (RC VE 6-2014), entitled "A pilot study to evaluate the prognostic potential of circulating tumour cells in dogs with metastatic breast cancer," (PI: MV); R.V. was supported by a fellowships funded by Ricerca Corrente (Italian Ministry of Health).
All the authors declare to have no competing interests, neither of a financial nor non-financial nature.
{#cancers-11-00163-f001}
{#cancers-11-00163-f002}
cancers-11-00163-t001_Table 1
######
Clinical-pathological characteristics of dogs' cohort.
Variable N CTC-Neg CTC-Pos *p*-Value
------------------------- ---- --------------------- --------------------- -----------
Breed
Purebred 18 11 (61.11%) 7 (38.89%) 0.448
Crossbreed 9 4 (44.44%) 5 (55.56%)
Age (years) 27 10.53 (SD \* 1.99) 11.16 (SD \* 2.72) 0.492
Weight (kg) 27 20.83 (SD \* 13.87) 17.23 (SD \* 11.33) 0.475
Previous surgery
Non-surgical candidates 6 3 (50.00%) 3 (50.00%) 1.000
Prior surgery 21 12 (57.14%) 9 (42.86%)
Neutering status
Intact 3 2 (66.67%) 1 (33.33%) 1.000
Neutered 24 13 (54.17%) 11 (45.83%)
Grading
1--2 8 3 (37.50%) 5 (62.50%) 0.204
3 16 11 (68.75%) 5 (31.25%)
Inflammatory carcinoma
No 14 11 (78.57%) 3 (21.43%) 0.035
Yes 10 3 (30.00%) 7 (70.00%)
Staging
III + IV 10 5 (50.00%) 5 (50.00%) 0.706
V 17 10 (58.82%) 7 (41.18%)
Metastatic sites
Single 12 6 (50.00%) 6 (50.00%) 1.000
Multiple 14 8 (57.14%) 6 (42.86%)
Oestrogen receptor
Negative 18 10 (55.56%) 8 (44.44%) 1.000
Positive 6 4 (66.67%) 2 (33.33%)
HER-2
Negative 18 11 (61.11%) 7 (38.89%) 0.665
Positive 6 3 (50.00%) 3 (50.00%)
Progesterone receptor
Negative 23 13 (56.52%) 10 (43.48%) na \*\*
Positive 1 1 (100.00%) 0 (0.00%)
\* SD: Standard Deviation; \*\* na: not applicable.
[^1]: These authors contributed equally to this work.
| {
"pile_set_name": "PubMed Central"
} |
Background
==========
Esophageal cancer, which originates in the esophageal mucosal epithelium, is characterized by strong invasiveness and high mortality \[[@b1-medscimonit-22-2195]\]. Approximately, 300 000 people die from esophageal cancer in the world annually \[[@b2-medscimonit-22-2195]\]. There is significant variability in morbidity and mortality rates of among different countries. China has one of the highest frequencies of esophageal cancer, with an average annual death rate of 150 000 \[[@b3-medscimonit-22-2195]\]. The pathology in more than 90% of these patients is esophageal squamous cell carcinoma (ESCC). Postoperative recurrence and metastasis are the main causes of death in patients with ESCC. Factors that lead to postoperative recurrence, invasion, and metastasis include activation of proto-oncogenes, inactivation of tumor suppressor genes, and down-regulation or abnormal expression of multiple proteins.
MicroRNAs (miRNAs) are post-transcriptional expression products of a series of regulatory genes that have both oncogene and tumor-suppressor roles. These micromolecules are crucial for the genesis and development of tumors \[[@b4-medscimonit-22-2195]\]. ESCC tissues have been shown to contain abnormally expressed miRNAs, which are closely related to the signaling pathways required for the initiation and development of the disease \[[@b5-medscimonit-22-2195]\]. miRNAs are responsible for regulating proliferation, differentiation, apoptosis, invasion, and metastasis in tumor cells. Approximately 1000 different miRNAs have been identified, of which miRNA-506 is an important regulator of tumorigenesis \[[@b6-medscimonit-22-2195]\]. miRNA-506 is abnormally expressed in multiple tumors, indicating that it is potentially oncogenic or tumor-suppressive. Currently, little is known about the association between miRNA-506 and ESCC. Therefore, we examined the expression of miRNA-506 in the plasma of ESCC patients using quantitative real-time polymerase chain reaction (qRT-PCR), and investigated the association between miRNA-506 expression and clinicopathological features of ESCC.
Material and Methods
====================
Patients and blood samples
--------------------------
The subjects in this study were 110 ESCC patients treated in The First Hospital of Lanzhou University from January 2009 to December 2011. All patients were diagnosed with ESCC, and complete clinical data for these patients were available. Among the 110 ESCC cases, 45 were female and 55 were male, with ages ranging from 37 to 74 years and an average age of 59.2±10.3 years. Fasting peripheral blood (5 mL) was drawn from each patient and placed in anticoagulative tubes at room temperature for 30 min, followed by centrifugation at 4000×*g* for 5 min at 4°C. The plasma supernatant was collected and stored at −80°C until use. None of the patients received anti-cancer treatment before the blood specimen collection and all provided informed consent. The blood samples were processed in accordance with medical ethics standards. The research was approved by the Ethics Committee of The First Hospital of Lanzhou University. The control group consisted of 40 healthy volunteers.
RNA isolation and qRT-PCR
-------------------------
Total RNA was isolated from the plasma samples in accordance with the instructions on the miRNAVana™ PARIST™ kit (Ambion, Austin, TX). The OD~260/280~ of total RNA was measured using the NanoDropND-1000 ultraviolet photometric machine (Thermo Scientific Wilmington, DE). The extracted RNA was reverse transcribed into cDNA according to the instructions of TaqMan miRNA reverse transcription reagent kit (Qiagen, Valencia, CA). The reverse transcription conditions were as follows: 30 min at 16°C, 60 min at 42°C, 5 min at 85°C, and a termination reaction at 4°C. PCR amplification was subsequently performed, and primers specific for miRNA-506 and an internal reference were used. The PCR conditions were as follows: 1 cycle at 95°C for 30 s; 40 cycles of 95°C for 5 s and 60°C for 30 s, and 1 cycle of 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s. A total volume of 10 μL was used and the reactions were performed in triplicate with U6 as an internal reference. Each experiment was repeated 3 times and data were quantitatively analyzed by comparing threshold cycle (Ct) values (2^−ΔΔCt^) \[[@b7-medscimonit-22-2195]\]. The relative expression (RQ) of the target gene in the sample was expressed as ^ΔΔ^Ct, which was calculated using the equation:
Δ
Δ
Ct
=
Δ
Ct
\-
Avg
.
Δ
Ct
=
(
Ct
miRNA
\-
506
\-
Ct
U
6
)
\-
Avg
(
Ct
miRNA
\-
506
\-
Ct
U
6
)
.
Statistical methods
-------------------
The clinical stage of patients was determined using the tumor node metastasis (TNM) stage system developed jointly by the American Joint Committee on Cancer (AJCC) and the Union for International Cancer Control (UICC) \[[@b8-medscimonit-22-2195]\]. The Mann-Whitney test was adopted to compare differences in plasma miRNA-506 expression between ESCC patients and healthy volunteers. Differences in plasma miRNA-506 levels among patients with ESCC of different stages were analyzed using the Mann-Whitney test (for 2 groups). The receiver operating characteristic curve (ROC curve) was produced to evaluate the utility of miRNA-506 for diagnosing ESCC. A chi-square test was applied to assess the relationship between the expression of plasma miRNA-506 and clinicopathological features of ESCC. The primary outcome indicators for ESCC patients were disease-free survival (DFS) and overall survival (OS). The Kaplan-Meier method was utilized for survival analysis, whereas the log-rank test was used to compare survival rates between the 2 groups. The joint effect analysis of all covariates was conducted using the Cox proportional hazard model. SPSS 19.0 software (SPSS Inc., Chicago, IL) was used for statistical analysis. A value of *P*\<0.05 was considered statistically significant.
Results
=======
miRNA-506 expression in plasma of ESCC patients
-----------------------------------------------
Based on qRT-PCR results, the average miRNA-506 expression was significantly higher in the plasma of ESCC patients than in healthy volunteers (*P*\<0.001; [Figure 1](#f1-medscimonit-22-2195){ref-type="fig"}). We conducted subgroup analysis of the 100 ESCC cases. Compared to that of patients with stage I or II ESCC, or with a tumor length of \<4 cm, the expression of miRNA-506 was higher in the plasma of patients with stage III ESCC or with a tumor length \>4 cm (*P*\<0.001, *P*=0.031, respectively; [Figures 2](#f2-medscimonit-22-2195){ref-type="fig"}, [3](#f3-medscimonit-22-2195){ref-type="fig"}). The 100 ESCC patients were divided into a high-expression group and a low-expression group based on the median expression level of plasma miRNA-506 (median 2^−ΔΔCt^ value). We then analyzed the relationship between the expression of plasma miRNA-506 and the clinicopathological features of ESCC; the results indicated that the expression level of plasma miRNA-506 was closely associated with lymph node status (*P*=0.004), TNM stage (*P*=0.031), and tumor length (*P*\<0.001). However, the expression of plasma miRNA-506 had no significant relationship with other clinicopathological features in ESCC patients ([Table 1](#t1-medscimonit-22-2195){ref-type="table"}).
Plasma miRNA-506 as diagnostic biomarker for ESCC
-------------------------------------------------
We then produced ROC curves for ESCC diagnosis by plasma miRNA-506 and calculated the area under the curve, as well as the sensitivity and specificity of all thresholds. The area under the curve for plasma miRNA-506 was 0.835, indicating that there was a statistically significant difference in ESCC diagnosis by plasma miRNA-506 (*P*\<0.001). At the best cutoff point, the sensitivity and specificity were 81.2% and 87.3%, respectively ([Figure 4](#f4-medscimonit-22-2195){ref-type="fig"}).
miRNA-506 expression in plasma and prognosis of patients with ESCC
------------------------------------------------------------------
To probe the relationship between the expression of plasma miRNA-506 and prognosis for ESCC patients, we conducted long-term follow-up of the 100 ESCC patients. We plotted the survival curves for these ESCC patients. Kaplan-Meier analysis showed that patients with high miRNA-506 expression had significantly shorter survival time (DFS and OS) than that of patients with low miRNA-506 expression. The survival curves for ESCC patients are shown in [Figures 5](#f5-medscimonit-22-2195){ref-type="fig"} and [6](#f6-medscimonit-22-2195){ref-type="fig"}. Univariate analysis showed that T stage, N stage, tumor length, and high miRNA-506 expression levels were significantly correlated with DFS and OS (*P*\<0.05; [Tables 2](#t2-medscimonit-22-2195){ref-type="table"}, [3](#t3-medscimonit-22-2195){ref-type="table"}). Multivariate analysis using the Cox regression model suggested that high miRNA-506 expression was an independent indicator of poor patient prognosis \[DFS: HR=2.647, 95% CI=(1.529--4.582), *P*=0.001; OS HR=2.351, 95% CI=(1.317--4.195), *P*=0.004\]. Other clinical and pathological features, as well as prognosis of ESCC patients, are summarized in [Table 3](#t3-medscimonit-22-2195){ref-type="table"}.
Discussion
==========
The miRNA-506 gene is located on the X chromosome (Xq27.3) \[[@b9-medscimonit-22-2195]\]. An increasing amount of evidence suggests that miRNA-506 inhibits the expression of its target genes through binding to the target mRNAs, and is involved in regulating and controlling cell proliferation, apoptosis, cell cycle arrest, cell aging, cell differentiation, epithelial-mesenchymal transition, cell invasion, and metastasis, as well as initiation and development of tumors \[[@b10-medscimonit-22-2195]--[@b12-medscimonit-22-2195]\]. *In vitro* experiments confirmed that miRNA-506 can directly bind to the 3′UTR of SNAI2, which results in increased expression of E-cadherin through inhibiting the expression of SNAI2; this inhibits epithelial-mesenchymal transition in ovarian carcinoma cells and the invasive capacity of tumor cells \[[@b13-medscimonit-22-2195],[@b14-medscimonit-22-2195]\]. Similarly, miRNA-506 has an anti-tumorigenic role in various malignant tumors such as breast cancer, colorectal cancer, gastric cancer, and liver cancer \[[@b15-medscimonit-22-2195]--[@b18-medscimonit-22-2195]\]. However, there is also research showing that miRNA-506 has an oncogenic role in lung cancer \[[@b19-medscimonit-22-2195]\]. The relationship between the expression of miRNA-506 and ESCC was previously unknown. In light of this conflicting evidence, we performed this research to probe the effects of miRNA-506 on the initiation and development of ESCC.
The plasma miRNA-506 level in ESCC patients was detected through qRT-PCR, and healthy volunteers served as control subjects. We discovered that the average miRNA-506 expression was remarkably higher in the plasma of ESCC patients compared to that of healthy volunteers. Subgroup analysis showed that the expression level of miRNA-506 was higher in the plasma of patients with stage III ESCC or those with a tumor length \>4 cm. We also discovered that the expression level of miRNA-506 in the plasma was closely associated with clinicopathological features of ESCC patients, such as lymph node status, TNM stage, and tumor length. This suggests that miRNA-506 might promote the growth, proliferation, and invasion of ESCC tumor cells. However, this hypothesis is based on *in vivo* results, and thus need to be verified through *in vitro* experimentation. Long-term follow-up of ESCC patients showed that compared to ESCC patients with low miRNA-506 expression, those with high miRNA-506 expression had shorter survival. Multi-factor regression analysis revealed that high miRNA-506 expression was an important, independent indicator for predicting poor prognosis in ESCC patients. In addition, the ROC curves of ESCC diagnosis by plasma miRNA-506 showed an area under the curve of 0.835, indicating that determination of plasma miRNA-506 levels was of high diagnostic value for ESCC.
The results of the present work are contrary to those of numerous earlier studies. We reviewed, analyzed, and summarized the relevant literature regarding the relationship between miRNA-506 and malignant tumors. The mechanism of action of miRNA-506 during ESCC progression might be as follows. First, miRNA-506 could directly bind to cyclin-dependent kinase4/6 (CDK4/6), and then to cyclinD, regulating cell cycle progression from G1 to S stage \[[@b20-medscimonit-22-2195]\]. Second, miRNA-506 would inhibit the expression of CDK4/6, which could stimulate proliferation of tumor cells in a feedback-mediated manner \[[@b19-medscimonit-22-2195],[@b20-medscimonit-22-2195]\]. Third, miRNA-506 could inhibit the expression of SNAI2 and consequently increase the expression of E-cadherin, which would confer adhesiveness to mobile tumor cells \[[@b21-medscimonit-22-2195]\]. Fourth, ETS1 protein is an important target of miR-506, and the combination of ETS1 and miR-506 could further influence tumor angiogenesis and the metastasis \[[@b22-medscimonit-22-2195]\]. In addition, the direct binding of miR-506 to NF-κB-p65 could inhibit the NF-κB pathway, induce the production of reactive oxygen species (ROS), activate p53, and result in a mutual positive feedback loop with p53 \[[@b19-medscimonit-22-2195],[@b23-medscimonit-22-2195]\].
Conclusions
===========
High miRNA-506 expression was found to be associated with poor prognosis in patients with ESCC. Therefore, miRNA-506 could serve as an important molecular marker for diagnosis and prognosis in ESCC. However, the precise mechanism remains unclear and further in-depth research is required. We believe that it is possible to delay tumor growth, proliferation, infiltration, and metastasis by blocking miRNA-506-mediated signal transduction pathways.
**Conflicts of interest**
The authors report no conflicts of interest in this work.
**Source of support:** Departmental sources
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{#f2-medscimonit-22-2195}
{#f3-medscimonit-22-2195}
{#f4-medscimonit-22-2195}
{#f5-medscimonit-22-2195}
{#f6-medscimonit-22-2195}
######
The plasma of miRNA-506 expression status and clinicopathological characteristics of patients with ESCC.
Features miRNA-506 *P* value
------------------ ----------- ----------- ---------
Age 0.654
\<65 25 20
≥65 33 22
Sex 0.439
Male 30 25
Female 28 17
Smoking 0.764
Never or light 30 23
Heavy 28 19
Drinking 0.832
Never or light 33 22
Heavy 25 20
Differentiation 0.756
Well 19 11
Moderate 25 19
Poor 14 12
T stage 0.20
T1--2 31 17
T3--4 27 25
N stage 0.004
N0--1 39 16
N2--3 19 26
TNM stage 0.031
I--II 32 14
III 26 28
Tumor length \<0.001
\<4 cm 44 17
≥4 cm 14 25
Site of tumor
Cervical 6 4 0.079
Upper thoracic 3 9
Middle thoracic 24 17
Low thoracic 25 12
######
Univariate and multivariate analyses of prognostic variables for disease-free survival.
Variable Univariate analysis Multivariate analysis
----------------- --------------------- ----------------------- -------
Sex 0.897 1.038 (0.441--2.446) 0.932
Age 0.577 1.007 (0.606--1.675) 0.978
Smoking 0.945 0.830 (0.379--1.818) 0.641
Drinking 0.697 1.086 (0.486--2.427) 0.841
T stage 0.037 1.775 (1.147--2.746) 0.010
N stage 0.032 1.753 (1.116--2.754) 0.015
Differentiation 0.884 1.022 (0.707--1.479) 0.907
Tumor length 0.027 1.334 (1.142--1.790) 0.012
Site of tumor 0.477 0.884 (0.667--1.171) 0.390
miRNA-506 0.001 2.647 (1.529--4.582) 0.001
######
Univariate and multivariate analyses of prognostic variables for overall survival.
Variable Univariate analysis Multivariate analysis
----------------- --------------------- ----------------------- -------
Sex 0.654 0.936 (0.379--2.314) 0.886
Age 0.591 0.960 (0.564--1.632) 0.879
Smoking 0.950 0.833 (0.364--1.911) 0.667
Drinking 0.722 0.951 (0.401--2.258) 0.910
T stage \<0.001 1.925 (1.195--3.101) 0.007
N stage \<0.001 1.699 (1.038--2.781) 0.035
Differentiation 0.777 0.991 (0.672--1.461) 0.962
Tumor length 0.004 1.380 (1.156--1.923) 0.033
Site of tumor 0.581 0.867 (0.645--1.165) 0.344
miRNA-506 0.008 2.351 (1.317--4.195) 0.004
[^1]: Study Design
[^2]: Data Collection
[^3]: Statistical Analysis
[^4]: Data Interpretation
[^5]: Manuscript Preparation
[^6]: Literature Search
[^7]: Funds Collection
| {
"pile_set_name": "PubMed Central"
} |
Background
==========
Strict maritime quarantine (with facility quarantine on land in some cases), appeared to effectively prevent the entry of the 1918--19 influenza pandemic into American Samoa and delayed its entry into mainland Australia, Tasmania and New Caledonia \[[@B1]\]. Quarantine measures during this pandemic also worked successfully in Yerba Buena, an island off San Francisco \[[@B2]\], and within parts of Iceland \[[@B3]\]. More generally, a systematic review has reported evidence that interventions that included quarantine (2 studies) and isolation (10 studies) were effective in containing respiratory virus epidemics \[[@B4]\]. An earlier review had suggested a limited use for quarantine but had focused on quarantine attempts in countries with porous land borders \[[@B5]\].
Since it appears that quarantine was successful in island settings from 1918--19, some Pacific island nations have included the option of border quarantine in their current influenza pandemic plans \[[@B6]\]. Theoretically, since small island nations will most likely experience introduction of pandemic influenza at just one airport or seaport alone, the use of border control would be one of the most important options to protect their communities from the pandemic. As an example, New Zealand consists of multiple islands and has a pandemic plan that includes significant detail about border control and quarantine \[[@B6],[@B7]\]. In addition, border quarantine is also included in the pandemic plans of some European countries \[[@B8]\].
However, detailed guidelines for effective use of quarantine have yet to be formulated. One of the key questions among infectious disease specialists and public health practitioners is how to optimise the duration of quarantine to achieve a desired level of effectiveness. Presently, there is no universal proposal for quarantine period following exposure to pandemic influenza cases. Although the etymological root of quarantine originates from 13th century public health practices requiring incoming ships to remain in port for 40 days \[[@B9]\], quarantine in the present day refers to compulsory physical separation for a defined period, including restriction of movement, of healthy individuals who have been potentially exposed to an infectious disease \[[@B10]\]. Since the restriction of movement often involves legal and ethical constraints, because it limits the freedom of quarantined individuals \[[@B11]\], the optimal length of quarantine needs to be clarified using scientifically sound approaches.
To suggest the optimal length of quarantine for pandemic influenza, we need to consider the detailed epidemiologic characteristics of this disease including the presence of asymptomatic infection \[[@B12]\]. The present study aimed to assess the potential effectiveness of quarantine, suggest an optimal length, and examine its potential performance for small island nations.
Methods
=======
Hypothetical setting
--------------------
To clarify the optimal length of quarantine, we first consider a hypothetical setting where infected travellers are flying from a nation with an epidemic (somewhere in Asia, given the data on the origin of seasonal influenza \[[@B13]\]) to a disease-free small island nation (e.g., New Zealand or smaller South Pacific and Caribbean islands). Specifically, we consider a situation when the disease-free country is fortunate enough to be informed about the possible emergence of the influenza pandemic at the source, sufficiently in advance of its arrival to implement border control measures. Given that the possible emergence is still uncertain and very recent news, we assume that the disease-free island nation is not ready or willing to completely shut down all its airports, but that quarantine is immediately instituted at the border. Before closing all the airports we assume that the island nation still permits the arrival of 20 aircraft with a total of 8000 incoming individuals (i.e., each with 400 individuals including airline staff on board) who were potentially exposed to influenza at the source country or on the aircraft. For this population of travellers we explore the question -- how long should we place them in quarantine?
We assume that all incoming individuals are placed into routine quarantine on arrival in the island nation and are monitored for onset of symptoms during the quarantine period. We also assume that all infected individuals who develop influenza symptoms are successfully detected (e.g., through self-report questionnaires, reporting by ground staff, specific interview assessment by trained health personnel and/or thermal scanning). The impact of imperfect detection on the effectiveness of quarantine is examined in the Appendix. Optimistically, symptomatic cases are assumed to be immediately isolated in a designated facility at symptom onset, and assumed not to result in any secondary transmissions \[[@B14]\]. Similarly, those who developed symptoms en-route are also assumed to be successfully isolated upon arrival (and we ignore these individuals in the following analyses as the detection is owing to the entry screening). We assume that quarantine security would be fully effective and that no secondary transmission would occur in the quarantine facility. Successful detection during quarantine relies largely on onset of influenza-like symptoms, but, as a possible option, we also consider adding rapid diagnostic testing to improve the sensitivity of case detection.
Epidemiologic characteristics of influenza
------------------------------------------
To theoretically and quantitatively examine the effectiveness of quarantine, we use several parameters describing the epidemiologic characteristics of seasonal influenza -- which we then use for considering pandemic influenza. The most important of these characteristics is the cumulative distribution of the incubation period (i.e., the time from infection to onset) of length *t*, *F*(*t*). The incubation period has been very useful in suggesting the optimal length of quarantine for many diseases \[[@B15]\], because arbitrarily taking the 95th or 99th percentile point as the quarantine period could ensure the absence of symptomatic infection with probability of 95% or 99% \[[@B12],[@B16]-[@B21]\]. However, it is difficult to directly apply this concept to influenza \[[@B12]\], because the conditional probability, *α*, of developing symptomatic disease (given infection) has been suggested to be 66.7% \[[@B22],[@B23]\], and detection through quarantine is not relevant for asymptomatic infected individuals who account for the remaining 33.3%. Thus, we consider the effectiveness of quarantine as the reduction of the risk of introducing \"infectious\" individuals into the community and, thus, additionally use the cumulative distribution of the generation time (i.e., the time from infection of a primary case to infection of a secondary case by the primary case) of length *t*, *G*(*t*). Further, to simulate the key ripple benefit of quarantine (the predicted number of secondary transmissions caused by released infectious individuals), we assume that the reproduction numbers of symptomatic (*R*~s~) and asymptomatic cases (*R*~a~), i.e., the average numbers of secondary transmissions caused by a single symptomatic case and an asymptomatic case are 2.0 and 1.0, respectively. The basic reproduction number, *R*~0~, is therefore *αR*~s~+(1-*α*)*R*~a~= 1.67 which corresponds to an estimate in a previous study \[[@B24]\]. Moreover, the estimate is also within the estimated range of community transmission in another study which explored various historical data \[[@B25]\].
Distribution of the incubation period, which was assumed to follow a gamma distribution, was extracted from a published dataset \[[@B26]\]. Since the original data showed daily frequency of onset only, we fitted the cumulative distribution of the incubation period to the observed data, minimising the sum of squared errors. We did not identify more detailed data and note that the obtained frequency did not deviate much from outbreak data on an aircraft \[[@B27],[@B28]\], a historical study of Spanish influenza \[[@B15],[@B29]\], and from data in a published meta-analysis \[[@B22]\]. Similarly, the generation time was retrieved from a previous study of volunteers infected with influenza \[[@B22]\], which assumed that infectiousness is proportional to viral shedding, and we obtained the parameter estimates by minimising the sum of squared errors. A lognormal distribution was employed to model the generation time. Strictly speaking, the viral shedding curve alone does not inform the generation time, but our outcome measure (i.e., the probability of releasing infectious individuals) is reasonably analysed using virological data (as we are dealing with infectiousness), assuming that the frequency of contact is independent of time since infection. Furthermore, we favoured the use of this dataset as it would give a more conservative result since the right-tail is fatter than those assumed previously \[[@B30],[@B31]\].
Effectiveness of quarantine
---------------------------
Although secondary transmission on aircraft is probably relatively rare due to the functioning of ventilation systems \[[@B32],[@B33]\], a previous transmission event has been reported in this setting \[[@B27]\]. Therefore, we use arrival time as the latest time of possible infection (i.e., *t*= 0). In other words, we conservatively argue the quarantine period as if all infected incoming individuals experienced this infection upon arrival. In reality, earlier acquisition of infection would increase the probability of non-infection after quarantine and therefore increase the effectiveness of quarantine. Although our worst case scenario potentially overestimates the optimal length of quarantine, a more realistic scenario requires the exact time of infection for all incoming infected individuals, which is in principle impractical (see Appendix for more detailed insights into this issue).
We considered the effectiveness of quarantine, *ε*(*t*), as a relative reduction of the risk of introducing infected individuals into the community as a function of time since infection *t*, i.e.,
$$\varepsilon(t) = 1 - \frac{r_{1}(t)}{r_{0}(t)}$$
where *r*~1~(*t*) and *r*~0~(*t*) are the risks of releasing infected individuals into a new community in the presence and absence of the quarantine measure, respectively. Since all infected individuals enter the community without quarantine, we assume *r*~0~(*t*) = 1 for any *t*. If the risk in the presence of quarantine, *r*~1~(*t*), is regarded as the risk of releasing \"symptomatic infected\" individuals (regardless of infectiousness) after quarantine of length *t*, *r*~1~(*t*) is given by 1-*F*(*t*). Therefore, only the incubation period determines the effectiveness, i.e., *ε*(*t*) = *F*(*t*), which has been the fundamental concept in previous studies \[[@B12],[@B15]-[@B21]\]. However, we further consider the infectiousness for influenza, emphasising the importance of asymptomatic infection, because the proportion 100×(1-*α*) is as large as 33.3%. We thus regard the risk *r*~1~(*t*) as the probability of releasing \"infectious\" individuals into the community after quarantine of *t*days.
To comprehensively discuss this issue we decompose *r*~1~(*t*) into the sum of symptomatic and asymptomatic individuals (denoted by *r*~1s~(*t*) and *r*~1a~(*t*), respectively). For those who will eventually develop symptoms, the probability of release, *r*~1s~(t), is
where *F*(*t*) and *G*(*t*) are, respectively, the cumulative distributions of the incubation period and generation time. Because of the absence of adequate data, we assume independence between the incubation period and generation time, which most likely yields conservative estimates of the effectiveness (compared to that explicitly addressing dependence between these two distributions). For those who remained asymptomatic throughout the entire course of infection, the probability *r*~1a~(t) is
because the incubation period is not relevant to the detection of asymptomatic infected individuals. Due to the absence of data, it should be noted that we assume that the length of generation time among asymptomatic individuals is identical to that among symptomatic cases, an assumption that has been used by others \[[@B24],[@B25]\]. As the assumption adds an uncertainty to the model prediction, we examine the potential impact of differing generation times between symptomatic and asymptomatic infected individuals (see Appendix). Consequently, the effectiveness of quarantine, *ε*(*t*), is given by subtracting *r*~1s~(*t*) and *r*~1a~(*t*) from 1: i.e.,
We further investigate the additional benefit of testing for the pandemic influenza virus using rapid diagnostic testing during quarantine. A key assumption made is that the currently available diagnostic tests would perform as well with the new pandemic strain of virus (and be supplied to the islands in time). We assume that the sensitivity (S~e~) and specificity (S~p~) of the rapid diagnostic test are 69.0% and 99.0%, respectively \[[@B34]\]. Since our effectiveness measure is conditioned on infected individuals, the risk of releasing infectious individuals in the presence of quarantine with use of rapid diagnostic testing is obtained by multiplying a factor (1-S~e~) to *r*~1~(t) which represents a proportion of cases that are missed even following rapid diagnostic testing. Thus, we get the effectiveness *ε*~d~(*t*) as
Due to the absence of more detailed data, we assume that both the sensitivity and specificity of the rapid diagnostic test are independent of time since infection. Considering that the sensitivity may well decline in later stages of illness (by implicitly assuming that the diagnostic test is correlated with viral load), it should be noted that the results associated with equation (5) are probably most valid only for those in the early stage of illness (which is consistent with our particular interest in quarantine period). We stress that the estimated effectiveness *ε*~d~(*t*) for a long quarantine period (e.g., longer than 8 days) should be treated cautiously. Since the sensitivity S~e~of asymptomatic infected individuals may be smaller than that among symptomatic cases (due to lower virus shedding titres among asymptomatic individuals), we examine the effectiveness of quarantine with differing S~e~between symptomatic and asymptomatic infected individuals (see Appendix).
Sensitivity analysis and preventive performance
-----------------------------------------------
We also examined the sensitivity of our effectiveness measures (4) and (5) to different lengths of quarantine and prevalence levels at the source by means of simulations. First, the sensitivity was assessed using the number of released infectious individuals after quarantine of length *t*. We examined plausible prevalence levels of 1%, 5% and 10% at the source, which respectively indicate that there were 80, 400 and 800 infected individuals among a total of 8000 incoming individuals. The highest prevalence, 10%, may represent transmission events within an airport of the country of origin or on an aircraft. The analysis was made by randomly simulating the incubation period (*F*), the generation time (*G*), the presence of any symptoms (*α*) and the sensitivity of the rapid diagnostic test (S~e~) where *F*and *G*randomly follow the assumed gamma and lognormal distributions, respectively. The two dichotomous variables (i.e., the presence of symptoms and sensitivity of the rapid diagnostic test) were randomly simulated with uniform distributions (i.e., drawing random real numbers from 0 to 1) and using cut-off points at *α*= 0.667 and S~e~= 0.690. The random sampling was performed for the number of infected individuals (80, 400 and 800 times) in each simulation, and the simulation was run 100 times for each length of quarantine and prevalence level. To show the ripple benefit, we also investigated the number of secondary transmissions caused by released infectious individuals. This estimate was achieved by further randomly simulating the numbers of symptomatic and asymptomatic secondary transmissions. Both numbers were assumed to follow Poisson distributions with mean *R*~s~(1-*G*(*t*~d~))(1-*F*(*t*~d~)) and *R*~a~(1-*G*(*t*~d~)), respectively, for each of the released symptomatic and asymptomatic infectious individuals after the quarantine of length *t*~d~days.
Finally, we examined the preventive performance of quarantine combined with rapid diagnostic testing. When the combination scheme is employed, those testing negative to the rapid diagnostic test following quarantine of length *t*would be the population of interest, as they are then released into the community. Let *p*be the prevalence level at the source (0 ≤ *p*≤ 1). Among infected individuals (who account for 100*p*% of the travellers), the fraction of those who are detected or lose infectiousness following quarantine of length *t*(i.e., true positives) is (1-*r*~1~(t)). Of the remaining infected individuals *r*~1~(t), the fraction of those testing positive, S~e~*r*~1~(t), to the rapid diagnostic test are placed into isolation and, thus, are added to the true positives. Consequently, the remaining fraction (1-S~e~)*r*~1~(t) are false negatives and are released into the community (Figure [1](#F1){ref-type="fig"}). Among uninfected individuals (i.e., 100(1-*p*)% of the travellers), the length of quarantine does not influence the preventive performance (because they are not infected and their quarantine is irrelevant to the loss of infectiousness). Thus, among the total number of incoming travellers, the fractions (1-*p*)(1-S~p~) and (1-*p*)S~p~will be testing positive (false positives) and negative (true negatives), respectively, to the rapid diagnostic test. Consequently, positive predictive value (PPV) of quarantine combined with rapid diagnostic testing is
{#F1}
$$\text{PPV} = \frac{p\lbrack 1 - (1 - \text{S}_{\text{e}})r_{1}(t)\rbrack}{p\lbrack 1 - (1 - \text{S}_{\text{e}})r_{1}(t)\rbrack + (1 - p)(1 - \text{S}_{\text{p}})}$$
whereas negative predictive value (NPV) is
$$\text{NPV} = \frac{(1 - p)\text{S}_{\text{p}}}{p(1 - \text{S}_{\text{e}})r_{1}(t) + (1 - p)\text{S}_{\text{p}}}$$
PPV measures the preventive performance of quarantine policy to correctly place infected individuals in quarantine (or isolation) during their infectious period (i.e., how efficiently are we placing infectious individuals in the quarantine facility, among a total of those who are diagnosed as positive either by quarantine of length *t*or rapid diagnostic testing). NPV measures the preventive performance of the release policy (i.e., how large is the fraction of true negatives among a total of those who are diagnosed as negative after the quarantine of length *t*and rapid diagnostic testing). We numerically computed both PPV and NPV for different prevalence levels (from 0--15%) and different lengths of quarantine (from 0 to 10 days). All analyses were made using the statistical software JMP ver. 7.0 (SAS Institute Inc., Cary, NC).
Results
=======
Intrinsic dynamics of influenza
-------------------------------
Figure [2](#F2){ref-type="fig"} shows density functions of the incubation period and generation time. The incubation period was similar to those reported previously \[[@B12],[@B27],[@B28]\]. Mean and variance of the incubation period were estimated as 1.43 days and 0.48 days^2^, respectively. The generation time in the figure includes the original datasets on different types of influenza virus (weighed by each sample size). The mean, median (25--75th percentile) and variance of the generation time were 2.92 days, 2.27 (1.41--3.67) days and 5.57 days^2^, respectively.
![**Probability density functions of the incubation period and generation time of influenza**. Gamma distribution was employed to model the incubation period (i.e., the time from infection to onset), whereas lognormal distribution was fitted to the generation time (i.e., the time from infection of a primary case to infection of a secondary case by the primary case). The mean and variance of the incubation period and generation time are estimated as 1.43 days and 0.48 days^2^and 2.92 days and 5.57 days^2^, respectively. For the original data see: \[[@B22]\] and \[[@B26]\].](1471-2334-9-27-2){#F2}
Effectiveness of quarantine
---------------------------
Figure [3](#F3){ref-type="fig"} shows the estimated effectiveness of quarantine as a function of time since infection (i.e., time since arrival). A different effectiveness measure (i.e., relative reduction of the risk of releasing \"symptomatic infected\" individuals regardless of infectiousness) is shown (dashed line) comparatively with the other two results showing the relative reduction of the risk of releasing \"infectious\" individuals in the presence and absence of the use of rapid diagnostic testing (thin and thick solid lines, respectively). It should be noted that the reduction of symptomatic infected individuals is based only on the incubation period, measuring a different concept of effectiveness from other two. The incubation period alone suggests that 95% effectiveness in preventing the release of symptomatic infected individuals is achieved by quarantine of 2.73 days.
![**Effectiveness of quarantine with and without use of rapid diagnostic testing as a function of time since infection (i.e., time since arrival)**. Different effectiveness measures of quarantine are comparatively shown. The dashed line represents the effectiveness of quarantine, measured as the relative reduction of the risk of releasing \"symptomatic infected\" individuals (regardless of infectiousness) based on the incubation period alone. The two continuous lines measure the effectiveness as the relative reduction of the risk of releasing \"infectious individuals\" into the community, based on the incubation period, generation time and probability of symptomatic disease, with (thin) and without (thick) use of rapid diagnostic testing. The sensitivity of the rapid diagnostic test was assumed to be 69.0% (based on current test performance for seasonal influenza A \[[@B34]\]).](1471-2334-9-27-3){#F3}
We predict that 95% and 99% effectiveness in preventing the release of infectious individuals is achieved with quarantine periods of longer than 4.74 and 8.62 days, respectively. As can be observed from Figure [3](#F3){ref-type="fig"}, the impact of using rapid diagnostic testing on effectiveness is larger for a short quarantine period. If a rapid diagnostic test was available and this performed to the current standard for detecting influenza A in the pre-pandemic setting, we estimated that this additional testing would result in quarantine periods for longer than 2.59 and 5.71 days having effectiveness of over 95% and 99% respectively (Figure [3](#F3){ref-type="fig"}).
Sensitivity analysis
--------------------
Given the above mentioned results, we investigated the sensitivity of quarantine effectiveness to four different lengths of quarantine (2.8, 4.8, 5.7 and 8.7 days) and to three different prevalence levels at the source (1%, 5% and 10%). The shortest length, 2.8 days, was suggested by the incubation period as being 95% effective in preventing the release of symptomatic infected individuals into the community. Two of the others (4.8 and 8.7 days) corresponded to 95% and 99% effectiveness in preventing release of infectious individuals by means of quarantine alone, and 5.7 days corresponded to 99% effectiveness when quarantine was combined with rapid diagnostic testing.
Figure [4](#F4){ref-type="fig"} shows the median (and 5--95th percentile) numbers of infectious individuals who are released into the community after quarantine of specified lengths. The quarantine for 2.8 days could miss as many as 11 (5--16), 56 (45--68) and 114 (92--129) infectious cases for the prevalence of 1%, 5% and 10%, respectively in the 8000 arriving travellers considered. However, these misses were reduced to 4 (1--7), 20 (13--27) and 39 (28--53) cases by the quarantine of length 4.8 days, to 3 (0--5), 13 (7--19) and 27 (16--36) by 5.7 days and, moreover, to 1 (0--2), 4 (1--8) and 8 (4--13) cases by 8.7 days. The additional diagnostic testing could greatly reduce the released number of infectious individuals (Figure [4B](#F4){ref-type="fig"}). For the quarantine lengths of 2.8 and 5.7 days with rapid diagnostic testing, 3 (1--7), 18 (10--25) and 34 (25--45) cases and 1 (0--2), 4 (1--8) and 8 (4--14) cases, respectively, were expected be released into the community for the prevalence of 1%, 5% and 10%. All values for the quarantine period of 5.7 days combined with use of a diagnostic test were less than 3% of the total number of incoming infected individuals.
{#F4}
Figure [5](#F5){ref-type="fig"} describes the ripple benefit of quarantine, expressed as the number of secondary transmissions caused by released infectious individuals. The qualitative patterns found were similar to those of Figure [4](#F4){ref-type="fig"}, but it should be noted that no secondary transmission was observed in the community in several scenarios. Quarantine of length 2.8 days, with or without rapid diagnostic testing, would lead to many secondary transmissions caused by released infectious individuals. When there was quarantine of 4.8 days without rapid diagnostic testing, we found 0 (0--2), 3 (1--7) and 5 (1--11) secondary transmissions. Extending quarantine to 8.7 days resulted in no secondary transmissions at prevalence levels of 1%, 5% and 10% (i.e., all were 0 except for 1 secondary transmission at the 95th percentile for all three prevalence levels). When diagnostic testing was combined with the quarantine period for 5.7 days, 0 (0--1), 0 (0--1) and 0 (0--2) secondary transmissions resulted. That is, even though quarantine alone for 8.7 days and quarantine combined with diagnostic testing for 5.7 days permit the release of several infectious individuals (up to 3% of the total number of incoming infected passengers), the majority of the released cases are at the late stage of infection and hardly cause secondary transmissions in the island nation (i.e. even in the worst case, only a few secondary transmissions would be expected).
{#F5}
Preventive performance of quarantine with rapid diagnostic testing
------------------------------------------------------------------
Figure [6](#F6){ref-type="fig"} shows contour plots of PPV and NPV of quarantine combined with rapid diagnostic testing as functions of the length of quarantine and prevalence of influenza at the source. Given a fixed prevalence, PPV was greater for shorter length of quarantine (especially, for *t*\< 1 day) due mainly to the relative increase in detection of true positives by rapid diagnostic testing (see equation (6)). However, it became less sensitive to the length of quarantine as the length became longer (for *t*\> 2 days) and depended almost only on the prevalence (Figure [6A](#F6){ref-type="fig"}). Figure [6B](#F6){ref-type="fig"} demonstrates that NPV was on the whole very high and sensitive to both the length of quarantine and prevalence at the source. For prevalence levels up to 10%, NPV with quarantine for longer than 2 days could be greater than 99.0%. In particular, at a quarantine length of 6 days, NPV was greater than 99.9% for prevalence levels up to 10%. In other words, within the range of interest for quarantine lengths, PPV was mainly determined by the prevalence level (i.e., longer quarantine with rapid diagnostic testing does not load too many additional false positives on isolation facilities compared with the use of shorter quarantine and testing). Also, NPV can be extremely high, indicating that the release policy can efficiently suggest that the released individuals are likely to be true negatives.
{#F6}
Discussion
==========
The present study provides theoretical support for border quarantine as a worthwhile pandemic influenza control measure for small island nations. Detailed advance planning for quarantine measures may therefore be justified during the pre-pandemic period. From our quantitative findings, we recommend a quarantine period of 9 days (rounding 8.7 days to the next integer) to reduce by more than 99% the risk of introducing infectious individuals and to ensure the absence of secondary transmissions caused by released infectious individuals in the community. If the use of rapid diagnostic testing can be combined with quarantine, the quarantine period could be shortened to 6 days (rounding 5.7 days to the next integer). To the best of our knowledge, the present study is the first to explicitly suggest an optimal length of quarantine for pandemic influenza derived from detailed epidemiologic characteristics of this infection. Although our recommendations are based on arbitrarily considering the specific percentiles of effectiveness, and although the absence of secondary transmissions depends also on the absolute number of incoming individuals, we believe that our findings (with realistic ranges of prevalence and the number of travellers) provide evidence-based estimates that can be used for pandemic planning. Quarantine might ultimately be unsuccessful in preventing importation of infected individuals \[[@B35]\]. However, delayed entry of the pandemic virus could provide time to introduce other social distancing and pharmaceutical interventions that may reduce the overall impact of a pandemic \[[@B1],[@B9],[@B30],[@B36]-[@B39]\].
In recent studies, the optimal length of quarantine was considered by using the incubation period distribution alone, identifying the 95th or 99th percentile point of the theoretical distribution \[[@B16]-[@B21]\]. For instance, 95th percentiles of the incubation period for severe acute respiratory syndrome (SARS) and smallpox were suggested to be 11--13 days \[[@B19],[@B21]\] and 16--17 days \[[@B16]\] since exposure, respectively. Direct application of this concept to pandemic influenza suggests that the optimal quarantine period for pandemic influenza is only 2.73 days since exposure, which is far shorter than those for SARS and smallpox. However, since influenza involves a non-negligible fraction of asymptomatic infections \[[@B12],[@B22]\], we also undertook the additional step of incorporating this feature into our assessment of quarantine effectiveness. This refinement permitted further elaboration of effectiveness estimates, which we believe contributes to theoretical considerations around the control of other infectious diseases. In addition, we reasonably showed the preventive performance of quarantine, expressed as the number of released infectious individuals and the ripple benefit expressed as number of secondary transmissions caused by them. Using further information on the contact structure in the island nation, our framework could be further extended to estimate the probability of extinction and the delay effect of epidemic spread imposed by quarantine, the latter of which was discussed by a recent study \[[@B35]\]. Although the recent study theoretically emphasises the difficulty of effective border control (including quarantine) \[[@B35]\], we stress that the epidemiologic characteristics of influenza (e.g., short incubation period and generation time) permit anticipating large ripple benefits from quarantine (given that importation may continue for only a short period of time before full border closure occurs).
Access to a highly sensitive test for pandemic influenza infection may increase the effectiveness of quarantine and shorten the quarantine period routinely required for incoming travellers. Preventive performance in finding true positives (i.e., PPV of quarantine combined with rapid diagnostic testing) appeared not to be very sensitive to the length of quarantine (for *t*\> 2 days). This result suggests that if diagnostic test kit supplies are plentiful, then testing should be done early in quarantine. But if a test kit sparing approach is used (i.e., avoiding testing of those who become symptomatic) then there is not much benefit in delaying testing until after day 2 in quarantine. A test with both high sensitivity and high specificity would also allow for better use of resources if the travellers who tested negative are released into the community. Since PPV is mainly determined by prevalence at the source, it should be noted that an exit screening process at the source lowers the prevalence as well as PPV. Nevertheless, the effectiveness of quarantine itself is independent of the prevalence, and moreover, lower prevalence among incoming individuals yields a higher chance of extinction (or greater ripple effect of quarantine (Figures [4](#F4){ref-type="fig"} and [5](#F5){ref-type="fig"})). NPV of quarantine combined with diagnostic testing would be extremely high with quarantine periods for lengths of 3 days or longer, supporting our suggestion to release quarantined individuals testing negative to the rapid diagnostic test into the community (if there was high confidence in test performance parameters for the emergent pandemic strain). In light of our findings, island countries may consider including influenza testing capacity and test kit stockpiles in their pandemic plans. The use of rapid diagnostic tests, if available through stockpiling in advance or rapid delivery after pandemic emergence, may permit more effective border control, with more efficient use of isolation facilities and shortening of the quarantine period.
The operation of quarantine would be most feasible for islands with low traveller numbers and with pre-existing facilities that could be used for quarantine (e.g., hotels). Our study was indeed motivated by the consideration of protecting small island nations (e.g., in the South Pacific and Caribbean), because use of border control at usually just one or two international airports would be the major way in which the introduction of pandemic influenza could be prevented in these islands. Yet the analysis could potentially hold for larger island nations such as Australia, whose pandemic plan also includes border quarantine \[[@B40]\]. The logistics of quarantine might be far more demanding in Australia with its multiple international airports, but which nonetheless used strict maritime quarantine to successfully delay the entry of the 1918 pandemic \[[@B41]\]. Evidence about the geographic spread of influenza highlights the importance of quarantine in multiple locations \[[@B42]-[@B44]\]. Small countries with land borders and limited entry points could also use these approaches to delay entry of pandemic influenza as occurred for Israel in the 1957 influenza pandemic \[[@B5]\]. Facility-based quarantine could also be supplemented with ongoing surveillance in the community of those released from quarantine.
Our analysis employed a number of simplifying assumptions, among which we should emphasise the most important one. The detailed natural history parameters for seasonal influenza are not well documented and, moreover, we of course do not know if the incubation period and generation time of an emergent pandemic strain would be close to those of seasonal influenza documented in the limited number of publications to date. It should be noted that our analysis is solely based on the available published evidence and that the effectiveness of quarantine would be overestimated if the emergent strain of pandemic influenza had a longer incubation period or a longer generation time than we have assumed. However, the incubation period for human infection with H5N1 appears to be similar to other sub-types infecting humans \[[@B45]\]. This issue applies not only to the incubation period but also to other parameters, the role of which for each can be inspected using equations (4) and (5). For example, a historical analysis suggests that only 9% of infections resulted in an asymptomatic infection \[[@B46]\], which would contribute to improved quarantine effectiveness (compared to our results). Given that our exercise indicates the critical importance of the incubation period and generation time, epidemiological investigations should be performed to better quantify these parameters and further inform evidence-based pandemic planning.
Extrinsic factors should also be more precisely quantified in future. As an indirect extrinsic effect, when infected individuals are released into the community and become infectious to others, recently quarantined individuals may be detected and isolated earlier than those who have not been quarantined \[[@B47]\]. Another issue of detection is that some island states may have access to laboratory-based PCR influenza tests which are far more sensitive and specific than rapid tests \[[@B48]\], which could offer the test results in a few hours and greatly shorten the length of quarantine.
To more appropriately quantify the effectiveness of quarantine, two other technical issues have to be discussed. The first is concerned with skewness of the offspring distribution (i.e. the distribution of the number of secondary transmissions caused by a single primary case). Although our study reasonably showed the absence of secondary transmissions for quarantine of certain lengths, we ignored the skewness (i.e., the presence of potential super-spreaders \[[@B49]\]), and thus, the uncertainty bounds might have been smaller than in reality. Although the mean and median of the predicted number of secondary transmissions are still valid, and even though the skewed offspring distribution was partly incorporated in the model with the right-skewed generation time distribution, super-spreading events played a key role in triggering the international spread during the epidemic of SARS, and in light of this, quantification of the dispersion parameter (of the offspring distribution) is needed in future studies. Another issue is related to our conservative assumption that all incoming individuals experienced infection upon arrival. Since it is impractical to know the time of infection for all incoming infected individuals (which should ideally be known when the quarantine is started at time *t*= 0), we adopted a worst case scenario where all infected individuals experience infection at *t*= 0 (see Appendix). This assumption could have overestimated the optimal length of quarantine. If further research demonstrates that influenza transmission on board flights is very rare, then it would be possible to set the quarantine period to begin at the start of the flight and therefore reduce its duration correspondingly following arrival. However, then we have to take into account the possible secondary transmissions during the quarantine period. Estimation of the effectiveness of imperfect quarantine (i.e., quarantine which allows secondary transmissions within the quarantine facility) would be far more complicated than our simpler model, and clarification on this point is a task for future research.
In addition to the present study, it should be noted that quarantine may be combined with reduction of travel volumes (e.g., even mandatory restrictions on non-essential travel) which would have a large effect if it occurred rapidly \[[@B35],[@B50],[@B51]\]. Substantial reductions of travel volumes could make the logistics of quarantine far more feasible for island nations and increase the probability of ensuring the absence of secondary transmissions (given the same prevalence level to that of a larger travel volume). Moreover, there is the potential usefulness of antiviral prophylaxis during the quarantine period which could theoretically reduce the number of infectious individuals. Despite the plausible reduction of infectiousness under antiviral prophylaxis, the probability of symptomatic infection will also likely be reduced, and thus, the detection of cases might be reduced. Unless the efficacy of antiviral prophylaxis and detection under this measure are well documented and promisingly high, it is difficult to determine if this countermeasure is likely to offer an overall positive impact on the success of quarantine, and this point should be clarified in future research. Another topic area to be clarified further is concerned with cost-effectiveness. Although we implicitly assumed that the governments of island nations may be willing to allocate quarantine facilities and spend sufficient money for diagnostic testing, these measures are economically demanding, especially for developing island nations. Extension of our method would permit estimating the required cost to achieve a specific ripple benefit (e.g., zero secondary cases for a certain period of time). Use of home-based quarantine (with health agency surveillance and support) is another cost-saving option that could be considered for islands with limited capacity for using facility-based quarantine (e.g., those with few hotels that could be requisitioned), but it should be noted that home-based quarantine might violate our assumption of ignoring secondary transmissions during the quarantine period. In practice, there may also be scenarios where it is not practical to separate all incoming travellers into separate quarters within a quarantine facility (e.g. parents with small children). In such cases, health workers may need to monitor such individuals especially closely and isolation may need to include a parent and infant when only one is symptomatic (all of which would increase costs).
Despite our simplifying assumptions, the present study reasonably suggests that use of quarantine has the potential to substantially reduce the risk of pandemic influenza arriving or at least significantly delay arrival, in small island nations. To ensure the absence of secondary transmissions for plausible ranges of prevalence at the source and a modest number of incoming travellers, we recommend quarantining the incoming individuals for 9 days if quarantine alone is implemented and 6 days if quarantine is combined with rapid diagnostic testing.
Conclusion
==========
To inform border control for pandemic influenza in small island nations we examined the potential effectiveness of quarantine using several parameters which describe the epidemiologic characteristics of influenza. In particular, our modelling approach accounted for asymptomatic infection which is deemed a key requirement for successful influenza control \[[@B52],[@B53]\]. The effectiveness was modelled as a relative reduction of the risk of introducing infectious individuals into the community as a function of time since arrival. We recommend a quarantine period of 9 days to reduce by more than 99% the risk of introducing infectious individuals and to ensure the absence of secondary transmissions. When rapid diagnostic testing is combined with quarantine, we recommend quarantine for 6 days to similarly prevent secondary transmissions.
Competing interests
===================
The authors declare that they have no competing interests.
Authors\' contributions
=======================
NW and MGB conceived of the study and participated in its design and coordination. HN developed methodological ideas and performed statistical analyses. HN and NW did most of the work on drafting the manuscript. All authors read and approved the final manuscript.
Appendix
========
Earlier infection before quarantine
-----------------------------------
For simplicity, we consider the impact of earlier exposure to infection on the effectiveness of quarantine in terms of the frequency of onset during the quarantine period, which is relevant to the determination of the incubation period conducted by Anderson Grey McKendrick \[[@B15],[@B29]\]. Let the length of quarantine be *t*. To account for earlier infections before starting quarantine at *t*= 0, we consider infection-age (i.e. the time since infection) for infected individuals, denoted by *τ*. Let *i*(*t*, *τ*) and *j*(*τ*), respectively, be the number of incubating infected individuals at quarantine period *t*and infection-age *τ*and the number of incubating infected individuals at infection-age *τ*at the beginning of quarantine *t*= 0 (i.e. *i*(0, *τ*) = *j*(*τ*)). *i*(*t*, *τ*) is written as
$$i(t,\tau) = j(\tau - t)\frac{\Gamma(\tau)}{\Gamma(\tau - t)}$$
for *τ*-*t*\> 0 where Γ (*τ*) informs the survivorship function of incubating individuals at infection-age *τ*, i.e.,
$$\Gamma(\tau) = \exp\left( {- {\int_{0}^{\tau}{\gamma(\sigma)d\sigma}}} \right)$$
where *γ*(*τ*) is the rate (or force) of onset at infection-age *τ*. Consequently, the density function of the incubation period, *f*(*τ*), is given by
Since we assume that there is no secondary transmission during quarantine period, *i*(*t*, *τ*) = 0 for *t*-*τ*\> 0. The number of new symptomatic cases at quarantine of length *t*, *n*(*t*), is
$$n(t) = {\int_{t}^{\infty}{\gamma(\tau)i(t,\tau)d\tau}}$$
Replacing the right-hand side of (A4) by that of (A1), we get
$$n(t) = {\int_{t}^{\infty}{\gamma(\tau)j(\tau - t)\frac{\Gamma(\tau)}{\Gamma(\tau - t)}d\tau}} = {\int_{0}^{\infty}{f(t + \sigma)\frac{j(\sigma)}{\Gamma(\sigma)}d\sigma}}$$
In our setting, all quarantined individuals have not experienced symptom onset before quarantine starts at *t*= 0. Assuming that all infected individuals eventually experience symptom onset (just for now), the total number of infected individuals satisfies
$${\int_{0}^{\infty}{n(t)dt}} = {\int_{0}^{\infty}{j(\tau)d\tau}}$$
Using (A5) and (A6), the density of symptom onset at quarantine period *t*(i.e. the frequency of symptom onset relative to the quarantine period *t*), *h*(*t*), is
$$h(t) = \frac{n(t)}{\int_{0}^{\infty}{n(t)dt}} = {\int_{0}^{\infty}{\frac{f(t + \sigma)}{\Gamma(\sigma)}\frac{j(\sigma)}{\int_{0}^{\infty}{j(s)ds}}d\sigma}}$$
Equation (A7) indicates the critical importance in understanding the earlier exposure in order to determine the optimal length of quarantine. That is, the density of symptom onset *h*(*t*) always depends on the infection-age distribution (which is informed by *j*(*τ*)) at the starting time point of quarantine (*t*= 0).
If the epidemic at the source country becomes endemic and reaches a stationary state with constant incidence *Q*, and if the infected travellers result from random sampling of infected individuals at the source country, we have *j*(*τ*) = *Q*Γ(*τ*), leading to
$$h(t) = \frac{\Gamma(t)}{\int_{0}^{\infty}{\Gamma(\tau)d\tau}}$$
which is equivalent to the survivorship of the incubating infected individuals (written as 1-*F*(*t*) in the main text using the cumulative distribution function of the incubation period *F*(*t*)). The simplification in (A8) holds only when a stationary state is the case at the source country, which is not likely to be observed in the event of an influenza pandemic. Thus, we need to use (A7) with some prior information of *j*(*τ*). Nevertheless, since the infection event is unobservable, we seldom know *j*(*τ*). Therefore, we recommend assuming that the start of quarantine *t*= 0 as the time of infection, which is the worst case scenario. Although the above mentioned arguments apply to symptomatic cases alone, we find exactly the same issue in the survivorship of infectiousness.
Differing parameters between symptomatic and asymptomatic cases
---------------------------------------------------------------
First, we consider the impact of differing generation times between symptomatic and asymptomatic cases on the effectiveness of quarantine. Although the generation time distribution of asymptomatic influenza infection has yet to be clarified, we at least theoretically separate the cumulative distributions *G*~s~(t) and *G*~a~(t), respectively, for symptomatic and asymptomatic cases. The equation (4) in the main text is replaced by
Since *G*~a~(t) is unknown, we examine the sensitivity of *ε*(*t*~*β*~), where the effectiveness is calculated as 100*β*% (i.e. *β*= 0.95 and 0.99), to different ratios of *G*~a~(*t*~*β*~) to *G*~s~(*t*~*β*~). Let *c*be *G*~a~(*t*~*β*~)/*G*~s~(*t*~*β*~). *G*~s~(*t*) is assumed to be equivalent to *G*(*t*) in the main text.
Figures [7A](#F7){ref-type="fig"} and [7B](#F7){ref-type="fig"} show the sensitivity of *ε*(*t*~*β*~) to different values of the ratio *c*with *t*~*β*~= 4.74 and 8.62 days. When *c*is smaller than 1 (i.e. when there are more asymptomatic infected individuals with extremely long generation times compared to symptomatic cases), the effectiveness measure (A9) becomes smaller than the baseline which we get from (4) in the main text. On the contrary, if the generation time of asymptomatic infected individuals is shorter than that of symptomatic infected individuals, the effectiveness rises up close to 100% with the assumed lengths of quarantine, suggesting the need to accumulate epidemiological evidence of the generation time.
{#F7}
Second, we investigate the impact of differing sensitivity of rapid diagnostic testing between symptomatic and asymptomatic cases on the effectiveness of quarantine. We theoretically separate the sensitivity S~e~into S~e,\ s~and S~e,\ a~for symptomatic and asymptomatic cases, respectively. Since asymptomatic cases may shed lower titres of virus, we suspect that the ratio S~e,\ a~to S~e,\ s~(*r*:= S~e,\ a~/S~e,\ s~) is smaller than 1. The equation (5) in the main text is replaced by
Figure [7C](#F7){ref-type="fig"} shows the sensitivity of *ε*~d~(*t*) to different values of the ratio *r*assuming that S~e,\ s~= 0.69. As the ratio *r*becomes smaller (i.e. as the diagnosis of asymptomatic infected individuals becomes more difficult than that of symptomatic cases), the effectiveness also becomes smaller. Although the difference in *ε*~d~(*t*) is greater for short quarantine periods, the effectiveness becomes less sensitive to *r*as the length of quarantine becomes longer. We estimated that 99.0% effectiveness in reducing the risk of introducing infectious individuals into the community is achieved with *t*= 5.71 days using the rapid diagnostic test of *r*= 1.0 in the main text. The effectiveness estimate with the same length of quarantine and *r*= 0.6 is still as large as 98.1%.
Imperfect case detection
------------------------
Although we considered perfect detection of symptomatic cases upon symptom onset during quarantine in the main text, here we examine the sensitivity of the effectiveness of quarantine to differing efficacy of case detection. Let the efficacy of case finding be *k*which we assumed as 1 in the main text. In reality, it might be difficult to detect all flu-like symptoms (i.e. *k*\< 1). The equation (4) in the main text is replaced by
It should be noted that *k*influences symptomatic cases alone, because the detection of symptoms does not apply to asymptomatic infected individuals. Figure [7D](#F7){ref-type="fig"} shows the sensitivity of *ε*(*t*) to different values of the ratio *k*which was assumed to lie in the range of 0.6 -- 1.0. As the ratio *k*becomes smaller (i.e. as the detection becomes less efficient), the effectiveness becomes smaller. The difference in *ε*(*t*) between different ratios *k*is particularly highlighted when the quarantine period is between 2 and 5 days. Nevertheless, for the shorter and longer quarantine periods, difference in *ε*(*t*) is almost negligible. In the main text, we estimated that quarantine for 8.62 days achieves 99.0% effectiveness of reducing the risk of releasing infectious individuals into the community with *k*= 1.0. The effectiveness estimate with the same length of quarantine and *k*= 0.6 is still as large as 98.2%.
Pre-publication history
=======================
The pre-publication history for this paper can be accessed here:
<http://www.biomedcentral.com/1471-2334/9/27/prepub>
Acknowledgements
================
We thank the Centers for Disease Control and Prevention (USA) for contributing to funding this research work on pandemic influenza control (via grant: 1 U01 CI000445-01). Early work on this topic was also supported by a research contract with the New Zealand Ministry of Health. The work of HN was supported by the Asian Neighbours Network Program of the Toyota Foundation and The Netherlands Organisation for Scientific Research (NWO).
| {
"pile_set_name": "PubMed Central"
} |
Introduction
============
Lipopolysaccharide (LPS), an essential component of the surface of Gram-negative bacteria \[[@r1]\], has potent proinflammatory properties in many cell types \[[@r2]-[@r4]\], including endothelial cells \[[@r5],[@r6]\]. A major consequence of the LPS action on endothelial cells is the upregulation of genes specifically involved in recruiting and adhering leukocytes \[[@r7]\].The firm adhesion of leukocytes to the vessel wall occurs via interaction of the CD11a/CD18 (β2) integrins to endothelial ligands such as intercellular adhesion molecule-1 (ICAM-1). ICAM-1, an inducible cell transmembrane glycoprotein, acts as a key component in inflammatory response for recruiting leukocytes to the sites of inflammation and is implicated in the pathogenesis of numerous inflammatory diseases such as rheumatoid arthritis \[[@r8]\], uveitis \[[@r9]\], and atherosclerosis \[[@r10],[@r11]\]. Thus, it is suggested that modulation of adhesion molecule expression and reduction of aberrant leukocyte adhesion to the endothelium may be an attractive approach for treating inflammation-related vascular complications, including inflammatory ocular disorders \[[@r12]\].
We previously demonstrated that the peptide GC31, which is derived from C-type lectin-like domain (CTLD) of human thrombomodulin (TM), has a potent anti-inflammatory effect on endotoxin-induced uveitis (EIU) by reducing leukocyte infiltration and proinflammatory mediator expression \[[@r13]\]. Uveitis is characterized by an increase in leukocyte rolling, sticking, and adhesion molecule expression, and breakdown of the blood--retinal barrier, which subsequently leads to transendothelial migration of leukocytes and recruitment of large numbers of cells to the retina \[[@r14]-[@r16]\]. GC31 intravitreal injection could reduce leukocyte counts in aqueous humor and leukocyte infiltration in the anterior chamber, iris-ciliary bodies, and posterior vitreous according to histological examination. In addition, it has been reported that TM CTLD dampens the inflammatory response by interfering with leukocytes adhesion through inhibiting adhesion molecule expression \[[@r17]\]. Despite those studies, it is still unknown whether the effect of GC31 on EIU is mediated by inhibiting leukocyte-endothelium adhesion. Thus, the aim of the present study was to investigate the ability of GC31 to modulate the expression of ICAM-1 in LPS-induced HUVECs and to identify the underlying mechanism(s).
Methods
=======
Reagents
--------
Rosewell Park Memorial Institute (RPMI) 1640, fetal bovine serum (FBS), and antibiotics were from Gibco-BRL (Grand Island, NY). LPS (*E. coli* 055:B5) was purchased from Sigma (St. Louis, MO). The polyclonal antibodies against p38 mitogen-activated protein kinase (MAPK), phospho-p38 (p-p38) MAPK, extracellular signal-regulated kinase-1/2 (ERK1/2), phospho-ERK1/2 (p-ERK1/2), inhibitor of nuclear factor kappa B alpha (IκBα), phospho-IκBα (p-IκBα), and phosphonuclear factor kappa B (NF-κB) p65 (p-NF-κB p65) were obtained from Cell Signaling Technology, Inc. (Beverly, MA). The rabbit monoclonal antibodies against ICAM-1 and Lamin A/C were obtained from Epitomics, Inc. (Burlingame, CA). Horseradish peroxidase (HRP)-conjugated monoclonal mouse antiglyceraldehyde-3-phosphate dehydrogenase (GAPDH) was from Kangchen Biotech (Shanghai, China). Goat antirabbit immunoglobulin G (Ig G) was from R&D Systems (Minneapolis, MN).
Synthesis of peptide
--------------------
Peptide GC31 and control peptide VP30 were synthesized using high-efficiency solid-phase peptide synthesis with an automatic peptide synthesizer (Symphony; Protein Technologies, Tucson, AZ) and performed by ChinaPeptides Co., Ltd. in Shanghai, PR China. The purity over 95% was characterized by analytical high-performance liquid chromatography and mass spectrometry. All the synthesized peptides were freeze-dried and stored at −20 °C until used. The peptides were dissolved in culture medium and sterilized by filtration through a 0.2 μm filter. All the synthesized peptides were freeze-dried and stored at −20 °C until used. The peptides were dissolved in culture medium and sterilized by filtration through a 0.2 μm filter.
Cell culture and treatment
--------------------------
Human umbilical vein endothelial cells (HUVECs) were obtained from ScienCell (San Diego, CA). The cells were maintained in endothelial cell medium (ECM; ScienCell) consisting of 500 ml of basal medium, 25 ml of fetal bovine serum, 5 ml of endothelial cell growth supplement, and 5 ml of penicillin/streptomycin in a humidified incubator at 37 °C with an atmosphere of 5% CO~2~. HUVECs were used at passages 3--8 and allowed to grow to subconfluence before treatment. Before the treatment with LPS or peptides, the culture medium was replaced by fresh medium, and LPS was added at a final concentration of 1 μg/ml. GC31 (0.1--10 μM) or VP30 (10 μM) peptides were added to the culture medium simultaneously with LPS. The monocyte U937 cell lines (human monocytic leukemia cell line U937) were purchased from the Cell Bank of Shanghai Institutes for Biologic Sciences (Shanghai, China). Cells were maintained in RPMI 1640 supplemented with 10% FBS, 100 IU/ml penicillin, and 100 μg/ml streptomycin in a humidified incubator at 37 °C with an atmosphere of 5% CO~2~.
Cell viability assay
--------------------
The cell viability of the HUVECs was assessed using the CellTiter 96 aqueous one solution cell proliferation assay (MTS) kit (Promega Corporation, Madison, WI). Briefly, 2×10^4^ cells in 100 μl of medium were seeded in each well of a 96-well plate and allowed to adhere for 48 h. Then cells were treated with various concentrations of peptides or LPS (1 μg/ml) in ECM with 0.1% FBS for 24 h. Cells in the control group were left untreated. Afterwards, 20 μl of MTS solution was added in each well and incubated for another 3 h at 37 °C. Absorbance was detected at 490 nm with a microplate reader (Model 680; Bio-Rad, Hercules, CA).
Monocytoid cell adhesion assays
-------------------------------
The effect of GC31 on the adhesion of U937 cells to LPS-activated HUVECs was evaluated as previously described \[[@r18],[@r19]\]. For the adhesion assays, HUVECs were grown to confluence in 24-well tissue culture plates, pretreated with various concentrations of GC31 or VP30 for 18 h, and then cells were treated with LPS with a final concentration of 1 μg/mL for an extra 6 h. U937 cells were suspended overnight in RPMI 1640 medium containing 0.1% FBS and freshly harvested and labeled with CM-H2DCFDA (10 µM, Invitrogen, Grand Island, NY) in 0.1% FBS RPMI 1640 medium at 37 °C for 30 min. Fluorescence-labeled U937 cells were washed three times with fresh RPMI 1640 medium, and then added at a density of 5×10^4^ cells/well onto the 24-well plate containing pretreated HUVECs. After incubation at 37 °C for 30 min, non-adhered U937 cells were removed by gentle washing with PBS (NaCl 8.0 g, KCl 0.2 g, Na~2~HPO~4~•12H~2~O 2.9 g, KH~2~PO~4~ 0.2 g, and distilled water 1 L), and the monolayers were fixed with 4% paraformaldehyde in PBS. The number of adhered U937 cells was counted in five random fields (10× objective) per well in a masked fashion and expressed as the number of adhered cells per field. The images were captured under a confocal fluorescence microscope (LSM 510; Carl Zeiss, Gottingen, Germany). Experiments were performed in triplicate and repeated at least three times.
Western blot analysis for intercellular adhesion molecule-1
-----------------------------------------------------------
Crude proteins were extracted from HUVECs treated with 1 μg/ml LPS in the presence or absence of GC31 (0.1--10 μM) or VP30 (10 μM) for 12 h. Protein concentration was determined using the Bio-Rad protein assay (Bio-Rad Lab). Proteins were then resolved on 10% sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS--PAGE; Bio-Rad) gels. After electrophoresis, the proteins were electrotransferred to polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA). The membrane was then blocked with 5% skim milk in Tween-20/PBS and incubated with primary antibodies: rabbit anti-ICAM-1 (1:1,000) and mouse anti-GAPDH (1:1,000). The blots were then incubated with HRP-conjugated secondary antibodies. The bands were visualized using the ECL system detection system (Pierce, Rockford, IL) and quantified with Image J software (NIH; Bethesda, MD) with a fixed rectangular box covering each band. The software automatically plots the lanes and provides an average intensity of pixels with a graphical peak. The band intensity was acquired after the peak area was closed off.
Real-time polymerase chain reaction
-----------------------------------
Confluent HUVECs in 6 cm plates were incubated with LPS (1 μg/ml) for 6 h in the presence or absence of GC31 (0.1--10 μM) or VP30 (10 μM). Total RNA was extracted with TRIzol reagent (Invitrogen, Grand Island, NY), according to the manufacturer's instructions. Total RNA (2 μg) of each sample was reverse-transcribed into cDNA, and the fluorescence quantitative real-time PCR (RT-PCR) was performed on the Rotor-Gene 3000 (Corbett Research, Mortlake, Australia) instrument with a One-Step SYBR PrimeScript RT-PCR kit (TaKaRa Bio Inc., Shiga, Japan) according to the protocol. Specific sense and antisense primers used were as follows: ICAM-1, sense: 5′-CAG TGA CCA TCT ACA GCT TTC CGG-3′, antisense: 5′-GCT GCT ACC ACA GTG ATG ATG ACA A-3′; GAPDH, sense: 5′-ACC ACA GTC CAT GCC ATC AC-3′; antisense: 5′-TCC ACC ACC CTG TTG CTG TA-3′. GAPDH was chosen as a housekeeping gene to compare the amount of total mRNA of each sample. The transcript number was calculated using a 2 −△CT method (relative) \[[@r20]\].
Western blot analysis for nuclear factor kappa B and mitogen-activated protein kinase pathways
----------------------------------------------------------------------------------------------
HUVECs were pretreated with GC31 (0.1--10 μM) or VP30 (10 μM) for 30 min and then incubated with LPS (1 μg/ml) for 30 min or 1 h. After treatment, cells were washed twice in PBS and rapidly harvested using a cell scraper. Nuclear and cytoplasmic extracts were prepared on ice with ProteoJET cytoplasmic and nuclear protein extraction kit (Fermentas Life Science, Opelstrasse, Germany). The whole cell lysates (40 μg) and nuclear extracts (30 μg) were separated with 10% SDS--PAGE and electrotransferred to PVDF membranes. After being blocked with 5% bovine serum albumin (BSA), the membranes were probed with primary antibodies, including IκBα, phospho-IκBα, p38, p-p38, ERK1/2, p-ERK1/2, or p-NFκB-p65 (Ser536). Each membrane was further incubated with HRP-conjugated goat antirabbit IgG (1:1000) secondary antibodies. Lamin A/C (1:1000) was chosen as a nuclear housekeeping protein in the nuclear extract. The bands were visualized using the ECL system detection system, and the band density was determined with Image J software.
Statistical analysis
--------------------
All experiments were repeated at least three times. The results were expressed as mean ± standard deviation (SD) and analyzed with one-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test using SPSS 16.0 (SPSS; Chicago, IL) software for multiple comparisons of mean values. p\<0.05 was considered statistically significant.
Results
=======
Few effects of GC31 on the viability of human umbilical vein endothelial cells
------------------------------------------------------------------------------
To evaluate the effects of GC31, VP30, and LPS treatment on the growth of HUVECs, cell viability upon different concentration treatment was analyzed with MTS assay. Twenty-four hours post treatment with GC31 (0.1--10 μM), VP30 (10 μM), or LPS (1 μg/ml), compared to the control group treated with culture medium only, the absorbance of all treated groups showed no significant difference, suggesting GC31, VP30, and LPS have few inhibitory effects on HUVECs (p\>0.05, [Figure 1](#f1){ref-type="fig"}). Since MTS assay can also be used to evaluate cell proliferation, the insignificant toxic effect also indicated that the modulatory activities of peptides on HUVECs were irrelevant to inhibiting cell proliferation.
{#f1}
GC31 treatment reduced U937 cell adhesion to lipopolysaccharide-induced human umbilical vein endothelial cells
--------------------------------------------------------------------------------------------------------------
Our previous study showed that GC31 treatment could effectively prevent leukocyte infiltration in EIU \[[@r13]\] and it was demonstrated that the earliest infiltrated inflammatory cells into ocular tissue of EIU rats were mononuclear cells \[[@r21]\]. Accordingly, we next examined the adhesion of monocytes to LPS-activated HUVECs pretreated with GC31. The U937 cell line is a human cell line established from a diffuse histiocytic lymphoma and displaying many monocytic characteristics. It serves as an in vitro model to study the behavior and differentiation of monocytes and macrophages. With 1 μg/ml of LPS treatment alone for 6 h, the number of U937 cell adhesion to HUVECs was significantly increased 12.45-fold compared to the control group (p\<0.01; [Figure 2A,B](#f2){ref-type="fig"}). When HUVECs were pretreated with indicated concentrations of GC31 for 18 h before exposure to LPS, the number of adhered U937 cells was strikingly decreased in a dose-dependent manner (0.1 μM, 0.77 fold, p\<0.05; 1 μM, 0.45 fold, p\<0.01; 10 μM, 0.28 fold, p\<0.01 compared with the LPS-treated group). Though VP30 (10 μM) pretreatment reduced the number of adhered U937 cells slightly, the difference was not statistically significant ([Figure 2B](#f2){ref-type="fig"}, p\>0.05).
{#f2}
GC31 suppressed lipopolysaccharide-induced intercellular adhesion molecule-1 expression in human umbilical vein endothelial cells
---------------------------------------------------------------------------------------------------------------------------------
To investigate the mechanism in which GC31 decreased the binding of monocytes to HUVECs, we examined the expression of ICAM-1 in LPS-activated HUVECs, which is well known to mediate the firm binding of monocytes \[[@r9],[@r22]\]. HUVECs were stimulated with 1 μg/ml of LPS in the presence or absence of GC31 or VP30 for 12 h. As indicated by the results of western blot analysis, exposure of cells to LPS (1 μg/ml) evoked a strong increase in ICAM-1 expression compared with unstimulated cells ([Figure 3A](#f3){ref-type="fig"}). However, GC31 treatment resulted in a significant reduction in LPS-induced ICAM-1 expression in a dose-dependent manner, indicating that peptide GC31 was effective in blocking ICAM-1 expression in LPS-activated HUVECs. To further explore the effect of GC31 on modulating the transcriptional level of adhesion molecules, total cellular RNAs were isolated and analyzed with RT--PCR. Similar results were obtained that treatment with GC31 significantly suppressed the induction of ICAM-1 mRNA in HUVECs stimulated with LPS for 6 h ([Figure 3B](#f3){ref-type="fig"}). Altogether, these results suggested that the ICAM-1 expression induced by LPS is post-transcriptionally prevented by GC31.
{#f3}
GC31 inhibited lipopolysaccharide-induced inhibitor of nuclear factor kappa B alpha degradation and nuclear factor kappa B p65 nuclear translocation
----------------------------------------------------------------------------------------------------------------------------------------------------
NF-κB is a crucial transcription factor for expression of adhesion molecules \[[@r23],[@r24]\]. The phosphorylation of NF-κB p65 (Ser536) plays an important role in regulating NF-κB activation and transcription of many inflammatory genes \[[@r24]\]. Therefore, we determined the effect of GC31 on NF-κB transcriptional activation. HUVECs were treated with various concentrations of GC31 for 30 min before stimulation with LPS for 1 h. Pretreatment with GC31 dose-dependently reduced the translocation of phosphorylated p65 NF-κB to the nuclear fraction compared to the LPS group ([Figure 4A](#f4){ref-type="fig"}). Translocation of NF-κB from cytoplasm to the nucleus was preceded by the phosphorylation, ubiquitination, and proteolytic degradation of IκBα \[[@r25]\]. To explore whether GC31 affected LPS-induced degradation of IκBα, western blot analysis was performed. As [Figure 4B](#f4){ref-type="fig"} shows, stimulation with LPS treatment for 30 min caused rapid phosphorylation and degradation of IκBα, while pretreatment with GC31 for 30 min inhibited the LPS-induced phosphorylation and degradation of IκBα in a dose-dependent fashion ([Figure 4B](#f4){ref-type="fig"}). Taken together, the data suggested that GC31 inhibited LPS-induced NF-κB activation, which might be associated with the blockade of LPS-induced adhesion molecule production by GC31.
{#f4}
GC31 reduced lipopolysaccharide-induced activation of mitogen-activated protein kinase pathways in human umbilical vein endothelial cells
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Of the signaling pathways activated by LPS in endothelial cells, the p44/42 MAPK and p38 MAPK pathways have been shown to be directly involved in producing cytokines and adhesion molecules \[[@r26],[@r27]\]. Hence, we examined the effect of GC31 on LPS-induced MAPK activation. As shown in [Figure 5](#f5){ref-type="fig"}, 30 min after LPS treatment, the levels of activated p38 MAPK and ERK1/2 in the untreated cells were markedly increased. However, GC31 pretreatment moderately inhibited p38 MAPK activity in a dose-dependent manner and mildly reduced ERK1/2 activity in the LPS-stimulated HUVECs ([Figure 5](#f5){ref-type="fig"}). These results suggested that GC31 may suppress LPS-induced ICAM-1 expression, at least in part, by reducing the activation of the p38 MAPK and ERK1/2 pathways.
{#f5}
Discussion
==========
Leukocyte-endothelial adhesion is an early step in many inflammatory disorders including sepsis \[[@r28]\], ischemia-reperfusion injury \[[@r29]\], atherosclerosis \[[@r30]\], and some ocular disorders including diabetic retinopathy \[[@r31],[@r32]\] and uveitis \[[@r9],[@r15]\]. The adhesion of monocytes to the endothelium is well established as a major early step in the development of atherosclerosis \[[@r33]\] as well as experimental uveitis \[[@r21]\]. In addition, it has been observed that preferential location of leukocyte adhesion occurs mainly in the endothelium of retinal veins and venules \[[@r14],[@r16]\] while the sensitivity of arterial endothelium on LPS and tumor necrosis factor-α induction is much slower than that of venous endothelium \[[@r34]\]. In the present study, we investigated the effect of GC31 on monocyte U937-cell adhesion to LPS-activated HUVECs. We demonstrated that GC31 inhibited the elevated mRNA and protein production of ICAM-1 in LPS-induced HUVECs and reduced monocyte adhesion to LPS-activated HUVECs. The effects of GC31 were possibly due to its blockade of the NF-κB, p38 MAPK, and ERK1/2 signaling pathways.
ICAM-1 and leukocyte function-associated antigen-1 have been shown to play an important role in the pathogenesis of EIU and experimental autoimmune uveitis (EAU) \[[@r9],[@r35]\]. It has also been reported that the expression of adhesion molecules increased progressively as EAU developed, with the increased ICAM-1 expression starting earlier and more closely associated with the sites of blood--retinal barrier breakdown \[[@r16]\]. In our present work, great overexpression of ICAM-1 was found in HUVECs treated with LPS (1 μg/mL). However, treatment with GC31 significantly and dose-dependently inhibited the mRNA and protein expression of ICAM-1. Meanwhile, similar results were obtained in the monocyte-endothelium adhesion assay. These results are in accordance with the observation that the recombinant lectin-like domain of TM can attenuate endothelial adhesion molecule expression and adhesion of polymorphonuclear leukocytes \[[@r17]\]. We have reported that GC31 peptide could reduce the level of monocyte chemoattractant protein (MCP)-1 in the aqueous humor of EIU rats \[[@r13]\]. MCP-1 is uniquely essential for monocyte recruitment and can direct migration of adherent cells across the endothelium \[[@r36],[@r37]\]. Taken together, all the results suggested that GC31 has an important effect on preventing leukocyte-endothelium interaction under inflammatory conditions.
The architecture of ICAM-1 promoter contains a large number of binding sites for inducible transcription factors, including AP1, C/EBP, TFIID, Ets, NF-κB, etc \[[@r38]\]. The proximal NF-κB binding site located about 200 bp upstream of the translation start site has been shown to be particularly important for the induction of ICAM-1 transcription \[[@r39]\]. In resting cells, NF-κB exists in its canonical form as a p50/p65 heterodimer bound to the inhibitor factor-κB (IκB) in the cytoplasm. In response to extracellular stimulation such as LPS and tumor necrosis factor-α, the degradation of IκB through proteolysis is initiated, which allows the translocation of NF-κB from the cytoplasm into the nucleus \[[@r23],[@r24]\]. Once translocated to the nucleus, p50/p65 binds to the κB sites in various genes, including elements in the E-selectin, vascular cell adhesion protein 1, and ICAM-1 promoters \[[@r23]\]. In the present study, GC31 pretreatment suppressed LPS-induced NF-κB activation by reducing the levels of NF-κB p65 in the nuclei of HUVECs and inhibiting the degeneration of IκBα in the cytoplasm, consistent with its effects on LPS-induced RAW264.7 cells \[[@r13]\]. Previous studies demonstrated that two different NF-κB inhibitors, PDTC and BAY11--7082, inhibited the expression of these adhesion molecules, thus preventing monocyte adhesion to endothelial cells \[[@r34],[@r40]\]. Therefore, we speculate that the inhibitory effect of GC31 on ICAM-1 mRNA and protein production and monocyte-endothelium interaction in LPS-induced HUVECs may result from the suppression of NF-κB activation.
It has been reported that the MAPK family is involved in LPS-mediated signaling in endothelial cells and ICAM-1 expression \[[@r26],[@r41]-[@r43]\]. Hence, we assessed the effect of GC31 on LPS-induced phosphorylation of p38 MAPK and ERK1/2. Our findings suggested that GC31 pretreatment mild to moderately suppressed the phosphorylation of both proinflammatory kinases in LPS-stimulated HUVECs, and the inhibitory effect on p38 MAPK activation is more prominent. In endothelial cells, p38 MAPK can mediate cellular responses through translocating to nucleus, where it phosphorylates and activates transcription factors, i.e., AP1 \[[@r44]\] and NF-κB \[[@r45]\]. Carter et al. \[[@r45]\] found that the p38 MAP kinase regulates NF-kB-dependent gene transcription, in part, by modulating activation of TATA-binding protein, and a dominant-negative p38 MAPK expression vector reduces NF-κB-dependent gene expression but has no impact on NF-κB activation at any levels. Thus, inhibition of p38 MAPK activation may further restrict other transcriptional factors' effect, such as NF-κB, and then prevent the inflammatory signaling cascade.
In some ocular surface inflammatory diseases, such as dry eye and pterygium, ICAM-1 was found to be upregulated on lymphocytes and/or vascular endothelial cells resulting in lymphocytic diapedesis to the lacrimal and conjunctival tissues, and may serve as a signaling molecule for predisposition of ocular surface inflammation \[[@r46],[@r47]\]. Because of its small size, the GC31 peptide may be promising for topical application. In addition, increased leukocyte adhesion and expression of proinflammatory cytokines and adhesion molecules have characterized several other ocular diseases, including diabetic retinopathy \[[@r48]\], retinal vessel occlusions, and ischemic-reperfusion injury \[[@r49]\]. The current findings about the novel peptide GC31 may be helpful in these conditions and deserves further investigation.
In conclusion, our results demonstrated that GC31 interfered with U937 monocyte adhesion to LPS-induced HUVECs by suppressing endothelial ICAM-1 mRNA and protein expression, through inhibiting the nucleus translocation of NF-κB, and partially by reducing the phosphorylation of p38 MAPK and ERK1/2. These findings underscore a role of GC31 in ameliorating endothelial dysfunction by exogenous insults and raise the possibility that GC31 plays a protective role in the pathophysiology of vascular inflammation and uveitis.
This work was supported by National Natural Science Foundation of China (No. 81273424) and National Specialized Science and Technology Projects for ''Significant New Drugs Creation'' (2011ZX09302--007--02).
| {
"pile_set_name": "PubMed Central"
} |
Background {#Sec1}
==========
A steady increase in the incidence rate of thyroid cancer has been noted in recent decades all over the world, and the causes of this increase are still controversial. Thyroid cancer is the most common endocrine malignancy (1.0--1.5% of all newly diagnosed cancers in the United States of America every year are originally thyroid). The increased frequency in thyroid cancer is almost exclusively due to the rise in the number of papillary cancers, with no significant changes in other histologic subtypes \[[@CR1], [@CR2]\]. The typical presentation is as small tumors, though there is a growing incidence of large tumors; it has been hypothesized that the rise in the incidence of thyroid cancer is mostly due to improved detection rather than to a real increase in frequency \[[@CR3]\]. Thyroid nodule can be defined as a discrete lesion within the thyroid gland that is radiologically distinct from the surrounding thyroid parenchyma. It may be solitary, multiple, solid, or cystic, and may or not be functional. Thyroid nodules are frequent among the general population and thyroid Ultrasound (US) has considerably increased the number of cases identified. Thyroid nodules may be palpated in about 4--8% of the general population (however, neck palpation is very imprecise in terms of determining the size and morphology). US identifies the presence of nodules in 19-67% of the cases, and is an accurate method for the detection of thyroid nodules; however, US has a low accuracy in differentiating between benign from malignant thyroid nodules \[[@CR4]\]. The sonographic characteristics of a thyroid nodule associated with a higher likelihood of malignancy include hypoechogenicity, increased intranodular vascularity, irregular margins, microcalcifications, absent halo, and a taller-than-wide shape measured in the transverse dimension. Thus, several benign and malignant ultrasound gray scale and Doppler features have emerged over the last ten years that may be used in different ways to assign probabilities, together with a method based on the Breast Imaging Reporting and Data System (BIRADS). Likewise, several US Thyroid Imaging Reporting and Data Systems (TIRADS) have been proposed for risk stratification of thyroid nodules \[[@CR5]\].
The nodules are usually divided into different categories based on TIRADS and are then referred for Fine-Needle Aspiration (FNA) Biopsy or follow-up, according to the variable risk of malignancy. The terminology of TIRADS was first used by Horvath et al. \[[@CR6]\]. They described 10 US patterns of thyroid nodules and related the rate of malignancy based on the pattern. The initial purpose of TIRADS was to improve patient management and cost-effectiveness by avoiding unnecessary FNA Biopsies in patients with thyroid nodules (Table [1](#Tab1){ref-type="table"}), with a sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of 88, 49, 49, 88, and 94%, respectively. However, its clinical use is still very limited and its practical application in clinical practice is questioned. Moreover, FNA Biopsy is the most accurate method for determining malignancy, and is a fundamental part of current thyroid nodule evaluation. The Bethesda System for Reporting Thyroid Cytopathology is a standardized reporting system for classifying thyroid FNA Biopsy results that comprises six diagnostic categories with unique risks of malignancy and recommendations for clinical management. Since its inception, the Bethesda System has been widely adopted, each category conveys a risk of malignancy and recommended next steps, though it is unclear if each category also predicts the type and extent of malignancy (Table [1](#Tab1){ref-type="table"}). Nevertheless, the implementation of this reporting system has shown significant diagnostic variability, both inter and intra pathologists, particularly when read as "atypical cells of undetermined significance, follicular lesion of undetermined significance, or follicular neoplasm" (also termed as Bethesda Category III, comprising a heterogeneous population of low-risk lesions that contain follicular cells exhibiting either architectural abnormalities or nuclear atypia that do not fit into other definitive cytological categories). A recent meta-analysis evaluated the validity of the Bethesda reporting system and found 97% sensitivity, 50.7% specificity and 68.8% diagnostic accuracy; the negative and positive predictive values were 96.3 and 55.9%, respectively \[[@CR7], [@CR8]\]. Notwithstanding the fact that both US and FNA biopsy are widely recommended procedures to study patients with thyroid nodules, the value of the existing concordance between the two methods has not been established. Consequently, the purpose of this study was to assess the existing concordance between the two diagnostic methods used in the initial evaluation of individuals with non-toxic thyroid nodule (TIRADS and Bethesda systems).Table 1Thyroid imaging reporting and data system (TIRADS) and the Bethesda System for Reporting Cytopathology (ref. 6, 8, 9)TIRADSBETHESDACategoriesFeaturesDiagnostic CategoriesRisk of malignancyTIRADS 1Normal thyroid gland.I. Nondiagnostic or unsatisfactory.-TIRADS 2Benign conditions (0% malignancy). Cyst fluid only.TIRADS 3Probably benign nodules (5% malignancy). Virtually acellular specimen.TIRADS 4Suspicious nodules (5--80% malignancy rate). A subdivision into 4a (malignancy between 5 and 10%) and 4b (malignancy between 10 and 80%) was optional. Other (obscuring blood, clotting artifact, etc.).TIRADS 5Probably malignant nodules (malignancy \>80%).II. Benign.0-3TIRADS 6Category included biopsy proven malignant nodules. Consistent with a benign follicular nodule (includes adenomatoid nodule, colloid nodule, etc.). Consistent with lymphocytic (Hashimoto) thyroiditis in the proper clinical context. Consistent with granulomatous (subacute) thyroiditis.III. Atypia of undetermined significance/follicular lesion of undetermined significance.5-15IV. Follicular neoplasm/\"suspicious\" for follicular neoplasm. Specify if Hürthle cell type.15-30V. Suspicious for malignancy.60-75 Suspicious for papillary carcinoma. Suspicious for medullary carcinoma. Suspicious for metastatic carcinoma. Suspicious for lymphoma.VI. Malignant.97-99 Papillary thyroid carcinoma. Poorly differentiated carcinoma. Medullary thyroid carcinoma. Undifferentiated (anaplastic) carcinoma. Squamous cell carcinoma. Carcinoma with mixed features. Metastatic.
Methods {#Sec2}
=======
The overall objective of the study was to determine the level of concordance between the ultrasound criteria established under TIRADS (The Thyroid Imaging Reporting and Data System for US of the thyroid); and the cytology criteria according to The Bethesda System for Reporting Thyroid Cytopathology \[[@CR9], [@CR10]\]. Additionally, the study population was characterized from the socio-demographic point of view, the concordance of the classification systems was estimated, and the heterogeneity of the factors influencing the consistency of the various classification systems was analyzed.
Ethics approval and consent to participate {#Sec3}
------------------------------------------
All personal data were confidential and managed exclusively by the principal investigator, according to the legal standards on the confidentiality of the medical record and adhering to the rules of the Institutional Review Committee of Human Ethics (reference number: 221--011). Universidad del Valle, Valle del Cauca-Colombia.
### Design of the study {#Sec4}
This was a cross-sectional study to evaluate the concordance between two diagnostic systems (TIRADS and Bethesda), administered simultaneously to the same individual. The population consisted of consecutive patients consulting the outpatient endocrinology, internal medicine, or general surgery departments at a high complexity referral center, with a diagnosis of nodular or non-nodular "thyroid dysfunction". The inclusion criteria were as follows: male and females aged 18 years and older, with a non-toxic thyroid nodule (ranges for normal thyroid tests were Thyrotrophin (TSH): 0.4 to 4 mIU/L; Free thyroxine: 0.8 to 1.8 ng/dL, according to the National Academy of Clinical Biochemistry) identified either clinically or through imaging \[[@CR11]\]. The exclusion criteria were: TIRADS 1 and Bethesda I (Table [1](#Tab1){ref-type="table"}); Graves-Basedow--associated hyperthyroidism, patients with toxic thyroid nodular disease, chronic hypothyroidism (with a minimum of six-months on treatment with levothyroxine sodium), iatrogenic hyperthyroidism resulting from high-dose sodium levothyroxine therapy regardless of the indication; a history of surgically resected thyroid cancer, and patients with a history of partial thyroidectomy (lobectomy) or subtotal/near total thyroidectomy under levothyroxine sodium therapy (the latter criterion is based on the fact that a constant high stimulus of thyroid hormones and the concomitant TSH suppression in patients with endogenous hyperthyroidism and levothyroxine management may impact the size of the thyroid nodules) \[[@CR12], [@CR13]\].
This study was supported by the Internal Medicine Department from The Faculty of medicina of the Universidad del Cauca (Popayán-Colombia), who provided funding to conduct the analysis and prepare the manuscript.
### Sample size estimate and sampling {#Sec5}
To estimate the sample size, matched categories in both reporting systems were considered. Based on the data from a pilot study with 32 subjects that met the above selection criteria, and using the formula below, the N value was established at 128 subjects:$$\documentclass[12pt]{minimal}
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\begin{document}$$ n=\frac{P_e}{e{e}^2\left(1-{P}_e\right)} $$\end{document}$$
Where:
P~e~: Expected percentage of random concordance
*Ee*: Kappa index standard error \[[@CR14]--[@CR16]\].
A consecutive non-probabilistic sampling was used based on an initial review of 217 medical records; however the final analysis was limited to 180 patients and 37 patients were excluded due to:Incomplete family history and missing socio-demographic information in 26 records.The echography was not reported according to TIRADS criteria in 4 records.The cytology results were not reported according to the Bethesda criteria in 5 cases.The ultrasound examination had been done at a different institution or by a different radiologist in 2 cases.
The source of the information in this study is a registry of consecutive data from an outpatient center for patients with a diagnosis of thyroid dysfunction. A standard form collected socio-demographic information, family and personal history of diseases, in addition to the data available from the medical record. Patients undergoing thyroid ultrasound imaging and FNA Biopsy due to non-toxic nodular thyroid disease were analyzed in accordance with the medical opinion of the institution's study group on thyroid disease (endocrinology, pathology, radiology and surgery). All patients were informed about the procedure and after signing the informed consent, the thyroid ultrasound was performed, and the node(s) were sampled according to Crockett's FNA Biopsy protocol \[[@CR17]\].
The same radiologist read all the tests. One out of every 20 patients was randomly selected to repeat the ultrasound examination. The principal researcher interpreted the results in accordance with the TIRADS criteria and if the second reading was inconsistent with the first, a second radiologist was asked for an opinion to arrive at a consensus between the two radiologists and establish a TIRADS-based ultrasonographic diagnosis. 9 of the 180 participants were randomly selected to assess the radiologists' agreement. One of the nine US results showed disagreement because the first radiologist reported TIRADS 3, while the second one reported TIRADS 2, based on the original classification. Upon further analysis the conclusion was TIRADS 3. The material obtained via the FNA Biopsy was placed on a glass slide previously impregnated with 96% alcohol and then a second glass slide was placed on top. The smear was again immersed in 96% alcohol and then stained using the Papanicolaou technique. To ensure the quality of the cytology specimens, the same experienced pathologist read the slides and reported a diagnosis based on the Bethesda criteria. One out every ten specimens was randomly selected to be analyzed by a second pathologist. In case of disagreement between the two pathologists, a pathologist meeting was convened (five pathologist). The second pathologist disagreed with two of the 18 specimens subject to a second evaluation; in both cases, the first pathologist classified the cytology specimen as Bethesda V, while the second pathologist classified the specimens as Bethesda VI. Both specimens were further evaluated at a pathologist meeting, and the final classification was Bethesda VI. The radiologists and the pathologists were blinded to the patients' medical record data for both the ultrasound examination and the FNA biopsy.
### Statistical analysis {#Sec6}
The weighted Kappa statistical method with a 95% confidence interval and the statistical Z-test were used to estimate the level of concordance between the two systems. In order to pursue the Kappa analysis, categories 5 and 6 of both the TIRADS and the Bethesda classification were combined since the highest risk for malignancy is usually described in these two categories. Category 1 in both classifications was excluded from the selection process because a TIRADS 1 ultrasound examination is considered normal, and Bethesda I is considered an unsatisfactory specimen. The purpose of excluding category 1 was to avoid invalidating further comparisons since category 1 is inconclusive, particularly Bethesda I. Consequently, the analysis categories are as follows:TIRADS 2: "BENIGN"TIRADS 3: "PROBABLY BENIGN"TIRADS 4: "SUSPICIOUS"TIRADS 5: "PROBABLY MALIGNANT"Bethesda II: "BENIGN".Bethesda III: "PROBABLY BENIGN".Bethesda IV: "SUSPICIOUS".Bethesda V: "PROBABLY MALIGNANT"
Weighted Kappa statistic with linear weight was used to estimate the level of agreement between the two systems; Kappa with quadratic weighting was used for comparative purposes. A descriptive analysis was used to indicate the distribution of the quantitative variables. Based on that distribution, the average represented the central trend and the scatter represented the standard deviation. The qualitative variables were defined in terms of percentages by category. A stratified analysis was performed to explore heterogeneity factors, resulting in a linear weighted Kappa for the following categories: Gender, age, nodule size, urban/non-urban origin, accelerated nodule growth, vocal folds paralysis, hard nodule, attached to underlying structures, history of head and neck radiation therapy, and family history of thyroid cancer. All the analyses used STATA 10.1
Results {#Sec7}
=======
The average age was 57 years old. Over 75% of the participants were females and 68.9% came from the urban area; however, there was a remarkable high frequency of risk factors for thyroid cancer. (Table [2](#Tab2){ref-type="table"}) The frequency distribution according to the scales was strikingly different for categories 2-II and 4-IV. The frequency of category II in Bethesda was 65/180 versus 45/180 in TIRADS 2. In contrast, the highest frequency in category 4-IV was 62/180 for TIRADS 4 versus 41/180 for Bethesda IV. (Table [3](#Tab3){ref-type="table"}) The highest concordance was found for categories TIRADS 2-Bethesda II (23.33%). None of the patients classified as TIRADS 2 were rated as Bethesda IV or V. In contrast, 4 subjects classified as Bethesda II were classified as TIRADS 4 (*n* = 2) or V (*n* = 2). Of the 35 patients classified as Bethesda V none were classified as TIRADS 2 or 3, but 3 of the 32 subjects with TIRADS 5 were classified as Bethesda II (*n* = 2) or III (*n* = 1). The weighted Kappa value according to the linear weights was 0.69 (95% CI: 0.59--0.79). The overall Kappa and the Kappa with quadratic weighting were also estimated for comparative purposes. (Table [4](#Tab4){ref-type="table"}) The heterogeneity analysis showed a trend towards a higher weighted kappa value in nodules ≥4 cm in males and individuals aged ≥50 years, with accelerated nodular growth, binding to adjacent structures, vocal folds paralysis, urban origin, and a history of head and neck radiation therapy (Tables [5](#Tab5){ref-type="table"} and [6](#Tab6){ref-type="table"}).Table 2Socio-demographic characteristics and risk factors for thyroid cancerCharacteristicFrequencyMean age57 y (SD:±14y)SexFem: 141 (78.3%)Male: 39 (21.7%)OriginUrban: 124 (68.9%).Non-Urban: 56 (31.1%).Thyroid cancer family backgroundsN (%) No119 (66.1%) Yes61 (33.9%)Accelerated Growth of the Thyroid Nodules No121 (67.2%) Yes59 (32.8%)Head Radiation No163 (90.6%) Yes17 (9.4%)Firm Nodule No94 (51.7%) Yes86 (47.8%)Adjacent structure Attachment No130 (72.2%) Yes50 (27.8%)Vocal Chord Paralysis No130 (72.2%) Yes50 (27.8%)≥4 cm nodule No109 (60.6%) Yes71 (39.4%) Table 3Joint distribution of BETHESDA & TIRADS categoriesDiagnostic categoriesTIRADSTotal2345BETHESDAII4219226523.33%10.56%1.11%1.11%III321141391.67%11.67%7.78%0.56%IV01337410%0.56%18.33%3.89%V001322350%0%7,22%12,22%Total45416232180 Table 4Kappa comparison according to the estimation methodKappaObserved AgreementExpected agreementKappaStandard errorIC 95%ZValue pGlobal65.56%25.27%0.53910.04250.46--0.6212.68\<0.001Weighted (linear weights)87.22%58.76%0.69010.05280.59--0.7913.07\<0.001Estimated (quadratic weights94.63%72.86%0.80210.07310.66--0.9410.97\<0.001 Table 5Stratification according to nodule size, sex, and age in order to assess heterogeneityStrataObserved AgreementExpected AgreementKappaStandard ErrorIC 95%Nodule Size \<4 cm85.63%65.15%0.58760.0670.46-0.72 ≥4 cm89.67%66.26%0.69380.0830.53-0.86Sex Men89.74%58.32%0.75390.1170.53-0.98 Women86.52%59.30%0.66890.0590.55-0.78Age \<50 years old82.49%59.91%0.56320.0920.38-0.74 ≥50 years old89.53%59.18%0.74350.0640.62-0.87 Table 6Heterogeneity assessment by stratifying the variables according to: thyroid cancer family history, accelerated growth of the nodules, firm nodule, underlying structure, vocal chords paralysis, origins, and history of radiationStrataObserved AgreementExpected AgreementKappaStandard Error95% CIThyroid Cancer Family history Yes87.98%60.71%0.6940.0860.53**--**0.86 No86.83%58.81%0.6800.0660.55**--**0.81Accelerated Growth of the nodule Yes90.40%60.12%0.7590.0900.58**--**0.94 No85.67%59.89%0.6430.0650.52**--**0.77Firm Nodule Yes86.82%58.55%0.6820.0760.53**--**0.83 No87.59%60.10%0.6890.0730.55**--**0.83Adjacent Structure Attachment Yes94.00%65.52%0.8260.0960.64**--**1.01 No84.62%59.42%0.6210.0620.50**--**0.74Vocal Chords Paralysis Yes94.67%66.11%0.8430.0960.65**--**1.03 No84.36%58.52%0.6230.0620.50**--**0.74Origin Urban (exclusive)88.70%58.99%0.72450.0650.60**--**0.85 Urban or Rural84.41%58.26%0.62650.08810.45**--**0.8Head and neck radiation therapy Yes96.08%61.48%0.89820.16890.57**--**1.23 No86.30%58.55%0.66950.05570.56**--**0.78
Discussion {#Sec8}
==========
This study evaluated the concordance between the TIRADS and the Bethesda reporting systems on the non-toxic thyroid nodule. The result showed a "good or substantial" concordance and the most frequent consistency was found for categories II and IV. The kappa index measures the level of inter-observer concordance, or as in this particular case, the concordance between two diagnostic methods rather than the "quality" of the observation, so it is not possible to establish the validity of the resulting classifications. This study addresses the level of discrepancy, the report categories, and which categories tend to exhibit a higher frequency of discrepancies between the two methods. When particular types of disagreements are more frequent, this information shall be kept in mind when developing the kappa index \[[@CR18], [@CR19]\]. For this reason, the weighted kappa analysis was used, without neglecting the fact that although using weights is logical and attractive, it introduces a component of subjectivity since assigning weights is subjective and may impact the interpretation of the data when used for a different population --the weights assigned may vary based on the frequency of the disease-. This is evidenced through the variation in the kappa estimates when weighing is used, and depends on the weighing method used. The weighted kappa estimate with linear weights assigned to the categories shows a value of 0.69. The weighted kappa value based on quadratic weights was higher than the overall kappa or the linear weighted kappa (the quadratic weighted kappa value was 0.80). The difference is based on the fact that the linear and quadratic methods are based on the relative separation among the classification categories but the quadratic approach uses square differences, while the linear approach uses absolute values \[[@CR20], [@CR21]\]. Consequently, quadratic weights tend to assign a higher weight to disagreements that were relatively few in this study; when the kappa interpretation is based on quadratic weights, the level of concordance remains unchanged versus the interpretation of the linear weighted kappa; but if analyzed as an absolute value, it is evidently overestimated. Since the kappa value is affected by the prevalence of the characteristic studied, caution is of the essence when generalizing the results of inter-observer comparisons in the presence of varying prevalence. The prevalence of malignancy based on cytology findings (Bethesda V in the matched scale) was reported at 19.4% (35/180); however, using the TIRADS scale (maximum value of 5 in the matched scale), the prevalence of malignancy was 17% (32/180), showing a non-significant difference between the two methods. This is extremely relevant when considering that a prevalence of close to 50% results in a higher kappa value for the same proportion of agreements observed \[[@CR22], [@CR23]\]. Thus, the interpretation of the kappa index requires identifying the value of the marginal frequencies on the table (prevalence observed per observer). Since the difference between the prevalence estimated by both methods is not significant, the conclusion is than that the prevalence of the event did not affect the kappa value reported. When evaluating heterogeneity based on characteristics such as gender, age, size of the nodule, place of origin, accelerated nodular growth, vocal folds paralysis, hard nodule, binding to adjacent structures, a history of head and neck radiation therapy, a family history of thyroid cancer, the trend indicates a stronger concordance (expressed as a weighted kappa value). This is also the case for variables such as nodule size ≥4 cm, male gender, and age ≥50 years. Despite this trend, the study failed to show statistically significant differences. The TIRADS classification attempts to improve the interpretation of the findings of a thyroid nodule by defining categories that in the end are exclusive, although the original classification indicates a risk of malignancy between 5-80% for TIRADS 4, and this fact makes it difficult to clinically define a follow-up and management strategy. Notwithstanding this consideration, from the clinical perspective, in a subject with low probability of having thyroid cancer (and a TIRADS 2 or 3) the US negative predictive value will be greatly enhanced. The best US diagnostic performance is probably with extreme results of the classification (TIRADS 2--3 and TIRADS 5--6 of the original classification). Depending on the clinical probability of malignancy, the US findings may be more or less useful and applicable \[[@CR24]\].
Previous studies have evaluated the diagnostic performance of both US and FNA Biopsy in the initial study of thyroid nodules. A recent study was aimed at developing a diagnostic algorithm using the data reported in the US (in accordance with a scoring system evaluating the risk of malignancy based on several US patterns) and the results of the FNA Biopsy (according to Bethesda). This study showed that classifying an individual in accordance with the presence of different US patterns as low, intermediate or high risk, together with the results of the FNA Biopsy, enables optimal clinical decision-making with regards to treatment strategies \[[@CR25]\]. Along the same lines, other studies classify the risk of malignancy in accordance with the US characteristics and based on such risk, establish the need to perform a FNA Biopsy. The higher the risk of malignancy (according to the US) the greater the need to do the FNA Biopsy, and vice-versa --the lower the risk of malignancy based on the US, the lower the indication for a FNA Biopsy-- \[[@CR26]--[@CR28]\].
Our study showed that the highest concordance was found among both the lowest risk (TIRADS 2 and Bethesda II) and the higher risk categories (TIRADS 4 and Bethesda IV), which is consistent with the previously described trials. This indicates that the US characteristics suggesting a higher or lower risk of malignancy, will be associated with higher or lower probability of malignancy according to the FNA Biopsy report (Bethesda), respectively.
Finally, the interpretation of the results in this study requires acknowledging that over two thirds of the subjects were women. Probably this trend is due to the fact that autoimmune thyroid disease is significantly more frequent in females than in males, so these patients with autoimmune thyroid disease visit the physician more often increasing the probability of detecting the nodules either through palpation or ultrasound; clinically this situation may be defined as a "medical surveillance bias" \[[@CR29], [@CR30]\]. The geographical distribution indicates that most of the patients were from urban areas and those from the rural areas were mostly from municipalities with accessible specialized care. The participants in the study had information about exposure/disease since they had been referred for a study of the thyroid nodule with a probable diagnosis of malignancy. In cross-section studies the participants may be more prone to participate based on their knowledge about exposure and disease and the convenience of their geographical location leading to a higher "selection bias" that in turn could overestimate the frequency of malignancies \[[@CR31], [@CR32]\]. This study highlights the high frequency of factors that have been historically associated with thyroid cancer. Those factors were evaluated with the survey administered to the study subjects that had been previously referred for tests to rule out malignancies, so these participants were more likely to recall past exposures (accurate or vague) potentially leading to a "recall bias" \[[@CR30], [@CR33]\]. Furthermore, since the data collection from the participants was not masked (they had been previously identified as nodular thyroid disease patients screened for malignancies), the interviewer's interest in evaluating the exposure factors could have resulted in an "interviewer bias" \[[@CR34], [@CR35]\].
Conclusions {#Sec9}
===========
The thyroid ultrasound report using the TIRADS criteria has a good concordance with the Bethesda cytology findings using FNA Biopsy. The ultrasound findings of benign pathology are aligned with the cytology results and vice-versa; ultrasound findings of malignancy shall be consistent with cytology-identified malignant disease. The correct interpretation of the two findings helps the clinician to reduce the risk of unnecessary invasive procedures in patients with a low probability of presenting thyroid cancer, while facilitating the identification of patients at higher risk of cancer. There is a need to develop study and monitoring protocols for cases classified as "discordant", particularly when extreme categories are identified (TIRADS 5-Bethesda II, TIRADS 2-Bethesda V).
BIRADS
: Breast Imaging Reporting and Data System
FNA
: Fine-Needle Aspiration
TIRADS
: The Thyroid Imaging Reporting and Data System for US of the thyroid
TSH
: Thyrotrophin
US
: Ultrasound
None.
Funding {#FPar1}
=======
This study was supported by the Internal Medicine Department from The Faculty of medicina of the Universidad del Cauca (Popayán-Colombia), who provided funding to conduct the analysis and prepare the manuscript.
Availability of data and materials {#FPar2}
==================================
We are reluctant to share this data in a publicly accessible repository as this would breech patient confidentiality according to the terms of governance approval for the study: Rules of the Institutional Review Committee of Human Ethics (reference number: 221--011). Universidad del Valle, Valle del Cauca-Colombia.
Authors' contributions {#FPar3}
======================
H V-U, I M-C and J H-Ch were involved in study design, acquisition of data, analysis and interpretation of data and drafting and revising the manuscript. H V-U, and I M-C were involved in data collection and analysis and manuscript drafting. All authors read and approved the final manuscript.
Competing interests {#FPar4}
===================
All authors declare no financial competing interests, nor any other type of conflicts of interest.
Consent for publication {#FPar5}
=======================
All authors gave their approval for the final version to be published and agree to be accountable for this work.
Ethics approval and consent to participate {#FPar6}
==========================================
All personal data were confidential and managed exclusively by the principal investigator, according to the legal standards on the confidentiality of the medical record and adhering to the rules of the Institutional Review Committee of Human Ethics (reference number: 221--011). Universidad del Valle, Valle del Cauca-Colombia.
| {
"pile_set_name": "PubMed Central"
} |
Introduction
============
OA is a common joint problem affecting older adults in Western populations \[[@B1]\]. Individuals with hand OA report significant pain and disability in their everyday life and consider hand OA to be a serious condition \[[@B2]\]. It has been suggested that OA in the thumb joints may represent a different syndrome of OA in comparison with other hand joints due to different causal mechanisms \[[@B3; @B4; @B5]\]. Previous studies of the thumb have only focused on the joints at the base of thumb: the first CMC and the trapezioscaphoid (TS) joints \[[@B6]\]. However, the joints of the thumb (thumb IP, first MCP, first CMC and TS joints) have been found to group together \[[@B7]\]. There is a paucity of research examining ROA of the whole thumb and associations with clinical characteristics of thumb OA, particularly in community-dwelling older adults.
The objectives of this study were to: (i) describe the frequency and patterns of thumb ROA in a cross-sectional study of community-dwelling older adults with hand pain or hand problems; and (ii) investigate associations of thumb pain, clinical features and clinical assessment results with the presence of thumb ROA.
Methods
=======
Study design and sample
-----------------------
The Clinical Assessment Study of the Hand (CAS-HA) is a prospective observational cohort study. All adults aged ≥50 years registered with two general practices in North Staffordshire were invited to take part in a postal survey. Respondents who indicated that they had experienced hand pain or hand problems in the past 12 months and consented to further contact were invited to attend a research clinic. This consisted of a clinical interview, physical examination, plain radiographs, anthropometric measurements and a brief self-complete questionnaire. Full details of the study design and methods have been previously reported \[[@B8]\]. The study was approved by the North Staffordshire Local Research Ethics Committee and all participants provided written informed consent (Project No. 1430).
Data collection
---------------
At the research clinics, participants were assessed by trained occupational therapists and physiotherapists. Thumbs were examined for the presence of deformity and enlargement in the thumb IPs, first MCP and the first CMC joints, nodes in the thumb IP joints and muscle wasting of the thenar eminence. The grind test was conducted to determine whether there was pain in the first CMC joint on movement of the joint under compression \[[@B9]\]. Finkelstein's test was performed to examine whether pain was located in the dorsal aspect of the wrist \[[@B10]\]. The Kapandji test assessed participants' ability to oppose the pulp of the thumb to the distal palmar crease \[[@B10]\]. Thumb extension was measured in degrees using a transparent 360° goniometer. Height and weight were measured at the clinic and used to calculate BMI. Reliability of the clinical features and tests has been established \[[@B11], [@B12]\].
In self-complete questionnaires, participants were asked to report whether they had experienced thumb pain during activity in the previous month, and to indicate on hand drawings the location of any aches or pains lasting a day or longer in the past month.
Radiographs
-----------
Posterior--anterior radiographs of the hands and wrists were taken according to a standardized protocol \[[@B8]\]. A single reader (M.M.) graded 16 joints in each hand: DIPs (*n* = 4), PIPs (*n* = 4), MCPs (*n* = 5), thumb IP, first CMC and TS joints for ROA using the Kellgren and Lawrence (K&L) grading system \[[@B13]\]. ROA for a joint was defined as the presence of K&L ≥ 2, and thumb ROA was defined as the presence of K&L ≥ 2 in one or more joints of the thumb (thumb IP, first MCP, first CMC and TS). Inter-observer reliability was found to be very good (unweighted mean κ 0.79, mean percentage agreement 95%).
Data analysis
-------------
Statistical analysis was performed using SPSS (version 14.0 SPSS, Chicago, IL, USA) and Confidence Interval analysis (version 2.0.0 Statistics with Confidence, second edition, BMJ Books, 2000). The number and percentage of participants with ROA in each joint and joint group were calculated. Combinations of involvement of the thumb joints with other joint groups were examined using a Venn diagram.
Univariable analysis was carried out to determine the magnitude and statistical significance of associations of the presence of ROA in any thumb joint with variables of thumb pain, clinical features and clinical assessment results. Thumb extension was measured as a continuous variable but was dichotomized for analysis using the median value in both thumbs (≥40° and \<40°). The analysis was repeated to compare those with and without ROA in the first CMC joint.
Multivariable logistic regression models were constructed using block-entered data: (i) demographic data (age, gender and BMI); (ii) thumb pain (during activity and as indicated on a hand diagram); (iii) clinical features (thenar muscle wasting, presence of nodes, enlargement or deformity in the thumb); and (iv) clinical assessments (grind test, Finkelstein's test, Kapandji index and thumb extension). Only items with a significance of \<0.1 were kept in the models. The amount of explained variance of the models was estimated by calculating Nagelkerke values and the discriminatory ability of the models was assessed by calculating the area under the receiver operating curves (AUCs).
Results
=======
Following exclusion for inflammatory arthritis and absence of hand X-rays, 592 participants were included in the analyses (62% females, mean age 64 years, 90% right handed).
Pattern and occurrence of ROA
-----------------------------
The first CMC and the thumb IP were the most frequently affected hand joints (45 and 33%, respectively). The joints of the thumb were the most commonly affected joint group: 70% (*n* = 412) of participants compared with 58, 33 and 24% for DIPs, PIPs and MCPs, respectively. Bilateral thumb involvement was present in 71% of the participants. There were 314 participants who had first CMC ROA, 61% of whom had bilateral involvement. The Venn diagram ([Fig. 1](#F1){ref-type="fig"}) shows that isolated thumb ROA was more common than ROA in any other isolated joint group. F[ig]{.smallcaps}. 1A Venn diagram demonstrating the frequency of ROA in different joint groups of the hand. OA = K&L≥2 in one or more joints in each joint group, ^a^DIP = 2nd--5th DIP joints, ^b^PIP = 2nd--5th PIP joints, ^c^MCP = 2nd--5th MCP joints, ^d^Thumb = thumb IP, first MCP, first CMC and FS joints.
Associations with pain, clinical features and tests
---------------------------------------------------
Univariable analysis found statistically significant associations for thumb pain during activity, thumb pain as indicated on a hand diagram, the presence of nodes, deformity or enlargement, muscle wasting of the thenar eminence and a positive grind test with the presence of ROA in the right ([Table 1](#T1){ref-type="table"}) and the left thumb (data not shown). Associations were found to be equally strong when the presence of ROA was based on involvement of the right first CMC ([Table 1](#T1){ref-type="table"}) and left first CMC joints (data not shown). Therefore, multivariable analysis was only undertaken to examine the wider definition of ROA in any of the thumb joints. T[able]{.smallcaps} 1Univariable and multivariable associations between thumb pain, clinical assessments and the presence of ROA in the thumb (right hand)Univariable OR (95% CI) Chi-square significanceMultivariable right thumb[^a^](#TF1){ref-type="table-fn"} ROAOutcomeFrequency, %Right first CMC ROARight thumb[^a^](#TF1){ref-type="table-fn"} ROAβ^b^*P*-value^c^OR (95% CI)Age group, years 50--6035.5Reference 1.0Reference 1.0 60--7037.31.6 (1.1, 2.5)1.9 (1.3, 2.9)0.60.0051.8 (1.2, 2.6) ≤7027.23.9 (2.5, 6.0)5.4 (3.4, 8.6)1.4\< 0.0014.1 (2.5, 6.7)*P* \< 0.001*P* \< 0.001DroppedGender61.81.4 (1.0, 1.9)1.5 (1.0, 2.0)*P* = 0.077*P* = 0.030BMI \<2525.6Reference 1.0 25.0--29.943.80.7 (0.5, 1.1)0.8 (0.6, 1.3)Dropped ≥30.030.60.8 (0.5, 1.3)1.1 (0.7, 1.7)*P* = 0.360*P* = 0.354Thumb pain during activity in the past month53.22.1 (1.5, 2.9)2.23 (1.6, 3.2)0.60.0011.8 (1.3, 2.6)*P* \< 0.001*P* \< 0.001Thumb pain in the past month as indicated on a hand diagram51.51.5 (1.0, 2.1)1.4 (1.0, 2.0)Dropped*P* = 0.018*P* = 0.023The presence of thenar muscle wasting20.83.0 (2.0, 4.6)3.1 (2.0, 5.0)0.60.0161.9 (1.1, 3.1)*P* \< 0.001*P* \< 0.001The presence of nodes, deformity or enlargement72.62.2 (1.5, 3.3)3.1 (2.1, 4.5)0.7\<0.0012.1 (1.4, 3.1)*P* \< 0.001*P* \< 0.001A positive grind test12.21.8 (1.1, 2.9)1.7 (1.0, 2.9)Dropped*P* = 0.015*P* = 0.027A positive Finkelstein's test16.81.4 (0.9, 2.2)1.2 (0.8, 1.9)Dropped*P* = 0.092*P* = 0.213Inability to achieve position 10 of the Kapandji index33.31.0 (0.7, 1.4)1.0 (0.7, 1.4)Dropped*P* = 0.511*P* = 0.535Thumb extension \<40 degrees55.31.3 (0.9, 1.8)1.2 (0.8, 1.6)Dropped*P* = 0.120*P* = 0.413[^1]
Multivariable regression analysis found that right thumb ROA was most strongly associated with a combination of older age; thumb pain during activity; thenar muscle wasting; and the presence of nodes, enlargement or deformity ([Table 1](#T1){ref-type="table"}). Gender, BMI and all four clinical assessments did not contribute significantly to the model. The amount of explained variance of the final model was 20% (Nagelkerke value = 0.203) and the AUC was 0.72 (95% CI 0.68, 0.76). The model obtained for the left thumb (data not shown) was identical to the model for right, except thumb pain as indicated on a hand diagram was included instead of thumb pain during activity. The amount of explained variance of the final model for the left thumb was 26% (Nagelkerke value = 0.261) and the AUC was 0.77 (95% CI 0.73, 0.81).
Discussion
==========
In this study, we examined the occurrence and patterns of involvement of ROA in the joints of the thumb in a community-dwelling population of older adults, and investigated associations between thumb pain, positive results of the clinical assessment findings and the presence of thumb ROA.
A high frequency of ROA was seen in the first CMC joint, which is consistent with the previous findings \[[@B14; @B15; @B16]\], but the frequent involvement of the thumb IP joint has only been described in a few studies \[[@B17], [@B18]\]. The lack of information about the involvement of this joint is partly due to its inconsistent inclusion and classification in studies of hand ROA \[[@B6]\]. When the thumb joints (thumb IP, first MCP, first CMC and TS) were assessed collectively they were found to be the most frequently affected joint group followed by the DIPs. This is inconsistent with previous research undertaken in a Dutch population where the DIP joints, including the thumb IP, were more frequently affected than the base of the thumb \[[@B16]\]. However, when data from our study were reanalysed using the same classification as the Dutch study, the same patterns were found. This indicates that the classification of the thumb IP joint can make substantial differences when examining the frequency of involvement in different joint groups within the hand.
Similar associations for thumb pain, thenar muscle wasting, and the presence of nodes, deformity or enlargement were found when defining thumb ROA as involvement of the first CMC joint, as when analysing involvement of any thumb joint. This could suggest that nothing is gained by including the other joints of the thumb. However, the group of participants with any thumb joint affected with ROA included an additional 98 participants who did not have first CMC involvement. If only the first CMC joints were examined, these individuals would not have been identified as having thumb ROA, even though they experienced similar levels of pain and disability.
Multivariable analysis found that the combination of age, thumb pain, muscle wasting, and the presence of nodes, deformity or enlargement best determined the presence of thumb ROA. None of the clinical assessments contributed to these models. Although a univariable association was found between thumb ROA and a positive grind test, in combination with other factors this test was not significantly associated with the presence of ROA. The frequency of a positive grind test in this sample was low. It has been suggested that this test may not be positive in milder cases, and therefore when combined with other clinical features of OA the grind test could add little information to the models \[[@B19]\].
Good discrimination between individuals with or without thumb ROA was found for the multivariable models. This study has shown that in combination, age, thumb pain, thenar muscle wasting, and the presence of nodes, deformity and enlargement are able to identify most individuals with thumb ROA. This could be useful in large population studies where radiography is not feasible. However, performance of these models could potentially be further improved, as several important items, such as severity of thumb pain and pain on palpation were not included in our study. Palpation of the first CMC joint may also indicate the presence of crepitus in the joint capsule, another clinical sign of OA \[[@B20]\].
Few studies have examined the thumb as a complex and the CAS-HA study provided a rich source of self-report, radiographic, physical examination and clinical assessment data to investigate associations of clinical signs and symptoms with the presence of thumb ROA. One limitation is that missing data may have led to bias as data were mainly missing from participants who were unable to adopt the starting position or complete a test due to the presence of pain. However, for each test the amount of missing data was small (\<3.6%). For the multivariable analysis, a complete case approach was taken, which meant that up to 10.2% of participants with any missing data were not included in these analyses. Finally, these findings need further testing in other populations to test their external validity.
In summary, this research has shown that the thumb IP and first CMC joints were frequently affected with ROA. Estimates of prevalence of thumb ROA may be underestimated if the thumb IP joints are not scored. ROA of both the left and right thumbs were most strongly associated with a combination of older age, thumb pain, thenar muscle wasting and the presence of nodes, deformity and enlargement. This model should be tested in other populations and future studies could improve on this model by including additional relevant features and clinical assessments.
The authors would like to acknowledge the contributions of Prof. Peter Croft, Prof. Elaine Hay, Dr George Peat, Dr Laurence Wood, Dr Elaine Thomas, June Handy, Charlotte Clements, Catherine Tyson, Prof. Chris Buckland-Wright and Prof. Iain McCall for aspects of the conception and design of the study and the acquisition of data. Dr Jacqueline Saklatvala, Carole Jackson, Julia Myatt, Janet Wisher, Sandra Yates, Krystina Wallbank and Jean Bamford from the Department of Radiography, Haywood Hospital have contributed specifically to the acquisition of radiographic data. The authors would also like to thank the administrative and health informatics staff at the Arthritis Research UK Primary Care Centre, Keele University, and the staff and patients of the participating general practices.
*Funding:* This work was supported by a Programme Grant awarded by the Medical Research Council, UK (Grant Code: G9900220) and by support for science funding secured by North Staffordshire Primary Care Research Consortium for National Health Service support costs. K.D. was supported by a grant from Arthritis Research UK. Funding to pay the Open Access publication charges for this article was provided by Arthritis Research UK Primary Care Centre.
*Disclosure statement*: The authors have declared no conflicts of interest.
[^1]: ^a^Thumb ROA is defined as K&L grade ≥2 in any thumb joint (thumb IP, first MCP, first CMC and TS), ^b^β is parameter estimate, ^c^*P*-value = significance level, missing data were \<10.2%.
| {
"pile_set_name": "PubMed Central"
} |
1. Introduction {#sec1-sensors-18-01336}
===============
According to a 2014 United States Census Bureau report, the population aged 65 and over has been projected to grow from 43.1 million in 2012 to 83.7 million in 2050 \[[@B1-sensors-18-01336]\]. One of the distinctive health states related to the aging process is frailty \[[@B2-sensors-18-01336]\]. The prevalence of frailty in the ambulatory population is about 15% and the prevalence of pre-frailty is about 45% \[[@B3-sensors-18-01336]\]. Frailty places older adults at risk for dramatic changes in physical and mental well-being following challenges to their health, such as an infection, injury, or medication interactions \[[@B4-sensors-18-01336]\]. Frailty is an independent predictor of adverse outcomes, including falls, delirium, hospitalization, and mortality \[[@B5-sensors-18-01336]\]. Identifying patients at risk for frailty (pre-frail adults) would enable healthcare providers to intervene from an early stage so as to mitigate some of the potential adverse sequelae \[[@B6-sensors-18-01336]\].
Although there is no consensus on the definition of frailty, recent efforts have focused on a standardization of the definition so as to enhance its application in clinical care \[[@B7-sensors-18-01336]\]. Geriatricians used to say, "I know it when I see it, but what I see may not be the same as what everyone else sees" \[[@B8-sensors-18-01336]\]. The definition of frailty has evolved from the stereotypical description of a "thin, stooped, slow octogenarian" person \[[@B9-sensors-18-01336]\]. Approaches introduced by Fried \[[@B10-sensors-18-01336]\] and Rockwood \[[@B11-sensors-18-01336]\] have had the strongest empirical and conceptual support.
Fried and colleagues developed a frailty phenotype theory based on mutually exacerbating cycles of negative energy balance, sarcopenia, and diminished strength and tolerance for exertion in the community dwelling geriatric population \[[@B7-sensors-18-01336]\]. In this theory, the frailty phenotype is associated with declining energy and reserve \[[@B10-sensors-18-01336]\]. Fried proposed five core clinical criteria for the impairment that is underlying frailty, namely, shrinking, exhaustion, inactivity, slowness, and weakness \[[@B10-sensors-18-01336]\]. Older adults are classified as frail, pre-frail, or robust. An individual meeting the threshold of impairment for three of these criteria are classified as frail. The individual meeting criteria for one or two components is classified as pre-frail. Those meeting none of the impairment criteria are classified as non-frail or robust. Individuals meeting the pre-frail criteria can potentially benefit more from clinical intervention \[[@B12-sensors-18-01336]\] compared with those meeting the frail and non-frail (robust) criteria \[[@B13-sensors-18-01336]\]. Furthermore, a greater variety of interventions are potentially available to pre-frail older adults, who require less supervision than frail older adults \[[@B14-sensors-18-01336]\].
The Fried frailty phenotype has several diagnostic limitations. The Fried approach has been described as impractical in a busy clinic settings \[[@B15-sensors-18-01336]\], not designed for inpatient or bed-bound older patients \[[@B16-sensors-18-01336]\], not sensitive to subtle physiological changes \[[@B17-sensors-18-01336]\], and as failing to account for the important domain of cognition function \[[@B18-sensors-18-01336]\]. Additionally, the approach's reliance on questionnaires to identify weight loss, exhaustion, and energy expenditure suffers from participant bias \[[@B19-sensors-18-01336],[@B20-sensors-18-01336],[@B21-sensors-18-01336],[@B22-sensors-18-01336]\].
To overcome limitations of the Fried frailty phenotype, researchers have proposed wearable sensors as an alternative to assessing the frailty phenotype \[[@B15-sensors-18-01336],[@B23-sensors-18-01336],[@B24-sensors-18-01336],[@B25-sensors-18-01336]\]. These wearable sensors can address the challenges in measuring frailty, such as feasibility, practicality, ease of use, accessibility, reproducibility, and reliability, without hindering daily activity in the outpatient or inpatient settings \[[@B15-sensors-18-01336],[@B23-sensors-18-01336],[@B24-sensors-18-01336]\]. Previous studies using a wrist sensor, by Lee et al., have demonstrated that a 20-s upper extremity test is capable of predicting frailty in the outpatient setting \[[@B26-sensors-18-01336]\] and in community dwelling settings \[[@B27-sensors-18-01336]\]. Schwenk et al. have shown that multiple sensor-based physical activity monitors, which measure posture (walking, standing, sitting, lying, and postural transition from sit-to-stand and stand-to-sit) and gait parameters (stride length, gait speed, gait velocity, and cadence), are capable of discriminating between non-frail, pre-frail, and frail patients \[[@B24-sensors-18-01336]\]. Other studies have shown that sensor-derived activity levels (sedentary behaviors, light and moderate-to-vigorous activity) have a high correlation with frailty status \[[@B28-sensors-18-01336]\] and are capable of discriminating between different frailty statuses \[[@B29-sensors-18-01336],[@B30-sensors-18-01336],[@B31-sensors-18-01336],[@B32-sensors-18-01336]\]. They found that an increase in the sedentary behavior and a decrease in the high intensity activity, such as moderate-to-vigorous activity, is a strong predictor of frailty progression. Interestingly, Theou et al. showed that a single parameter, the number of steps, which is derived from a wearable sensor is significantly correlated with the progression of frailty \[[@B31-sensors-18-01336]\]. Furthermore, studies of sensor-based in-home sleep monitors have found an association between sleep disruptions and \[[@B33-sensors-18-01336]\] the existence of frailty \[[@B34-sensors-18-01336]\].
While few studies proposed daily step measurement for in-place monitoring frailty status, to our knowledge, no prior studies have examined fine-grain characteristics of daily physical activities, such as activity behavior (e.g., sedentary), activity postures (e.g., sitting, standing, lying, and walking), and walking characteristics (e.g., number of taken steps), which are measured by a single senor into a cohesive model. Such models are potentially valuable because they would provide a clinical and technical validation of these sensor-derived parameters, and serve as a basis for future studies developing predictive models of change between frailty categories. In particular, there are very few studies that are enabled to identify pre-frailty using wearable-based activity monitoring. Pre-frailty is considered as the early stage of frailty \[[@B35-sensors-18-01336],[@B36-sensors-18-01336],[@B37-sensors-18-01336]\]. While several studies have suggested that frailty is not an irreversible process, it has been hypothesized that the early detection of a pre-frail status may provide a window of opportunity for timely preventive or therapeutic interventions, which may delay the progression of frailty and even reverse it \[[@B35-sensors-18-01336],[@B36-sensors-18-01336],[@B37-sensors-18-01336]\]. Thus, the early detection of pre-frailty may provide a unique opportunity to provide a timely intervention and is desperately needed. Therefore, the purpose of this study is to examine the ability of a practical wearable platform (a pendant accelerometer), to remotely monitor the frailty stages using daily activity monitoring, with an emphasis on distinguishing pre-frailty. Specifically, our first aim was to determine which sensor-derived parameters---including walking characteristics (e.g., daily number of taken steps); activity patterns, including postures (i.e., sitting, standing, and lying) and walking durations; activity patterns, including sedentary, light, and moderate-to-vigorous activities; and sleep parameters, including total sleep time and sleep efficiency---are capable of discriminating between the three frailty categories. The second aim was to identify the most significant independent parameters in order to discriminate the pre-frail from other groups. Finally, our third aim was to build a composite model that would have a promising performance so as to discriminate the pre-frail stage from non-frail and frail stages.
2. Materials and Methods {#sec2-sensors-18-01336}
========================
2.1. Participants and Assessment {#sec2dot1-sensors-18-01336}
--------------------------------
### 2.1.1. Participants Recruitment {#sec2dot1dot1-sensors-18-01336}
We recruited ambulatory older adults that were ≥60 years of age, who were able to walk 15 feet (\~4.5 m) independently, with or without aid. Participants were enrolled from outpatient clinics or community dwelling settings. Exclusion criteria were severe cognitive impairment (a Mini-Mental State Examination \[MMSE\] score ≤16) \[[@B38-sensors-18-01336]\] and those unable/unwilling to consent. Participants who met the eligibility criteria signed written consent form. This study was approved by the local institutional review boards.
### 2.1.2. Demographic and Clinical Characteristics {#sec2dot1dot2-sensors-18-01336}
Trained clinical coordinators collected patient demographic and clinical characteristics. The measures were history of falls, height, weight, and fear of falling, which was assessed by the Fall Efficacy Scale-International (FES-I) \[[@B39-sensors-18-01336]\]. Participants' depression scale was measured using the Center for Epidemiologic Studies Depression Scale (CES-D) \[[@B40-sensors-18-01336]\].
### 2.1.3. Frailty Assessment {#sec2dot1dot3-sensors-18-01336}
We used the Fried frailty phenotype assessment to stratify the participants into three groups, namely, non-frail, pre-frail, and frail \[[@B10-sensors-18-01336]\]. The Fried frailty assessment consisted of five phenotypes, namely, shrinking (losing more than 10 lb. in prior year unintentionally), exhaustion (self-reported questionnaire), inactivity (self-reported questionnaire), slowness (prolonged performance during 15-feet walk test), and weakness (decreased grip strength) \[[@B10-sensors-18-01336]\]. If the participants' performance placed them in the lowest quartile for a phenotype, they received one point for that phenotype. For the final score, the sum of all of the one points (*SUM*) was calculated and the subject was classified into one of three groups:$$Frailty~Status = ~\left\{ \begin{matrix}
{nonFrail/Robust:~} & {SUM = 0} \\
{preFrail:} & {0~ < ~SUM~ \leq 2} \\
{Frail:} & {SUM \geq 3} \\
\end{matrix} \right.$$
2.2. Sensor Based Assessment {#sec2dot2-sensors-18-01336}
----------------------------
We used a pendant sensor (PAMSys™, BioSensics LLC, Watertown, MA, USA), which was placed at the sternum ([Figure 1](#sensors-18-01336-f001){ref-type="fig"}). The participants were instructed to keep the sensor on for 48 h and then return it to the center, through either a paid envelope or collection by the study coordinators. The PAMSys had three dimensional accelerations that recorded the gravity and inertial accelerations, with a sampling frequency of 50 Hz. The sensor had a built-in memory that allowed for the saving of data and also downloading it to the computer via the company software that was provided. We used PAMWare™ software (BioSensics, Watertown, MA, USA) to download, calibrate, and normalize (to gravity or g) the data. All of the physical activity and sleep parameters were extracted from the pendant sensor. However, two different validated algorithms were used to extract the physical activities and sleep parameters using chest acceleration, as described in our previous studies \[[@B41-sensors-18-01336],[@B42-sensors-18-01336],[@B43-sensors-18-01336],[@B44-sensors-18-01336]\].
### 2.2.1. Physical Activity Behavior Parameters {#sec2dot2dot1-sensors-18-01336}
For the purpose of this study, the physical activity behavior parameters that were considered were sedentary behavior (Sed), light activity (Lgt), and moderate-to-vigorous activity (MtV). Sedentary behavior was defined as an activity with less than 1.5 metabolic equivalent (MET), such as sitting or lying \[[@B45-sensors-18-01336],[@B46-sensors-18-01336]\]. Light activity was defined as an activity between ≥1.5 MET and MET \<3.0, such as hanging out the washing, ironing and dusting, and working at a standing workstation \[[@B46-sensors-18-01336]\]. Moderate-to-vigorous activity referred to an activity demanding ≥3.0 MET, such as brisk walking, recreational activities, climbing stairs, etc. \[[@B46-sensors-18-01336]\].
To measure the physical activity levels in each category, we calculated the mean amplitude deviation (MAD) \[[@B47-sensors-18-01336]\]. Before we calculated the MAD, several steps were taken. Firstly, we preprocessed the data in order to remove the high frequency activities that had not originated from human body \[[@B41-sensors-18-01336]\]. We used a wavelet filter bank (Daubechies \[[@B48-sensors-18-01336]\]) with a cut-off at 12.5 Hz. The wavelet filter was used, as it has been shown to keep the morphology of signal better than the other filters \[[@B49-sensors-18-01336]\]. Then, we calculated the following:$$r_{i} = \sqrt{x_{i}^{2} + y_{i}^{2} + z_{i}^{2}}$$
Here, $r_{i}$ is the norm acceleration containing the static and dynamic component of the body accelerations for each sample (*i*). The $\left( {x_{i},y_{i},z_{i}} \right)$ are the three-dimensional accelerations. For each 6-s epoch, the average $r_{i}$ ($R_{ave}$) of the 300 samples (=6 s × 50 Hz) was calculated as follows:$$R_{ave} = \frac{1}{N}\sum_{i = 1}^{N = 6~s}r_{i}$$
The MAD value for each epoch was calculated as the absolute sum of distance from $R_{ave}$ as follows:$${MAD} = \frac{1}{N}\sum_{i = 1}^{N = 300}\left| {r_{i} - R_{ave}} \right|$$
The MADs for all of the possible epochs were calculated. The unit of MAD is in the milligravity, where 1 g is equal to 1000 mg. We used three cut-points to classify activity level into sedentary (MAD \< 20), light (20 ≤ MAD \< 90) and moderate-to-vigorous (MtV: MAD ≥ 90). This method had, on average, a very high sensitivity and specificity of 98% and 96%, respectively, in order to detect the physical activity levels \[[@B47-sensors-18-01336]\].
### 2.2.2. Non-Wear Time and Valid Day of Monitoring {#sec2dot2dot2-sensors-18-01336}
We excluded the intervals when participants did not wear the sensors. These non-wear periods occurred during aquatic activities, such as bathing, or because the participant forgot to wear the sensor. We used a method that was validated in older adults \[[@B50-sensors-18-01336]\], which defined non-wear periods as ≥90 min with no MAD (allowing for 2 interrupted minutes with MAD of \<20).
A valid day of monitoring was defined as ≥8 hours of wear \[[@B45-sensors-18-01336],[@B50-sensors-18-01336]\]. We used the valid day with the wear time annotation to report the physical activity parameters. The average of the activity parameters over the 2 days were reported to have reached the highest inter-class correlation (ICC) \[[@B51-sensors-18-01336]\].
For each valid day of monitoring, we calculated the following parameters:Total activity: the sum of the all of the specific activity (Sed, Lgt, and MtV).Percentage activity: the total activity duration of a specific activity, divided by the total duration of wear time, excluding the nocturnal time in bed.Median activity: the 50th percentile of the bout of the specific activity.Health and Human Services (HHS) guideline, %: The percentage of participants who met the U.S. Department of HSS recommendations that an adult should have at least 300 min of moderate-to-vigorous activity per week \[[@B52-sensors-18-01336]\]. To calculate this parameter, we estimated those who had at least 42 min (300 min/7 days = \~42 min) of moderate-to-vigorous physical activities per day.
The bout of activity was the consecutive, continuous interval of an activity without any interruption, such as Sed, Lgt, or MtV.
### 2.2.3. Physical Activity Pattern and Stepping Parameters {#sec2dot2dot3-sensors-18-01336}
The postural parameters that were calculated from the PAMSys sensor's raw data included lying, sitting, standing, walking, and the number of steps. The algorithm first detected the episodes of walking, which was three consecutive steps with less than specific time intervals \[[@B42-sensors-18-01336],[@B43-sensors-18-01336],[@B44-sensors-18-01336],[@B53-sensors-18-01336]\]. The steps were determined by the peaks in vertical acceleration, where the signal passed through a wavelet-based band pass filter, with absolute values greater than a certain threshold. Standing, sitting, and lying were considered non-walking intervals. Lying intervals were identified when the vertical acceleration was close to zero gravity. In the other words, during the lying intervals, the vertical vector was at a right angle with the frontal plain. Sitting and standing were identified through the pattern changes in frontal-vertical vectors. The sensitivity (87% to 99%) and specificity (87% to 99%) of the algorithm was reported previously \[[@B43-sensors-18-01336]\].
The postural data was reported for each 24 h period and the average was calculated for the final outcomes, as follows:Posture, %: the duration of each posture (lying, sitting, standing, walking) in 24 h.Total steps: the total number of steps per day.Longest unbroken posture, s: the maximum duration of an unbroken bout for each posture.Median posture, s: the median duration of a bout for each posture.Longest unbroken stepping bout: the number of steps during the longest bout of stepping without interruption.Median stepping bout: the number of steps in the median bout of stepping without interruption.
### 2.2.4. Sleep Quantity Parameters {#sec2dot2dot4-sensors-18-01336}
Using the physical activity algorithm \[[@B41-sensors-18-01336],[@B44-sensors-18-01336]\], the start and end of sleep during night time were recorded in order to estimate the time spent in the bed and out of the bed. The sleep algorithm was applied only during the time in the bed. The method for extracting sleep parameters of interest, using a chest accelerometer, was described in detail in our previous study \[[@B41-sensors-18-01336]\]. In summary, firstly, the acceleration data passed through a band pass filter, then a vector magnitude/norm of acceleration was built and a minute wise signal was calculated. Next, a feature vector, which consisted of an activity intensity in the moment and a standard deviation of the activities as well as any sleep position changes, was built for each minute and fed to a model. Finally, the model estimated the sleep/wake conditions. From the sleep/wake signal, the sleep quantity parameters were extracted as follows:Time in bed (TiB), hours: the total duration of a participant's time in bed.Total sleep time (TST), hours: the total duration of nocturnal sleep.Sleep onset latency (SOL), min: the total interval of the time to fall asleep, from the beginning of TiB.Wake after sleep onset (WASO), min: the total duration of the time awake, after sleep onset until sleep offset.Sleep efficiency (SE), %: the percentage of TST to onset of sleep to last offset of sleep.Supine, %: the total duration of supine during TiB.Prone, %: the total duration of prone during TiB.Sides, %: the total duration of side lying (left or right) during TiB.
2.3. Statistical Analysis {#sec2dot3-sensors-18-01336}
-------------------------
We used the Fisher exact test to evaluate the differences between the categorical variables (demographic or clinical characteristics). We used the ANCOVA with the Tukey LSD post hoc test, which was performed on the SPSS (IBM, V24.0.0), in order to test the significance level between the three groups of non-frail, pre-frail, and frail. We also estimated the Cohen's d effect size (*d*), where *d* ≈ 0.2, 0.5, and 0.8 were considered as small, medium, and large, respectively.
We selected independent variables in two of the steps \[[@B54-sensors-18-01336]\]. In the first step (filter method \[[@B54-sensors-18-01336]\]), we chose parameters from the sensor-derived parameters that had a *p*-value less than 0.05 and a *d* ≥ 0.4. In the second step (embedded method \[[@B54-sensors-18-01336]\]), these independent predictors were fed to a model in order to discriminate the pre-frail from the two other groups (non-frail and frail). The Receiver Operating Characteristic (ROC) curve, performance (sensitivity, specificity, and accuracy), and area under the curve (AUC), were calculated based on the one-vs-rest method \[[@B55-sensors-18-01336]\]. Of the independent predictors, those with an AUC greater than 0.7 were used to develop discrimination models. To select the independent predictors, the whole dataset was used \[[@B56-sensors-18-01336],[@B57-sensors-18-01336]\].
We developed four models as follows: (1) the step model: using step parameters, such as the total number of steps; (2) the physical activity pattern (PAP) model: PAP parameters such as the total walking and postures duration; (3) the physical activity behavior (PAB) model: PAB parameters, such as sedentary; and (4) the combined model: all of the parameters such as total number of steps, total walking, and sedentary. To train and test the model, we used a k-fold cross validation (k = 5). In this method, the dataset was randomly partitioned into five subsamples \[[@B58-sensors-18-01336],[@B59-sensors-18-01336]\]. Four partitions were used to train each model and one partition, which was not used for training, was used for validating each model. This step performed for five times. The average and standard deviation of the performance parameters for the validation phase were reported. The performance parameters that were measured for each model were sensitivity, specificity, accuracy, and the AUC \[[@B59-sensors-18-01336]\].
3. Results {#sec3-sensors-18-01336}
==========
3.1. Demographic and Clinical Characteristics {#sec3dot1-sensors-18-01336}
---------------------------------------------
Originally, 163 participants had consented to participant in this study. Data from 10 participants was excluded because of low wear-time (*n* = 3), less than two days of recording (*n* = 5), and forgetting to put on the sensor (*n* = 2). The remaining 153 participants (75 ± 10 years and 79% female) were included in the study, where 42 (27%) were considered as non-frail, 78 (51%) pre-frail, and 33 (22%) frail ([Table 1](#sensors-18-01336-t001){ref-type="table"}). In the progression of the frailty status among the participants, we observed a trend in several demographic characteristics, such as BMI, depression, fear of falling, cognitive dysfunction, number of the prescribed medication, and number of comorbidities. The pre-frail group had a significantly higher BMI than the non-frail group (*p*-value ≤ 0.001). Depression in the frail group was significantly higher than in the pre-frail group (*p*-value = 0.002). A fear of falling in the pre-frail group was lower than that in the frail group (*p*-value = 0.006) ([Table 2](#sensors-18-01336-t002){ref-type="table"}).
3.2. Sleep Quantity Parameters {#sec3dot2-sensors-18-01336}
------------------------------
In the sleep parameters, we observed a trend of reduction in TiB and TST, and a trend of increase in SOL in the progression of frailty. Specifically, TiB (*p*-value = 0.010, *d* = 0.50) and TST (*p*-value = 0.027, *d* = 0.45) differed significantly in non-frail and pre-frail groups ([Table 2](#sensors-18-01336-t002){ref-type="table"}). Interestingly, the sleep side position (*p*-value = 0.001, *d* = 0.65) was significantly different in the pre-frail and frail group. No sleep quantity parameters were capable of discriminating between the three groups of frailty statuses.
3.3. Physical Activity Pattern Parameters {#sec3dot3-sensors-18-01336}
-----------------------------------------
In the physical activity pattern parameters, we observed a trend of reduction in standing and walking, and a trend of increase in the lying duration ([Table 2](#sensors-18-01336-t002){ref-type="table"}). Specifically, the standing duration was significantly different between the pre-frail and non-frail (*p*-value = 0.003, *d* = 0.57). The total duration of walking, longest unbroken walking bout, and the median walking bout were capable of discriminating between the comparisons group of groups. When each parameter was fed into the model in order to identify the pre-frail group, only the total walking duration and longest unbroken walking bout had an AUC of \>0.7, while the median walking bout showed an AUC of \<0.7, and specificity, less than 50% ([Table 3](#sensors-18-01336-t003){ref-type="table"}).
3.4. Stepping Parameters {#sec3dot4-sensors-18-01336}
------------------------
All of the stepping parameters showed a trend of decline by frailty progression ([Table 2](#sensors-18-01336-t002){ref-type="table"}). The total number of steps and the longest unbroken stepping bout were significantly different between the non-frail vs. pre-frail, and the pre-frail vs. frail groups, and they showed a significant independent predictor with an AUC \> 0.7 for pre-frail status ([Table 3](#sensors-18-01336-t003){ref-type="table"}). The median stepping bout was not significant between the groups and was also rejected when it was independently fed to the model, for an AUC \< 0.7 ([Table 3](#sensors-18-01336-t003){ref-type="table"}).
3.5. Physical Activity Behavior Parameters {#sec3dot5-sensors-18-01336}
------------------------------------------
In the overall physical activity behavior parameters, we observed a reduction trend (from non-frail to frail) in the duration of light activity and moderate-to-vigorous activity, and a trend of increase in sedentary behavior ([Table 2](#sensors-18-01336-t002){ref-type="table"}). Specifically, the percentage of sedentary behavior (*p*-value \< 0.001, *d* = 0.98), duration of light activity (*p*-value = 0.001, *d* = 0.62), percentage of light activity (*p*-value \< 0.001, *d* = 0.79), and percentage of MtV activity (*p*-value \< 0.001, *d* = 1.13), differed significantly between the non-frail and pre-frail groups. Among the parameters, the total duration of sedentary behavior, median light activity, and total duration of MtV, differed significantly between the groups. The median light activity had a very low specificity and AUC; therefore, it was not considered for building the model so as to discriminate the pre-frail from other groups. However, the total sedentary and MtV was used in building this model. Also, we observed a trend of reduction in the percentage of participants in each group who met the physical activity recommendation from the HHS. The odds of meeting the HHS guidelines in the non-frail and pre-frail groups varied significantly (*p*-value \< 0.001)
3.6. Performance of Models for Discriminating Pre-Frail Status {#sec3dot6-sensors-18-01336}
--------------------------------------------------------------
Among the non-combined models, the stepping model and the physical activity pattern (PAP) model had the same level of high sensitivity (88.6%), while the specificity of physical activity behavior (PAB) was the highest (77.9%). The accuracy of PAB and PAP were slightly (less than 2%) higher than the stepping model ([Table 4](#sensors-18-01336-t004){ref-type="table"}). Overall, the four models showed a large AUC of ≥0.8 ([Table 4](#sensors-18-01336-t004){ref-type="table"}). The combined model was a composite of all of the sensory parameters that were independently predictive of pre-frail status (see [Table 3](#sensors-18-01336-t003){ref-type="table"}). This combined model had the highest sensitivity, specificity, accuracy, and AUC (91.8%, 81.4%, 84.7%, and 0.88, respectively) for identifying the pre-frail status ([Table 4](#sensors-18-01336-t004){ref-type="table"}).
4. Discussion {#sec4-sensors-18-01336}
=============
This study examined the association between the measurable physical activities, from a pendant accelerometer-based sensor, and the different frailty stages. Prior frailty studies, which had used sensor-derived parameters, were often based on the supervised assessment of motor performances (e.g., gait assessment, balance, Timed Up & Go, etc. \[[@B24-sensors-18-01336],[@B60-sensors-18-01336],[@B61-sensors-18-01336],[@B62-sensors-18-01336]\]), which were unsuitable for the remote monitoring of the frailty stages. There were few studies that attempted to determine the frailty stages based on activity monitoring \[[@B32-sensors-18-01336]\]. However, to our knowledge, none of the prior studies took into account both the daytime and nighttime (e.g., sleep) activities in order to distinguish the pre-frailty stage. The current study used and determined the most sensitive and independent metrics that were measurable from a single pendant sensor, including the physical activity pattern/stepping, physical activity behaviors, and sleep parameters, in order to discriminate among the frailty categories in community-dwelling older adults. Furthermore, we examined which activity-derived parameters were the most sensitive in order to distinguish pre-frailty, which was known as a potentially reversible frailty stage \[[@B35-sensors-18-01336],[@B36-sensors-18-01336],[@B37-sensors-18-01336]\]. From a model construction standpoint, we not only used uni-variate, multi-variable analysis, and embedded feature selections, but we also applied a decision trees model, which had been shown to be a more robust model than conventional multi-variable models (e.g., the linear regression of logistic regression model) \[[@B62-sensors-18-01336],[@B63-sensors-18-01336]\]. Together, the proposed approach allowed for distinguishing the pre-frailty stage from the other stages during activities of daily living, via a simple and practical wearable platform. More specifically, the results suggested that the most sensitive descriptors of the pre-frailty stage were total sedentary duration, total moderate-to-vigorous activity duration, total walking duration as a percentage of 24 h activities, longest unbroken walking bout, total daily steps, and longest unbroken steps.
While several instruments were proposed for assessing frailty (e.g., the frailty index, proposed by Rockwood et al. \[[@B11-sensors-18-01336]\], and the frailty phenotypes, proposed by Fried et al. \[[@B10-sensors-18-01336]\]), they were unsuitable for in-place and remote monitoring of the frailty stages, because they often required a supervised administration of the test, relied on subjective or semi-objective data obtained from self-reported inactivity and/or availability of patient health records, and were often insensitive to change over time \[[@B26-sensors-18-01336],[@B64-sensors-18-01336]\]. The proposed model/platform and its practical form factor (using a pendant instead of securing a sensor to the chest) might have addressed these limitations and thus could have facilitated the development of a telehealth platform, based on wearables and activity monitoring. Most importantly, the results of this study suggested that a single pendant sensor could distinguish the pre-frail stage from other frailty stages. In addition, we previously demonstrated that two days of activity monitoring would be enough to determine the frailty stages \[[@B51-sensors-18-01336]\]. This in turn, might have allowed for the tracking of changes in the frailty stages, with a relatively high time resolution (48 h), which would have provided a window of opportunity for timely preventive or therapeutic interventions that might have delayed the progression of frailty and identifying modifiable factors. This might have contributed to the deteriorating resilience (e.g., medication adverse effect, depression, immobility, etc.).
Our results were in agreement with previous studies, which suggested that total number of steps, amount of sedentary behaviors, and moderate-to-vigorous activity were associated with the progression of the frailty stages \[[@B31-sensors-18-01336],[@B32-sensors-18-01336],[@B60-sensors-18-01336],[@B61-sensors-18-01336]\]. However, to our knowledge, this was the first study that integrated a greater variety of sensor-based measurable physical activity metrics, including steps, sleep, activity pattern, and activity behavior, into a cohesive model in order to determine the independent descriptors of the frailty stages. In addition, our study was able to demonstrate which activity related parameters, which were measurable by a pendant sensor, allowed for determining the pre-frailty stage. Our results suggested that in order to more accurately discriminate between the pre-frail and non-frail stages, a more comprehensive set of measurable physical activity categories, including sleep, activity pattern, stepping parameters, and activity patterns, could enable a significant discrimination, with effect sizes ranging from medium to large. The largest effect sizes were observed for the total walk duration, as a percentage of 24 h activities; total daily number of steps; and MtV behavior (Cohen's effect, size *d* \> 1.00). The discrimination between the pre-frail and frail was, however, more challenging. Nevertheless, the moderate effect sizes were observed when the total walk, total step, longest unbroken steps number, median light bout activity, or total MtV activities were considered (*d* \> 0.50). Using the univariate analysis, none of the sleep parameters were enabled to simultaneously distinguish the pre-frail from other groups, and thus were excluded from the model design. Among the remaining parameters, the most sensitive parameters were the total sedentary duration, total MtV duration, and total walk duration, which were able to identify the pre-frail from the other groups with an AUC of greater than 0.90.
Overall, we found that the frail group had the highest sedentary behaviors, which was an indicator of functional disability, as was reported in previous studies \[[@B65-sensors-18-01336]\]. Furthermore, as previous literature had mentioned, we observed that the frail group had the highest sedentary duration, which might have led to a higher comorbidity \[[@B66-sensors-18-01336]\]. The HSS guidelines emphasized the importance of meeting the physical activity requirements, namely, having more than 300 min per week of moderate-to-vigorous activity. We observed that the odds of meeting the guideline recommendation were significantly lower in the frail group, which might have increased the risk of adverse health outcomes \[[@B67-sensors-18-01336],[@B68-sensors-18-01336]\].
Further investigation would be needed into the association between frailty status and light activity, which included domestic chores like instrumented activity of daily living (e.g., cooking, household tasks, etc.). In our study, light activity was unable to discriminate between the frail and pre-frail, but it did enable the distinguishing of the pre-frail from non-frail stage. A study on older females with Parkinson's disease reported an association between light activity duration and cognitive dysfunction \[[@B69-sensors-18-01336]\]. Thus, light activity might have been representative of instrumental activities of daily living or cognitive function. On the other hand, recent studies suggested that the combination of frailty and cognitive impairment (cognitive frailty) could have better determined the prospective decline in motor and cognitive performance \[[@B70-sensors-18-01336],[@B71-sensors-18-01336],[@B72-sensors-18-01336],[@B73-sensors-18-01336]\]. Our study did not incorporate cognitive function into the model, because it was based on the Fried frailty phenotypes, which did not include cognitive performance. Thus, further exploration would be warranted to better understand the association between light activity and frailty phenotype progression, mediated by measures of cognitive function and changes in cognitive function. Indeed, future studies investigating sensory-derived data as measures for cognitive function that integrate physical performance-based models (as presented in the current study) could provide a more holistic understanding of the progression of the frailty stages in older adults.
We observed a reduction in nocturnal sleep parameters, such as total sleep time and time in bed, and an increase in sleep onset latency in the advancing frailty stages. The same observation was reported in a previous cohort of older community-dwelling men (*n* = 3133), where the odds of sleep disturbances had increased by the risk of frailty \[[@B34-sensors-18-01336]\]. In our study, the non-frail group had significantly lower sleep disturbances, but group comparison between the pre-frail and frail did not achieve a statistically significant level in our sample.
Finally, in order to examine the robustness of a predictive multiple variables model, so as to identify the pre-frail group among other groups, we used k-fold cross validation (k = 5) method, in which a 20% randomly selected dataset were used for the validation of the model. Using this approach, namely, stepping; the physical activity pattern; and physical activity behavior models were able to distinguish between the pre-frail from the others groups, with an AUC of 0.87, 0.85, and 0.85, respectively. The combination model improved on the discriminative power, with an AUC of 0.88.
To improve the level of comfort and mimic the telehealth platforms, which often incorporated a pendant sensor (e.g., personal emergency response system \[PERS\], such as pendant automatic fall detectors), we used a pendant accelerometer to monitor sleep and activities instead of securing the sensor on the chest, which had been used in previous studies \[[@B41-sensors-18-01336],[@B43-sensors-18-01336]\]. This approach might have affected the accuracy of the activity detection, as well as the estimation of the sleep parameters of interest. Despite this potential limitation, the measured parameters achieved a statistically significant level so as to distinguish the pre-frailty stages, thus creating a more realistic sensor-based method in order to monitor the frailty stages and their fluctuation over time, without hindering the everyday activities of daily living. In addition, the proposed study design could have facilitated the integration of the designed model in the currently available pendant PERS platforms.
5. Limitations {#sec5-sensors-18-01336}
==============
This study had several limitations. The sample size (*n* = 163) was relatively small and may be insufficient to represent the general older adults population. In addition, the feature selection was done based on the entire sample, and the sample size might have been insufficient for the purpose of the k-fold cross validation model. However, as recommended by the prior literature, this approach was shown to be more robust than the conventional approaches for relatively small sample size studies \[[@B56-sensors-18-01336],[@B57-sensors-18-01336]\].To better examine the validity and reliability of the proposed model, another study was needed to confirm that the results remained the same when using an independent and larger dataset. Therefore, the results needed to be confirmed in a larger sample, in order to be generalized. As this was a cross-sectional study, the sensitivity to change over time for the proposed model was unclear and needed to be verified in another study. In addition, the ability of the proposed model to predict the prospective adverse health outcomes, including mortality or loss of independency, should have been examined in another study. We used the Fried physical phenotypes criteria to determine the frailty stages, which carried some limitations, including a lack of consideration for cognitive function and using the categorical stages (non-frail, pre-frail, and frail) instead of a continuous scale. Fine tuning the model outputs in comparison with other well-established frailty assessment tools, such as the frailty index (an alternative frailty conceptual model that measures accumulation of deficits and provides a continuous scale instead of categorical), might have been useful for designing a more sensitive to change metric for the purpose of longitudinal studies. Two days of continuous monitoring (48 h) might not have been sufficient in order to represent the overall in-place activities of older adults. However, as suggested in our previous study \[[@B51-sensors-18-01336]\], two days of continuous monitoring yielded a reliable representation of daily physical activities in a geriatric population, in particular among those with the frailty status, because of the reduction in the activities complexities or day-to-day variation, as suggested by previous studies \[[@B74-sensors-18-01336],[@B75-sensors-18-01336],[@B76-sensors-18-01336]\]. On the other hand, in order to determine the causal factors that might have led to physical frailty, for instance in response to medication, a high time resolution, to determine frailty phenotypes, might have been considered as an advantage of the proposed approach. However, future studies were needed that would examine whether the proposed frailty model was sensitive to change and could track changes in the frailty stages over time.
6. Conclusions {#sec6-sensors-18-01336}
==============
We demonstrated that a single pendant accelerometer enables determining the frailty stages, including pre-frailty, via an in-place monitoring of the spontaneous daily physical activity, including the day time and night time. Among the measurable parameters, using a single pendant accelerometer-based device, a combination of step parameters (e.g., number of daily taken steps, longest unbroken steps), activity behavior (e.g., moderate-to-vigorous and sedentary activities), and postures (e.g., duration of standing, walking, and longest unbroken walking bout duration) enables the distinguishing of the pre-frailty stage among non-frail and frail stages, with AUC of 0.88. The proposed model and the form factor of the sensor that was used (pendant instead of securing sensor to the skin) provide advantages, compared with the conventional frailty assessment tools, for the purpose of in-place and prolonged screening (over days and months). In addition, it doesn't require a supervised administration of testing (unsupervised monitoring of frailty stages); it is objective; and does not need patient health records, demographics, or self-report, which makes it easy and cost-effective for deployment for in-place monitoring platforms. It can also facilitate in the development of a telehealth platform, based on wearable technology, to determine the modifiable factors that are significant for the advancing frailty stages (e.g., use of medication, which may negatively impact subject resilient; sleep deprivation; depression; cognitive decline; etc.). These potential applications, however, need to be validated in future studies.
J.R. is the Post-doctoral research fellow at the Center for Innovations in Quality, Effectiveness and Safety (CIN 13-413), Michael E. DeBakey VA Medical Center, Houston, TX, USA and he receives support from the Big Data-Scientist Training Enhancement Program (BD-STEP). The content is solely the responsibility of the authors and does not necessarily represent the official views of the sponsors. The authors would like to thank Kimberly Macellaro, a member of the Baylor College of Medicine Michael E. DeBakey Department of Surgery Research Core Team, for her editorial assistance during the preparation of this manuscript.
B.N., A.D.N., M.E.K., A.S. and J.R. helped with the study design and interpretation of the data. J.R., H.Z. and M.A. helped on data collection and analyzing. All of the authors read the manuscript and participated in writing the manuscript.
This research was partly funded by the National Institute of Health/National Institute of Aging (Award number: 2R42AG032748-04) and the U.S. Department of Veterans Affairs, Veterans Health Administration, Health Services Research and Development Service. J.R. was also funded by receives support from the Big Data-Scientist Training Enhancement Program (BD-STEP).
While the overlap with this study is minimal, using activity monitoring to determine frailty is protected by a patent pending (US20150272511 A1). The patent is owned by University of Arizona, and B.N. is listed as a co-inventor on this patent pending. Other author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
{#sensors-18-01336-f001}
sensors-18-01336-t001_Table 1
######
Demographic and clinical characteristics reported by mean ± standard deviation.
Parameter Non-Frail (N) Pre-Frail (P) Frail (F) N vs. P (*p*-Value) P vs. F (*p*-Value)
------------------------------------------ --------------- --------------- --------------- --------------------- ---------------------
Number of participants 42 78 33
Age, years 74.02 ± 7.37 75.25 ± 11.53 78.03 ± 11.20 0.527 0.191
BMI, kg/m^2^ 25.21 ± 5.64 29.76 ± 6.88 31.18 ± 7.01 0.000 0.289
Depression (CES-D) 6.62 ± 5.64 8.75 ± 7.14 12.89 ± 6.33 0.083 0.002
Concern for fall (SFES-I) 20.69 ± 4.22 23.43 ± 11.14 29.69 ± 15.89 0.182 0.006
Gender (female), % 82% 54% 51% 0.001 0.247
Number of prescribed medication, N 2.5 ± 1.8 4.1 ± 3.8 6.0 ± 3.4 0.530 0.520
Number of over the counter medication, N 3.0 ± 2.5 2.7 ± 2.2 2.5 ± 2.5 0.150 0.080
Number of comorbidities, N 2.3 ± 1.8 3.7 ± 2.2 4.7 ± 1.7 0.710 0.490
Fall history:
0 falls 71% 69% 69% 0.177 0.425
1--3 falls 24% 30% 23% 0.090 0.800
\>3 falls 4% 1% 9% 0.368 0.020
BMI---body mass index; CES-D---Center for Epidemiologic Studies Depression; FES-I---Fall Efficacy Scale-International.
sensors-18-01336-t002_Table 2
######
Results of sensor derived parameters assessment, stratified by frailty status.
Parameters Non-Frail (N) † Pre-Frail (P) † Frail (F) † *p*-Value (Effect Size ‡)
-------------------------------------- ----------------- ----------------- ----------------- --------------------------- --------------
**Sleep Quantity**
Time in bed, min 494.3 ± 114.4 434.7 ± 125.3 402.4 ± 127.6 0.010 (0.50) 0.209 (0.26)
Sleep onset latency, min 16.9 ± 7.5 18.5 ± 7.9 20.0 ± 8.4 0.290 (0.20) 0.369 (0.18)
Total sleep time, min 367.5 ± 86.2 321.9 ± 116.5 300.9 ± 119.4 0.027 (0.45) 0.360 (0.18)
Wake after sleep onset, min 103.7 ± 48.0 89.4 ± 41.7 75.5 ± 43.0 0.081 (0.32) 0.129 (0.33)
Sleep efficiency, % 78.4 ± 9.3 77.5 ± 10.9 79.6 ± 11.6 0.650 (0.09) 0.333 (0.19)
Sleep supine position, % 43.6 ± 21.0 41.2 ± 23.2 47.5 ± 31.4 0.594 (0.11) 0.222 (0.23)
Sleep prone position, % 14.7 ± 17.5 12.3 ± 16.7 18.8 ± 23.3 0.484 (0.14) 0.091 (0.32)
Sleep side position, % 34.6 ± 17.1 34.6 ± 24.2 19.7 ± 21.1 0.994 (0.00) 0.001 (0.65)
**Physical Activity Pattern**
Total sit, % 43.3 ± 15.6 46.9 ± 17.1 45.7 ± 17.1 0.253 (0.22) 0.734 (0.07)
Total stand, % 16.9 ± 5.8 13.6 ± 5.8 11.3 ± 5.7 0.003 (0.57) 0.060 (0.40)
Total walk, % \* 8.7 ± 3.9 5.1 ± 3.3 3.2 ± 3.2 0.000 (1.02) 0.012 (0.57)
Total Lye, % 30.9 ± 15.6 34.3 ± 20.4 39.7 ± 20.9 0.348 (0.19) 0.188 (0.26)
Longest unbroken sitting bout, s 5356.7 ± 3499.1 5351.3 ± 3042.0 5576.5 ± 3927.4 0.993 (0.00) 0.749 (0.06)
Median sitting bout, s 96.2 ± 72.9 89.6 ± 105.2 81.1 ± 92.0 0.712 (0.07) 0.666 (0.09)
Longest unbroken standing bout, s 385.3 ± 387.3 550.6 ± 723.8 553.2 ± 360.5 0.132 (0.28) 0.983 (0.00)
Median standing bout, s 15.0 ± 3.2 13.3 ± 7.5 13.3 ± 11.7 0.247 (0.29) 0.983 (0.00)
Longest unbroken walking bout, s \* 351.3 ± 347.9 187.9 ± 223.9 110.3 ± 132.4 0.001 (0.56) 0.002 (0.42)
Median walking bout, s \* 9.7 ± 1.0 9.0 ± 1.5 8.3 ± 1.9 0.020 (0.51) 0.018 (0.43)
**Stepping Parameters**
Total step, N/1000 \* 12.2 ± 6.1 6.7 ± 4.2 4.3 ± 4.3 0.000 (1.04) 0.018 (0.57)
Longest unbroken stepping bout, N \* 694.3 ± 743.0 322.9 ± 411.0 162.5 ± 184.2 0.000 (0.62) 0.006 (0.57)
Median stepping bout, N 13.5 ± 2.2 10.8 ± 4.7 8.8 ± 5.2 0.001 (0.75) 0.113 (0.38)
**Physical Activity Behavior**
Median Sedentary bout, s 323.8 ± 2044.6 491.1 ± 4243.4 29.7 ± 16.7 0.783 (0.05) 0.497 (0.15)
Total sedentary, h \* 9.6 ± 2.6 11.7 ± 3.2 13.2 ± 4.2 0.001 (0.73) 0.029 (0.40)
Total sedentary, % 70.4 ± 12.7 81.1 ± 8.9 84.9 ± 7.0 0.000 (0.98) 0.066 (0.47)
Median light bout, s \* 10.8 ± 2.2 9.6 ± 2.8 8.3 ± 2.5 0.011 (0.50) 0.013 (0.51)
Total light, h 3.2 ± 1.3 2.4 ± 1.2 2.1 ± 0.9 0.001 (0.62) 0.206 (0.29)
Total light, prc 23.7 ± 9.9 16.7 ± 7.7 13.9 ± 6.2 0.000 (0.79) 0.105 (0.39)
Median MtV, s 6.8 ± 1.9 6.8 ± 2.4 6.3 ± 1.8 0.990 (0.00) 0.267 (0.24)
Total MtV, min \* 47.7 ± 30.7 19.6.3 ± 20.5 11.2 ± 14.6 0.000 (1.08) 0.047 (0.47)
Total MtV, % \* 6.0 ± 4.0 2.2 ± 2.4 1.2 ± 1.5 0.000 (1.13) 0.066 (0.50)
HHS guideline, % (N) 50.0 16.0 3.1 0.000 (-) 0.109 (-)
†---average ± standard deviation; ‡---effect size calculated based on Cohen's *d* effect size for normal distribution (*d*) or for those who reject the normality (*r*), \*---parameters that *p*-value \< 0.05 and *d/r* ≥ 0.4; MtV---moderate-to-vigorous activity; bout---consecutive interval of a physical activity behavior without interrupt; HHS guideline---U.S. Department of Health and Human Services recommended guideline for the MtV duration.
sensors-18-01336-t003_Table 3
######
The performance of each parameter to discriminate the pre-frail group from the non-frail and frail groups.
Sensitivity †, % Specificity †, % Accuracy †, % AUC
-------------------------------------- ------------------ ------------------ --------------- -------------
**Physical activity behaviors**
Total sedentary, h \* 91.9 ± 3.8 66.7 ± 4.4 76.3 ± 1.9 0.91 ± 0.03
Median light bout, s 100.0 ± 0.0 0.0 ± 0.0 51.9 ± 0.2 0.50 ± 0.00
Total MtV, min \* 85.4 ± 3.3 77.8 ± 4.9 78.8 ± 1.7 0.92 ± 0.02
**physical activity patterns**
Total walk, % \* 86.1 ± 4.5 73.7 ± 6.7 78.0 ± 1.9 0.90 ± 0.02
Longest unbroken walking bout, s \* 84.2 ± 5.6 67.9 ± 6.8 73.0 ± 1.6 0.87 ± 0.02
Median walking bout, s 85.3 ± 8.1 32.4 ± 13.2 59.0 ± 2.2 0.64 ± 0.02
**stepping parameters**
Total step, N/1000 \* 88.9 ± 3.0 74.0 ± 3.4 78.7 ± 1.2 0.89 ± 0.02
Longest unbroken stepping bout, N \* 85.0 ± 5.7 75.8 ± 4.3 75.3 ± 1.9 0.88 ± 0.03
†---average ± standard deviation; AUC---area under the curve; \*---parameters with asterisks were used later to develop the model.
sensors-18-01336-t004_Table 4
######
The performance of models to separate the pre-frail group from the rest of the groups (non-frail and frail).
Models Sensitivity †, % Specificity †, % Accuracy †, % AUC †
---------------------------------- ------------------ ------------------ --------------- -------------
Step Model 88.6 ± 3.8 77.5 ± 1.3 79.7 ± 0.5 0.87 ± 0.02
Physical Activity Pattern Model 88.6 ± 3.3 74.2 ± 1.7 80.6 ± 0.4 0.85 ± 0.02
Physical Activity Behavior Model 87.1 ± 6.1 77.9 ± 2.0 80.4 ± 0.6 0.85 ± 0.03
Combined Model 91.8 ± 4.2 81.4 ± 2.2 84.7 ± 0.4 0.88 ± 0.03
AUC---area under the curve; †---the mean ± standard deviation reported for the validation datasets based on a 5-fold cross validation; Step Model---the total number of steps and longest unbroken stepping bout; Physical Activity Pattern Model---the total walk and longest unbroken walking bout; Physical Activity Behavior Model---the total steps and longest unbroken stepping bout; Combined Model---all of the mentioned parameters.
| {
"pile_set_name": "PubMed Central"
} |
Introduction
============
microRNAs (miRNAs) constitute a new class of small noncoding RNAs that control post-transcriptionally the expression of gene products, either modulating directly protein translation or regulating the stability of messenger RNA. There is increasing evidence of the role that miRNAs play in regulating breast cancer gene expression. The main objective of this study is to evaluate the diagnostic utility of the expression of a panel of miRNAs in breast cancer and compare their expression with the expression of the proteins they regulate.
Methods
=======
miRNA expression was analyzed by RT-qPCR using TaqMan Arrays (Applied Biosystems). We compared the expression of 667 miRNAs on 19 fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) matched breast cancer samples. Regarding protein expression, we have developed and evaluated different protocols for protein extraction from FFPE samples. Next, we studied the applicability of these protein extracts to classical and new high-performance proteomics techniques.
Results
=======
After proper normalization, 123 out of 671 miRNAs showed a good correlation of their expression data between FFPE and FF tissue, and sufficient analytical robustness (they are expressed in at least one-third of FFPE samples). In addition, we analyzed the expression of various markers with diagnostic value in breast cancer. As regards high-performance proteomics, the protocols developed generated over 6,000 MS/MS spectra, enabling the identification of hundreds of proteins in each sample.
Conclusion
==========
We have selected the most appropriate assays to study miRNA expression in breast cancer FFPE archived samples. The protocols developed allow proteome analysis of FFPE samples using the latest mass spectrometry equipment. The technologies implemented during the development of this project allow one to compare the expression data at both miRNA and protein levels to study breast cancer from an authentic system-biology perspective.
| {
"pile_set_name": "PubMed Central"
} |
Background
==========
Linear growth retardation or low height-for-age, commonly known as stunting is a useful anthropometric measure for children in terms of its positive correlation with social and economic deprivation. Stunting is now acknowledged as the best proxy measure for child health inequalities \[[@B1],[@B2]\]. This is because stunting captures the multiple dimensions of children\'s health, development and the environment where they live. Stunting is attributable to a wide range of factors \[[@B3]\] including low birth weight \[[@B4]\], inadequate care and stimulation \[[@B5]\], insufficient nutrition and recurrent infections \[[@B6]\], and other environmental determinants.
Stunting is conveniently used because empirical evidence suggests that the distribution of healthy children\'s height is not affected by ethnicity and race for the first five years of life \[[@B7]\]. Any variation between populations or ethnic groups below five years of age is due to varying degree of the growth faltering caused by factors other than genetic predisposition. The only exception is the sex difference. Thus, among well-to-do children there is a normal pattern of dimorphism where males will tend to be taller and heavier than females.
Besides studies in Asia which show higher female vulnerability \[[@B8]\], several studies in low-income countries have indicated that male children are more likely to be stunted than their female counterparts, most of them in sub-Saharan Africa \[[@B4],[@B9]-[@B12]\]. One of our recent studies disaggregated stunting prevalence rates by sex and socio-economic status (SES) \[[@B9]\]. In that study it was revealed that in poorer households more boys were stunted than girls, and that the sex differences in stunting rates did not exist among children belonging to socio-economically better off groups.
In the current study we hypothesised that in many low-income countries in sub-Saharan Africa where the standard of living is generally considered low, male children will be more stunted than their female counterparts. We used data sets of 16 demographic health surveys derived from 10 sub-Saharan African countries. The aim was first, to investigate whether there exists systematic sex differences in the overall prevalence of stunting among children less than 5 years of age and second, to evaluate whether sex differences in stunting vary with household socio-economic status.
Methods
=======
The Demographic and Health Survey programme provides data on child anthropometric status and household-level information on mothers\' education and ownership of assets for about 60 low- and middle-income countries. Population sampling frames are used for data collection, which makes the data sets nationally representative. In most countries, between 3,000 and 10,000 children below the age of 60 months are assessed for their growth status using anthropometric measurements. These data sets are in the public domain and are available from the MACRO International web-site \[[@B13]\].
We obtained data sets across sub-Saharan Africa fulfilling the following criteria: containing information on height-for-age measurements; English-speaking country, for ease of review of DHS reports; country with experience of more than one DHS study; recent surveys (conducted between 1995--2003); and data available as of September 2004. A total of 16 studies were obtained from 10 countries including Cameroon, Ghana, Kenya, Malawi, Namibia, Nigeria, Tanzania, Uganda, Zambia and Zimbabwe.
Creation of socio-economic indices
----------------------------------
When constructing indices for socio-economic status, one of the basic decisions concerns the domains of variables to use and methods of score. In the DHS data sets, data are available on the following domains of household wealth: characteristics of the dwelling (floor, walls, and roof material), availability of electricity, water and sanitation services, ownership of household durable goods, and parental education. Other domains that one might expect such as income or occupation are not contained in the DHS data sets.
Two variables of household SES were created reflecting the education level (mothers\' education); and ownership of durable household assets and characteristics of the dwelling structure (asset index). An asset index is a good proxy for household income or expenditure \[[@B14]\]. An index for mothers\' education was deliberately created separate from other domains because it has a known association with child health inequalities that is independent of other socio-economic indicators \[[@B9]\]; compared to household assets it was also considered more feasible for intervention. In addition, use of two such variables supports evidence that colinearity of many SES indicators is often too low to render them as adequate proxies for one another \[[@B9],[@B15]\].
The indices were constructed separately for each country. The DHS variable on mothers\' education was re-categorised by maintaining categories of \"no education\" (zero years of schooling) and \"primary education\" (not more than 7 or 8 years of schooling, depending on country). However, we merged the categories of \"higher education\" and \"secondary education\" into one category of \"secondary education\" (more than 7 or 8 years of schooling) because of the low numbers in the former category that would not allow for any realistic analyses. A new variable with 3 categories was thus created, (0) no education, (1) primary education and (2) secondary education.
The asset index was developed by use of principal components analysis \[[@B14]\] with variables on asset ownership (bicycle, radio, television, motorcycle, car/truck) and materials of the dwelling structure (floor, wall, roof) as appropriate. Regression factor scores generated from the first principal component were ranked in ascending order and then categorised into quintiles (1) poorest, to (5) least poor, Table [1](#T1){ref-type="table"}, similarly presented elsewhere \[[@B16]\]. However, the covariance among asset variables for Tanzania 1996 and Zambia 2001/02 studies was too high to permit auto-categorisation into quintiles, instead quartiles were generated.
Data Analysis
-------------
Statistical analyses were performed with SPSS 12.0 and STATA 8.0. Stunting was defined as height-for-age Z-score less than -2 standard deviations of the WHO/NCHS reference standards \[[@B17]\]. Anthropometric data were missing on approximately 25% of children. This is because children whose months and year of birth are not known for certain reasons or parents who refuse to have their children measured are excluded from anthropometric analyses in the DHS data sets. Children with incomplete data on stunting plus the flagged cases were therefore excluded from our analyses. Student\'s t-test, Fisher\'s exact test or *χ*^2^test, as appropriate, and logistic regressions were used to compare the outcome between male and female children. The test of homogeneity between studies was conducted and the Cochran\'s statistic was also reported. Results of fixed effects models are presented. Interaction between SES and gender with respect to the stunting outcome was assessed by simultaneously controlling for the main effects and the product of SES and gender in logistic regression. The level of statistical significance for all analyses was set at p \< 0.05 with two-tailed comparisons.
Results
=======
The two SES indicators that were used -- asset index and mothers\' education -- were almost similar in demonstrating the span and magnitude of stunting in socio-economic groupings. Generally they both showed a dose-response relationship of stunting with SES (Figure [1](#F1){ref-type="fig"}). However for many studies, this relationship featured a skewed pattern with poorer categories disproportionately more affected. Among the better off or the \"least poor\" the lowest prevalence of stunting was observed for Ghana 1998 (12%) and Namibia 2000 (13%), but was otherwise below 30% in all studies except for Nigeria 2000 (38%), Malawi 2000 (34%) and Tanzania 1996 (30%). Among quintiles and quartiles for the \"most poor\" the lowest prevalence of stunting was observed for Namibia 2000 (31%); and was above 50% in six studies (Malawi 2000, Nigeria 1999, Tanzania 1996 and 1999, and Zambia 1996 and 2001/02). Among mothers with no formal education the prevalence rate of stunting averaged 44% while it was 24% in those with secondary education.
The proportion of male and female children included in the analysis was nearly equal (Table [2](#T2){ref-type="table"}). The mean z-scores for males were consistently lower than for females with the differences statistically significant in 12 out of 16 studies. The pooled mean z-scores (standard deviation) were -1.59 (1.56) for boys and -1.46 (1.57) for girls, and the mean difference was significant (p \< 0.001).
The average prevalence of stunting was also higher in male than in female children in all the studies. The corresponding odds ratios (OR) for the prevalence of stunting among males compared to females were statistically significant in 11 of the 16 studies (Figure [2](#F2){ref-type="fig"}). In the pooled analysis the prevalence of stunting amongst males (40%) remained significantly greater than for females 36% (p \< 0.001), OR 1.16 and 95% confidence interval (CI) 1.12 -- 1.20. When the age of child and individual study/country were controlled for in the analysis, the adjusted OR was 1.18, CI 1.14 -1.22; and the test of homogeneity for the different studies was not significant (p = 0.15), Cochran\'s statistic *χ*^2^of 85.5 (p \< 0.001); implying that studies were similar or random and fixed effects models are indistinguishable.
The magnitude of stunting prevalence in both sexes varied systematically and inversely with SES. The gradient depicted highly significant p-values for tests of trend in both sexes (Table [3](#T3){ref-type="table"}). There was a unique pattern that was observed, although not entirely consistent across countries, for sex differences in stunting to be more pronounced among children in the poorest 2 asset quintiles and in children of mothers with no education or primary education. Figure [3](#F3){ref-type="fig"}, depicts graphically four examples of studies with the sex difference being more pronounced in the poorest quintiles or quartiles whereas there is no difference in socio-economically better off groups. However, sex differentials of stunting with SES did not follow a similar pattern in studies of the same country. For example the trend for Zambia 2001/2 in Figure [3](#F3){ref-type="fig"}, does not apply to Zambia 1996, and the trend for Tanzania 1996 does not apply to Tanzania 1999. Additionally, evaluation of the interaction term between sex and SES in relation to stunting was not statistically significant for the individual study and for the pooled analysis.
Discussion
==========
This systematic analysis of nationally representative datasets included 16 studies from 10 countries with a total of 64,000 children. This is the first systematic analysis of sex differences in stunting among children less than 5 years of age. Our findings demonstrate that across the 10 countries in sub-Saharan Africa, male children are consistently more likely to become stunted than their female counterparts. Secondly, in several of the studies, sex differences in stunting were more pronounced, albeit inconsistent, in the lower socio-economic strata.
In meta-analysis, two main assumptions could be employed in interpreting the effect size or the systematic difference in stunting between sexes. First, studies were drawn from a common population, and therefore share a common effect size (fixed effects model). Second, studies were drawn from populations that differ from each other in ways that could impact on effect sizes (random effects model). In the former scenario, effect size varies from one study to the next due to random error inherent in each study. In the latter scenario it varies due to both random error and true variation in effect size from one study to the next. If studies are homogeneous (significant p-value implies no homogeneity), it implies that fixed effects and random effects models are similar and not statistically different. This further implies that similar studies done in similar set-ups would yield statistically similar results with any study-to-study dispersion attributable to random error.
Turning to our findings the studies were homogeneous (p = 0.15), thus similar studies done in similar set-ups would likely yield similar results. So the next question is which countries are similar to these 10 Anglophone countries? Our understanding is that these countries share a lot of communalities with the rest of the countries in sub-Saharan Africa.
So then, was it an accidental observation or a reality that the pattern of stunting differentials between sexes across socio-economic strata was not consistent in all surveys? We believe this is a reality. First, it seems that the effect of SES on the pattern of stunting between sexes is no longer present in better off populations like Namibia. Second, it could be attributable to potential biases. On average 25% of data on stunting was missing in all the studies. Therefore, the tendency for more boys than girls being stunted in the 2^nd^quintile rather than in the 1^st^quintile (poorest 20%) as observed for many studies in Table [3](#T3){ref-type="table"}, could be attributable to bias due to non-random loss of participants -- implying that among the flagged and missing data cases, the proportion of stunted boys might have been higher than that for girls. Another possibility is misclassification of SES in some surveys leading to a dilution of the associations.
Theoretically, there could be other sources of bias in the study. First, systematic errors with the measure could lead to the observed systematic sex differences. The NCHS/WHO growth reference \[[@B17]\] has separate references for males and females, thus observed sex difference might be related in some way to the reference itself. If this were true however, it would be difficult to understand why the inequality differentials of stunting with sex disappear in the socio-economically better off groups in many of the studies. In future it would be interesting to repeat such a study using the newly developed WHO international growth standard \[[@B18]\].
Second, analysis of this study was based on a single age group (0--5 years) although growth and nutritional profiles are drastically changing over this period of time. For instance, wasting is often peaking early in the second year of life, while stunting is often increasing over the whole age period. In a separate analysis we checked for differences in sex ratio in five different age categories (0--5 years), but no significant difference was found (data not shown). Thus, the potential bias of having more boys in older ages which could artificially increase the prevalence of stunting in boys in the group was dispelled.
Third, several statistical tests were employed in the current study thereby inflating the likelihood of finding spurious associations. This limitation should be borne in mind especially when making inferences based upon the reported p-values.
Although there is paucity of studies which have systematically addressed differentials of sex with respect to health inequality in the early childhood period, sex differences in anthropometry with females having an advantage over males have been previously reported \[[@B9]-[@B12],[@B19],[@B20]\]. Speculation on the observed sex differences in these studies mainly centres on behavioural patterns. For instance in an extensive analysis of gender bias in undernutrition in sub-Saharan Africa, Svedberg proposed that the slight anthropometric advantage shown by girls, women, or both in many countries may suggest a historical pattern of preferential treatment of females due to the high value placed on women\'s agricultural labour \[[@B19]\]. On the basis of a study of gender biases among the Mukogodo of Kenya, Cronk \[[@B20]\] suggested that favouritism towards daughters occurred as a result of lowered socio-economic status. However, there are also studies that report greater social valorisation of sons at the detriment of daughters \[[@B21]\], including dietary discrimination \[[@B22]\], thereby dispelling conclusions of a nutritionally advantaged position of female over male children.
An alternative hypothesis of the cause of the difference is a biological explanation. Epidemiological studies in neonatology and in cohorts of pre-term infants and children, depict both morbidity and mortality to be consistently higher in males than females in early life, with the differences persisting after adjusting for gestational age and body size, and being more marked in the pre-term subjects \[[@B23]-[@B26]\]. Aside from the specific sex-chromosome factors, the underlying mechanisms to why male gender is associated with increased neonatal mortality and morbidity is poorly understood \[[@B26],[@B27]\]. However, the reported male predominance in both symptomatic and asymptomatic morbidity \[[@B27]\] suggest that boys generally, are more vulnerable, which could partly explain our findings.
In evolutionary theory, selective male mortality has been previously linked to biased sex ratios at birth as modelled by Trivers and Willard \[[@B28]\]. The theory has been expanded by Wells \[[@B29]\] to explore its significance for differential sex morbidity and mortality among the under-fives. According to the expanded theory, in its summarised and simple form, natural selection favours a sex ratio of 1.0. Since there is an excess of males at conception the theory predicts mortality and morbidity to remain greater in males than in females for any given degree of environmental stress in the first 4 years of life. This theory in some way predicts our findings.
Conclusion
==========
This study reveals that in 10 countries in sub-Saharan Africa, male children below five years of age are more likely to become stunted than their female counterparts. An inconsistent pattern was observed where sex differences in stunting tended to be more pronounced in the poorest, socio-economically. The study also indirectly reaffirms that stunting, a proxy for child health inequalities, is as well a proxy for socio-economic inequalities. Even though the study advances knowledge on the understanding of early childhood health inequalities, it raises interesting issues that mandate further research.
The research agenda
-------------------
The sex difference in stunting with regard to SES seems not to be uniform across populations and the determinants for its variation are unknown at this stage. Further research is therefore needed to confirm and/or obtain explanation regarding sex differentials with stunting across socio-economic strata. As demonstrated in one of our previous papers \[[@B9]\], SES is not unidimensional. At present we do not know the number of dimensions that are critical for stunting in different contexts. In order to have effective interventions along the socio-economic pathway, researchers are urged to use a number of socio-economic indicators such as parents education, income, household dependency ratio, land and asset ownership independently, rather bunching them together as each could have its unique contribution \[[@B15],[@B30]\]. Studies therefore need to decompose SES in order to identify which components are most associated with stunting differentials of sex in the different contexts.
In addition, the study findings need to be corroborated with findings from other regions. For instance many Asian populations actually suffer severe stunting in young age. Unfortunately many DHS data sets from Asia lack information on height-for-age, for example Indonesia 1997 and Philippines 1998. Even where information on height-for-age exists in the datasets such as Pakistan and Sri Lanka, missing information goes as high as 50%. It was therefore not possible to make any comparisons.
This first systematic analysis of sex differences in stunting might also serve as an eye opener for an attempt to map out vulnerability of different sexes across the lifecycle. Functional and long-term consequences associated with early male vulnerability need to be explored. This is especially important in studies for early nutrition conditions and later risk of disease, especially cardio-vascular diseases and diabetes \[[@B31]\], life expectancy and certain behaviours that are particularly known to be more prevalent amongst males than females. Finally, a question that mandates further research following findings of this study is the biological explanation as to why male children should be worse off compared to female children!
Competing interests
===================
The author(s) declare that they have no competing interests.
Authors\' contributions
=======================
HW performed the statistical analysis and drafted the manuscript. All authors participated in the design of the study, read and approved the final manuscript.
Pre-publication history
=======================
The pre-publication history for this paper can be accessed here:
<http://www.biomedcentral.com/1471-2431/7/17/prepub>
Acknowledgements
================
This study was financially supported by the NUFU funded project \"Essential Nutrition and Child Health in Uganda\", the NORAD Fellowship Program and the Norwegian Quota Program.
Figures and Tables
==================
{#F1}
{#F2}
{#F3}
######
Asset quintiles and quartiles generated from scores of the first principal component
Socio-economic status Zimbabwe 1999 Zambia 2001/2 Zambia 1996 Uganda 2000/1 Uganda 1995/6 Tanzania 1999 Tanzania 1996 Nigeria 2003
----------------------- --------------- --------------- ------------- --------------- --------------- --------------- --------------- ---------------
1^st^/poorest 624 (24%) 1826 (34%) 624 (11%) 1026 (20%) 993 (22%) 455 (20%) 1769 (34%) 918 (22%)
2^nd^ 607 (23%) 1690 (31%) 974 (19%) 883 (20%) 469 (20%) 688 (16%)
3^rd^ 286 (11%) 1547 (29%) 687 (13%) 1248 (25%) 709 (16%) 460 (20%) 1421 (28%) 1003 (23%)
4^th^ 655 (25%) 936 (17%) 1503 (28%) 915 (18%) 1026 (23%) 474 (20%) 850 (17%) 710 (17%)
5^th^/wealthy 457 (17%) 1092 (20%) 949 (17%) 938 (18%) 861 (19%) 474 (20%) 1078 (21%) 917 (22%)
Total 2629 5401 5453 5101 4772 2332 5118 4236
Nigeria 1999 Namibia 2000 Malawi 2000 Kenya 2003 Kenya 1998 Ghana 2003 Ghana 1998 Cameroon 1998
1^st^/poorest 201 (15%) 586 (20%) 2187 (24%) 920 (20%) 573 (20%) 457 (15%) 528 (20%) 324 (20%)
2^nd^ 283 (20%) 575 (20%) 1095 (12%) 1258 (27%) 553 (19%) 852 (28%) 798 (30%) 361 (21%)
3^rd^ 244 (17%) 473 (16%) 1605 (18%) 899 (19%) 644 (23%) 590 (19%) 190 (08%) 321 (19%)
4^th^ 316 (23%) 678 (24%) 2307 (25%) 764 (16%) 598 (21%) 590 (19%) 621 (24%) 328 (20%)
5^th^/wealthy 356 (25%) 577 (20%) 1932 (21%) 848 (18%) 496 (17%) 597 (19%) 482 (18%) 329 (20%)
Total 1400 2889 9126 4689 2864 3086 2619 1663
######
The 16 Demographic and Health Surveys (DHS) studied; with country, year of study, age groups and total number of children included in this analysis.
Country Year of Study Age in months [Total^a^]{.ul} [Mean Z-scores^a^]{.ul} P-value
------------------- -------------------- --------------- ----------------- ------------------------- ------------------- ------------------- ----------------
Zimbabwe 1999 0--59 1335 1297 -1.16 (1.5) -0.97 (1.6) 0.002
Zambia 2001/02 0--59 2723 2707 -1.94 (1.5) -1.84 (1.6) 0.011
Zambia 1996 0--59 2688 2815 -1.80 (1.5) -1.74 (1.5) 0.147
Uganda 2000/01 0--59 2548 2597 -1.62 (1.5) -1.55 (1.4) 0.058
Uganda 1995/6 0--47 2204 2315 -1.57 (1.5) -1.38 (1.5) \< 0.001
Tanzania 1999 0--59 1272 1242 -1.71 (1.3) -1.64 (1.3) 0.170
Tanzania 1996 0--59 2668 2558 -1.83 (1.4) -1.70 (1.5) 0.002
Nigeria 2003 0--59 2165 2128 -1.55 (1.8) -1.39 (1.8) 0.002
Nigeria 1999 0--35 739 717 -1.76 (2.1) -1.50 (2.1) 0.017
Namibia 2000 0--59 1472 1437 -1.03 (1.4) -0.91 (1.4) 0.019
Malawi 2000 0--59 4557 4605 -1.88 (1.6) -1.75 (1.6) \< 0.001
Kenya 2003 0--59 2366 2353 -1.29 (1.5) -1.08 (1.6) \< 0.001
Kenya 1998 0--35 1451 1448 -1.29 (1.7) -1.15 (1.6) 0.026
Ghana 2003 0--59 1567 1527 -1.42 (1.4) -1.20 (1.5) \< 0.001
Ghana 1998 0--59 1288 1338 -1.26 (1.5) -1.12 (1.5) 0.026
Cameroon 1998 0--59 905 879 -1.17 (1.6) -1.03 (1.5) 0.056
***All studies*** ***1995 to 2003*** ***0--59*** ***31948*** ***31963*** ***-1.59 (1.6)*** ***-1.46 (1.6)*** ***\< 0.001***
^a^Values include only children with complete data on height and age, and who were not flagged
In addition a comparison of the mean z scores (standard deviation) for height-for-age between male and female children, and p-values for the difference is indicated.
######
A comparison of the proportion of stunted children among males and females in each of the asset index quintile from 1^st^(poorest) to 5^th^(least poor) and in each of the mothers\' education groups.
Country (year of study) Sex Percentage stunted by household asset index quintile p-value for test of trend Percentage stunted by mothers education status p-value for test of trend
------------------------- ----- ------------------------------------------------------ --------------------------- ------------------------------------------------ --------------------------- -------- ------------ ----------- ----------- ----------- ------------
Zimbabwe (1999) M 33.1 28.4 27.3 24.2 24.3 *0.007* 31.0 31.9 22.7 *0.001*
F 30.6 28.9 23.7 23.8 14.5 *0.001* 36.9 25.9 21.7 *0.001*
*Pearson χ*^2^*test* *0.49* *0.90* *0.46* *0.91* *0.01* *0.35* *0.02* *0.67*
Zambia (2001/02)^b^ M 57.0 51.5 47.9 34.1 *\< 0.001* 53.8 52.4 36.1 *\< 0.001*
F 52.0 46.2 44.5 36.1 *\< 0.001* 52.2 47.5 35.9 *\< 0.001*
*Pearson χ*^2^*test* *0.03* *0.04* *0.30* *0.48* *0.64* *\< 0.01* *0.95*
Zambia (1996) M 49.0 51.9 47.0 43.5 28.3 *\< 0.001* 50.0 46.5 33.4 *\< 0.001*
F 54.1 52.4 44.3 39.3 25.2 *\< 0.001* 51.1 45.4 30.9 *\< 0.001*
*Pearson χ*^2^*test* *0.20* *0.83* *0.48* *0.11* *0.28* *0.76* *0.53* *0.37*
Uganda (2000/01) M 46.9 45.9 44.0 30.8 26.7 *\< 0.001* 46.7 39.4 26.7 *\< 0.001*
F 44.2 41.1 39.5 33.1 22.5 *\< 0.001* 46.7 34.9 25.7 *\< 0.001*
*Pearson χ*^2^*test* *0.38* *0.13* *0.11* *0.45* *0.14* *0.99* *\< 0.01* *0.77*
Uganda (1995/6) M 44.7 44.9 42.2 35.9 21.6 *\< 0.001* 44.6 38.9 23.2 *\< 0.001*
F 42.1 38.1 32.4 35.8 16.9 *\< 0.001* 41.6 32.9 19.3 *\< 0.001*
*Pearson χ*^2^*test* *0.40* *0.04* *\< 0.01* *1.00* *0.08* *0.30* *\< 0.01* *0.21*
Tanzania (1999) M 54.7 47.3 37.8 36.9 22.6 *\< 0.001* 44.9 42.4 19.7 *\< 0.001*
F 53.1 41.9 44.9 32.6 19.0 *\< 0.001* 47.0 38.1 19.9 *\< 0.001*
*Pearson χ*^2^*test* *0.67* *0.31* *0.23* *0.28* *0.35* *0.59* *0.09* *0.98*
Tanzania (1996)^b^ M 56.2 47.2 45.5 29.6 *\< 0.001* 52.9 44.3 31.0 *\< 0.001*
F 50.5 44.0 40.7 30.5 *\< 0.001* 48.1 41.7 24.2 *\< 0.001*
*Pearson χ*^2^*test* *0.01* *0.22* *0.16* *0.74* *0.06* *0.12* *0.22*
Nigeria (2003) M 52.4 51.8 42.8 33.1 24.1 *\< 0.001* 52.7 39.6 21.6 *\< 0.001*
F 44.2 44.0 36.2 33.7 19.6 *\< 0.001* 45.9 32.1 19.6 *\< 0.001*
*Pearson χ*^2^*test* *0.01* *0.04* *0.03* *0.85* *0.11* *\< 0.01* *0.01* *0.38*
Nigeria (1999) M 58.2 54.4 47.2 46.8 41.1 *0.002* 57.4 43.2 43.4 *0.002*
F 52.4 53.6 44.4 34.8 36.2 *0.001* 55.2 36.6 34.6 *0.001*
*Pearson χ*^2^*test* *0.41* *0.88* *0.66* *0.03* *0.34* *0.68* *0.17* *0.04*
Namibia (2000) M 30.7 23.0 23.8 20.7 13.9 *\< 0.001* 29.8 26.7 17.0 *\< 0.001*
F 31.4 24.3 17.2 20.3 10.3 *\< 0.001* 30.7 24.3 14.4 *\< 0.001*
*Pearson χ*^2^*test* *0.87* *0.72* *0.08* *0.89* *0.19* *0.84* *0.38* *0.20*
Malawi (2000) M 57.0 56.2 48.8 48.2 34.6 *\< 0.001* 53.7 48.3 28.7 *\< 0.001*
F 53.2 48.9 47.1 46.4 34.0 *\< 0.001* 52.2 45.1 26.7 *\< 0.001*
*Pearson χ*^2^*test* *0.07* *0.01* *0.48* *0.40* *0.78* *0.44* *\< 0.01* *0.57*
Kenya (2003) M 39.6 35.7 34.4 25.8 24.0 *\< 0.001* 36.2 35.7 20.8 *\< 0.005*
F 35.2 29.3 29.1 24.1 11.5 *\< 0.001* 31.8 29.8 13.7 *\< 0.001*
*Pearson χ*^2^*test* *0.18* *0.02* *0.08* *0.58* *0.01* *0.17* *\< 0.01* *\< 0.01*
Kenya (1998) M 46.0 42.3 28.2 31.7 25.5 *\< 0.001* 44.5 37.2 24.0 *\< 0.001*
F 37.0 35.9 25.3 27.9 15.3 *\< 0.001* 42.9 30.9 16.0 *\< 0.001*
*Pearson χ*^2^*test* *0.03* *0.10* *0.41* *0.31* *0.01* *0.76* *\< 0.01* *\< 0.01*
Ghana (2003) M 39.2 40.6 29.7 36.0 20.7 *\< 0.001* 41.0 29.1 25.1 *\< 0.001*
F 36.3 33.3 24.1 31.0 15.4 *\< 0.001* 36.3 20.8 21.8 *\< 0.001*
*Pearson χ*^2^*test* 0.52 0.03 0.13 0.20 0.11 0.06 0.01 0.12
Ghana (1998) M 37.8 35.1 39.4 28.9 13.8 *\< 0.001* 35.8 32.8 22.2 *\< 0.001*
F 34.4 27.0 27.5 25.2 12.2 *\< 0.001* 31.9 24.5 18.2 *\< 0.001*
*Pearson χ*^2^*test* *0.42* *0.01* *0.08* *0.31* *0.60* *0.15* *0.04* *0.12*
Cameroon (1998) M 38.2 35.5 36.5 25.6 14.9 *\< 0.001* 41.3 28.3 20.7 *\< 0.001*
F 32.0 32.6 22.5 18.9 18.2 *\< 0.001* 35.0 24.4 17.6 *\< 0.001*
*Pearson χ*^2^*test* *0.24* *0.56* *0.01* *0.14* *0.42* 0.13 0.23 0.37
M = Male; F = Female; ^b^The asset index in this survey was categorised in quartiles
Pearson chi square test for each male-female comparison and p-values for the test of trend across quintiles or mothers education groups is indicated.
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#Sec1}
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Let $\documentclass[12pt]{minimal}
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\begin{document}$${\mathbb {R}}^{n+1} (n=1,2)$$\end{document}$ evolving in time. In this paper we consider a finite element approach for solving the parabolic surface PDE equation$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \partial _t^{\bullet } u + u \nabla _{\varGamma } \cdot {\varvec{v}}- \varDelta _{\varGamma } u= & {} f \qquad \text{ on } S_T \end{aligned}$$\end{document}$$ $$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} u(\cdot ,0)= & {} u_0 \quad \; \text{ on } \varGamma (0), \end{aligned}$$\end{document}$$which models advection and diffusion of a surface quantity *u* with $\documentclass[12pt]{minimal}
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\begin{document}$$u(\cdot ,t):\varGamma (t) \rightarrow {\mathbb {R}}$$\end{document}$. Here, $\documentclass[12pt]{minimal}
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\begin{document}$$S_T= \bigcup _{t \in (0,T)} \bigl ( \varGamma (t) \times \lbrace t \rbrace \bigr )$$\end{document}$ and $\documentclass[12pt]{minimal}
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\begin{document}$${\varvec{v}}: \overline{S_T} \rightarrow {\mathbb {R}}^{n+1}$$\end{document}$ denotes a given velocity field. Furthermore, $\documentclass[12pt]{minimal}
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\begin{document}$$\nabla _{\varGamma }$$\end{document}$ is the tangential gradient, $\documentclass[12pt]{minimal}
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\begin{document}$$\varDelta _{\varGamma } = \nabla _{\varGamma } \cdot \nabla _{\varGamma }$$\end{document}$ the Laplace Beltrami operator and $\documentclass[12pt]{minimal}
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\begin{document}$$\partial _t^{\bullet }=\partial _t + {\varvec{v}}\cdot \nabla $$\end{document}$ denotes the material derivative.
Parabolic surface PDEs of the form ([1](#Equ1){ref-type=""}) have applications in fluid dynamics and materials science, such as the transport and diffusion of surfactants on a fluid/fluid interface, \[[@CR25]\] or diffusion-induced grain boundary motion, \[[@CR5]\]. In these as in several other applications the velocity $\documentclass[12pt]{minimal}
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\begin{document}$${\varvec{v}}$$\end{document}$ is not given but determined through an additional equation so that ([1](#Equ1){ref-type=""}) becomes a subproblem of a more complicated system in which the variable *u* is coupled to other variables. The analysis and the numerical solution of such systems then naturally requires the development of corresponding methods for ([1](#Equ1){ref-type=""}). We refer to \[[@CR13]\] for a comprehensive overview of finite element methods for solving PDEs on stationary and evolving surfaces.
Concerning the numerical methods that have been proposed for ([1](#Equ1){ref-type=""}) one may distinguish between Lagrangian and Eulerian type schemes. The first approach has been pursued by Dziuk and Elliott within their evolving surface finite element method, \[[@CR8]\], which uses polyhedral approximations of the evolving hypersurfaces $\documentclass[12pt]{minimal}
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\begin{document}$$\varGamma (t)$$\end{document}$. While \[[@CR8]\] contains an error analysis in the spatially discrete case, the fully discrete case is investigated in \[[@CR11], [@CR14]\] and \[[@CR19]\]. Optimal $\documentclass[12pt]{minimal}
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\begin{document}$$L^2$$\end{document}$-error bounds are obtained in \[[@CR12]\] and a corresponding finite volume approach is proposed and analyzed in \[[@CR18]\]. Since the mesh for the discretization of ([1](#Equ1){ref-type=""}) is fitted to the hypersurface $\documentclass[12pt]{minimal}
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\begin{document}$$\varGamma (t)$$\end{document}$, a coupling to a bulk equation is not straightforward. This difficulty is not present in Eulerian type schemes, in which $\documentclass[12pt]{minimal}
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\begin{document}$$\varGamma (t)$$\end{document}$ is typically described via a level set function defined in an open neighbourhood of $\documentclass[12pt]{minimal}
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\begin{document}$$\varGamma (t)$$\end{document}$. In order to discretize the surface PDE in this setting it has been proposed in \[[@CR1], [@CR3]\] and \[[@CR27]\] to extend the surface quantity *u* to a band around $\documentclass[12pt]{minimal}
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\begin{document}$$\varGamma (t)$$\end{document}$ and to solve a suitable (weakly) parabolic PDE in that bulk region using a finite difference method. In \[[@CR9]\] and \[[@CR10]\], the same idea is used in a finite element context for which the underlying variational formulation is derived with the help of a transport identity. An Eulerian finite element approach that doesn't use an extended PDE is proposed and analyzed in \[[@CR20]\] and \[[@CR21]\]. The method is based on a weak formulation on the space-time manifold and the finite element space is obtained by taking traces of the corresponding bulk finite elements. The approximation of $\documentclass[12pt]{minimal}
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\begin{document}$$\varGamma (t)$$\end{document}$ on which these spaces are defined usually arises from a suitable interpolation of the given level set function describing $\documentclass[12pt]{minimal}
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\begin{document}$$\varGamma (t)$$\end{document}$. The resulting discrete hypersurface will in general cut arbitrarily through the background mesh and its location forms one of the main difficulties in implementing the scheme. A different approach of generating the discrete hypersurfaces is pursued in \[[@CR17]\], where a discretization of ([4](#Equ4){ref-type=""}) below is combined with the cut finite element technique. Finally, Section 5 in \[[@CR7]\] proposes a hybrid method that employs the above--mentioned idea of trace finite elements together with a narrow band technique for the elliptic part of the PDE.
In this paper we are concerned with the diffuse interface approach for solving ([1](#Equ1){ref-type=""}), which was introduced in \[[@CR22]\] for a stationary surface and in \[[@CR16], [@CR23]\] and \[[@CR26]\] for evolving surfaces. As in some of the methods described above, the surface quantity *u* is extended to a bulk quantity satisfying a suitable parabolic PDE in a neighbourhood of $\documentclass[12pt]{minimal}
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\begin{document}$$\varGamma (t)$$\end{document}$ and the bulk equation is then localized to a thin layer of thickness $\documentclass[12pt]{minimal}
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\begin{document}$$\epsilon $$\end{document}$ with the help of a phase field function (see \[[@CR15]\] for a corresponding convergence analysis). Since we are interested in using finite elements, the localized PDE needs to be written in a suitable variational form. Following \[[@CR16]\] this is achieved with the help of a transport identity and results in a discretization by linear finite elements in space and a backward Euler scheme in time. The detailed derivation along with an existence result for the discrete solution will be given in Sect. [3](#Sec5){ref-type="sec"}. As the main new contribution of our paper we shall derive conditions relating the interface width $\documentclass[12pt]{minimal}
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\begin{document}$$\epsilon $$\end{document}$, the spatial grid size *h* and the time step $\documentclass[12pt]{minimal}
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\begin{document}$$\tau $$\end{document}$ which allow for a rigorous stability and error analysis. More precisely, we shall prove that the numerical solution is bounded uniformly in $\documentclass[12pt]{minimal}
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\begin{document}$$L^{\infty }(L^2)$$\end{document}$ and $\documentclass[12pt]{minimal}
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\begin{document}$$L^2(H^1)$$\end{document}$ over the diffuse interface (see Theorem [1](#FPar16){ref-type="sec"} in Sect. [4](#Sec8){ref-type="sec"}) and that it converges with respect to these norms both over the diffuse interface and on the sharp interface with an order $\documentclass[12pt]{minimal}
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\begin{document}$$O(\epsilon )$$\end{document}$ provided that$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} h \le c_1 \epsilon , \; \tau \le c_2 \epsilon ^2, \end{aligned}$$\end{document}$$see Theorem [2](#FPar20){ref-type="sec"} and Corollary [1](#FPar22){ref-type="sec"} in Sect. [5](#Sec9){ref-type="sec"} respectively. In Sect. [6](#Sec10){ref-type="sec"} we report on results of numerical tests both for $\documentclass[12pt]{minimal}
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\begin{document}$$n=1$$\end{document}$ and $\documentclass[12pt]{minimal}
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\begin{document}$$n=2$$\end{document}$.
An advantage of our approach is that in the implementation the evolution of the hypersurfaces is easily incorporated by evaluating the phase field function. We shall employ a function with compact support, namely $\documentclass[12pt]{minimal}
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\begin{document}$$\rho (x,t):= g(\frac{\phi (x,t)}{\epsilon })$$\end{document}$, where $\documentclass[12pt]{minimal}
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\begin{document}$$\varGamma (t)$$\end{document}$ is the zero level set of $\documentclass[12pt]{minimal}
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\begin{document}$$\phi (\cdot ,t)$$\end{document}$ and$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} g(r)= \left\{ \begin{array}{ll} \cos ^2(r), &{} | r | \le \frac{\pi }{2}, \\ 0, &{} |r| > \frac{\pi }{2}. \end{array} \right. \end{aligned}$$\end{document}$$In view of the evolution of the hypersurfaces the numerical scheme then naturally contains terms in which $\documentclass[12pt]{minimal}
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\begin{document}$$\rho $$\end{document}$ is evaluated at different times. One of the main challenges in the analysis is to handle the corresponding differences, for which one has to bound integrals that are multiplied by a negative power of $\documentclass[12pt]{minimal}
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\begin{document}$$\epsilon $$\end{document}$ (arising from derivatives of $\documentclass[12pt]{minimal}
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\begin{document}$$\rho $$\end{document}$) as well as integrals that are not weighted with $\documentclass[12pt]{minimal}
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\begin{document}$$\rho $$\end{document}$. We shall deal with these difficulties by introducing an additional stabilization term with extended support that is also used for proving the well--posedness of the scheme.
Let us finally remark that a phase field approach involving a phase field function with noncompact support and finite elements has been proposed in \[[@CR4]\] for an elliptic surface PDE. Theorem 7 in \[[@CR4]\] provides an error estimate in terms of an approximation error and an error due to the phase field representation. The latter decays at a rate $\documentclass[12pt]{minimal}
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\begin{document}$$O(\epsilon ^p)$$\end{document}$ for some $\documentclass[12pt]{minimal}
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\begin{document}$$p<1$$\end{document}$, while a coupling between $\documentclass[12pt]{minimal}
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\begin{document}$$\epsilon $$\end{document}$ and the grid size *h* is not discussed.
Preliminaries {#Sec2}
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Surface representation and surface derivatives {#Sec3}
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\begin{document}$$t \in [0,T]$$\end{document}$ let $\documentclass[12pt]{minimal}
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\begin{document}$$\varGamma (t) \subset {\mathbb {R}}^{n+1} \, (n=1,2)$$\end{document}$ be a connected, compact and orientable hypersurface without boundary. We suppose that $\documentclass[12pt]{minimal}
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\begin{document}$${\varvec{v}}: \overline{S_T} \rightarrow {\mathbb {R}}^{n+1}$$\end{document}$ is a prescribed velocity field of the form$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} {\varvec{v}}= V \nu + {\varvec{v_\tau }}, \quad \text{ with } ({\varvec{v_\tau }}, \nu )=0. \end{aligned}$$\end{document}$$Here, $\documentclass[12pt]{minimal}
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\begin{document}$$\nu $$\end{document}$ is a unit normal and *V* the corresponding normal velocity of $\documentclass[12pt]{minimal}
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\begin{document}$$(\cdot , \cdot )$$\end{document}$ denotes the Euclidian scalar product in $\documentclass[12pt]{minimal}
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\begin{document}$${\mathbb {R}}^{n+1}$$\end{document}$. Note that the normal part $\documentclass[12pt]{minimal}
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\begin{document}$$V\nu $$\end{document}$ is responsible for the geometric motion of $\documentclass[12pt]{minimal}
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\begin{document}$$\varGamma (t)$$\end{document}$, while the tangential part $\documentclass[12pt]{minimal}
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\begin{document}$${\varvec{v_\tau }}$$\end{document}$ is associated with the transport of material along the surface. We assume that there exists a smooth map $\documentclass[12pt]{minimal}
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\begin{document}$$\varPsi :\varGamma (0) \times [0,T] \rightarrow {\mathbb {R}}^{n+1}$$\end{document}$ such that $\documentclass[12pt]{minimal}
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\begin{document}$$\varPsi (\cdot ,t)$$\end{document}$ is a diffeomorphism from $\documentclass[12pt]{minimal}
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\begin{document}$$\varGamma (0)$$\end{document}$ onto $\documentclass[12pt]{minimal}
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\begin{document}$$\varGamma (t)$$\end{document}$ for every $\documentclass[12pt]{minimal}
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\begin{document}$$t \in [0,T]$$\end{document}$ satisfying$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \frac{\partial \varPsi }{\partial t}(P,t)= & {} {\varvec{v}}(\varPsi (P,t),t), \quad P \in \varGamma (0), t \in (0,T]; \end{aligned}$$\end{document}$$ $$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \varPsi (P,0)= & {} P, \qquad \qquad \quad \; \, P \in \varGamma (0). \end{aligned}$$\end{document}$$Let us next introduce the differential operators which are required to formulate our PDE. To begin, for fixed *t* and a function $\documentclass[12pt]{minimal}
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\begin{document}$$\eta : \varGamma (t) \rightarrow {\mathbb {R}}$$\end{document}$ we denote by $\documentclass[12pt]{minimal}
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\begin{document}$$\nabla _{\varGamma } \eta =(\underline{D}_1 \eta ,\ldots , \underline{D}_{n+1}\eta )$$\end{document}$ its tangential gradient. If $\documentclass[12pt]{minimal}
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\begin{document}$$\bar{\eta }$$\end{document}$ is an extension of $\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \nabla _{\varGamma } \eta (x) = \bigl ( I- \nu (x,t) \otimes \nu (x,t) \bigr ) \nabla \bar{\eta }(x), \quad x \in \varGamma (t). \end{aligned}$$\end{document}$$Furthermore, $\documentclass[12pt]{minimal}
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\begin{document}$$\varDelta _{\varGamma } \eta = \nabla _{\varGamma } \cdot \nabla _{\varGamma } \eta = \sum _{i=1}^{n+1} \underline{D}_i \underline{D}_i \eta $$\end{document}$ denotes the Laplace-Beltrami operator.
Next, for a smooth function $\documentclass[12pt]{minimal}
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\begin{document}$$\eta $$\end{document}$ on $\documentclass[12pt]{minimal}
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\begin{document}$$S_T$$\end{document}$ we define the material derivative of $\documentclass[12pt]{minimal}
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\begin{document}$$(x,t)=(\varPsi (P,t),t)$$\end{document}$ by $\documentclass[12pt]{minimal}
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\begin{document}$$\partial _t^{\bullet }\eta (x,t):= \frac{d}{dt} [ \eta (\varPsi (P,t),t)]$$\end{document}$. If $\documentclass[12pt]{minimal}
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\begin{document}$$\bar{\eta }$$\end{document}$ is an extension of $\documentclass[12pt]{minimal}
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\begin{document}$$\eta $$\end{document}$ to an open space-time neighbourhood, then$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \partial _t^{\bullet } \eta (x,t) = \bar{\eta }_t(x,t) + ({\varvec{v}}(x,t),\nabla \bar{\eta }(x,t)), \quad (x,t) \in S_T. \end{aligned}$$\end{document}$$Our numerical approach will be based on an implicit representation of $\documentclass[12pt]{minimal}
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\begin{document}$$\varGamma (t)$$\end{document}$, so that we suppose in what follows that there exists a smooth function $\documentclass[12pt]{minimal}
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\begin{document}$$\phi : \varOmega \times [0,T] \rightarrow {\mathbb {R}}$$\end{document}$ such that for $\documentclass[12pt]{minimal}
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\begin{document}$$0 \le t \le T$$\end{document}$$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \displaystyle \varGamma (t) = \lbrace x \in \varOmega \, | \, \phi (x,t) = 0 \rbrace \quad \text{ and } \quad \nabla \phi (x,t) \ne 0, \, x \in \varGamma (t). \end{aligned}$$\end{document}$$Here, $\documentclass[12pt]{minimal}
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\begin{document}$$\varOmega \subset {\mathbb {R}}^{n+1}$$\end{document}$ is a bounded domain with $\documentclass[12pt]{minimal}
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\begin{document}$$\varGamma (t) \subset \varOmega $$\end{document}$ for all $\documentclass[12pt]{minimal}
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\begin{document}$$t \in [0,T]$$\end{document}$. For later use we introduce for $\documentclass[12pt]{minimal}
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\begin{document}$$t \in [0,T], \, r>0$$\end{document}$ the sets$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} U_r(t):= \lbrace x \in \varOmega \, | \, | \phi (x,t) | <r \rbrace \quad \text{ and } \quad {\mathcal {U}}_{r,T}:= \bigcup _{t \in [0,T]} \bigl ( U_r(t) \times \lbrace t \rbrace \bigr ). \end{aligned}$$\end{document}$$In view of ([7](#Equ7){ref-type=""}) there exist $\documentclass[12pt]{minimal}
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\begin{document}$$\delta _0>0, 0< c_0 \le c_1, \, c_2>0$$\end{document}$ such that $\documentclass[12pt]{minimal}
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\begin{document}$$\overline{U_{\delta _0}(t)} \subset \varOmega , 0 \le t \le T$$\end{document}$ and$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \displaystyle c_0 \le | \nabla \phi (x,t) | \le c_1, \; | D^2 \phi (x,t) |, \, | \phi _t(x,t) |, \, | \phi _{tt}(x,t) | \le c_2, \quad (x,t) \in {\mathcal {U}}_{\delta _0,T}. \end{aligned}$$\end{document}$$
Extension {#Sec4}
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Our next aim is to extend functions defined on $\documentclass[12pt]{minimal}
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\begin{document}$$S_T$$\end{document}$ to a space-time neighbourhood. A common approach which is well suited to a description of $\documentclass[12pt]{minimal}
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\begin{document}$$\varGamma (t)$$\end{document}$ via the signed distance function consists in extending constantly in the normal direction. In what follows we shall introduce a suitable generalization to the case ([7](#Equ7){ref-type=""}). Consider for $\documentclass[12pt]{minimal}
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\begin{document}$$P \in \varGamma (0)$$\end{document}$ and $\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \gamma _{P,t}'(s) = \frac{\nabla \phi (\gamma _{P,t}(s),t)}{| \nabla \phi (\gamma _{P,t}(s),t) |^2}, \quad \gamma _{P,t}(0)= \varPsi (P,t). \end{aligned}$$\end{document}$$Using a compactness argument it can be shown that there exists $\documentclass[12pt]{minimal}
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\begin{document}$$0< \delta < \delta _0$$\end{document}$ so that the solution $\documentclass[12pt]{minimal}
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\begin{document}$$\gamma _{P,t}$$\end{document}$ of ([9](#Equ9){ref-type=""}) exists uniquely on $\documentclass[12pt]{minimal}
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\begin{document}$$(-\delta ,\delta )$$\end{document}$ uniformly in $\documentclass[12pt]{minimal}
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\begin{document}$$P \in \varGamma (0), t \in [0,T]$$\end{document}$. Thus we can define the smooth mapping $\documentclass[12pt]{minimal}
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\begin{document}$$F_t:\varGamma (0) \times (-\delta ,\delta ) \rightarrow {\mathbb {R}}^{n+1}$$\end{document}$ by$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} F_t(P,s):=\gamma _{P,t}(s), \quad P \in \varGamma (0), |s| < \delta . \end{aligned}$$\end{document}$$In view of the chain rule and ([9](#Equ9){ref-type=""}) we immediately see that $\documentclass[12pt]{minimal}
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\begin{document}$$\frac{d}{ds} \phi (\gamma _{P,t}(s),t) = 1$$\end{document}$, which implies that $\documentclass[12pt]{minimal}
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\begin{document}$$\phi (\gamma _{P,t}(s),t)=s, |s| < \delta $$\end{document}$ since $\documentclass[12pt]{minimal}
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\begin{document}$$\gamma _{P,t}(0)=\varPsi (P,t) \in \varGamma (t)$$\end{document}$. In particular, $\documentclass[12pt]{minimal}
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\begin{document}$$x=F_t(P,s)$$\end{document}$ yields that $\documentclass[12pt]{minimal}
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\begin{document}$$| \phi (x,t) | < \delta $$\end{document}$ and it is not difficult to verify that $\documentclass[12pt]{minimal}
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\begin{document}$$F_t$$\end{document}$ is a diffeomorphism of $\documentclass[12pt]{minimal}
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\begin{document}$$\varGamma (0) \times (-\delta ,\delta )$$\end{document}$ onto $\documentclass[12pt]{minimal}
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\begin{document}$$U_{\delta }(t)$$\end{document}$ for $\documentclass[12pt]{minimal}
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\begin{document}$$t \in [0,T]$$\end{document}$, whose inverse has the form$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} F_t^{-1}(x)= (p(x,t),\phi (x,t)), \quad x \in U_{\delta }(t). \end{aligned}$$\end{document}$$Here, $\documentclass[12pt]{minimal}
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\begin{document}$$p: {\mathcal {U}}_{\delta ,T} \rightarrow {\mathbb {R}}^{n+1}$$\end{document}$ satisfies $\documentclass[12pt]{minimal}
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\begin{document}$$p(x,t) \in \varGamma (0), x \in U_{\delta }(t)$$\end{document}$. Furthermore, since $\documentclass[12pt]{minimal}
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\begin{document}$$\phi (F_t(P,s),t)=s$$\end{document}$ we deduce from ([11](#Equ11){ref-type=""}) that$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \displaystyle p(x,t) = P, \quad \text{ if } x= F_t(P,s) \in U_{\delta }(t). \end{aligned}$$\end{document}$$The function $\documentclass[12pt]{minimal}
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\begin{document}$$\tilde{p}: {\mathcal {U}}_{\delta ,T} \rightarrow {\mathbb {R}}^{n+1}, \, \tilde{p}(x,t):= \varPsi (p(x,t),t)$$\end{document}$ then is smooth and satisfies $\documentclass[12pt]{minimal}
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\begin{document}$$\tilde{p}(x,t) \in \varGamma (t), 0 \le t \le T$$\end{document}$. In addition we claim that$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \displaystyle \tilde{p}(x,t) = x, \quad x \in \varGamma (t). \end{aligned}$$\end{document}$$To see this, let $\documentclass[12pt]{minimal}
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\begin{document}$$x \in \varGamma (t)$$\end{document}$, say $\documentclass[12pt]{minimal}
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\begin{document}$$x=\varPsi (P,t)=\gamma _{P,t}(0)=F_t(P,0)$$\end{document}$ for some $\documentclass[12pt]{minimal}
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\begin{document}$$P \in \varGamma (0)$$\end{document}$. Using ([12](#Equ12){ref-type=""}) with $\documentclass[12pt]{minimal}
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\begin{document}$$s=0$$\end{document}$ we deduce that$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \tilde{p}(x,t)= \varPsi (p(x,t),t) = \varPsi (P,t) = x, \end{aligned}$$\end{document}$$proving ([13](#Equ13){ref-type=""}). Let us next use $\documentclass[12pt]{minimal}
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\begin{document}$$\tilde{p}$$\end{document}$ in order to extend a function $\documentclass[12pt]{minimal}
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\begin{document}$$z: \overline{S_T} \rightarrow {\mathbb {R}}$$\end{document}$ to $\documentclass[12pt]{minimal}
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\begin{document}$${\mathcal {U}}_{\delta ,T}$$\end{document}$ by setting$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \displaystyle z^e(x,t):= z(\tilde{p}(x,t),t), \quad (x,t) \in {\mathcal {U}}_{\delta ,T}. \end{aligned}$$\end{document}$$Clearly, $\documentclass[12pt]{minimal}
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\begin{document}$$z^e(\cdot ,t)=z(\cdot ,t)$$\end{document}$ on $\documentclass[12pt]{minimal}
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\begin{document}$$\varGamma (t)$$\end{document}$ by ([13](#Equ13){ref-type=""}). Moreover, ([12](#Equ12){ref-type=""}) implies for $\documentclass[12pt]{minimal}
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\begin{document}$$P \in \varGamma (0), |s| < \delta $$\end{document}$$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} z^e(F_t(P,s),t) = z \bigl ( \tilde{p}(F_t(P,s),t),t \bigr )= z \bigl (\varPsi (p(F_t(P,s),t),t),t \bigr ) = z(\varPsi (P,t),t), \end{aligned}$$\end{document}$$from which we obtain by differentiating with respect to *s* and using ([9](#Equ9){ref-type=""}), ([10](#Equ10){ref-type=""}) that$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \displaystyle \bigl ( \nabla z^e(x,t),\nabla \phi (x,t) \bigr ) = 0, \quad (x,t) \in {\mathcal {U}}_{\delta ,T}. \end{aligned}$$\end{document}$$
### Lemma 1 {#FPar1}
Let $\documentclass[12pt]{minimal}
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\begin{document}$$z^e$$\end{document}$ be defined by ([14](#Equ14){ref-type=""}). Then we have for $\documentclass[12pt]{minimal}
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\begin{document}$$t \in [0,T], \, 0< r < \delta $$\end{document}$ and $\documentclass[12pt]{minimal}
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\begin{document}$$| \alpha | =k \in \lbrace 0,1,2 \rbrace $$\end{document}$:$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \Vert D_x^{\alpha } z^e(\cdot ,t) \Vert _{L^2(U_r(t))}\le & {} C \sqrt{r} \Vert z(\cdot ,t) \Vert _{H^k(\varGamma (t))}; \end{aligned}$$\end{document}$$ $$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \Vert D_x^{\alpha } z^e_t(\cdot ,t) \Vert _{L^2(U_r(t))}\le & {} C \sqrt{r} \bigl ( \Vert \partial ^{\bullet }_t z (\cdot ,t) \Vert _{H^k(\varGamma (t))} + \Vert z(\cdot ,t) \Vert _{H^{k+1}(\varGamma (t))} \bigr ). \end{aligned}$$\end{document}$$
### Proof {#FPar2}
Let us recall that $\documentclass[12pt]{minimal}
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\begin{document}$$F_t$$\end{document}$ is a diffeomorphism from $\documentclass[12pt]{minimal}
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\begin{document}$$\varGamma (0) \times (-r,r)$$\end{document}$ onto $\documentclass[12pt]{minimal}
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\begin{document}$$U_r(t)$$\end{document}$ while $\documentclass[12pt]{minimal}
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\begin{document}$$\varPsi (\cdot ,t)$$\end{document}$ is a diffeomorphism from $\documentclass[12pt]{minimal}
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\begin{document}$$\varGamma (0)$$\end{document}$ onto $\documentclass[12pt]{minimal}
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\begin{document}$$\varGamma (t)$$\end{document}$. We deduce from ([12](#Equ12){ref-type=""}) and the definition of $\documentclass[12pt]{minimal}
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\begin{document}$$\tilde{p}$$\end{document}$ that $\documentclass[12pt]{minimal}
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\begin{document}$$\tilde{p}(F_t(P,s),t)=\varPsi (P,t), P \in \varGamma (0), | s| < r$$\end{document}$ so that we obtain with the help of the transformation rule$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \displaystyle \int _{U_r(t)} | z^e(x,t) |^2 dx= & {} \int _{U_r(t)} | z(\tilde{p}(x,t),t) |^2 dx \le c \int _{-r}^r \int _{\varGamma (0)} | z(\varPsi (P,t),t) |^2 do_P ds \nonumber \\\le & {} c r \int _{\varGamma (t)} | z(Q,t) |^2 do_Q \end{aligned}$$\end{document}$$which is ([16](#Equ16){ref-type=""}) for $\documentclass[12pt]{minimal}
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\begin{document}$$k=0$$\end{document}$. Next, differentiating the identity $\documentclass[12pt]{minimal}
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\begin{document}$$\phi (\tilde{p}(x,t),t)=0$$\end{document}$ with respect to $\documentclass[12pt]{minimal}
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\begin{document}$$x_i$$\end{document}$ we infer that $\documentclass[12pt]{minimal}
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\begin{document}$$(\nabla \phi (\tilde{p}(x,t),t),\tilde{p}_{x_i}(x,t))=0,i=1,\ldots ,n+1$$\end{document}$. Hence we obtain from ([14](#Equ14){ref-type=""}) and ([6](#Equ6){ref-type=""}) that$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} z^e_{x_i}(x,t)= & {} \sum _{k=1}^{n+1} z^e_{x_k}(\tilde{p}(x,t),t) \tilde{p}_{k,x_i}(x,t) = \sum _{k=1}^{n+1} \underline{D}_k z(\tilde{p}(x,t),t) \tilde{p}_{k,x_i}(x,t), \end{aligned}$$\end{document}$$ $$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} z^e_{x_i x_j}(x,t)= & {} \sum _{k,l=1}^{n+1} \underline{D}_l \underline{D}_k z(\tilde{p}(x,t),t) \tilde{p}_{k,x_i}(x,t) \tilde{p}_{l,x_j}(x,t)\nonumber \\&+ \sum _{k=1}^{n+1} \underline{D}_k z(\tilde{p}(x,t),t) \tilde{p}_{k,x_i x_j}(x,t). \end{aligned}$$\end{document}$$Similarly, $\documentclass[12pt]{minimal}
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\begin{document}$$(\nabla \phi (\tilde{p}(x,t),t),\tilde{p}_t(x,t)) = - \phi _t(\tilde{p}(x,t),t)=(\nabla \phi (\tilde{p}(x,t),t), {\varvec{v}}(\tilde{p}(x,t),t))$$\end{document}$ by ([24](#Equ24){ref-type=""}) below, so that$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} z^e_t(x,t)= & {} z^e_t(\tilde{p}(x,t),t) + (\nabla z^e(\tilde{p}(x,t),t), \tilde{p}_t(x,t)) \nonumber \\= & {} \partial ^{\bullet }_t z(\tilde{p}(x,t),t) + \sum _{k=1}^{n+1} \underline{D}_k z(\tilde{p}(x,t),t) \bigl ( \tilde{p}_{k,t}(x,t) - {\varvec{v}}_k(\tilde{p}(x,t),t) \bigr ). \qquad \end{aligned}$$\end{document}$$Combining ([19](#Equ19){ref-type=""}), ([20](#Equ20){ref-type=""}) with the argument in ([18](#Equ18){ref-type=""}) we obtain ([16](#Equ16){ref-type=""}). The estimate ([17](#Equ17){ref-type=""}) follows in a similar way if one starts from ([21](#Equ21){ref-type=""}). $\documentclass[12pt]{minimal}
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\begin{document}$$\square $$\end{document}$
Let us next extend the surface differential operators $\documentclass[12pt]{minimal}
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\begin{document}$$\nabla _{\varGamma }$$\end{document}$ and $\documentclass[12pt]{minimal}
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\begin{document}$$\partial ^{\bullet }_t$$\end{document}$. By reversing the orientation of $\documentclass[12pt]{minimal}
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\begin{document}$$\varGamma (t)$$\end{document}$ if necessary we may assume that the functions $\documentclass[12pt]{minimal}
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\begin{document}$$\nu : {\mathcal {U}}_{\delta ,T} \rightarrow {\mathbb {R}}^{n+1}$$\end{document}$, $\documentclass[12pt]{minimal}
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\begin{document}$$V: {\mathcal {U}}_{\delta ,T} \rightarrow {\mathbb {R}}$$\end{document}$ defined by$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \nu (x,t):= \frac{ \nabla \phi (x,t)}{| \nabla \phi (x,t) |}, \quad V(x,t):= - \frac{\phi _t(x,t)}{| \nabla \phi (x,t) |}, \quad (x,t) \in {\mathcal {U}}_{\delta ,T} \end{aligned}$$\end{document}$$are extensions of the unit normal and the normal velocity respectively. In particular, we define for a function $\documentclass[12pt]{minimal}
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\begin{document}$$\eta \in C^1(U_{\delta }(t))$$\end{document}$ its Eulerian tangential gradient by$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \displaystyle \nabla _{\phi } \eta (x):= \bigl ( I - \nu (x,t) \otimes \nu (x,t) \bigr ) \nabla \eta (x), \quad x \in U_{\delta }(t) \end{aligned}$$\end{document}$$and remark that $\documentclass[12pt]{minimal}
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\begin{document}$$(\nabla _{\phi } \eta )_{| \varGamma (t)} = \nabla _{\varGamma } [\eta _{| \varGamma (t)}]$$\end{document}$. Furthermore, it follows from Lemma 2 in \[[@CR10]\] that for $\documentclass[12pt]{minimal}
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\begin{document}$$\eta \in C^1_0(\varOmega )$$\end{document}$ with $\documentclass[12pt]{minimal}
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\begin{document}$$\text{ supp }\eta \subset U_{\delta }(t)$$\end{document}$$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \displaystyle \int _{\varOmega } \nabla _{\phi } \eta \, | \nabla \phi | = - \int _{\varOmega } \eta H \nu \, | \nabla \phi |, \quad \text{ where } H= - \nabla \cdot \nu . \end{aligned}$$\end{document}$$Note that $\documentclass[12pt]{minimal}
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\begin{document}$$H_{| \varGamma (t)}$$\end{document}$ is the mean curvature of $\documentclass[12pt]{minimal}
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\begin{document}$$\varGamma (t)$$\end{document}$.
Let us also extend the velocity field $\documentclass[12pt]{minimal}
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\begin{document}$${\varvec{v}}$$\end{document}$ to $\documentclass[12pt]{minimal}
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\begin{document}$${\mathcal {U}}_{\delta ,T}$$\end{document}$. We first extend its tangential part by setting$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} {\tilde{\mathbf{v}}_{\tau }}(x,t):= (I - \nu (x,t) \otimes \nu (x,t)) {\varvec{v_\tau }}^e(x,t), \quad (x,t) \in {\mathcal {U}}_{\delta ,T}. \end{aligned}$$\end{document}$$In view of ([3](#Equ3){ref-type=""}) the function $\documentclass[12pt]{minimal}
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\begin{document}$${\varvec{v}}(x,t):= V(x,t) \nu (x,t) + {\tilde{\mathbf{v}}_{\tau }}(x,t)$$\end{document}$ extends the given velocity field from $\documentclass[12pt]{minimal}
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\begin{document}$$\overline{S_T}$$\end{document}$ to $\documentclass[12pt]{minimal}
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\begin{document}$${\mathcal {U}}_{\delta ,T}$$\end{document}$ and satisfies$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \displaystyle \phi _t + ( {\varvec{v}}, \nabla \phi ) =0 \quad \text{ in } {\mathcal {U}}_{\delta ,T}. \end{aligned}$$\end{document}$$In particular, we can use the extended velocity $\documentclass[12pt]{minimal}
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\begin{document}$${\varvec{v}}$$\end{document}$ to define the material derivative for a function $\documentclass[12pt]{minimal}
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\begin{document}$$\eta $$\end{document}$ on $\documentclass[12pt]{minimal}
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\begin{document}$${\mathcal {U}}_{\delta ,T}$$\end{document}$ by setting$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \partial _t^{\bullet } \eta (x,t):= \eta _t(x,t) + ({\varvec{v}}(x,t),\nabla \eta (x,t)), \quad (x,t) \in {\mathcal {U}}_{\delta ,T}. \end{aligned}$$\end{document}$$
Weak formulation and numerical scheme {#Sec5}
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Phase field approach {#Sec6}
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Consider for $\documentclass[12pt]{minimal}
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\begin{document}$$0< \epsilon < \frac{2 \delta }{\pi }$$\end{document}$ the function$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \rho (x,t):= g \left( \frac{\phi (x,t)}{\epsilon } \right) , \end{aligned}$$\end{document}$$where $\documentclass[12pt]{minimal}
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\begin{document}$$g \in C^{1,1}({\mathbb {R}})$$\end{document}$ is given by$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} g(r)= \left\{ \begin{array}{ll} \cos ^2(r), &{}\quad | r | \le \frac{\pi }{2}, \\ 0, &{}\quad |r| > \frac{\pi }{2}. \end{array} \right. \end{aligned}$$\end{document}$$Note that $\documentclass[12pt]{minimal}
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\begin{document}$$\text{ supp }[\rho (\cdot ,t)] = \overline{U_{\frac{\epsilon \pi }{2}}(t)} \subset U_{\delta }(t)$$\end{document}$. Furthermore, we obtain from the definition of $\documentclass[12pt]{minimal}
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\begin{document}$$\nabla _{\phi }$$\end{document}$ and ([24](#Equ24){ref-type=""})$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \nabla _{\phi } \rho= & {} \frac{1}{\epsilon } g'\left( \frac{\phi }{\epsilon }\right) \nabla _{\phi } \phi =0, \end{aligned}$$\end{document}$$ $$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \partial _t^{\bullet } \rho= & {} \frac{1}{\epsilon } g'\left( \frac{\phi }{\epsilon }\right) \bigl ( \phi _t + ({\varvec{v}},\nabla \phi ) \bigr )=0. \end{aligned}$$\end{document}$$The phase field function $\documentclass[12pt]{minimal}
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\begin{document}$$\rho $$\end{document}$ allows us to approximate the integration over a surface $\documentclass[12pt]{minimal}
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\begin{document}$$\varGamma (t)$$\end{document}$ in terms of a volume integral over the diffuse interface. More precisely, for fixed $\documentclass[12pt]{minimal}
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\begin{document}$$t \in [0,T]$$\end{document}$, the coarea formula implies for $\documentclass[12pt]{minimal}
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\begin{document}$$\eta \in L^1(\varOmega )$$\end{document}$$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \int _{\varOmega } \eta \, \rho (\cdot ,t) \, | \nabla \phi (\cdot ,t) | \, dx = \int _{- \frac{\epsilon \pi }{2}}^{ \frac{\epsilon \pi }{2}} g \left( \frac{s}{\epsilon } \right) \int _{\lbrace \phi (\cdot ,t)=s \rbrace } \eta \, d {\mathcal {H}}^n ds \approx \frac{\epsilon \pi }{2} \int _{\lbrace \phi (\cdot ,t)=0 \rbrace } \eta \, d {\mathcal {H}}^n \end{aligned}$$\end{document}$$for small $\documentclass[12pt]{minimal}
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\begin{document}$$\epsilon >0$$\end{document}$, so that we can view $\documentclass[12pt]{minimal}
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\begin{document}$$\frac{2}{\epsilon \pi } \int _{\varOmega } \eta \, \rho (\cdot ,t) \, | \nabla \phi (\cdot ,t) | \, dx$$\end{document}$ as an approximation of $\documentclass[12pt]{minimal}
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\begin{document}$$\int _{\varGamma (t)} \eta \, d {\mathcal {H}}^n$$\end{document}$. This formula explains the appearance of the weight $\documentclass[12pt]{minimal}
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\begin{document}$$\rho (\cdot ,t) \, | \nabla \phi (\cdot ,t) |$$\end{document}$ in subsequent volume integrals.
In what follows we shall make use of the following continuity properties of $\documentclass[12pt]{minimal}
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\begin{document}$$\rho $$\end{document}$.
### Lemma 2 {#FPar3}
Let $\documentclass[12pt]{minimal}
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\begin{document}$$s,t \in [0,T]$$\end{document}$ with $\documentclass[12pt]{minimal}
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\begin{document}$$| s-t | < \frac{\pi }{4 c_2} \epsilon $$\end{document}$, $\documentclass[12pt]{minimal}
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\begin{document}$$c_2$$\end{document}$ as in ([8](#Equ8){ref-type=""}). Then $\documentclass[12pt]{minimal}
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\begin{document}$$\text{ supp }[ \rho (\cdot ,s)] \subset U_{\frac{3 \epsilon \pi }{4}}(t)$$\end{document}$ and$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} | \rho (\cdot ,t) - \rho (\cdot ,s) |\le & {} C \frac{|t-s|}{\epsilon } \sqrt{\rho (\cdot ,t)} + C \frac{(t-s)^2}{\epsilon ^2} \chi _{U_{\frac{3\epsilon \pi }{4}}(t)} \quad \text{ in } \varOmega ; \end{aligned}$$\end{document}$$ $$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} | \rho _t(\cdot ,t) - \rho _t(\cdot ,s) |\le & {} C \frac{|t-s|}{\varepsilon ^2} \chi _{U_{\frac{3\epsilon \pi }{4}}(t)} \quad \text{ in } \varOmega . \end{aligned}$$\end{document}$$
### Proof {#FPar4}
Let $\documentclass[12pt]{minimal}
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\begin{document}$$s, t \in [0,T]$$\end{document}$ with $\documentclass[12pt]{minimal}
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\begin{document}$$| s-t| < \frac{\pi }{4 c_2} \epsilon $$\end{document}$ and $\documentclass[12pt]{minimal}
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\begin{document}$$x \in \text{ supp }[ \rho (\cdot ,s)]= \overline{U_{\frac{\epsilon \pi }{2}}(s)}$$\end{document}$. Using the mean value theorem and ([8](#Equ8){ref-type=""}) we then have$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} | \phi (x, t)| \le | \phi (x,s)| + | \phi _t(x,\xi ) | \, | t - s| \le \frac{\epsilon \pi }{2}+ c_2 | t-s| < \frac{3 \epsilon \pi }{4}, \end{aligned}$$\end{document}$$i.e. $\documentclass[12pt]{minimal}
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\begin{document}$$x \in U_{\frac{3 \epsilon \pi }{4}}(t)$$\end{document}$. In order to prove ([27](#Equ27){ref-type=""}) and ([28](#Equ28){ref-type=""}) we first observe that it is enough to verify the estimates for $\documentclass[12pt]{minimal}
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\begin{document}$$x \in U_{\frac{3\epsilon \pi }{4}}(t)$$\end{document}$ in view of what we have just shown. There exists $\documentclass[12pt]{minimal}
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\begin{document}$$\xi $$\end{document}$ between *s* and *t* such that$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} | \rho (x,t) - \rho (x,s) |= & {} | \rho _t(x,\xi ) | \, | t-s| = \frac{1}{\epsilon } | \phi _t(x,\xi ) | \, \left| g'\left( \frac{\phi (x,\xi )}{\varepsilon }\right) \right| \, | t-s| \nonumber \\\le & {} \frac{c_2 |t-s|}{\epsilon } \left| g'\left( \frac{\phi (x,\xi )}{\varepsilon }\right) \right| \end{aligned}$$\end{document}$$by ([8](#Equ8){ref-type=""}). Furthermore, since$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} g'(r)= \left\{ \begin{array}{ll} -2 \sin (r) \cos (r), &{}\quad | r | \le \frac{\pi }{2}, \\ 0, &{}\quad |r| > \frac{\pi }{2} \end{array} \right. \end{aligned}$$\end{document}$$we see immediately that$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \displaystyle | g'(r) | \le 2 \sqrt{g(r)}, \quad | g'(r) - g'(\tilde{r})| \le 2 | r - \tilde{r}|, \quad r, \tilde{r} \in {\mathbb {R}}. \end{aligned}$$\end{document}$$As a result,$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \left| g'\left( \frac{\phi (x,\xi )}{\varepsilon }\right) \right| \le \left| g'\left( \frac{\phi (x,t)}{\varepsilon }\right) \right| + \frac{2}{\epsilon } | \phi (x,\xi ) - \phi (x,t)| \le 2 \sqrt{\rho (x,t)} + \frac{2 c_2 |t-s| }{\epsilon }. \end{aligned}$$\end{document}$$Inserting this bound into ([29](#Equ29){ref-type=""}) yields ([27](#Equ27){ref-type=""}). Finally, using again ([30](#Equ30){ref-type=""}) and ([8](#Equ8){ref-type=""}) we obtain for $\documentclass[12pt]{minimal}
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\begin{document}$$x \in U_{\frac{3\epsilon \pi }{4}}(t)$$\end{document}$$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} | \rho _t(x,t) - \rho _t(x,s) |\le & {} \frac{1}{\epsilon } \left| g'\left( \frac{\phi (x,t)}{\epsilon } \right) - g'\left( \frac{\phi (x,s)}{\epsilon } \right) \right| \, | \phi _t(x,t) | \\&\quad + \frac{1}{\varepsilon } \left| g'\left( \frac{\phi (x,s)}{\epsilon } \right) \right| \, | \phi _t(x,t) - \phi _t(x,s)| \\\le & {} \frac{C}{\epsilon ^2} | \phi (x,t) - \phi (x,s) | + \frac{C}{\varepsilon } | \phi _t(x,t) - \phi _t(x,s) | \le \frac{C}{\varepsilon ^2} | t-s|. \end{aligned}$$\end{document}$$$\documentclass[12pt]{minimal}
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\begin{document}$$\square $$\end{document}$
### Remark 1 {#FPar5}
Our analysis will work for other profile functions *g* than the one chosen above as long as they satisfy $\documentclass[12pt]{minimal}
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\begin{document}$$g \in C^{1,1}({\mathbb {R}})$$\end{document}$ and $\documentclass[12pt]{minimal}
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\begin{document}$$g(r)>0$$\end{document}$ if $\documentclass[12pt]{minimal}
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\begin{document}$$|r| <R$$\end{document}$, $\documentclass[12pt]{minimal}
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\begin{document}$$g(r)=0$$\end{document}$ if $\documentclass[12pt]{minimal}
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\begin{document}$$|r| \ge R$$\end{document}$ as well as $\documentclass[12pt]{minimal}
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\begin{document}$$|g'(r)| \le C \sqrt{g(r)}$$\end{document}$ for suitable $\documentclass[12pt]{minimal}
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\begin{document}$$R,C>0$$\end{document}$. Profile functions with noncompact support have been used in \[[@CR4], [@CR22]\] and \[[@CR26]\]. However it is not obvious how to extend the analysis presented below to that setting.
Discretization {#Sec7}
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Suppose that *u* is a smooth solution of ([1](#Equ1){ref-type=""}). It is shown in Lemma [8](#FPar28){ref-type="sec"} of the "Appendix" that its extension $\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \partial _t^{\bullet } u^e + u^e \, \nabla _{\phi } \cdot {\varvec{v}}- \frac{1}{| \nabla \phi | } \nabla \cdot \bigl ( | \nabla \phi | \, \nabla u^e \bigr ) = f^e + \phi \, R \quad \text{ in } {\mathcal {U}}_{\delta ,T}, \end{aligned}$$\end{document}$$where$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \displaystyle R(x,t)= & {} \sum _{k,l=1}^{n+1} b_{lk}(x,t) \underline{D}_l \underline{D}_k u(\tilde{p}(x,t),t) + \sum _{k=1}^{n+1} c_k(x,t) \underline{D}_k u(\tilde{p}(x,t),t) \nonumber \\&\quad +\,d(x,t) u(\tilde{p}(x,t),t) \end{aligned}$$\end{document}$$and $\documentclass[12pt]{minimal}
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\begin{document}$$b_{lk},c_k,d$$\end{document}$ are smooth functions depending on $\documentclass[12pt]{minimal}
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\begin{document}$$\phi $$\end{document}$ and $\documentclass[12pt]{minimal}
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\begin{document}$${\varvec{v}}$$\end{document}$.
In order to associate with ([31](#Equ31){ref-type=""}) a suitable variational formulation we adapt an idea from \[[@CR16]\], which uses an Eulerian transport identity. More precisely, we infer with the help of Lemma 3 in \[[@CR10]\], ([26](#Equ26){ref-type=""}) and ([31](#Equ31){ref-type=""}) that for every $\documentclass[12pt]{minimal}
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\begin{document}$$\eta \in H^1(\varOmega )$$\end{document}$$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \frac{d}{dt}\int _\varOmega u^e\eta \, \rho \, | \nabla \phi |= & {} \int _\varOmega \bigl ( \partial _t^{\bullet }( u^e\eta \rho ) + u^e\eta \rho \, \nabla _{\phi } \cdot {\varvec{v}}\bigr ) | \nabla \phi |\nonumber \\= & {} \int _\varOmega \eta \bigl ( \partial _t^{\bullet } u^e + u^e \, \nabla _{\phi }\cdot {\varvec{v}}\bigr ) \rho \, | \nabla \phi |+ \int _\varOmega u^e \partial _t^{\bullet } \eta \, \rho \, | \nabla \phi |\nonumber \\= & {} \int _\varOmega \eta \, \nabla \cdot \bigl ( | \nabla \phi |\, \nabla u^e\bigr ) \rho + \int _\varOmega \eta \bigl ( f^e+\phi R \bigr ) \rho \, | \nabla \phi |\nonumber \\&+ \int _\varOmega u^e\partial _t^{\bullet } \eta \, \rho \, | \nabla \phi |=-\int _\varOmega (\nabla u^e, \nabla \eta ) \rho \, | \nabla \phi |+\int _\varOmega f^e\eta \, \rho \, | \nabla \phi |\nonumber \\&+\int _\varOmega u^e({\varvec{v}},\nabla \eta ) \rho \, | \nabla \phi |+\int _\varOmega \phi \, R \, \eta \, \rho \, | \nabla \phi |. \end{aligned}$$\end{document}$$Here, the last equality follows from integration by parts together with the fact that $\documentclass[12pt]{minimal}
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\begin{document}$$(\nabla u^e, \nabla \rho )= \frac{1}{\epsilon } g'\left( \frac{\phi }{\epsilon } \right) (\nabla u^e,\nabla \phi )=0$$\end{document}$ in view of ([15](#Equ15){ref-type=""}).
Let us first discretize with respect to time and denote by $\documentclass[12pt]{minimal}
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\begin{document}$$0=t_0< t_1< \cdots < t_M = T$$\end{document}$ a partioning of \[0, *T*\] with time steps $\documentclass[12pt]{minimal}
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\begin{document}$$\tau _m:=t_m - t_{m-1}$$\end{document}$ and $\documentclass[12pt]{minimal}
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\begin{document}$$\tau := \max _{m=1,\ldots ,M} \tau _m$$\end{document}$. For a function $\documentclass[12pt]{minimal}
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\begin{document}$$f=f(x,t)$$\end{document}$ we shall write $\documentclass[12pt]{minimal}
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\begin{document}$$f^m(x)=f(x,t_m)$$\end{document}$. Integrating ([33](#Equ33){ref-type=""}) with respect to $\documentclass[12pt]{minimal}
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\begin{document}$$t \in (t_{m-1},t_m)$$\end{document}$ we obtain for $\documentclass[12pt]{minimal}
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\begin{document}$$\eta \in H^1(\varOmega )$$\end{document}$$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned}&\int _\varOmega u^{e,m} \eta \rho ^m|\nabla \phi ^{m}|-\int _\varOmega u^{e,m-1} \eta \rho ^{m-1}| \nabla \phi ^{m-1}|+\int _{t_{m-1}}^{t_m}\int _\varOmega (\nabla u^e, \nabla \eta ) \rho \, | \nabla \phi |\nonumber \\&\quad - \int _{t_{m-1}}^{t_m}\int _\varOmega u^e ({\varvec{v}},\nabla \eta ) \rho \, | \nabla \phi |= \int _{t_{m-1}}^{t_m}\int _\varOmega f^e\, \eta \, \rho \, | \nabla \phi |+ \int _{t_{m-1}}^{t_m}\int _\varOmega \phi \, R \, \eta \, \rho \, | \nabla \phi |.\nonumber \\ \end{aligned}$$\end{document}$$Under a suitable regularity assumption on *u* we have that $\documentclass[12pt]{minimal}
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\begin{document}$$| \phi \, R | \le C \epsilon $$\end{document}$ on $\documentclass[12pt]{minimal}
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\begin{document}$$\text{ supp } \rho $$\end{document}$ so that we neglect the corresponding term when now deriving the spatial discretization from ([34](#Equ34){ref-type=""}).
In what follows we assume that $\documentclass[12pt]{minimal}
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\begin{document}$$\varOmega $$\end{document}$ is polyhedral and consider a family $\documentclass[12pt]{minimal}
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\begin{document}$$({\mathcal {T}}_h)_{0 < h \le h_0}$$\end{document}$ of triangulations of $\documentclass[12pt]{minimal}
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\begin{document}$$\varOmega $$\end{document}$ with mesh size $\documentclass[12pt]{minimal}
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\begin{document}$$h= \max _{T \in {\mathcal {T}}_h}h_T, \; h_T=\text{ diam }(T)$$\end{document}$. We assume that the family is regular in the sense that there exists $\documentclass[12pt]{minimal}
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\begin{document}$$\sigma >0$$\end{document}$ with$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} r_T \ge \sigma h_T \qquad \forall T \in {\mathcal {T}}_h \quad \forall 0 < h \le h_0, \end{aligned}$$\end{document}$$where $\documentclass[12pt]{minimal}
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\begin{document}$$r_T$$\end{document}$ is the radius of the largest ball contained in *T*. Let us denote by $\documentclass[12pt]{minimal}
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\begin{document}$${\mathcal {N}}_h$$\end{document}$ the set of vertices of the triangulation $\documentclass[12pt]{minimal}
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\begin{document}$${\mathcal {T}}_h$$\end{document}$. In order to formulate our scheme we require a second phase field function with a slightly larger support, namely$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \tilde{\rho }(x,t)=g \left( \frac{\phi (x,t)}{2 \epsilon } \right) , \quad 0<\epsilon < \frac{\delta }{\pi }. \end{aligned}$$\end{document}$$For $\documentclass[12pt]{minimal}
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\begin{document}$$0 \le m \le M$$\end{document}$ we then define$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} {\mathcal {T}}^m_h:= \lbrace T \in {\mathcal {T}}_h \, | \, \tilde{\rho }^m(x)>0 \text{ for } \text{ some } x \in T \cap {\mathcal {N}}_h \rbrace \quad \text{ and } \quad D^m_h:= \bigcup _{T \in {\mathcal {T}}^m_h} T \end{aligned}$$\end{document}$$as well as the finite element space$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} V^m_h:= \lbrace v_h \in C^0(D^m_h) \, | \, v_{h|T} \text{ is } \text{ a } \text{ linear } \text{ polynomial } \text{ on } \text{ each } T \in {\mathcal {T}}^m_h \rbrace . \end{aligned}$$\end{document}$$We denote by $\documentclass[12pt]{minimal}
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\begin{document}$$I^m_h: C^0(D^m_h) \rightarrow V^m_h$$\end{document}$ the standard Lagrange interpolation operator, i.e. $\documentclass[12pt]{minimal}
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\begin{document}$$[I^m_h f](x)=f(x), x \in D^m_h \cap {\mathcal {N}}_h$$\end{document}$. Note that $\documentclass[12pt]{minimal}
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\begin{document}$$D^m_h = \text{ supp } I^m_h \tilde{\rho }^m$$\end{document}$.
### Lemma 3 {#FPar6}
Suppose that$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \displaystyle h \le \frac{\cos ^2\left( \frac{3 \pi }{8}\right) }{2 c_1} \epsilon , \, \tau \le \frac{\cos ^2\left( \frac{3 \pi }{8}\right) }{2 c_2} \epsilon . \end{aligned}$$\end{document}$$Then$\documentclass[12pt]{minimal}
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\begin{document}$$U_{\frac{3 \epsilon \pi }{4}}(t) \subset D^m_h \subset U_{\frac{3 \epsilon \pi }{2}}(s)$$\end{document}$ for all $\documentclass[12pt]{minimal}
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\begin{document}$$s, t \in [\max (t_{m-1},0),\min (t_{m+1},T)], 0 \le m \le M$$\end{document}$;$\documentclass[12pt]{minimal}
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\begin{document}$$[I^m_h \tilde{\rho }^m](x) \ge \frac{1}{2} \cos ^2\left( \frac{3 \pi }{8}\right) , \, x \in U_{\frac{3 \epsilon \pi }{4}}(t_m), 0 \le m \le M$$\end{document}$.
### Proof {#FPar7}
a\) Let $\documentclass[12pt]{minimal}
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\begin{document}$$x \in D^m_h$$\end{document}$, so that there exists $\documentclass[12pt]{minimal}
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\begin{document}$$y \in {\mathcal {N}}_h$$\end{document}$ such that $\documentclass[12pt]{minimal}
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\begin{document}$$|y-x| \le h$$\end{document}$ and $\documentclass[12pt]{minimal}
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\begin{document}$$\tilde{\rho }^m(y)>0$$\end{document}$. Hence $\documentclass[12pt]{minimal}
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\begin{document}$$| \phi ^m(y) | < \epsilon \pi $$\end{document}$ and the mean value theorem together with ([8](#Equ8){ref-type=""}) yields for $\documentclass[12pt]{minimal}
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\begin{document}$$s \in [\max (t_{m-1},0),\min (t_{m+1},T)]$$\end{document}$$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} | \phi (x,s) |\le & {} | \phi (x,s) - \phi ^m(x) | + | \phi ^m(x) - \phi ^m(y) | + | \phi ^m(y) | \\< & {} | \phi _t(x,\xi ) | \, | s-t_m| + | \nabla \phi ^m(\eta ) | \, | x-y | + \epsilon \pi \\\le & {} c_2 \tau + c_1 h + \epsilon \pi \le \cos ^2\left( \frac{3 \pi }{8}\right) \epsilon + \epsilon \pi \le \frac{3 \epsilon \pi }{2} \end{aligned}$$\end{document}$$in view of ([36](#Equ36){ref-type=""}). Hence, $\documentclass[12pt]{minimal}
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\begin{document}$$x \in U_{\frac{3 \epsilon \pi }{2}}(s)$$\end{document}$. Next, let $\documentclass[12pt]{minimal}
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\begin{document}$$x \in U_{\frac{3 \epsilon \pi }{4}}(t)$$\end{document}$ for some $\documentclass[12pt]{minimal}
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\begin{document}$$t \in [\max (t_{m-1},0),\min (t_{m+1},T)]$$\end{document}$. Then $\documentclass[12pt]{minimal}
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\begin{document}$$\tilde{\rho }(x,t) \ge \cos ^2(\frac{3 \pi }{8})$$\end{document}$ and we obtain similarly as above$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} {[}I^m_h \tilde{\rho }^m{]}(x)\ge & {} \tilde{\rho }(x,t) - | \tilde{\rho }(x,t) - \tilde{\rho }^m(x)| - | \tilde{\rho }^m(x) - {[}I^m_h \tilde{\rho }^m{]}(x) | \\\ge & {} \cos ^2\left( \frac{3 \pi }{8}\right) - | \tilde{\rho }_t(x,\xi ) | \, | t - t_m| - h \max _{y \in \overline{U_{\delta }(t_m)}} | \nabla \tilde{\rho }^m(y) | \\\ge & {} \cos ^2\left( \frac{3 \pi }{8}\right) - c_2 \frac{\tau }{2 \epsilon } - c_1 \frac{h}{ 2 \epsilon } \ge \frac{1}{2} \cos ^2\left( \frac{3 \pi }{8}\right) . \end{aligned}$$\end{document}$$In particular, $\documentclass[12pt]{minimal}
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\begin{document}$$[I^m_h \tilde{\rho }^m](x)>0$$\end{document}$, so that $\documentclass[12pt]{minimal}
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\begin{document}$$x \in D^m_h$$\end{document}$. Using the above inequality for $\documentclass[12pt]{minimal}
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\begin{document}$$t=t_m$$\end{document}$ implies b). $\documentclass[12pt]{minimal}
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\begin{document}$$\square $$\end{document}$
Our finite element approximation of ([1](#Equ1){ref-type=""}), ([2](#Equ2){ref-type=""}) now reads: For $\documentclass[12pt]{minimal}
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\begin{document}$$m=1,2,\ldots ,M$$\end{document}$ find $\documentclass[12pt]{minimal}
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\begin{document}$$u^m_h \in V^m_h$$\end{document}$ such that for all $\documentclass[12pt]{minimal}
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\begin{document}$$v_h \in V^m_h$$\end{document}$$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned}&\int _{\varOmega } u^m_h \, v_h \, \rho ^m \, | \nabla \phi ^m | - \int _{\varOmega } u^{m-1}_h \, v_h \, \rho ^{m-1} \, | \nabla \phi ^{m-1} | + \tau _m \, \int _{\varOmega } (\nabla u^m_h, \nabla v_h) \, \rho ^m \, | \nabla \phi ^m | \nonumber \\&\qquad - \tau _m \, \int _{\varOmega } u^m_h \, ({\varvec{v}}^m,\nabla v_h) \, \rho ^m \, | \nabla \phi ^m | + \gamma \tau _m^2 \, \int _{\varOmega } I^m_h \tilde{\rho }^m ( \nabla u^m_h,\nabla v_h)\nonumber \\&\quad = \tau _m \, \int _{\varOmega } f^{e,m} \, v_h \, \rho ^m \, | \nabla \phi ^m |. \end{aligned}$$\end{document}$$Here, $\documentclass[12pt]{minimal}
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\begin{document}$$u^0_h \in V^0_h$$\end{document}$ is defined as an $\documentclass[12pt]{minimal}
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\begin{document}$$L^2$$\end{document}$ projection of $\documentclass[12pt]{minimal}
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\begin{document}$$u_0^e(x):=u_0(\tilde{p}(x,0)), x \in U_{\delta }(0)$$\end{document}$, more precisely$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \displaystyle \int _{D^0_h} u^0_h \, v_h = \int _{D^0_h} u_0^e \, v_h \qquad \forall v_h \in V^0_h. \end{aligned}$$\end{document}$$Furthermore, $\documentclass[12pt]{minimal}
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\begin{document}$$f^{e,m}(x):=f(\tilde{p}(x,t_m),t_m), x \in U_{\delta }(t_m), 1 \le m \le M$$\end{document}$. The parameter $\documentclass[12pt]{minimal}
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\begin{document}$$\gamma >0$$\end{document}$ will be chosen in such a way as to ensure existence and stability for the scheme, see Lemma [5](#FPar11){ref-type="sec"} and Theorem [1](#FPar16){ref-type="sec"} below.
### Remark 2 {#FPar8}
a\) Lemma [3](#FPar6){ref-type="sec"} a) implies that $\documentclass[12pt]{minimal}
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\begin{document}$$\text{ supp } \rho ^m, \text{ supp } \rho ^{m-1} \subset D^m_h = \text{ supp } I^m_h \tilde{\rho }^m$$\end{document}$, so that all integrals appearing in ([37](#Equ37){ref-type=""}) are taken only over $\documentclass[12pt]{minimal}
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\begin{document}$$D^m_h$$\end{document}$. In particular, if $\documentclass[12pt]{minimal}
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\begin{document}$$f \equiv 0$$\end{document}$ we see from the choice $\documentclass[12pt]{minimal}
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\begin{document}$$v_h \equiv 1$$\end{document}$ on $\documentclass[12pt]{minimal}
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\begin{document}$$D^m_h$$\end{document}$ that the scheme is mass conserving in the sense that$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \int _\varOmega u^m_h \, \rho ^m \, | \nabla \phi ^m | = \int _\varOmega u^0_h \, \rho ^0 \, | \nabla \phi ^0|, \qquad m=1,\ldots ,M. \end{aligned}$$\end{document}$$b) The term $\documentclass[12pt]{minimal}
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\begin{document}$$\gamma \tau _m^2 \, \int _{\varOmega } I^m_h \tilde{\rho }^m ( \nabla u^m_h,\nabla v_h)$$\end{document}$ introduces artificial diffusion into the scheme and will play a crucial role in our analyis. A different form of stabilization is used in \[[@CR16]\], Section 2.5.
c\) Unlike the schemes introduced in \[[@CR16]\] our method is not fully practical because we assume that the integrals are evaluated exactly. In Sect. [6](#Sec10){ref-type="sec"} we shall follow \[[@CR16]\] in using numerical integration to obtain a fully practical scheme. A nice feature of the resulting method is that the evolution of the hypersurfaces is tracked in a simple way via the evaluation of $\documentclass[12pt]{minimal}
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\begin{document}$$\rho $$\end{document}$.
In what follows we shall be concerned with the existence, stability and error bounds for ([37](#Equ37){ref-type=""}). The extension of our analysis to the fully practical method mentioned above is currently out of reach and left for future research. However, the test calculations in Sect. [6](#Sec10){ref-type="sec"} show that the parameter choices suggested by the analysis work well also for the fully practical scheme.
### Lemma 4 {#FPar9}
There exists $\documentclass[12pt]{minimal}
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\begin{document}$$0 < h_1 \le h_0$$\end{document}$ such that $\documentclass[12pt]{minimal}
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\begin{document}$$D^m_h$$\end{document}$ is connected for all $\documentclass[12pt]{minimal}
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\begin{document}$$0< h \le h_1$$\end{document}$ and $\documentclass[12pt]{minimal}
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\begin{document}$$0 \le m \le M$$\end{document}$.
### Proof {#FPar10}
To begin, we remark that there exists $\documentclass[12pt]{minimal}
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\begin{document}$$0<h_1 \le h_0$$\end{document}$ and $\documentclass[12pt]{minimal}
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\begin{document}$$\mu >0$$\end{document}$ only depending on $\documentclass[12pt]{minimal}
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\begin{document}$$\sigma , c_0,c_1, c_2$$\end{document}$ such that for every $\documentclass[12pt]{minimal}
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\begin{document}$$a \in {\mathcal {N}}_h \cap \overline{U_{\delta }(t)}$$\end{document}$ there exists a neighbour $\documentclass[12pt]{minimal}
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\begin{document}$$b \in {\mathcal {N}}_h$$\end{document}$ with$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} | \phi (a,t) - \phi (b,t) | \ge \mu h_T \qquad \text{ where } a,b, \in T \end{aligned}$$\end{document}$$for all $\documentclass[12pt]{minimal}
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\begin{document}$$t \in [0,T], 0 < h \le h_1$$\end{document}$. Since $\documentclass[12pt]{minimal}
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\begin{document}$$\varGamma (t_m)$$\end{document}$ is connected it is sufficient to show that for every $\documentclass[12pt]{minimal}
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\begin{document}$$y \in D^m_h$$\end{document}$ there exists $\documentclass[12pt]{minimal}
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\begin{document}$$z \in \varGamma (t_m)$$\end{document}$ and a path in $\documentclass[12pt]{minimal}
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\begin{document}$$D^m_h$$\end{document}$ connecting *y* to *z*. Let us fix $\documentclass[12pt]{minimal}
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\begin{document}$$y \in D^m_h$$\end{document}$, say $\documentclass[12pt]{minimal}
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\begin{document}$$y \in T$$\end{document}$, where $\documentclass[12pt]{minimal}
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\begin{document}$$\tilde{\rho }^m(x)>0$$\end{document}$ for some $\documentclass[12pt]{minimal}
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\begin{document}$$x \in T \cap {\mathcal {N}}_h$$\end{document}$. We assume w.l.o.g. that $\documentclass[12pt]{minimal}
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\begin{document}$$0< \phi ^m(x) < \epsilon \pi $$\end{document}$. In view of ([39](#Equ39){ref-type=""}) there exists a neighbour $\documentclass[12pt]{minimal}
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\begin{document}$$x_1 \in {\mathcal {N}}_h$$\end{document}$ of *x* such that $\documentclass[12pt]{minimal}
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\begin{document}$$\phi ^m(x_1) \le \phi ^m(x) - \mu h_{\tilde{T}}$$\end{document}$, where $\documentclass[12pt]{minimal}
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\begin{document}$$x,x_1 \in \tilde{T}$$\end{document}$. If $\documentclass[12pt]{minimal}
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\begin{document}$$\phi ^m(x_1) \le 0$$\end{document}$ then there is $\documentclass[12pt]{minimal}
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\begin{document}$$z \in [x,x_1]$$\end{document}$ with $\documentclass[12pt]{minimal}
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\begin{document}$$\phi ^m(z)=0$$\end{document}$. Hence, $\documentclass[12pt]{minimal}
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\begin{document}$$z \in \varGamma (t_m)$$\end{document}$ and the union of the segments \[*y*, *x*\] and \[*x*, *z*\] is a path in $\documentclass[12pt]{minimal}
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\begin{document}$$D^m_h$$\end{document}$ connecting *y* to *z*. If $\documentclass[12pt]{minimal}
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\begin{document}$$\phi ^m(x_1)>0$$\end{document}$, then $\documentclass[12pt]{minimal}
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\begin{document}$$\tilde{\rho }^m(x_1)>0$$\end{document}$ so that $\documentclass[12pt]{minimal}
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\begin{document}$$[x,x_1] \subset D^m_h$$\end{document}$ and we may repeat the above argument with *x* replaced by $\documentclass[12pt]{minimal}
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\begin{document}$$x_1$$\end{document}$ and so on, until we reach $\documentclass[12pt]{minimal}
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\begin{document}$$\varGamma (t_m)$$\end{document}$ in a finite number of steps. $\documentclass[12pt]{minimal}
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\begin{document}$$\square $$\end{document}$
### Lemma 5 {#FPar11}
(Existence) Let $\documentclass[12pt]{minimal}
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\begin{document}$$0 < h \le h_1$$\end{document}$. There exists $\documentclass[12pt]{minimal}
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\begin{document}$$\tau _0>0$$\end{document}$ such that the scheme ([37](#Equ37){ref-type=""}) has a unique solution $\documentclass[12pt]{minimal}
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\begin{document}$$u^m_h \in V^m_h$$\end{document}$ provided that $\documentclass[12pt]{minimal}
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\begin{document}$$0 < \tau \le \tau _0$$\end{document}$.
### Proof {#FPar12}
Since ([37](#Equ37){ref-type=""}) is equivalent to solving a linear system with a quadratic coefficient matrix, it is sufficient to prove that the following problem only has the trivial solution: find $\documentclass[12pt]{minimal}
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\begin{document}$$u_h \in V^m_h$$\end{document}$ such that for all $\documentclass[12pt]{minimal}
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\begin{document}$$v_h \in V^m_h$$\end{document}$$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned}&\int _{\varOmega } u_h \, v_h \, \rho ^m \, | \nabla \phi ^m | + \tau _m \int _{\varOmega } (\nabla u_h, \nabla v_h) \, \rho ^m \, | \nabla \phi ^m | - \tau _m \int _{\varOmega } u_h \, ({\varvec{v}}^m,\nabla v_h) \, \rho ^m \, | \nabla \phi ^m | \\&\quad + \gamma \tau _m^2 \, \int _{\varOmega } I^m_h \tilde{\rho }^m ( \nabla u_h, \nabla v_h) =0. \end{aligned}$$\end{document}$$Inserting $\documentclass[12pt]{minimal}
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\begin{document}$$v_h=u_h$$\end{document}$ we infer$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned}&\int _{\varOmega } (u_h)^2 \rho ^m \, | \nabla \phi ^m | + \tau _m \int _{\varOmega } | \nabla u_h |^2 \, \rho ^m \, | \nabla \phi ^m | + \gamma \tau _m^2 \, \int _{\varOmega } I^m_h \tilde{\rho }^m | \nabla u_h |^2 \\&\quad = \tau _m \int _{\varOmega } u_h \, ({\varvec{v}}^m,\nabla u_h) \, \rho ^m \, | \nabla \phi ^m | \le \tau _m \, \max _{x \in \overline{U_{\delta }(t_m)}} | {\varvec{v}}^m(x) | \, \int _\varOmega | u_h | \, | \nabla u_h | \, \rho ^m \, | \nabla \phi ^m | \\&\quad \le \frac{1}{2} \int _{\varOmega } (u_h)^2 \, \rho ^m \, | \nabla \phi ^m | + \frac{1}{2} \tau \, \left( \max _{x \in \overline{U_{\delta }(t_m)}} | {\varvec{v}}^m(x) |\right) ^2 \tau _m \int _{\varOmega } | \nabla u_h |^2 \, \rho ^m \, | \nabla \phi ^m |. \end{aligned}$$\end{document}$$If we choose $\documentclass[12pt]{minimal}
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\begin{document}$$\tau _0>0$$\end{document}$ so small that $\documentclass[12pt]{minimal}
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\begin{document}$$\frac{1}{2} \tau \, \bigl ( \max _{x \in \overline{U_{\delta }(t_m)}} | {\varvec{v}}^m(x) | \bigr )^2 \le 1$$\end{document}$ we deduce that$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \int _{\varOmega } (u_h)^2 \, \rho ^m \, | \nabla \phi ^m | = \int _{\varOmega } I^m_h \tilde{\rho }^m | \nabla u_h |^2=0, \end{aligned}$$\end{document}$$which implies that $\documentclass[12pt]{minimal}
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\begin{document}$$u_h \equiv 0$$\end{document}$ on $\documentclass[12pt]{minimal}
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\begin{document}$$\varGamma (t_m)$$\end{document}$ and $\documentclass[12pt]{minimal}
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\begin{document}$$\nabla u_h \equiv 0$$\end{document}$ in $\documentclass[12pt]{minimal}
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\begin{document}$$D^m_h$$\end{document}$. According to Lemma [4](#FPar9){ref-type="sec"}, $\documentclass[12pt]{minimal}
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\begin{document}$$D^m_h$$\end{document}$ is connected, so that we conclude that $\documentclass[12pt]{minimal}
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\begin{document}$$u_h \equiv 0$$\end{document}$. $\documentclass[12pt]{minimal}
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\begin{document}$$\square $$\end{document}$
Stability bound {#Sec8}
===============
The following lemma will be useful in estimating $\documentclass[12pt]{minimal}
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\begin{document}$$L^2$$\end{document}$-integrals that are not weighted by $\documentclass[12pt]{minimal}
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\begin{document}$$\rho $$\end{document}$.
Lemma 6 {#FPar13}
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There exists $\documentclass[12pt]{minimal}
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\begin{document}$$C\ge 0$$\end{document}$ such that for $\documentclass[12pt]{minimal}
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\begin{document}$$t \in [0,T]$$\end{document}$:$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \int _{U_{\frac{3 \epsilon \pi }{4}}(t)} f^2 \le C \int _\varOmega f^2 \rho (\cdot ,t) | \nabla \phi (\cdot ,t)| +C\varepsilon ^2\int _{U_{\frac{3 \epsilon \pi }{4}}(t)} | \nabla f |^2 \qquad \text{ for } \text{ all } f \in H^1(\varOmega ). \end{aligned}$$\end{document}$$
Remark 3 {#FPar14}
--------
Note that Lemma [3](#FPar6){ref-type="sec"} b) implies that$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \displaystyle \int _{U_{\frac{3 \epsilon \pi }{4}}(t_m)} | \nabla f |^2 \le \frac{2}{\cos ^2(\frac{3 \pi }{8})} \int _\varOmega I^m_h \tilde{\rho }^m | \nabla f |^2, \quad f \in H^1(\varOmega ), m=0,\ldots ,M. \end{aligned}$$\end{document}$$
Proof {#FPar15}
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We may assume that *f* is smooth, the general case then follows with the help of an approximation argument. Since $\documentclass[12pt]{minimal}
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\begin{document}$$F_t$$\end{document}$ is a diffeomorphism from $\documentclass[12pt]{minimal}
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\begin{document}$$\varGamma (0) \times (-\frac{3 \epsilon \pi }{4}, \frac{3 \epsilon \pi }{4})$$\end{document}$ onto $\documentclass[12pt]{minimal}
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\begin{document}$$U_{\frac{3 \epsilon \pi }{4}}(t)$$\end{document}$, the transformation rule yields$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \displaystyle c_1 \int _{U_{\frac{3\epsilon \pi }{4}}(t)} f(x)^2 dx \le \int _{-\frac{3 \varepsilon \pi }{4}}^{\frac{3 \varepsilon \pi }{4}}\int _{\varGamma (0)} f(F_t(P,s))^2 do_P ds \le c_2 \int _{U_{\frac{3\epsilon \pi }{4}}(t)} f(x)^2 dx. \end{aligned}$$\end{document}$$The definition of $\documentclass[12pt]{minimal}
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\begin{document}$$F_t$$\end{document}$ together with ([9](#Equ9){ref-type=""}) implies for $\documentclass[12pt]{minimal}
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\begin{document}$$|s|\le \frac{3 \varepsilon \pi }{4}$$\end{document}$, $\documentclass[12pt]{minimal}
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\begin{document}$$|\tilde{s} | \le \frac{\varepsilon \pi }{4}$$\end{document}$$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} f(F_t(P,s))= & {} f(F_t(P,\tilde{s}))+ \int _{\tilde{s}}^s \left( \nabla f(F_t(P,r)), \frac{\partial F_t}{\partial r}(P,r) \right) dr \\= & {} f(F_t(P,\tilde{s}))+\int _{\tilde{s}}^s \left( \nabla f(F_t(P,r)), \frac{\nabla \phi (F_t(P,r),t)}{|\nabla \phi (F_t(P,r),t)|^2}\right) dr \end{aligned}$$\end{document}$$and therefore$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} f(F_t(P,s))^2\le & {} 2 f(F_t(P,\tilde{s}))^2 + C \varepsilon \int _{-\frac{3 \varepsilon \pi }{4}}^{\frac{3 \varepsilon \pi }{4}} | \nabla f (F_t(p,r)) |^2 dr \\\le & {} C f(F_t(P,\tilde{s}))^2 \rho (F_t(P,\tilde{s}),t) +C\varepsilon \int _{-\frac{3 \varepsilon \pi }{4}}^{\frac{3 \varepsilon \pi }{4}} | \nabla f (F_t(p,r)) |^2 dr, \end{aligned}$$\end{document}$$since $\documentclass[12pt]{minimal}
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\begin{document}$$\rho (F_t(P,\tilde{s}),t) = \cos ^2 \bigl (\frac{\phi (F_t(P,\tilde{s}),t)}{\epsilon } \bigr ) = \cos ^2(\frac{\tilde{s}}{\epsilon }) \ge \cos ^2(\frac{\pi }{4}), | \tilde{s} | \le \frac{\epsilon \pi }{4}$$\end{document}$. Integrating with respect to $\documentclass[12pt]{minimal}
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\begin{document}$$P \in \varGamma (0), s \in (-\frac{3 \varepsilon \pi }{4},\frac{3 \varepsilon \pi }{4})$$\end{document}$ and recalling ([42](#Equ42){ref-type=""}) we obtain for $\documentclass[12pt]{minimal}
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\begin{document}$$|\tilde{s} | \le \frac{\varepsilon \pi }{4}$$\end{document}$$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \int _{U_{\frac{3 \epsilon \pi }{4}}(t)} f(x)^2 dx\le & {} C \epsilon \int _{\varGamma (0)} f(F_t(P,\tilde{s}))^2 \rho (F_t(P,\tilde{s}),t) do_P \\&+ C \epsilon ^2 \int _{-\frac{3 \varepsilon \pi }{4}}^{\frac{3 \varepsilon \pi }{4}} \int _{\varGamma (0)} | \nabla f (F_t(p,r)) |^2 do_P dr \\\le & {} C \epsilon \int _{\varGamma (0)} f(F_t(P,\tilde{s}))^2 \rho (F_t(P,\tilde{s}),t) do_P \\&+ C\varepsilon ^2 \int _{U_{\frac{3 \epsilon \pi }{4}}(t)} | \nabla f(x) |^2 dx. \end{aligned}$$\end{document}$$If we integrate with respect to $\documentclass[12pt]{minimal}
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\begin{document}$$\tilde{s} \in (-\frac{\varepsilon \pi }{4},\frac{\varepsilon \pi }{4})$$\end{document}$, divide by $\documentclass[12pt]{minimal}
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\begin{document}$$\epsilon $$\end{document}$ and recall ([8](#Equ8){ref-type=""}) we obtain the assertion. $\documentclass[12pt]{minimal}
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\begin{document}$$\square $$\end{document}$
It follows from Theorem 4.4 in \[[@CR8]\] (extended in a straightforward way to the case of a nontrivial *f*) that ([1](#Equ1){ref-type=""}), ([2](#Equ2){ref-type=""}) has a unique solution *u* which satisfies$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned}&\sup _{(0,T)} \Vert u(\cdot ,t) \Vert _{L^2(\varGamma (t))}^2 + \int _0^T \Vert \nabla _{\varGamma } u(\cdot ,t) \Vert _{L^2(\varGamma (t))}^2 dt \le c \left( \Vert u_0 \Vert _{L^2(\varGamma (0))}^2 \right. \\&\left. \quad + \int _0^T \Vert f(\cdot ,t) \Vert _{L^2(\varGamma (t))}^2 dt \right) . \end{aligned}$$\end{document}$$The following theorem gives a discrete version of this estimate in the phase field setting.
Theorem 1 {#FPar16}
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Suppose that ([36](#Equ36){ref-type=""}) holds. There exist $\documentclass[12pt]{minimal}
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\begin{document}$$\gamma _1 >0$$\end{document}$ and $\documentclass[12pt]{minimal}
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\begin{document}$$\tau _1 \le \tau _0$$\end{document}$ such that$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned}&\max _{m=1,\ldots ,M} \frac{2}{\epsilon \pi } \int _{\varOmega } (u^m_h)^2 \, \rho ^m \, | \nabla \phi ^m | + \, \sum _{m=1}^M \tau _m \, \frac{2}{\epsilon \pi } \int _{\varOmega } | \nabla u^m_h |^2 \rho ^m \, | \nabla \phi ^m | \\&\quad \le C \left( \int _{\varGamma (0)} (u_0)^2 + \sum _{m=1}^M \tau _m \int _{\varGamma (t_m)} (f^m)^2 \right) , \end{aligned}$$\end{document}$$provided that $\documentclass[12pt]{minimal}
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\begin{document}$$\gamma \ge \gamma _1$$\end{document}$ and $\documentclass[12pt]{minimal}
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\begin{document}$$\tau \le \min \bigl ( \tau _1, \epsilon ^2 \bigr ) $$\end{document}$.
Proof {#FPar17}
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Setting $\documentclass[12pt]{minimal}
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\begin{document}$$v_h=u^m_h$$\end{document}$ in ([37](#Equ37){ref-type=""}) we find after a straighforward calculation$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned}&\frac{1}{2}\int _\varOmega (u^m_h)^2 \, \rho ^m\, |\nabla \phi ^{m}|- \frac{1}{2}\int _\varOmega (u^{m-1}_h)^2 \, \rho ^{m-1}\, | \nabla \phi ^{m-1}|\nonumber \\&\qquad +\frac{1}{2}\int _\varOmega (u^m_h-u^{m-1}_h)^2 \, \rho ^{m-1}\, | \nabla \phi ^{m-1}|\nonumber \\&\qquad + \tau _m \int _\varOmega |\nabla u^m_h|^2 \, \rho ^m\, |\nabla \phi ^{m}|+ \gamma \tau _m^2 \int _\varOmega I^m_h \tilde{\rho }^m | \nabla u^m_h|^2 \nonumber \\&\quad = - \frac{1}{2}\int _\varOmega (u^m_h)^2 \, (\rho ^m-\rho ^{m-1}) \, | \nabla \phi ^{m-1}|+ \frac{1}{2}\int _\varOmega (u^m_h)^2 \, \rho ^m\, (| \nabla \phi ^{m-1}|-|\nabla \phi ^{m}|)\nonumber \\&\qquad + \tau _m \int _\varOmega u^m_h({\varvec{v}}^{m},\nabla u^m_h) \, \rho ^m\, |\nabla \phi ^{m}|+ \tau _m \int _\varOmega f^{e,m} u^m_h\, \rho ^m\, |\nabla \phi ^{m}|\nonumber \\&\quad := I + II + III + IV. \end{aligned}$$\end{document}$$Clearly,$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} I = - \frac{1}{2}\int _{t_{m-1}}^{t_m} \int _\varOmega (u^m_h)^2 \, \rho _t(\cdot ,s) \, | \nabla \phi ^{m-1}|ds, \end{aligned}$$\end{document}$$while$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} II= & {} - \frac{1}{2}\tau _m \int _\varOmega (u^m_h)^2 \, \rho ^m\, (\nabla \phi _t^m, \nu ^m)\nonumber \\&+\frac{1}{2} \int _\varOmega (u^m_h)^2 \, \rho ^m\, \bigl (| \nabla \phi ^{m-1}|- |\nabla \phi ^{m}|+\tau _m (\nabla \phi ^m_t,\nu ^m) \bigr ) = II_1 + II_2. \end{aligned}$$\end{document}$$Integrating by parts and abbreviating $\documentclass[12pt]{minimal}
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\begin{document}$$H^m = - \nabla \cdot \nu ^m$$\end{document}$ we obtain$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} II_1= & {} \frac{1}{2} \tau _m \int _\varOmega (u^m_h)^2 \, (\nabla \rho ^m, \nu ^m) \, \phi _t^m + \tau _m \int _\varOmega u^m_h(\nabla u^m_h,\nu ^m) \rho ^m\, \phi _t^m\\&+ \frac{1}{2} \tau _m \int _\varOmega (u^m_h)^2 \, \nabla \cdot \nu ^m \, \rho ^m\, \phi _t^m \\= & {} \frac{1}{2} \tau _m \int _\varOmega (u^m_h)^2 \, \rho _t^m \, |\nabla \phi ^{m}|+ \tau _m \int _\varOmega u^m_h(\nabla u^m_h,\nu ^m) \rho ^m\, \phi _t^m \\&- \frac{1}{2} \tau _m \int _\varOmega (u^m_h)^2 H^m \, \rho ^m\, \phi _t^m, \end{aligned}$$\end{document}$$since$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} ( \nabla \rho ^m, \nu ^m) \phi ^m_t = \frac{1}{\epsilon } g'\left( \frac{\phi ^m}{\epsilon }\right) \, \phi ^m_t \, ( \nabla \phi ^m,\nu ^m) = \rho ^m_t \, | \nabla \phi ^m |. \end{aligned}$$\end{document}$$In order to rewrite *III* we first observe that in view of ([22](#Equ22){ref-type=""}) and ([24](#Equ24){ref-type=""})$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} ({\varvec{v}}^{m}, \nabla u^m_h)= & {} ({\varvec{v}}^{m}, \nabla _{\phi ^m} u^m_h) + (\nabla u^m_h, \nu ^m) ({\varvec{v}}^{m}, \nu ^m) \\= & {} ({\varvec{v}}^{m}, \nabla _{\phi ^m} u^m_h) - (\nabla u^m_h, \nu ^m) \frac{ \phi ^m_t}{|\nabla \phi ^{m}|}, \end{aligned}$$\end{document}$$so that ([23](#Equ23){ref-type=""}), ([25](#Equ25){ref-type=""}) and again ([24](#Equ24){ref-type=""}) imply$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} III= & {} \frac{1}{2} \tau _m \int _\varOmega ({\varvec{v}}^{m}, \nabla _{\phi ^m}(u^m_h)^2) \rho ^m\, |\nabla \phi ^{m}|- \tau _m \int _\varOmega u^m_h(\nabla u^m_h, \nu ^m) \rho ^m\, \phi _t^m \nonumber \\= & {} - \frac{1}{2} \tau _m \int _\varOmega \nabla _{\phi ^m } \cdot {\varvec{v}}^{m}(u^m_h)^2 \, \rho ^m\, |\nabla \phi ^{m}|- \frac{1}{2} \tau _m \int _\varOmega H^m ( {\varvec{v}}^{m}, \nu ^m) (u^m_h)^2 \, \rho ^m\, |\nabla \phi ^{m}|\nonumber \\&- \tau _m \int _\varOmega u^m_h(\nabla u^m_h, \nu ^m) \rho ^m\, \phi _t^m = - \frac{1}{2} \tau _m \int _\varOmega \nabla _{\phi ^m} \cdot {\varvec{v}}^{m}(u^m_h)^2 \, \rho ^m\, |\nabla \phi ^{m}|\nonumber \\&+ \frac{1}{2} \tau _m \int _\varOmega (u^m_h)^2H^m \, \rho ^m\, \phi _t^m - \tau _m \int _\varOmega u^m_h(\nabla u^m_h, \nu ^m) \, \rho ^m\, \phi _t^m. \end{aligned}$$\end{document}$$Inserting ([44](#Equ44){ref-type=""})--([46](#Equ46){ref-type=""}) into ([43](#Equ43){ref-type=""}) we infer that$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned}&\frac{1}{2}\int _\varOmega (u^m_h)^2 \, \rho ^m\,|\nabla \phi ^{m}|- \frac{1}{2}\int _\varOmega (u^{m-1}_h)^2 \, \rho ^{m-1}\, | \nabla \phi ^{m-1}|+ \tau _m \int _\varOmega |\nabla u^m_h|^2 \, \rho ^m\, |\nabla \phi ^{m}|\nonumber \\&\quad + \gamma \tau _m^2 \int _\varOmega I^m_h \tilde{\rho }^m | \nabla u^m_h|^2 \quad \le \frac{1}{2}\int _{t_{m-1}}^{t_m}\int _\varOmega (u^m_h)^2 \left( \rho _t^m |\nabla \phi ^{m}|-\rho _t(.,s)| \nabla \phi ^{m-1}|\right) \nonumber \\&\quad -\frac{1}{2} \tau _m \int _\varOmega \nabla _{\phi ^m}\cdot {\varvec{v}}^{m}(u^m_h)^2 \, \rho ^m\, |\nabla \phi ^{m}|+ \frac{1}{2} \int _\varOmega (u^m_h)^2 \, \rho ^m\, \bigl ( | \nabla \phi ^{m-1}|\nonumber \\&\quad - |\nabla \phi ^{m}|+\tau _m (\nabla \phi ^m_t,\nu ^m) \bigr ) + \tau _m \int _\varOmega f^{e,m} u^m_h\, \rho ^m\,|\nabla \phi ^{m}|. \end{aligned}$$\end{document}$$We deduce from ([28](#Equ28){ref-type=""}), Lemma [6](#FPar13){ref-type="sec"}, ([41](#Equ41){ref-type=""}) and the assumption $\documentclass[12pt]{minimal}
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\begin{document}$$\tau \le \epsilon ^2$$\end{document}$ that$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned}&\left| \frac{1}{2} \int _{t_{m-1}}^{t_m}\int _\varOmega (u^m_h)^2 (\rho ^m_t|\nabla \phi ^{m}|-\rho _t(.,s)| \nabla \phi ^{m-1}|) \right| \\&\quad \le C \int _{t_{m-1}}^{t_m}\int _\varOmega (u^m_h)^2 \bigl ( | \rho ^m_t - \rho _t(.,s) | + | \rho _t(.,s) | \, | |\nabla \phi ^{m}|-| \nabla \phi ^{m-1}|| \bigr ) \\&\quad \le C \frac{\tau _m^2}{\epsilon ^2} \int _{U_{\frac{3 \epsilon \pi }{4}}(t_m)} (u^m_h)^2 \le C \frac{\tau _m^2}{\epsilon ^2} \int _\varOmega (u^m_h)^2 \, \rho ^m|\nabla \phi ^{m}|+ C \tau _m^2 \int _{U_{\frac{3 \epsilon \pi }{4}}(t_m)} | \nabla u^m_h|^2\\&\quad \le C \tau _m \int _\varOmega (u^m_h)^2 \, \rho ^m|\nabla \phi ^{m}|+ (\gamma -1) \tau _m^2 \int _\varOmega I_h \tilde{\rho }^m \, | \nabla u^m_h|^2 \end{aligned}$$\end{document}$$if we choose $\documentclass[12pt]{minimal}
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\begin{document}$$\gamma \ge \gamma _1:=C+1$$\end{document}$. Finally, using Taylor expansion and ([8](#Equ8){ref-type=""}) we infer that$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned}&\left| -\frac{1}{2} \tau _m \int _\varOmega \nabla _{\phi ^m}\cdot {\varvec{v}}^{m}(u^m_h)^2 \, \rho ^m\, |\nabla \phi ^{m}|\right. \\&\quad \left. + \frac{1}{2} \int _\varOmega (u^m_h)^2 \, \rho ^m\, \bigl ( | \nabla \phi ^{m-1}|- |\nabla \phi ^{m}|+\tau _m (\nabla \phi ^m_t,\nu ^m) \bigr ) \right| \\&\quad \le C \tau _m \int _\varOmega (u^m_h)^2 \, \rho ^m|\nabla \phi ^{m}|+ C \tau _m^2 \int (u^m_h)^2 \rho ^m \le C \tau _m \int _\varOmega (u^m_h)^2 \rho ^m |\nabla \phi ^{m}|. \end{aligned}$$\end{document}$$Inserting the above estimates into ([47](#Equ47){ref-type=""}) we find$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned}&\frac{1}{2}\int _\varOmega (u^m_h)^2 \, \rho ^m\, |\nabla \phi ^{m}|+ \tau _m \int _\varOmega |\nabla u^m_h|^2 \, \rho ^m\, |\nabla \phi ^{m}|+ \tau _m^2 \int _\varOmega I^m_h \tilde{\rho }^m | \nabla u^m_h|^2 \nonumber \\&\qquad \le \frac{1}{2}\int _\varOmega (u^{m-1}_h)^2 \, \rho ^{m-1}\, | \nabla \phi ^{m-1}|+ C \tau _m \int _\varOmega (u^m_h)^2 \, \rho ^m \, |\nabla \phi ^{m}|\nonumber \\&\qquad + \tau _m \int _\varOmega (f^{e,m})^2 \, \rho ^m \, |\nabla \phi ^{m}|. \end{aligned}$$\end{document}$$If $\documentclass[12pt]{minimal}
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\begin{document}$$\tau _1 \le \tau _0$$\end{document}$ is sufficiently small we therefore deduce for $\documentclass[12pt]{minimal}
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\begin{document}$$\tau \le \tau _1$$\end{document}$$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned}&\int _\varOmega (u^m_h)^2 \, \rho ^m\, |\nabla \phi ^{m}|+ \tau _m \int _\varOmega |\nabla u^m_h|^2 \, \rho ^m\, |\nabla \phi ^{m}|\\&\quad \le (1+ C \tau _m) \int _\varOmega (u^{m-1}_h)^2 \, \rho ^{m-1}\, | \nabla \phi ^{m-1}|+ C \tau _m \int _\varOmega (f^{e,m})^2 \, \rho ^m \, |\nabla \phi ^{m}|, \end{aligned}$$\end{document}$$from which we obtain after summation from $\documentclass[12pt]{minimal}
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\begin{document}$$m=1,\ldots ,l$$\end{document}$ and division by $\documentclass[12pt]{minimal}
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\begin{document}$$\epsilon $$\end{document}$ that$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned}&\frac{1}{\epsilon } \int _\varOmega (u_h^l)^2 \, \rho ^l \, | \nabla \phi ^l | + \sum _{m=1}^l \tau _m \frac{1}{\epsilon } \int _\varOmega |\nabla u^m_h|^2 \, \rho ^m\, |\nabla \phi ^{m}|\\&\quad \le \frac{1}{\epsilon } \int _\varOmega (u_h^0)^2 \, \rho ^0 \, | \nabla \phi ^0 | + C \sum _{m=0}^{l-1} \tau _{m+1} \frac{1}{\epsilon } \int _\varOmega (u^m_h)^2 \, \rho ^m\, |\nabla \phi ^{m}|\\&\quad +C \sum _{m=1}^l \tau _m \frac{1}{\epsilon } \int _\varOmega (f^{e,m})^2 \, \rho ^m \, |\nabla \phi ^{m}|. \end{aligned}$$\end{document}$$Using Lemma [3](#FPar6){ref-type="sec"} a), ([38](#Equ38){ref-type=""}) and ([16](#Equ16){ref-type=""}) we may estimate$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \frac{1}{\epsilon } \int _\varOmega (u_h^0)^2 \, \rho ^0 \, | \nabla \phi ^0 |\le & {} \frac{C}{\epsilon } \int _{D^0_h} (u^0_h)^2 \le \frac{C}{\epsilon } \int _{D^0_h} (u^e_0)^2 \le \frac{C}{\epsilon } \int _{U_{\frac{3 \epsilon \pi }{2}}(0)} (u^e_0)^2\\\le & {} C \int _{\varGamma (0)} (u_0)^2. \end{aligned}$$\end{document}$$Arguing in a similar way for the term involving $\documentclass[12pt]{minimal}
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\begin{document}$$f^{e,m}$$\end{document}$ we derive$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned}&\frac{1}{\epsilon } \int _\varOmega (u_h^l)^2 \, \rho ^l \, | \nabla \phi ^l | + \sum _{m=1}^l \tau _m \frac{1}{\epsilon } \int _\varOmega |\nabla u^m_h|^2 \, \rho ^m\, |\nabla \phi ^{m}|\nonumber \\&\quad \le C \sum _{m=0}^{l-1} \tau _{m+1} \frac{1}{\epsilon } \int _\varOmega (u^m_h)^2 \, \rho ^m\, |\nabla \phi ^{m}|+ C \left( \int _{\varGamma (0)} (u_0)^2\right. \nonumber \\&\left. \quad + \sum _{m=1}^l \tau _m \int _{\varGamma (t_m)} (f^m)^2 \right) . \end{aligned}$$\end{document}$$The discrete Gronwall inequality yields the bound on $\documentclass[12pt]{minimal}
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\begin{document}$$\max _{m=1,\ldots ,M} \frac{1}{\epsilon } \int _{\varOmega } (u^m_h)^2 \, \rho ^m \, | \nabla \phi ^m |$$\end{document}$, which combined with ([49](#Equ49){ref-type=""}) implies the second inequality. $\documentclass[12pt]{minimal}
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\begin{document}$$\square $$\end{document}$
Error estimate {#Sec9}
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Before we formulate our error bound we derive interpolation estimates that are adapted to our setting.
Lemma 7 {#FPar18}
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Suppose that ([36](#Equ36){ref-type=""}) holds and let $\documentclass[12pt]{minimal}
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\begin{document}$$z^e$$\end{document}$ be defined by ([14](#Equ14){ref-type=""}). Then we have for $\documentclass[12pt]{minimal}
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\begin{document}$$m=1,\ldots ,M$$\end{document}$ and $\documentclass[12pt]{minimal}
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\begin{document}$$t \in [t_{m-1},t_m]$$\end{document}$:$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned}&\int _{D^m_h} | (z^e - I^m_h z^e)(\cdot ,t) |^2 + h^2 \int _{D^m_h} | \nabla (z^e - I^m_h z^e)(\cdot ,t) |^2 \le C \epsilon h^4 \Vert z(\cdot ,t) \Vert _{H^2(\varGamma (t))}^2, \\&\quad \int _{D^m_h} | (z^e_t - I^m_h z^e_t)(\cdot ,t) |^2 \le C \epsilon h^4 \bigl ( \Vert \partial _t^{\bullet } z(\cdot ,t) \Vert _{H^2(\varGamma (t))}^2 + \Vert z(\cdot ,t) \Vert _{H^3(\varGamma (t))}^2 \bigr ). \end{aligned}$$\end{document}$$
Proof {#FPar19}
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Let $\documentclass[12pt]{minimal}
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\begin{document}$$t \in [t_{m-1},t_m]$$\end{document}$. Standard interpolation theory together with Lemma [3](#FPar6){ref-type="sec"} a) and ([16](#Equ16){ref-type=""}) implies that$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned}&\int _{D^m_h} | (z^e - I^m_h z^e)(\cdot ,t) |^2 + h^2 \int _{D^m_h} | \nabla (z^e - I^m_h z^e)(\cdot ,t) |^2 \\&\quad \le c h^4 \int _{D^m_h} | D^2 z^e(\cdot ,t) |^2 \le c h^4 \int _{U_{\frac{3 \epsilon \pi }{2}}(t)} | D^2 z^e(\cdot ,t) |^2 \le C \epsilon h^4 \Vert z(\cdot ,t) \Vert _{H^2(\varGamma (t))}^2. \end{aligned}$$\end{document}$$The second bound follows in the same way using ([17](#Equ17){ref-type=""}). $\documentclass[12pt]{minimal}
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\begin{document}$$\square $$\end{document}$
Theorem 2 {#FPar20}
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Suppose that the solution of ([1](#Equ1){ref-type=""}), ([2](#Equ2){ref-type=""}) satisfies$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \displaystyle \max _{t \in [0,T]} \Vert u(\cdot ,t) \Vert _{W^{2,\infty }(\varGamma (t))}^2 + \int _0^T \bigl ( \Vert u(\cdot ,t) \Vert _{H^3(\varGamma (t))}^2 + \Vert \partial _t^{\bullet } u(\cdot ,t) \Vert _{H^2(\varGamma (t))}^2 \bigr ) dt < \infty . \end{aligned}$$\end{document}$$Then there exists $\documentclass[12pt]{minimal}
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\begin{document}$$0< \tau _2 \le \tau _1$$\end{document}$ and a constant $\documentclass[12pt]{minimal}
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\begin{document}$$C\ge 0$$\end{document}$ such that$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned}&\max _{m=1,\ldots ,M} \frac{2}{\epsilon \pi } \int _{\varOmega } \, | u^{e,m} - u^m_h|^2 \, \rho ^m \, | \nabla \phi ^m | \\&\quad + \sum _{m=1}^M \tau _m \frac{2}{\epsilon \pi } \int _{\varOmega } | \nabla (u^{e,m}- u^m_h) |^2 \rho ^m \, | \nabla \phi ^m | \le C \epsilon ^2, \end{aligned}$$\end{document}$$provided that $\documentclass[12pt]{minimal}
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\begin{document}$$\tau \le \min (\epsilon ^2,\tau _2)$$\end{document}$, $\documentclass[12pt]{minimal}
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\begin{document}$$\gamma \ge \gamma _1$$\end{document}$ and ([36](#Equ36){ref-type=""}) hold.
Proof {#FPar21}
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Let us write$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} u^{e,m} - u^m_h = (u^{e,m} - I^m_h u^{e,m}) + (I^m_h u^{e,m} - u^m_h)=: d^m + e^m_h. \end{aligned}$$\end{document}$$If we combine ([34](#Equ34){ref-type=""}) for $\documentclass[12pt]{minimal}
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\begin{document}$$\eta = v_h \in V^m_h$$\end{document}$ with ([37](#Equ37){ref-type=""}) we find$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned}&\int _\varOmega e_h^{m}v_h \, \rho ^m|\nabla \phi ^{m}|-\int _\varOmega e_h^{m-1}v_h \, \rho ^{m-1}| \nabla \phi ^{m-1}|+ \tau _m \int _\varOmega (\nabla e_h^{m},\nabla v_h) \rho ^m|\nabla \phi ^{m}|\\&\qquad -\tau _m \int _\varOmega e_h^{m}({\varvec{v}}^{m},\nabla v_h) \rho ^m|\nabla \phi ^{m}|+ \gamma \tau _m^2\int _\varOmega I^m_h \tilde{\rho }^m (\nabla e_h^{m},\nabla v_h) \\&\quad = \left[ - \int _\varOmega d^{m}v_h \, \rho ^m|\nabla \phi ^{m}|+ \int _\varOmega d^{m-1}v_h \, \rho ^{m-1}| \nabla \phi ^{m-1}|\right] \\&\qquad - \tau _m \int _\varOmega (\nabla d^{m},\nabla v_h) \rho ^m|\nabla \phi ^{m}|+ \tau _m \int _\varOmega d^{m}({\varvec{v}}^{m},\nabla v_h) \rho ^m|\nabla \phi ^{m}|\\&\qquad + \gamma \tau _m^2\int _\varOmega I^m_h \tilde{\rho }^m (\nabla I_h u^{e,m},\nabla v_h) + \int _{t_{m-1}}^{t_m}\int _\varOmega \left[ (\nabla u^{e,m}, \nabla v_h) \rho ^m|\nabla \phi ^{m}|\right. \\&\qquad \left. - (\nabla u^e,\nabla v_h) \rho \, | \nabla \phi |\right] \! +\!\! \int _{t_{m-1}}^{t_m}\int _\varOmega \left[ u^e ({\varvec{v}}, \nabla v_h) \rho \, | \nabla \phi |- \! u^{e,m} ({\varvec{v}}^{m}, \nabla v_h) \rho ^m|\nabla \phi ^{m}|\right] \\&\qquad +\int _{t_{m-1}}^{t_m}\int _\varOmega \left[ f^ev_h \, \rho \, | \nabla \phi |- f^{e,m} v_h \, \rho ^m|\nabla \phi ^{m}|\right] +\int _{t_{m-1}}^{t_m}\int _\varOmega \phi \, R \, v_h \, \rho \, | \nabla \phi |\\&\quad =: \sum _{i=1}^8 \langle S_i^m, v_h \rangle . \end{aligned}$$\end{document}$$Inserting $\documentclass[12pt]{minimal}
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\begin{document}$$v_h= e^m_h$$\end{document}$ and following the argument in the proof of Theorem [1](#FPar16){ref-type="sec"} leading to ([48](#Equ48){ref-type=""}) we obtain$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned}&\frac{1}{2}\int _\varOmega (e_h^{m})^2 \rho ^m|\nabla \phi ^{m}|+ \tau _m \int _\varOmega |\nabla e_h^{m}|^2 \rho ^m|\nabla \phi ^{m}|+ \tau _m^2 \int _\varOmega I^m_h \tilde{\rho }^m | \nabla e_h^{m}| ^2 \nonumber \\&\quad \le \frac{1}{2}\int _\varOmega (e_h^{m-1})^2 \rho ^{m-1}| \nabla \phi ^{m-1}|+ C \tau _m \int _\varOmega (e_h^{m})^2 \rho ^m |\nabla \phi ^{m}|+ \sum _{i=1}^8 \langle S^m_i,e_h^{m}\rangle .\nonumber \\ \end{aligned}$$\end{document}$$We now deal individually with the terms $\documentclass[12pt]{minimal}
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\begin{document}$$\langle S^m_i,e_h^{m}\rangle , i =1,\ldots ,8$$\end{document}$ in ([51](#Equ51){ref-type=""}). Clearly,$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned}&| \langle S_1^m, e_h^{m}\rangle | \le C \int _\varOmega | d^m - d^{m-1} | \, | e_h^{m}| \, \rho ^m+ C \int _\varOmega | d^{m-1} | \, | e_h^{m}| \, | \nabla ( \phi ^m - \phi ^{m-1}) | \, \rho ^m\\&\qquad + C \int _\varOmega | d^{m-1} | \, | e_h^{m}| \, | \rho ^m- \rho ^{m-1}| \equiv I + II + III. \end{aligned}$$\end{document}$$In order to estimate *I* we first deduce from Lemma [3](#FPar6){ref-type="sec"} a) that every $\documentclass[12pt]{minimal}
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\begin{document}$$T \in {\mathcal {T}}_h$$\end{document}$ with $\documentclass[12pt]{minimal}
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\begin{document}$$T \cap \text{ supp } \rho ^m \ne \emptyset $$\end{document}$ satisfies $\documentclass[12pt]{minimal}
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\begin{document}$$T \in {\mathcal {T}}^{m-1}_h \cap {\mathcal {T}}^m_h$$\end{document}$. Therefore $\documentclass[12pt]{minimal}
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\begin{document}$$I^{m-1}_h u^{e,m-1} = I^m_h u^{e,m-1}$$\end{document}$ on $\documentclass[12pt]{minimal}
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\begin{document}$$\text{ supp } \rho ^m$$\end{document}$, which yields$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} d^m - d^{m-1}= & {} [u^{e,m}- u^{e,m-1}]- I^m_h[u^{e,m}- u^{e,m-1}] \\= & {} \int _{t_{m-1}}^{t_m} (u^e_t - I^m_h u^e_t)(\cdot ,t) \quad \text{ on } \text{ supp } \rho ^m. \end{aligned}$$\end{document}$$Hence, Lemma [3](#FPar6){ref-type="sec"} a), Lemma [7](#FPar18){ref-type="sec"} and ([8](#Equ8){ref-type=""}) imply that$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} | I |\le & {} C \int _\varOmega \int _{t_{m-1}}^{t_m} | u^e_t - I^m_h u^e_t | \, | e_h^{m}| \, \rho ^m\\\le & {} C \sqrt{\tau _m} \left( \int _\varOmega (e_h^{m})^2 \rho ^m\right) ^{\frac{1}{2}} \left( \int _{t_{m-1}}^{t_m} \int _{D^m_h} | u^e_t - I^m_h u^e_t |^2 \right) ^{\frac{1}{2}} \\\le & {} \tau _m \int _\varOmega (e^m_h)^2 \rho ^m|\nabla \phi ^{m}|+ C \epsilon h^4 \int _{t_{m-1}}^{t_m} \left( \Vert \partial ^{\bullet }_t u(\cdot ,t) \Vert _{H^2(\varGamma (t))}^2 \right. \\&\left. +\Vert u(\cdot ,t) \Vert _{H^3(\varGamma (t))}^2 \right) dt \end{aligned}$$\end{document}$$and similarly,$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} | II |\le & {} C \tau _m \int _\varOmega | d^{m-1} | \, | e_h^{m}| \, \rho ^m\le C \tau _m \left( \int _\varOmega (e_h^{m})^2 \rho ^m\right) ^{\frac{1}{2}} \left( \int _{D^{m-1}_h} | d^{m-1} |^2 \right) ^{\frac{1}{2}} \\\le & {} \tau _m \int _\varOmega (e^m_h)^2 \rho ^m|\nabla \phi ^{m}|+ C \epsilon h^4 \tau _m \Vert u^{m-1} \Vert _{H^2(\varGamma (t_{m-1}))}^2. \end{aligned}$$\end{document}$$Next, we deduce from ([27](#Equ27){ref-type=""}), Lemma [3](#FPar6){ref-type="sec"} a), ([8](#Equ8){ref-type=""}), Lemma [7](#FPar18){ref-type="sec"}, Lemma [6](#FPar13){ref-type="sec"} and ([41](#Equ41){ref-type=""}) that$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} | III |\le & {} C \frac{\tau _m}{\epsilon } \int _\varOmega | d^{m-1} \, | e_h^{m}| \, \sqrt{\rho ^m} + C \frac{\tau _m^2}{\epsilon ^2} \int _{U_{\frac{3 \epsilon \pi }{4}}(t_m)} | d^{m-1} | \, | e_h^{m}| \\\le & {} \tau _m \int _\varOmega (e_h^{m})^2 \rho ^m + C \frac{\tau _m}{\epsilon ^2} \Vert d^{m-1} \Vert _{L^2(D^{m-1}_h)}^2 \\&\quad + C \frac{\tau _m^2}{\epsilon ^2} \left( \int _{ U_{\frac{3 \epsilon \pi }{4}}(t_m)} ( e_h^{m})^2 \right) ^{\frac{1}{2}} \Vert d^{m-1} \Vert _{L^2(D^{m-1}_h)} \\\le & {} C \tau _m \int _\varOmega (e_h^{m})^2 \rho ^m |\nabla \phi ^{m}|+ C \frac{\tau _m h^4}{\epsilon } \Vert u^{m-1} \Vert _{H^2(\varGamma (t_{m-1}))}^2 \\&\quad + C \frac{\tau _m^2 h^2}{\epsilon ^{\frac{3}{2}}} \Vert u^{m-1} \Vert _{H^2(\varGamma (t_{m-1}))} \left( \int _\varOmega ( e_h^{m})^2 \rho ^m|\nabla \phi ^{m}|\! +\! \epsilon ^2 \int _{ U_{\frac{3 \epsilon \pi }{4}}(t_m)} | \nabla e^m_h |^2 \right) ^{\frac{1}{2}} \\\le & {} C \tau _m \int _\varOmega (e^m_h)^2 \rho ^m|\nabla \phi ^{m}|+ \frac{\tau _m^2}{8} \int _\varOmega I^m_h \tilde{\rho }^m | \nabla e^m_h |^2 \!+\! C \frac{\tau _m h^4}{\epsilon } \Vert u^{m-1} \Vert _{H^2(\varGamma (t_{m-1}))}^2, \end{aligned}$$\end{document}$$where we used that $\documentclass[12pt]{minimal}
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\begin{document}$$\tau \le \epsilon ^2$$\end{document}$. Again by Lemma [7](#FPar18){ref-type="sec"} we have$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} | \langle S^m_2,e_h^{m}\rangle |\le & {} \tau _m \left( \int _\varOmega | \nabla e_h^{m}|^2 \rho ^m|\nabla \phi ^{m}|\right) ^{\frac{1}{2}} \left( \int _{D^m_h} | \nabla d^m |^2 \right) ^{\frac{1}{2}} \\\le & {} \frac{1}{8} \tau _m \int _\varOmega | \nabla e_h^{m}|^2 \rho ^m|\nabla \phi ^{m}|+ C \tau _m \epsilon h^2 \Vert u^m \Vert _{H^2(\varGamma (t_m))}^2, \end{aligned}$$\end{document}$$while$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} | \langle S^m_3,e_h^{m}\rangle |\le & {} C \tau _m \int _\varOmega | d^m | \, | \nabla e_h^{m}| \, \rho ^m|\nabla \phi ^{m}|\\\le & {} C \tau _m \left( \int _\varOmega | \nabla e_h^{m}|^2 \rho ^m|\nabla \phi ^{m}|\right) ^{\frac{1}{2}} \left( \int _{D^m_h} | d^m|^2 \right) ^{\frac{1}{2}} \\\le & {} \frac{1}{8} \tau _m \int _\varOmega | \nabla e_h^{m}|^2 \rho ^m|\nabla \phi ^{m}|+ C \tau _m \epsilon h^4 \Vert u^m \Vert _{H^2(\varGamma (t_m))}^2. \end{aligned}$$\end{document}$$Lemma [3](#FPar6){ref-type="sec"} a), ([16](#Equ16){ref-type=""}) and Lemma [7](#FPar18){ref-type="sec"} yield$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} | \langle S^m_4,e_h^{m}\rangle |\le & {} C \tau _m^2 \left( \int _\varOmega I^m_h \tilde{\rho }^m | \nabla e_h^{m}|^2 \right) ^{\frac{1}{2}} \left( \int _{D^m_h} | \nabla I^m_h u^{e,m} |^2 \right) ^{\frac{1}{2}} \\\le & {} \frac{\tau _m^2}{8} \int _\varOmega I^m_h \tilde{\rho }^m | \nabla e_h^{m}|^2 + C \tau _m^2 \int _{D^m_h} \bigl ( | \nabla u^{e,m} |^2 + | \nabla d^m |^2 \bigr ) \\\le & {} \frac{\tau _m^2}{8} \int _\varOmega I^m_h \tilde{\rho }^m | \nabla e_h^{m}|^2 + C \tau _m^2 \epsilon \Vert u^m \Vert _{H^2(\varGamma (t_m))}^2. \end{aligned}$$\end{document}$$We deduce from ([27](#Equ27){ref-type=""}), Lemma [3](#FPar6){ref-type="sec"} a), Lemma [1](#FPar1){ref-type="sec"} and ([41](#Equ41){ref-type=""}) that$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned}&| \langle S^m_5,e_h^{m}\rangle | \le C \int _{t_{m-1}}^{t_m} \int _\varOmega \left[ | \nabla (u^{e,m} - u^e) | \, \rho ^m \right. \\&\qquad \left. + | \nabla u^e | \, | \nabla (\phi ^m - \phi ) | \, \rho ^m + | \nabla u^e | \, | \rho ^m - \rho | \right] | \nabla e_h^{m}| \\&\quad \le C \tau _m \int _{t_{m-1}}^{t_m} \int _\varOmega | \nabla u^e_t | \, | \nabla e_h^{m}| \rho ^m + C \tau _m \int _{t_{m-1}}^{t_m} \int _\varOmega | \nabla u^e | \, | \nabla e_h^{m}| \rho ^m \\&\qquad + C \frac{\tau _m}{\epsilon } \int _{t_{m-1}}^{t_m} \int _\varOmega | \nabla u^e | \, | \nabla e_h^{m}| \sqrt{\rho ^m} + C \frac{\tau _m^2}{\epsilon ^2} \int _{t_{m-1}}^{t_m} \int _{U_{\frac{3 \epsilon \pi }{4}}(t_m)} | \nabla u^e | \, | \nabla e_h^{m}| \\&\quad \le C \left[ \tau _m^{\frac{3}{2}} \left( \int _{t_{m-1}}^{t_m} \Vert u^e_t(\cdot ,t) \Vert ^2_{H^1(U_{\frac{3 \epsilon \pi }{4}}(t))} \right) ^{\frac{1}{2}}\right. \\&\qquad \left. + \frac{\tau _m^2}{\epsilon } \max _{t_{m-1} \le t \le t_m} \Vert u^e(\cdot ,t) \Vert _{H^1(U_{\frac{3 \epsilon \pi }{4}}(t))} \right] \left( \int _\varOmega | \nabla e_h^{m}|^2 \rho ^m \right) ^{\frac{1}{2}} \\&\qquad + C \frac{\tau _m^3}{\epsilon ^2} \max _{t_{m-1} \le t \le t_m} \Vert u^e(\cdot ,t) \Vert _{H^1(U_{\frac{3 \epsilon \pi }{2}}(t))} \left( \int _{U_{\frac{3 \epsilon \pi }{4}}(t_m)} | \nabla e_h^{m}|^2 \right) ^{\frac{1}{2}} \\&\quad \le \frac{\tau _m}{8} \int _\varOmega | \nabla e_h^{m}|^2 \rho ^m |\nabla \phi ^{m}|+ C \tau _m^2 \epsilon \int _{t_{m-1}}^{t_m} \bigl ( \Vert \partial _t^{\bullet } u(\cdot ,t) \Vert _{H^1(\varGamma (t))}^2 \\&\qquad + \Vert u(\cdot ,t) \Vert _{H^2(\varGamma (t))}^2 \bigr ) dt \!+\! \frac{\tau _m^2}{8} \int _\varOmega I^m_h \tilde{\rho }^m | \nabla e_h^{m}|^2 \!+\! C \tau _m^2 \epsilon \max _{t_{m-1} \le t \le t_m} \Vert u(\cdot ,t) \Vert _{H^1(\varGamma (t))}^2. \end{aligned}$$\end{document}$$Here we have used again that $\documentclass[12pt]{minimal}
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\begin{document}$$\tau _m \le \tau \le \epsilon ^2$$\end{document}$. In a similar way we obtain$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned}&| \langle S^m_6,e_h^{m}\rangle | \le C \int _{t_{m-1}}^{t_m} \int _\varOmega \left[ | u^{e,m} - u^e| \, \rho ^m + | u^e | \, | {\varvec{v}}^m | \nabla \phi ^m | \right. \\&\left. \quad - {\varvec{v}}| \nabla \phi | | \, \rho ^m + | u^e | \, | \rho ^m - \rho | \right] | \nabla e_h^{m}| \le \frac{\tau _m}{8} \int _\varOmega | \nabla e_h^{m}|^2 \rho ^m |\nabla \phi ^{m}|\\&\quad +C \tau _m^2 \epsilon \int _{t_{m-1}}^{t_m} \left( \Vert \partial _t^{\bullet } u(\cdot ,t) \Vert _{L^2(\varGamma (t))}^2 + \Vert u(\cdot ,t) \Vert _{H^1(\varGamma (t))}^2 \right) dt \\&\quad + \frac{\tau _m^2}{8} \int _\varOmega I^m_h \tilde{\rho }^m | \nabla e_h^{m}|^2 + C \tau _m^2 \epsilon \max _{t_{m-1} \le t \le t_m} \Vert u(\cdot ,t) \Vert _{L^2(\varGamma (t))}^2 \end{aligned}$$\end{document}$$as well as$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} | \langle S^m_7,e_h^{m}\rangle |\le & {} C \int _{t_{m-1}}^{t_m} \int _\varOmega \left[ | f^e | \nabla \phi | - f^{e,m} | \nabla \phi ^m | \, | \, | e_h^{m}| \, \rho ^m+ | f^{e,m} | \, | e_h^{m}| \, | \rho - \rho ^m| \right] \\\le & {} C \tau _m^2 \int _\varOmega | e_h^{m}| \, \rho ^m+ C \frac{\tau _m^2}{\epsilon } \int _\varOmega | e_h^{m}| \, \sqrt{\rho ^m} + C \frac{\tau _m^3}{\epsilon ^2} \int _{U_{\frac{3 \epsilon \pi }{4}}(t_m)} | e_h^{m}| \\\le & {} \tau _m \int _\varOmega ( e_h^{m})^2 \rho ^m \!+\! C \frac{\tau _m^3}{\epsilon } + C \frac{\tau _m^3}{\epsilon ^{\frac{3}{2}}} \left( \int _\varOmega ( e_h^{m})^2 \rho ^m + \epsilon ^2 \int _{U_{\frac{3 \epsilon \pi }{4}}(t_m)} | \nabla e_h^{m}|^2 \right) ^{\frac{1}{2}} \\\le & {} C \tau _m \int _\varOmega (e_h^{m})^2 \rho ^m|\nabla \phi ^{m}|+ \frac{\tau _m^2}{8} \int _\varOmega I^m_h \tilde{\rho }^m | \nabla e_h^{m}|^2 + C \frac{\tau _m^3}{\epsilon }, \end{aligned}$$\end{document}$$where we have used that $\documentclass[12pt]{minimal}
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\begin{document}$$| U_{\frac{3 \epsilon \pi }{4}}(t_m) | \le C \epsilon $$\end{document}$ and again the fact that $\documentclass[12pt]{minimal}
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\begin{document}$$\tau \le \epsilon ^2$$\end{document}$. Finally, ([32](#Equ32){ref-type=""}) and the definition of $\documentclass[12pt]{minimal}
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\begin{document}$$\rho $$\end{document}$ imply that$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} | R(\cdot ,t) | \le C \Vert u(\cdot ,t)\Vert _{W^{2,\infty }(\varGamma (t))} \text{ and } | \phi (\cdot ,t) | \le c \epsilon \text{ a.e. } \text{ on } \text{ supp } \rho (\cdot ,t), \, t \in [t_{m-1},t_m] \end{aligned}$$\end{document}$$so that we may estimate with the help of ([27](#Equ27){ref-type=""}), ([50](#Equ50){ref-type=""}) and Lemma [6](#FPar13){ref-type="sec"}$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} | \langle S^m_8,e_h^{m}\rangle |\le & {} C \int _{t_{m-1}}^{t_m} \int _\varOmega \left[ | \phi | \, | e_h^{m}| \, \rho ^m+ | \phi | \, | e_h^{m}| \, | \rho - \rho ^m| \right] \\\le & {} C \epsilon \tau _m \int _\varOmega | e_h^{m}| \, \rho ^m+ C \tau _m^2 \int _\varOmega | e_h^{m}| \, \sqrt{\rho ^m} + C \frac{\tau _m^3}{\epsilon } \int _{U_{\frac{3 \epsilon \pi }{4}}(t_m)} | e_h^{m}| \\\le & {} \tau _m \int _\varOmega ( e_h^{m})^2 \rho ^m + C \tau _m \epsilon ^3 + C \tau _m^3 \epsilon \\&\quad + C \frac{\tau _m^3}{\sqrt{\epsilon }} \left( \int _\varOmega ( e_h^{m})^2 \rho ^m + \epsilon ^2 \int _{U_{\frac{3 \epsilon \pi }{4}}(t_m)} | \nabla e_h^{m}|^2 \right) ^{\frac{1}{2}} \\\le & {} C \tau _m \int _\varOmega (e_h^{m})^2 \rho ^m|\nabla \phi ^{m}|+ \frac{\tau _m^2}{8} \int _\varOmega I^m_h \tilde{\rho }^m | \nabla e_h^{m}|^2 + C \tau _m \epsilon ^3 + C \tau _m^3 \epsilon . \end{aligned}$$\end{document}$$Inserting the above estimates into ([51](#Equ51){ref-type=""}) we obtain$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned}&\frac{1}{2}\int _\varOmega (e_h^{m})^2 \rho ^m|\nabla \phi ^{m}|+ \frac{\tau _m}{2} \int _\varOmega |\nabla e_h^{m}|^2 \rho ^m|\nabla \phi ^{m}|+ \frac{\tau _m^2}{4} \int _\varOmega I^m_h \tilde{\rho }^m | \nabla e_h^{m}|^2 \\&\quad \le \frac{1}{2}\int _\varOmega (e_h^{m-1})^2 \rho ^{m-1}| \nabla \phi ^{m-1}|+ C \tau _m \int _\varOmega (e_h^{m})^2 \rho ^m |\nabla \phi ^{m}|+ C \left( \frac{\tau _m^3}{\epsilon } + \tau _m \epsilon ^3 \right) \\&\quad \quad + C \tau _m \, \epsilon \left( h^2 + \frac{h^4}{\epsilon ^2} + \tau \right) \max _{t_{m-1} \le t \le t_m} \Vert u(\cdot ,t) \Vert _{H^2(\varGamma (t))}^2 \\&\quad \quad + C \epsilon \bigl ( h^4 + \tau ^2 \bigr ) \int _{t_{m-1}}^{t_m} \left( \Vert \partial ^{\bullet }_t u(\cdot ,t) \Vert _{H^2(\varGamma (t))}^2 + \Vert u(\cdot ,t) \Vert _{H^3(\varGamma (t))}^2 \right) dt. \end{aligned}$$\end{document}$$Choosing $\documentclass[12pt]{minimal}
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\begin{document}$$\tau _2 \le \tau _1$$\end{document}$ small enough and using ([36](#Equ36){ref-type=""}) as well as $\documentclass[12pt]{minimal}
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\begin{document}$$\tau \le \epsilon ^2$$\end{document}$ we infer$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned}&\int _\varOmega (e_h^{m})^2 \rho ^m|\nabla \phi ^{m}|+ \tau _m \int _\varOmega |\nabla e_h^{m}|^2 \rho ^m|\nabla \phi ^{m}|\\&\quad \le (1+ C \tau _m) \int _\varOmega (e_h^{m-1})^2 \rho ^{m-1}| \nabla \phi ^{m-1}|+ C \epsilon ^3 \tau _m \max _{t_{m-1} \le t \le t_m} \Vert u(\cdot ,t) \Vert _{H^2(\varGamma (t))}^2 \\&\quad + C \epsilon ^5 \int _{t_{m-1}}^{t_m} \bigl ( \Vert \partial ^{\bullet }_t u(\cdot ,t) \Vert _{H^2(\varGamma (t))}^2 + \Vert u(\cdot ,t) \Vert _{H^3(\varGamma (t))}^2 \bigr ) dt + C \tau _m \epsilon ^3. \end{aligned}$$\end{document}$$Summing from $\documentclass[12pt]{minimal}
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\begin{document}$$m=1,\ldots ,l$$\end{document}$, dividing by $\documentclass[12pt]{minimal}
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\begin{document}$$\epsilon $$\end{document}$ and recalling ([50](#Equ50){ref-type=""}) we derive$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned}&\frac{1}{\varepsilon }\int _\varOmega (e^l_h)^2 \rho ^l |\nabla \phi ^l| + \sum _{m=1}^l \tau _m \frac{1}{\epsilon } \int _\varOmega |\nabla e_h^{m}|^2 \rho ^m|\nabla \phi ^{m}|\\&\quad \le \frac{1}{\varepsilon }\int _\varOmega (e^0_h)^2\rho ^0 \, | \nabla \phi ^0 | + C \sum _{m=0}^{l-1} \tau _{m+1} \frac{1}{\varepsilon }\int _\varOmega (e_h^{m})^2 \rho ^m\, |\nabla \phi ^{m}|+ C \epsilon ^2. \end{aligned}$$\end{document}$$In order to estimate the first term on the right hand side we write $\documentclass[12pt]{minimal}
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\begin{document}$$e^0_h = (I^0_h u^e_0 - u^e_0) + (u^e_0- u_h^0)$$\end{document}$ and recall the definition ([38](#Equ38){ref-type=""}) of $\documentclass[12pt]{minimal}
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\begin{document}$$u^0_h$$\end{document}$ as an $\documentclass[12pt]{minimal}
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\begin{document}$$L^2$$\end{document}$ projection:$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \int _\varOmega (e^0_h)^2 \rho ^0 \, | \nabla \phi ^0 | \le C \int _{D^0_h} (e^0_h)^2 \le C \int _{D^0_h} | u^e_0 - I^0_h u^e_0 |^2 \le C \epsilon h^4 \Vert u_0 \Vert _{H^2(\varGamma (0))}^2 \end{aligned}$$\end{document}$$by Lemma [7](#FPar18){ref-type="sec"}. Thus$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned}&\displaystyle \frac{1}{\varepsilon }\int _\varOmega (e^l_h)^2 \rho ^l |\nabla \phi ^l| + \sum _{m=1}^l \tau _m \frac{1}{\epsilon } \int _\varOmega |\nabla e_h^{m}|^2 \rho ^m|\nabla \phi ^{m}|\nonumber \\&\quad \le C \sum _{m=0}^{l-1} \tau _{m+1} \frac{1}{\varepsilon }\int _\varOmega (e_h^{m})^2 \rho ^m|\nabla \phi ^{m}|+ C \epsilon ^2 \end{aligned}$$\end{document}$$and the discrete Gronwall lemma gives$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \displaystyle \max _{m=1,\ldots ,M} \frac{1}{\varepsilon }\int _\varOmega (e_h^{m})^2 \rho ^m|\nabla \phi ^{m}|\le C \epsilon ^2. \end{aligned}$$\end{document}$$The remainder of the proof follows from ([52](#Equ52){ref-type=""}) and Lemma [7](#FPar18){ref-type="sec"}. $\documentclass[12pt]{minimal}
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\begin{document}$$\square $$\end{document}$
Using the result of Theorem [2](#FPar20){ref-type="sec"} we can now also derive an error bound on the surface.
Corollary 1 {#FPar22}
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In addition to the assumptions of Theorem [2](#FPar20){ref-type="sec"} suppose that there exists $\documentclass[12pt]{minimal}
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\begin{document}$$\alpha >0$$\end{document}$ such that $\documentclass[12pt]{minimal}
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\begin{document}$$h_T \ge \alpha \epsilon $$\end{document}$ for all $\documentclass[12pt]{minimal}
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\begin{document}$$T \in {\mathcal {T}}_h$$\end{document}$ with $\documentclass[12pt]{minimal}
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\begin{document}$$| T \cap \varGamma (t) |>0, t \in [0,T]$$\end{document}$. Then$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \max _{m=1,\ldots ,M} \int _{\varGamma (t_m)} | u^m - u^m_h |^2 + \sum _{m=1}^M \tau _m \int _{\varGamma (t_m)} | \nabla _{\varGamma } ( u^m - u^m_h ) |^2 \le C \epsilon ^2. \end{aligned}$$\end{document}$$
Proof {#FPar23}
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Let us fix $\documentclass[12pt]{minimal}
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\begin{document}$$m \in \lbrace 1,\ldots ,M \rbrace $$\end{document}$ and define $\documentclass[12pt]{minimal}
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\begin{document}$${\mathcal {T}}^m_{\varGamma ,h}:= \lbrace T \in {\mathcal {T}}_h \, | \, | T \cap \varGamma (t_m) |>0 \rbrace $$\end{document}$. Hence, given $\documentclass[12pt]{minimal}
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\begin{document}$$T \in {\mathcal {T}}^m_{\varGamma ,h}$$\end{document}$, there exists $\documentclass[12pt]{minimal}
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\begin{document}$$x_T \in \varGamma (t_m)$$\end{document}$ with $\documentclass[12pt]{minimal}
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\begin{document}$$\phi ^m(x_T)=0$$\end{document}$. We infer from ([8](#Equ8){ref-type=""}) and ([36](#Equ36){ref-type=""}) that for arbitrary $\documentclass[12pt]{minimal}
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\begin{document}$$x \in T$$\end{document}$$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} | \phi ^m(x) | = | \phi ^m(x) - \phi ^m(x_T) | \le c_1 | x - x_T| \le c_1 h_T \le \frac{\epsilon }{2} \cos ^2 \left( \frac{3 \pi }{8} \right) \le \frac{\epsilon \pi }{4}, \end{aligned}$$\end{document}$$and therefore$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \rho ^m(x) \ge \frac{1}{2} \quad \text{ for } \text{ all } x \in T, \, T \in {\mathcal {T}}^m_{\varGamma ,h}. \end{aligned}$$\end{document}$$We now argue in a similar way as in \[[@CR6]\], page 368. Using an interpolation inequality and an inverse estimate we infer that$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \int _{\varGamma (t_m)} | u^m - u^m_h |^2= & {} \sum _{T \in {\mathcal {T}}^m_{\varGamma ,h}} \int _{T \cap \varGamma (t_m)} | u^m - u^m_h |^2 \\\le & {} 2 \sum _{T \in {\mathcal {T}}^m_{\varGamma ,h}} | T \cap \varGamma (t_m) | \left( \Vert d^m \Vert _{L^{\infty }(T)}^2 + \Vert e^m_h \Vert _{L^{\infty }(T)}^2 \right) \\\le & {} C \sum _{T \in {\mathcal {T}}^m_{\varGamma ,h} } | T \cap \varGamma (t_m) | \, h_T^2 \Vert \nabla u^{e,m} \Vert _{W^{1,\infty }(T)}^2 \\&+ C \sum _{T \in {\mathcal {T}}^m_{\varGamma ,h} } h_T^n h_T^{-(n+1)} \Vert e^m_h \Vert _{L^2(T)}^2 \\\le & {} C h^2 | \varGamma (t_m) | \Vert u^m \Vert _{W^{1,\infty }(\varGamma (t_m))}^2 + C \epsilon ^{-1} \sum _{T \in {\mathcal {T}}^m_{\varGamma ,h} } \int _T | e^m_h |^2 \rho ^m \, | \nabla \phi ^m |, \end{aligned}$$\end{document}$$where the last inequality follows from ([54](#Equ54){ref-type=""}), ([8](#Equ8){ref-type=""}) and the assumption that $\documentclass[12pt]{minimal}
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\begin{document}$$h_T \ge \alpha \epsilon , T \in {\mathcal {T}}^m_{\varGamma ,h}$$\end{document}$. In a similar way we obtain$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned}&\int _{\varGamma (t_m)} | \nabla _{\varGamma } (u^m-u^m_h) |^2 \le C h^2 | \varGamma (t_m) | \Vert u^m \Vert _{W^{2,\infty }(\varGamma (t_m))}^2 \\&\quad + C \epsilon ^{-1} \sum _{T \in {\mathcal {T}}^m_{\varGamma ,h} } \int _T | \nabla e^m_h |^2 \rho ^m \, | \nabla \phi ^m |. \end{aligned}$$\end{document}$$Thus,$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned}&\max _{m=1,\ldots ,M} \int _{\varGamma (t_m)} | u^m - u^m_h |^2 + \sum _{m=1}^M \tau _m \int _{\varGamma (t_m)} | \nabla _{\varGamma } ( u^m - u^m_h ) |^2 \\&\quad \le C h^2 \max _{t \in [0,T]} \Vert u(\cdot ,t) \Vert _{W^{2,\infty }(\varGamma (t))}^2 \\&\qquad + C \epsilon ^{-1} \max _{m=1,\ldots ,M} \int _{\varOmega } | e^m_h |^2 \rho ^m | \nabla \phi ^m | \!+\! C \epsilon ^{-1} \sum _{m=1}^M \tau _m \int _{\varOmega } | \nabla e^m_h |^2 \rho ^m | \nabla \phi ^m | \le C \epsilon ^2, \end{aligned}$$\end{document}$$by ([36](#Equ36){ref-type=""}), ([53](#Equ53){ref-type=""}) and ([52](#Equ52){ref-type=""}). $\documentclass[12pt]{minimal}
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\begin{document}$$\square $$\end{document}$
Numerical results {#Sec10}
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As already mentioned in Remark [2](#FPar8){ref-type="sec"} c), the scheme ([37](#Equ37){ref-type=""}), ([38](#Equ38){ref-type=""}) is not fully practical. Therefore, our implementation uses the following modification: Find $\documentclass[12pt]{minimal}
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\begin{document}$$u^m_h \in V^m_h,$$\end{document}$ such that$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned}&\int _{\varOmega } u^m_h \, v_h \, I^m_h \rho ^m \, | \nabla I^m_h \phi ^m | - \int _{\varOmega } u^{m-1}_h \, v_h \, I^{m-1}_h \rho ^{m-1} \, | \nabla I^{m-1}_h \phi ^{m-1} | \nonumber \\&\quad + \tau _m \, \int _{\varOmega } (\nabla u^m_h, \nabla v_h) \, I^m_h \rho ^m \, | \nabla I^m_h \phi ^m | - \tau _m \, \int _{\varOmega } u^m_h \, (I_h^m \hat{{\varvec{v}}}^m,\nabla v_h) \, I^m_h \rho ^m \, | \nabla I^m_h \phi ^m | \nonumber \\&\quad + \gamma \tau _m^2 \, \int _{\varOmega } I^m_h \tilde{\rho }^m ( \nabla u^m_h,\nabla v_h) = \tau _m \, \int _{\varOmega } I_h^m\hat{f}^m \, v_h \, I^m_h \rho ^m \, | \nabla I^m_h \phi ^m | \end{aligned}$$\end{document}$$for all $\documentclass[12pt]{minimal}
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\begin{document}$$v_h \in V^m_h$$\end{document}$ and $\documentclass[12pt]{minimal}
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\begin{document}$$1 \le m \le M$$\end{document}$. Here, $\documentclass[12pt]{minimal}
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\begin{document}$$\hat{{\varvec{v}}}^m(x):= {\varvec{v}}(\hat{p}(x,t_m),t_m)$$\end{document}$, $\documentclass[12pt]{minimal}
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\begin{document}$$\hat{f}^m(x)= f(\hat{p}(x,t_m),t_m)$$\end{document}$, where $\documentclass[12pt]{minimal}
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\begin{document}$$\hat{p}(x,t)$$\end{document}$ denotes the closest point projection of a point *x* onto $\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \displaystyle \int _{D^0_h} \hat{u}^0_h \, v_h = \int _{D^0_h} I^0_h \hat{u}_0 \, v_h \qquad \forall v_h \in V^0_h. \end{aligned}$$\end{document}$$Let us remark that the evaluation of $\documentclass[12pt]{minimal}
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\begin{document}$$\hat{p}(x,t)$$\end{document}$ is easier compared to $\documentclass[12pt]{minimal}
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\begin{document}$$\tilde{p}(x,t)$$\end{document}$, which has been used to extend the data for the scheme ([37](#Equ37){ref-type=""}), ([38](#Equ38){ref-type=""}). However, we claim that$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \tilde{p}(x,t) - \hat{p}(x,t) = O(\phi (x,t)^2). \end{aligned}$$\end{document}$$To see this, we first observe that $\documentclass[12pt]{minimal}
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\begin{document}$$\hat{p}(x,t)$$\end{document}$ is characterized by the conditions$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \phi (\hat{p}(x,t),t)=0 \quad \text{ and } \quad x - \hat{p}(x,t) \perp \varGamma (t) \text{ at } \hat{p}(x,t). \end{aligned}$$\end{document}$$Therefore, it is not difficult to verify with the help of Taylor expansion that$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} x \!-\! \hat{p}(x,t) \!=\! \lambda (x,t) \nabla \phi (\hat{p}(x,t),t), \quad \text{ with } \lambda (x,t)\! =\! \frac{\phi (x,t)}{| \nabla \phi (\hat{p}(x,t),t)|^2}\! +\! O(\phi (x,t)^2). \end{aligned}$$\end{document}$$Combining this relation with ([58](#Equ58){ref-type=""}) in the "Appendix" we find that$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \tilde{p}(x,t) - \hat{p}(x,t)= & {} \phi (x,t) \left[ \frac{\nabla \phi (\hat{p}(x,t),t)}{| \nabla \phi (\hat{p}(x,t),t) |^2} - \frac{ \nabla \phi (x,t)}{ | \nabla \phi (x,t) |^2} \right] \\&+ O(\phi (x,t)^2) = O(\phi (x,t)^2). \end{aligned}$$\end{document}$$In particular, we infer from ([57](#Equ57){ref-type=""}) that replacing $\documentclass[12pt]{minimal}
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\begin{document}$$\tilde{p}$$\end{document}$ by $\documentclass[12pt]{minimal}
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\begin{document}$${\varvec{v}},f$$\end{document}$ and $\documentclass[12pt]{minimal}
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\begin{document}$$u_0$$\end{document}$ will not affect the result of Theorem [2](#FPar20){ref-type="sec"}. In contrast, it is not straightforward to handle the interpolation terms $\documentclass[12pt]{minimal}
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\begin{document}$$I^m_h \rho ^m$$\end{document}$ and $\documentclass[12pt]{minimal}
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\begin{document}$$I^{m-1}_h \rho ^{m-1}$$\end{document}$ in ([55](#Equ55){ref-type=""}). Applying a standard interpolation estimate to $\documentclass[12pt]{minimal}
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\begin{document}$$\rho ^m - I^m_h \rho ^m$$\end{document}$ will result in a term of the form $\documentclass[12pt]{minimal}
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\begin{document}$$h^2 \Vert \rho ^m \Vert _{H^2} \approx \frac{h^2}{\epsilon ^2}$$\end{document}$, which we are currently not able to analyze. The results of our test calculations below however show that the use of the interpolation operator in ([55](#Equ55){ref-type=""}), ([56](#Equ56){ref-type=""}) does not lead to reduced convergence rates. More precisely we investigate the experimental order of convergence (eoc) for the following errors:$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \mathcal {E}_1= & {} \max _{m=1,\ldots ,M} \frac{2}{\epsilon \pi } \int _{\varOmega } \, | I_h^m\hat{u}^m - u^m_h|^2 \, I^m_h \rho ^m\, | \nabla I^m_h \phi ^m |, \\ \mathcal {E}_2= & {} \frac{2}{\epsilon \pi } \sum _{m=1}^M \tau _m \int _{\varOmega } | \nabla (I_h^m \hat{u}^m- u^m_h) |^2 I^m_h \rho ^m \, | \nabla I^m_h \phi ^m |, \end{aligned}$$\end{document}$$where $\documentclass[12pt]{minimal}
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\begin{document}$$\hat{u}^m(x)=u(\hat{p}(x,t_m),t_m)$$\end{document}$. We use the finite element toolbox Alberta 2.0, \[[@CR24]\], and implement a similar mesh refinement strategy to that in \[[@CR2]\] with a fine mesh constructed in $\documentclass[12pt]{minimal}
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\begin{document}$$D_h^m$$\end{document}$ and a coarser mesh in $\documentclass[12pt]{minimal}
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\begin{document}$$\varOmega \backslash D_h^m$$\end{document}$. The linear systems appearing in each time step were solved using GMRES together with diagonal preconditioning. The values of *h* given below are such that $\documentclass[12pt]{minimal}
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\begin{document}$$h:=\max _{T\in D_h^m}h_T$$\end{document}$, $\documentclass[12pt]{minimal}
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\begin{document}$$h_T=\text{ diam }(T)$$\end{document}$.
Remark 4 {#FPar24}
--------
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\begin{document}$$\gamma >0$$\end{document}$, the method works with $\documentclass[12pt]{minimal}
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\begin{document}$$\gamma =0$$\end{document}$ and produces very similar eocs to the ones displayed in the tables below for $\documentclass[12pt]{minimal}
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\begin{document}$$\gamma =0.01$$\end{document}$.
2D examples {#Sec11}
-----------
We set $\documentclass[12pt]{minimal}
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\begin{document}$$\varOmega = (-2.4, 2.4)^2$$\end{document}$, $\documentclass[12pt]{minimal}
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\begin{document}$$T=0.1$$\end{document}$, and choose $\documentclass[12pt]{minimal}
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\begin{document}$$\gamma =0.01, \, \epsilon =85.33 \, h$$\end{document}$ as well as a uniform time step $\documentclass[12pt]{minimal}
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\begin{document}$$\tau _m = 0.0025\varepsilon ^2, m=1,\ldots ,M$$\end{document}$. In all our examples below $\documentclass[12pt]{minimal}
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\begin{document}$$\varGamma (t)$$\end{document}$ will be a circle $\documentclass[12pt]{minimal}
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\begin{document}$$\varGamma (t)= \lbrace x \in {\mathbb {R}}^2 \, | \, | x - m(t) |= 1 \rbrace $$\end{document}$ of radius 1 with center $\documentclass[12pt]{minimal}
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\begin{document}$$m(t) \in {\mathbb {R}}^2$$\end{document}$. In addition to $\documentclass[12pt]{minimal}
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\begin{document}$${\mathcal {E}}_1, {\mathcal {E}}_2$$\end{document}$ we shall also investigate the errors appearing in Corollary [1](#FPar22){ref-type="sec"}. To do so we choose $\documentclass[12pt]{minimal}
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\begin{document}$$L>0$$\end{document}$ and define the following quadrature points$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} x_l(t):= m(t)+ \left( \cos \left( \frac{2 \pi l}{L}\right) , \sin \left( \frac{2 \pi l}{L}\right) \right) ^T, \quad l=0,\ldots ,L-1 \end{aligned}$$\end{document}$$as well as$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \mathcal {E}_3= & {} \max _{m=1,\ldots ,M} \sum _{l=0}^{L-1}\frac{2\pi }{L}| u(x_l(t_m),t_m) - u^{m}_h(x_l(t_m)) |^2,\\ \mathcal {E}_4= & {} \sum _{m=1}^M \tau _m\sum _{l=0}^{L-1}\frac{2\pi }{L}| \nabla _{\varGamma }u(x_l(t_m),t_m) - \nabla _{\varGamma } u^{m}_h(x_l(t_m)) |^2. \end{aligned}$$\end{document}$$In our computations $\documentclass[12pt]{minimal}
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\begin{document}$$L=200$$\end{document}$ turned out to be sufficient.
### Example 1 {#FPar25}
For our first example we consider the stationary unit circle $\documentclass[12pt]{minimal}
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\begin{document}$$\varGamma (t)=\varGamma = S^1, t \in [0,T]$$\end{document}$ described as the zero level set of the function $\documentclass[12pt]{minimal}
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\begin{document}$$\phi (x):= x_1^2+x_2^2-1$$\end{document}$.
The function $\documentclass[12pt]{minimal}
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\begin{document}$$u(x,t):= e^{-4t} \left[ x_1 x_2 \cos (\pi t) + \frac{1}{2}(x_1^2 - x_2^2) \sin (\pi t) \right] $$\end{document}$ is a solution of ([1](#Equ1){ref-type=""}), ([2](#Equ2){ref-type=""}) for the velocity field $\documentclass[12pt]{minimal}
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\begin{document}$${\varvec{v}}({x}) = \frac{\pi }{2} (x_2, -x_1)^T, f=0$$\end{document}$ and the initial data $\documentclass[12pt]{minimal}
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\begin{document}$$u_0(x)=x_1 x_2$$\end{document}$. A similar choice of velocity appears in Example [3](#FPar27){ref-type="sec"} in \[[@CR10]\]. In Tables [1](#Tab1){ref-type="table"} and [2](#Tab2){ref-type="table"} we display the values of $\documentclass[12pt]{minimal}
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\begin{document}$$\mathcal {E}_i$$\end{document}$, $\documentclass[12pt]{minimal}
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\begin{document}$$i=1\rightarrow 4$$\end{document}$, together with the eocs.
Table 1Errors and experimental orders of convergence for Example [1](#FPar25){ref-type="sec"}*h*$\documentclass[12pt]{minimal}
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\begin{document}$$\varepsilon $$\end{document}$$\documentclass[12pt]{minimal}
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\begin{document}$$\mathcal {E}_1$$\end{document}$$\documentclass[12pt]{minimal}
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\begin{document}$$eoc_1$$\end{document}$$\documentclass[12pt]{minimal}
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\begin{document}$$\mathcal {E}_2$$\end{document}$$\documentclass[12pt]{minimal}
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\begin{document}$$eoc_2$$\end{document}$4.6875e−030.42.0565e−04--1.0763e−03--3.3146e−03$\documentclass[12pt]{minimal}
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\begin{document}$$0.2\sqrt{2}$$\end{document}$3.2822e−055.2952.7030e−043.9872.3437e−030.26.5608e−064.6456.7864e−053.9881.6573e−03$\documentclass[12pt]{minimal}
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\begin{document}$$0.1\sqrt{2}$$\end{document}$1.4513e−064.3531.7017e−053.9911.1719e−030.13.4022e−074.1864.2668e−063.991 Table 2Errors and experimental orders of convergence for Example [1](#FPar25){ref-type="sec"}*h*$\documentclass[12pt]{minimal}
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\begin{document}$$\varepsilon $$\end{document}$$\documentclass[12pt]{minimal}
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\begin{document}$$\mathcal {E}_3$$\end{document}$$\documentclass[12pt]{minimal}
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\begin{document}$$eoc_3$$\end{document}$$\documentclass[12pt]{minimal}
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\begin{document}$$\mathcal {E}_4$$\end{document}$$\documentclass[12pt]{minimal}
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\begin{document}$$eoc_4$$\end{document}$4.6875e−030.42.7651e−05--4.3137e−06--3.3146e−03$\documentclass[12pt]{minimal}
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\begin{document}$$0.2\sqrt{2}$$\end{document}$8.1077e−063.5401.6031e−062.8562.3437e−030.22.1848e−063.7845.9541e−072.8581.6573e−03$\documentclass[12pt]{minimal}
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\begin{document}$$0.1\sqrt{2}$$\end{document}$5.6637e−073.8952.3962e−072.6261.1719e−030.11.4412e−073.9499.6590e−082.622
### Example 2 {#FPar26}
(cf. \[[@CR16], Section 3.1\], \[[@CR26]\], Example 5.2) We consider the family of unit circles $\documentclass[12pt]{minimal}
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\begin{document}$$\varGamma (t) = \lbrace x \in {\mathbb {R}}^2 \, | \, (x_1 + \frac{1}{2} -2t)^2 + x_2^2 =1 \rbrace $$\end{document}$ described as the zero level set of $\documentclass[12pt]{minimal}
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\begin{document}$$\phi (x,t)=(x_1 + \frac{1}{2}-2t)^2+x_2^2-1$$\end{document}$. The function $\documentclass[12pt]{minimal}
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\begin{document}$$u:S_T \rightarrow {\mathbb {R}}$$\end{document}$, $\documentclass[12pt]{minimal}
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\begin{document}$$u(x,t)= e^{-4t}(x_1 + \frac{1}{2}-2t) x_2$$\end{document}$ is a solution of ([1](#Equ1){ref-type=""}), ([2](#Equ2){ref-type=""}) for the velocity field $\documentclass[12pt]{minimal}
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\begin{document}$${\varvec{v}}(x,t) = (2,0)^T,f=0$$\end{document}$ and the initial data $\documentclass[12pt]{minimal}
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\begin{document}$$u_0(x)=(x_1+\frac{1}{2})x_2$$\end{document}$. The results are displayed in Tables [3](#Tab3){ref-type="table"} and [4](#Tab4){ref-type="table"} where we see eocs that are similar to the ones in Tables [1](#Tab1){ref-type="table"} and [2](#Tab2){ref-type="table"}.
We see that the eoc for $\documentclass[12pt]{minimal}
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\begin{document}$$\mathcal {E}_1$$\end{document}$ is reducing towards 4, the eocs for $\documentclass[12pt]{minimal}
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\begin{document}$$\mathcal {E}_2$$\end{document}$ and $\documentclass[12pt]{minimal}
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\begin{document}$$\mathcal {E}_3$$\end{document}$ are close to 4 and the eoc for $\documentclass[12pt]{minimal}
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\begin{document}$$\mathcal {E}_4$$\end{document}$ is between 2 and 3 which is better than Theorem [2](#FPar20){ref-type="sec"} predicts. Since $\documentclass[12pt]{minimal}
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\begin{document}$$\mathcal {E}_1$$\end{document}$ and $\documentclass[12pt]{minimal}
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\begin{document}$$\mathcal {E}_3$$\end{document}$ approximate $\documentclass[12pt]{minimal}
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\begin{document}$$L^2$$\end{document}$--errors, higher eocs can be expected although a corresponding proof is by no means straightforward and beyond the scope of this paper. The higher eoc for $\documentclass[12pt]{minimal}
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\begin{document}$$\mathcal {E}_2$$\end{document}$ presumably reflects a superconvergence effect because we consider $\documentclass[12pt]{minimal}
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\begin{document}$$\nabla (I^m_h \hat{u}^m - u^m_h)$$\end{document}$ rather than $\documentclass[12pt]{minimal}
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\begin{document}$$\nabla ( \hat{u}^m - u^m_h)$$\end{document}$. We expect that $\documentclass[12pt]{minimal}
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\begin{document}$$\mathcal {E}_4$$\end{document}$ will tend towards 2 if $\documentclass[12pt]{minimal}
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\begin{document}$$\varepsilon ,~h$$\end{document}$ and $\documentclass[12pt]{minimal}
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\begin{document}$$\tau $$\end{document}$ are reduced further.Table 3Errors and experimental orders of convergence for Example [2](#FPar26){ref-type="sec"}*h*$\documentclass[12pt]{minimal}
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\begin{document}$$\varepsilon $$\end{document}$$\documentclass[12pt]{minimal}
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\begin{document}$$\mathcal {E}_1$$\end{document}$$\documentclass[12pt]{minimal}
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\begin{document}$$eoc_1$$\end{document}$$\documentclass[12pt]{minimal}
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\begin{document}$$\mathcal {E}_2$$\end{document}$$\documentclass[12pt]{minimal}
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\begin{document}$$eoc_2$$\end{document}$4.6875e−030.41.5537e−04--9.3201e−04--3.3146e−03$\documentclass[12pt]{minimal}
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\begin{document}$$0.2\sqrt{2}$$\end{document}$2.5206e−055.2482.3280e−044.0022.3437e−030.24.8726e−064.7425.8500e−053.9851.6573e−03$\documentclass[12pt]{minimal}
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\begin{document}$$0.1\sqrt{2}$$\end{document}$1.0558e−064.4131.4776e−053.9701.1719e−030.12.4507e−074.2143.7865e−063.929 Table 4Errors and experimental orders of convergence for Example [2](#FPar26){ref-type="sec"}*h*$\documentclass[12pt]{minimal}
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\begin{document}$$\varepsilon $$\end{document}$$\documentclass[12pt]{minimal}
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\begin{document}$$\mathcal {E}_3$$\end{document}$$\documentclass[12pt]{minimal}
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\begin{document}$$eoc_3$$\end{document}$$\documentclass[12pt]{minimal}
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\begin{document}$$\mathcal {E}_4$$\end{document}$$\documentclass[12pt]{minimal}
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\begin{document}$$eoc_4$$\end{document}$4.6875e−030.41.8431e−05--3.0082e−06--3.3146e−03$\documentclass[12pt]{minimal}
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\begin{document}$$0.2\sqrt{2}$$\end{document}$5.6312e−063.4211.2489e−062.5372.3437e−030.21.5443e−063.7334.8015e−072.7581.6573e−03$\documentclass[12pt]{minimal}
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\begin{document}$$0.1\sqrt{2}$$\end{document}$4.0396e−073.8691.9389e−072.6161.1719e−030.11.0350e−073.9298.1747e−082.492
3D example {#Sec12}
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### Example 3 {#FPar27}
Here we consider the first example in Section 7 of \[[@CR18]\] in which a family of expanding and collapsing spheres is considered such that $\documentclass[12pt]{minimal}
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\begin{document}$$\varGamma (t)= \lbrace x \in {\mathbb {R}}^2 \, | \, |x|=r(t) \rbrace $$\end{document}$ where $\documentclass[12pt]{minimal}
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\begin{document}$$r(t) = 1 + \sin ^2(\pi t)$$\end{document}$, described as the zero level set of $\documentclass[12pt]{minimal}
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\begin{document}$$\phi (x,t)=x_1^2+x_2^2+x_3^2-r(t)^2$$\end{document}$. The function $\documentclass[12pt]{minimal}
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\begin{document}$$u:S_T \rightarrow {\mathbb {R}}, u(x,t)= \frac{2}{r(t)^2|x|^2} e^{-6\int _0^t\frac{1}{r^2(t)}} x_1 x_3$$\end{document}$ is a solution of ([1](#Equ1){ref-type=""}), ([2](#Equ2){ref-type=""}) for the velocity field $\documentclass[12pt]{minimal}
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\begin{document}$${\varvec{v}}(x,t) = \frac{r'(t)}{|x|} x, f=0$$\end{document}$ and the initial data $\documentclass[12pt]{minimal}
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\begin{document}$$u_0(x)=\frac{2}{|x|^2}x_1 x_3$$\end{document}$. We set $\documentclass[12pt]{minimal}
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\begin{document}$$\varOmega =(-4,4)^3$$\end{document}$, $\documentclass[12pt]{minimal}
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\begin{document}$$T=0.1$$\end{document}$ and choose $\documentclass[12pt]{minimal}
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\begin{document}$$\gamma =0.01, \, \epsilon = 1.85 \, h$$\end{document}$ as well as a uniform time step $\documentclass[12pt]{minimal}
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\begin{document}$$\tau _m = 0.5h^2, m=1,\ldots ,M$$\end{document}$. For this example we only display the errors on the surfaces which are in this case approximated by the quadrature rules$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \mathcal {E}_3= & {} \max _{m=1,\ldots ,M} \sum _{k=0}^{2L-1} \sum _{l=0}^{L-1} \left( \frac{\pi }{L}\right) ^2 | u(x_{k,l}(t_m),t_m) - u^{m}_h(x_{k,l}(t_m)) |^2 \, \sin \left( \frac{l \pi }{L}\right) ,\\ \mathcal {E}_4= & {} \sum _{m=1}^M \tau _m \sum _{k=0}^{2L-1} \sum _{l=0}^{L-1} \left( \frac{\pi }{L}\right) ^2 | \nabla _{\varGamma }u(x_{k,l}(t_m),t_m) - \nabla _{\varGamma } u^{m}_h(x_{k,l}(t_m)) |^2 \, \sin \left( \frac{l \pi }{L}\right) , \end{aligned}$$\end{document}$$
where$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} x_{k,l}(t)= & {} r(t) \left( \cos \left( \frac{k \pi }{L}\right) \sin \left( \frac{l \pi }{L}\right) , \sin \left( \frac{k \pi }{L}\right) \sin \left( \frac{l \pi }{L}\right) , \cos \left( \frac{l \pi }{L}\right) \right) ^T, \\&\quad k=0,\ldots ,2L-1, l=0,\ldots ,L-1. \end{aligned}$$\end{document}$$For the choice $\documentclass[12pt]{minimal}
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\begin{document}$$L=200$$\end{document}$ the results are displayed in Table [5](#Tab5){ref-type="table"}, where we see eocs close to 4 for $\documentclass[12pt]{minimal}
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\begin{document}$$\mathcal {E}_3$$\end{document}$ and eocs close to 2 for $\documentclass[12pt]{minimal}
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\begin{document}$$\mathcal {E}_4$$\end{document}$.
We conclude with Fig. [1](#Fig1){ref-type="fig"} in which we present the approximate $\documentclass[12pt]{minimal}
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\begin{document}$$u_h^m$$\end{document}$ at times $\documentclass[12pt]{minimal}
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\begin{document}$$t_m=0, 0.2,0.4$$\end{document}$ plotted on the zero level surface of $\documentclass[12pt]{minimal}
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\begin{document}$$\phi _h^m$$\end{document}$.Table 5Errors and experimental orders of convergence for Example [3](#FPar27){ref-type="sec"}*h*$\documentclass[12pt]{minimal}
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\begin{document}$$\varepsilon $$\end{document}$$\documentclass[12pt]{minimal}
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\begin{document}$$\mathcal {E}_3$$\end{document}$$\documentclass[12pt]{minimal}
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\begin{document}$$eoc_3$$\end{document}$$\documentclass[12pt]{minimal}
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\begin{document}$$\mathcal {E}_4$$\end{document}$$\documentclass[12pt]{minimal}
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\begin{document}$$eoc_4$$\end{document}$2.1651e−010.45.2016e−05--2.5203e−03--1.5309e−01$\documentclass[12pt]{minimal}
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\begin{document}$$0.2\sqrt{2}$$\end{document}$1.1008e−054.4811.3058e−031.8971.0825e−010.22.8535e−063.8966.8447e−041.8647.6547e−02$\documentclass[12pt]{minimal}
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\begin{document}$$0.1\sqrt{2}$$\end{document}$6.9422e−074.0793.4543e−041.973
Fig. 1Computational results from Example [3](#FPar27){ref-type="sec"}: $\documentclass[12pt]{minimal}
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\begin{document}$$u_h^m$$\end{document}$ at times $\documentclass[12pt]{minimal}
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\begin{document}$$t_m=0, 0.2,0.4$$\end{document}$ plotted on the zero level surface of $\documentclass[12pt]{minimal}
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\begin{document}$$\phi _h^m$$\end{document}$
Appendix {#Sec13}
========
Lemma 8 {#FPar28}
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Suppose that *u* is a smooth solution of ([1](#Equ1){ref-type=""}) and denote by $\documentclass[12pt]{minimal}
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\begin{document}$$u^e$$\end{document}$ the extension defined in ([14](#Equ14){ref-type=""}). Then $\documentclass[12pt]{minimal}
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\begin{document}$$u^e$$\end{document}$ is a solution of ([31](#Equ31){ref-type=""}) with *R* satisfying ([32](#Equ32){ref-type=""}).
Proof {#FPar29}
-----
We use the notation introduced in Sect. [2.2](#Sec4){ref-type="sec"} and begin by deriving a formula for $\documentclass[12pt]{minimal}
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\begin{document}$$\tilde{p}(x,t)$$\end{document}$ for $\documentclass[12pt]{minimal}
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\begin{document}$$x \in U_{\delta }(t), t \in [0,T]$$\end{document}$. Define$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \eta (\tau ) := F_t(p(x,t), (1-\tau ) \phi (x,t)), \quad \tau \in [0,1]. \end{aligned}$$\end{document}$$Recalling ([9](#Equ9){ref-type=""}) and the definition of $\documentclass[12pt]{minimal}
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\begin{document}$$F_t$$\end{document}$ we have$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \eta '(\tau ) = -\phi (x,t) \frac{ \nabla \phi ( \gamma _{p(x,t),t} ((1-\tau ) \phi (x,t)),t)}{ \left| \nabla \phi ( \gamma _{p(x,t),t} ( ( 1 - \tau ) \phi (x,t)),t) \right| ^2 }. \end{aligned}$$\end{document}$$Observing that $\documentclass[12pt]{minimal}
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\begin{document}$$\gamma _{p(x,t),t} ( \phi (x,t) ) = F_t( p(x,t), \phi (x,t) ) = x$$\end{document}$ and using similar arguments to calculate $\documentclass[12pt]{minimal}
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\begin{document}$$\eta ''(\tau )$$\end{document}$ we find for $\documentclass[12pt]{minimal}
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\begin{document}$$k=1,\ldots ,n+1$$\end{document}$ that$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \eta _k'(0)= & {} - \phi (x,t) \frac{ \phi _{x_k}(x,t)}{ \left| \nabla \phi (x,t) \right| ^2}, \\ \eta _k''(0)= & {} \phi (x,t)^2 \, \sum _{l,r=1}^{n+1} \left( \delta _{kr} - \frac{2 \phi _{x_k}(x,t) \phi _{x_r}(x,t)}{ \left| \nabla \phi (x,t) \right| ^2} \right) \frac{ \phi _{x_l}(x,t) \phi _{x_l x_r}(x,t)}{\left| \nabla \phi (x,t) \right| ^4}. \end{aligned}$$\end{document}$$Since $\documentclass[12pt]{minimal}
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\begin{document}$$\eta (1)=F_t(p(x,t),0)=\varPsi (p(x,t),t)= \tilde{p}(x,t)$$\end{document}$, $\documentclass[12pt]{minimal}
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\begin{document}$$\eta (0)=x$$\end{document}$ we deduce with the help of Taylor's theorem that for $\documentclass[12pt]{minimal}
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\begin{document}$$k=1,\ldots ,n+1$$\end{document}$$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned}&\tilde{p}_k(x,t) = x_k - \phi (x,t) \frac{\phi _{x_k}(x,t)}{| \nabla \phi (x,t) |^2} + \frac{1}{2} \phi (x,t)^2 \sum _{l,r=1}^{n+1} \Bigl ( \delta _{kr}\nonumber \\&\qquad - \frac{2 \phi _{x_k}(x,t) \phi _{x_r}(x,t)}{| \nabla \phi (x,t) |^2} \Bigr ) \frac{\phi _{x_l}(x,t) \phi _{x_l x_r}(x,t)}{| \nabla \phi (x,t) |^4} + \phi (x,t)^3 r_k(x,t), \end{aligned}$$\end{document}$$where $\documentclass[12pt]{minimal}
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\begin{document}$$r_k$$\end{document}$ are smooth functions. Starting from ([58](#Equ58){ref-type=""}) it is not difficult to derive formulae for $\documentclass[12pt]{minimal}
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\begin{document}$$\tilde{p}_{x_i}, \tilde{p}_{x_i x_j}$$\end{document}$ (cf. (2.9), (2.10) in \[[@CR7]\]) and hence to deduce from ([19](#Equ19){ref-type=""}) and ([20](#Equ20){ref-type=""}) that$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned}&\nabla u^e(x,t) = (I+\phi (x,t) A(x,t)) \nabla _{\varGamma }u( \tilde{p}(x,t),t) \end{aligned}$$\end{document}$$ $$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned}&\frac{1}{| \nabla \phi (x,t)|} \nabla \cdot \bigl ( | \nabla \phi (x,t)| \nabla u^e(x,t) \bigr ) = (\varDelta _{\varGamma } u)(\tilde{p}(x,t),t) \nonumber \\&\qquad +\, \phi (x,t) \left( \sum _{k,l=1}^{n+1} b_{lk}(x,t) \underline{D}_l \underline{D}_k u(\tilde{p}(x,t),t) + \sum _{k=1}^{n+1} \tilde{c}_k(x,t) \underline{D}_k u(\tilde{p}(x,t),t) \right) ,\nonumber \\ \end{aligned}$$\end{document}$$where $\documentclass[12pt]{minimal}
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\begin{document}$$A=(a_{ik}),b_{lk}$$\end{document}$ and $\documentclass[12pt]{minimal}
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\begin{document}$$\tilde{c}_k$$\end{document}$ are smooth. Furthermore, differentiating ([58](#Equ58){ref-type=""}) with respect to *t* we find that$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \tilde{p}_t(x,t)= & {} - \frac{\phi _t(x,t)}{| \nabla \phi (x,t) |^2} \nabla \phi (x,t) + \phi (x,t) q(x,t)\\= & {} V(x,t) \nu (x,t) + \phi (x,t) q(x,t), \quad q \text{ smooth } \end{aligned}$$\end{document}$$so that we infer from ([59](#Equ59){ref-type=""}), ([21](#Equ21){ref-type=""}) and ([6](#Equ6){ref-type=""}) that$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned}&\partial ^{\bullet }_t u^e(x,t) = u^e_t(x,t) + \bigl ( {\varvec{v}}(x,t), \nabla u^e(x,t) \bigr ) = u^e_t(x,t) \nonumber \\&\qquad + \bigl ( {\varvec{v}}(x,t), (I+\phi (x,t) A(x,t)) \nabla _{\varGamma }u( \tilde{p}(x,t),t) \bigr )\nonumber \\&\quad = \partial ^{\bullet }_t u (\tilde{p}(x,t),t) + \bigl ( ({\varvec{v}}(x,t) - {\varvec{v}}(\tilde{p}(x,t),t)), \nabla _{\varGamma } u(\tilde{p}(x,t),t) \bigr ) \nonumber \\&\qquad + \,V(x,t) \bigl ( (\nu (x,t) - \nu (\tilde{p}(x,t),t)), \nabla _{\varGamma } u(\tilde{p}(x,t),t) \bigr )\nonumber \\&\qquad +\, \phi (x,t) \bigl ( q(x,t) + A(x,t)^T {\varvec{v}}(x,t), \nabla _{\varGamma } u(\tilde{p}(x,t),t) \bigr ). \end{aligned}$$\end{document}$$The fundamental theorem of calculus together with ([58](#Equ58){ref-type=""}) implies that$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} {\varvec{v}}(x,t) - {\varvec{v}}(\tilde{p}(x,t),t)= & {} \int _0^1 D {\varvec{v}}(sx +(1-s) \tilde{p}(x,t),t) ds \, (x - \tilde{p}(x,t)) \\= & {} \phi (x,t) \, \tilde{q}(x,t) \end{aligned}$$\end{document}$$for some smooth $\documentclass[12pt]{minimal}
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\begin{document}$$\tilde{q}$$\end{document}$. Arguing in the same way for the corresponding difference involving $\documentclass[12pt]{minimal}
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\begin{document}$$\nu $$\end{document}$ we infer from ([61](#Equ61){ref-type=""})$$\documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \displaystyle \partial ^{\bullet }_t u^e(x,t) = \partial ^{\bullet }_t u (\tilde{p}(x,t),t) + \phi (x,t) \sum _{k=1}^{n+1} \hat{c}_k(x,t) \underline{D}_k u(\tilde{p}(x,t),t), \end{aligned}$$\end{document}$$where $\documentclass[12pt]{minimal}
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\begin{document}$$\hat{c}_k$$\end{document}$ are smooth. Finally, since $\documentclass[12pt]{minimal}
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\begin{document}$$\nabla _{\phi } \cdot {\varvec{v}}(\tilde{p}(x,t),t) = \nabla _{\varGamma } \cdot {\varvec{v}}(\tilde{p}(x,t),t)$$\end{document}$ we have$$\documentclass[12pt]{minimal}
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The authors would like to thank the Isaac Newton Institute for Mathematical Sciences for its hospitality during the programme *Coupling Geometric PDEs with Physics for Cell Morphology, Motility and Pattern Formation* supported by EPSRC Grant Number EP/K032208/1 and VS gratefully acknowledges the support of the Leverhulme Trust Research Project Grant (RPG-2014-149).
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INTRODUCTION
============
Cervical cancer is the second most common type of cancer among women in Brazil ([@b1-cln_68p809],[@b2-cln_68p809]), and in 2012, it was estimated that 17,540 new cases of cervical cancer would occur in Brazil (approximately 17 cases in every 100,000 women) ([@b3-cln_68p809]). Human papilloma virus (HPV) has been identified as a key factor in the development of cervical cancer ([@b4-cln_68p809]-[@b6-cln_68p809]). Among the HPV types classified as high-risk, HPV 16 and HPV 18 are responsible for the largest percentage of cervical cancer cases ([@b6-cln_68p809],[@b7-cln_68p809]).
Many prognostic factors for cervical cancer have been established, including clinical staging, pelvic lymph node involvement, parametrial involvement and lymphovascular space invasion ([@b8-cln_68p809]-[@b11-cln_68p809]).
Cervical cancer screening studies have reported that the prevalence of HPV infection in Brazil ranges from 15% to 27%, according to hybrid capture (HC) or polymerase chain reaction (PCR) assays ([@b12-cln_68p809],[@b13-cln_68p809]). In patients with cervical cancer, HPV DNA has been detected in 55.2% to 91% of patients, depending on the type of biological material and the method used ([@b14-cln_68p809],[@b15-cln_68p809]).
For almost 2 decades, studies have indicated the possibility that HPV 18 may negatively affect the prognosis of cervical cancer patients ([@b7-cln_68p809],[@b11-cln_68p809]),. Furthermore, a significant association was found between lymphovascular space invasion and lymph node involvement and the presence of both HPV 16 and 18 ([@b17-cln_68p809]). Nevertheless, other studies have reported varying results; some have implicated HPV 16 as an unfavorable factor, while others have failed to detect any differences between these 2 virus types ([@b17-cln_68p809],[@b19-cln_68p809]). Attempts have also been made to correlate the viral load with prognosis, and different studies have produced conflicting reports ([@b20-cln_68p809]).
Using immunohistochemistry, research has shown that activation of the epidermal growth factor receptor (EGFR) is associated with chemoradiotherapy resistance in cases of advanced cervical cancer. As a result, EGFR activation is also associated with a poor prognosis ([@b21-cln_68p809]).
Investigation of the EGFR status in early stage tumors has revealed that the lack of expression of the phosphatase and tensin (PTEN) tumor suppressor gene is associated with metastases to pelvic lymph nodes ([@b22-cln_68p809]). This same line of research demonstrated that PTEN expression decreases progressively from normal cervical tissue to cervical intraepithelial neoplasia to squamous cell carcinoma. On the other hand, the expression of survivin, a protein encoded by an anti-apoptotic gene, was shown to increase as the neoplasia progresses. Thus, PTEN and survivin expression levels may serve as indices for evaluating prognosis ([@b23-cln_68p809]).
When an invasive tumor is confined to the cervix, i.e., stage IB cervical cancer, it is often treated using the classic surgical technique known as the Wertheim-Meigs hysterectomy, a radical hysterectomy with pelvic lymphadenectomy ([@b11-cln_68p809]). Nevertheless, radiotherapy also produces results of similar efficacy ([@b24-cln_68p809]).
The objective of this study was to evaluate the prevalence of HPV types 16 and 18 in women with stage IB cervical cancer who underwent a radical hysterectomy with pelvic lymphadenectomy and to establish a correlation between HPV type and cancer prognosis.
MATERIALS AND METHODS
=====================
Sample selection
----------------
A cohort study was conducted in the Araújo Jorge Hospital in Goiânia, Goiás, Brazil. The charts of 160 women with stage I invasive cervical cancer who underwent a radical hysterectomy with lymphadenectomy between 1992 and 2003 were reviewed. This study was designed to include only those patients at clinical stage IB who had received a radical hysterectomy with pelvic lymphadenectomy. All of the patients were treated at a single institution in the city of Goiânia, Goiás, Brazil. The clinical and pathological data were analyzed according to HPV type to evaluate their effects on tumor recurrence and overall survival. The study was approved by the institution\'s internal review board (approval letter number 027/07). To analyze overall survival, an active attempt was made to contact the patients by telephone and telegram with the objective of reducing the rates of censoring due to loss to follow-up.
Samples
-------
A total of 92 biopsies of the cervix (samples fixed in formalin and embedded in paraffin) were selected from the hospital\'s anatomopathology department and tested for HPV using PCR. The molecular analysis was performed at the Oncology Research Institute (IPON) of the Federal University of Triângulo Mineiro (UFTM), Uberaba, Minas Gerais, Brazil.
DNA extraction from paraffin-embedded samples
---------------------------------------------
The paraffin-embedded blocks were cut into 5-μm-thick sections and placed in 2-ml Eppendorf tubes. They were then submitted to the following deparaffinization process. Briefly, 1 ml of 97% xylol was added to each microtube, and the mixture was homogenized in a vortex mixer, heated in an oven at 60°C for 10 minutes and centrifuged at 2,500 rpm for 10 minutes. The excess xylol was removed, and the quality of the DNA was verified using β-actin. Next, 200 μL of chloroform was added for each 1.0 ml of TRIZOL®. The mixture was then vortexed for 15 seconds, incubated at room temperature for 3 minutes and centrifuged at 12,000 g for 15 minutes at 4°C. Next, the pellets were washed twice in 300 μl of 100% ethanol, and then 1 ml of 75% ethanol was added. The material was placed in the refrigerator and allowed to dry for 12 hours before posterior amplification of the sample. At the time of use, this precipitate was again suspended in Tris-acetate-EDTA buffer.
HPV genotyping and DNA amplification cycle
------------------------------------------
To identify molecular HPV and β-actin, PCR amplification was performed. The reaction mixture contained 5.0 μl 10× buffer, 1.0 μl dNTPs (10 mM), 1.5 μl 50 mM MgCl~2~, 0.2 μl Taq DNA polymerase and distilled H~2~O to a final volume of 50.0 μl. The reaction mixture was then added to a tube containing 1.0 μl primer and 4.0 μl of DNA to reach a final volume of 50.0 μl. Type-specific primers for HPV 16 (5\' = 5\'ACCGAAACCGGTTAGTATAAAAGC3\' and 3\' = 5\'ATAACTGTGGTAACTTTCTGGGTC3\') with a product of 477 base pairs (bp) and primers for HPV 18 (5\' = 5\'CGGTCGGGACCGAAAACGGTG3\' and 3\' = 5\'CGTGTTGGATCCTCAAAGCGCGCC3) with a product of 422 bp were used. β-actin primers (5\' = 5\'GTGGGGCGCCCCAGGCACCA3\' and 3\' = 5\'CTCCTTAATGTCACGCACGATTTC3\') with a product of 295 bp were used as an internal control. Annealing was performed at 56°C for each of these 3 primers ([@b25-cln_68p809],[@b26-cln_68p809]). The reaction was initiated at 94°C for 1 minute for denaturation, followed by 30 cycles of 2 minutes at 50°C for annealing and 3 minutes at 72°C for polymerization. The reaction was amplified using an Eppendorf thermal cycler.
Statistical analysis
--------------------
Various clinical and pathological characteristics were analyzed, including the age of the patient at the time of cancer diagnosis, the number of pregnancies and deliveries she had prior to her cancer diagnosis and the histological type of the tumor (based on the World Health Organization\'s (WHO) classifications). In addition, the following factors were taken into consideration: the degree of anaplasia according to the WHO classifications (grades I, II, III or undifferentiated), whether there was lymphovascular invasion and whether the pelvic lymph nodes were affected. Because the study objective was to characterize the sample of patients with cervical cancer, descriptive analyses rather than statistical analyses were initially performed on the study variables mentioned above. The differences in parameters between the groups were assessed using the chi-square test and Fisher\'s exact test, as appropriate. To calculate survival, the Kaplan-Meier method was used, while the log-rank test was applied to compare the mean survival rates associated with different possible prognostic factors for cervical cancer. In calculating the overall survival, all deaths were taken into consideration, regardless of their cause. For cancer-associated survival, the criterion applied was the event (i.e., the recurrence of locoregional or metastatic disease). The patients who were alive at the time of the last medical follow-up visit or who died after 60 or more months of follow-up were considered censured. P-values \<0.05 were considered statistically significant for all tests. The Statistical Package for the Social Sciences (SPSS), version 15.0 for Windows (SPSS®, Chicago, IL, USA), was used for all statistical analyses.
RESULTS
=======
The ages of the patients in this study ranged from 26 to 64 years, with a mean of 40±8.95 years (standard deviation \[SD\]). The mean duration of follow-up was 67 months (range 4-134 months; median 73 months; SD 44.14 months).
Of the 86 patients studied, 8 suffered a disease recurrence, and 4 of these patients died during the study. Only 1 patient had a diagnosis of vaginal intraepithelial neoplasia (VAIN) III during the clinical follow-up period, and this condition regressed spontaneously. The VAIN III diagnosis occurred 2 years after the patient had been treated for a recurrence of the pelvic tumor. This recurrence was treated with radiotherapy, and there is currently no sign of disease in this patient. The demographic, clinical and pathological characteristics of the patients who tested positive for HPV 16 and 18 are shown in [Table 1](#t1-cln_68p809){ref-type="table"}.
With respect to genotype, HPV 16 alone was found in 30 samples, whereas HPV 18 alone was detected in only 5 samples. The concomitant presence of both types of HPV was detected in 21 samples. In 22 samples, neither of these viral types was detected.
With regard to the histological type, HPV 18 was detected in 84.6% of squamous cell carcinomas (22 cases), in 7.7% of adenocarcinomas (2 cases) and in 7.7% of adenosquamous cell carcinomas (2 cases). HPV 18 was not detected in any cases of undifferentiated carcinoma. HPV 16 was detected in 85.2% of squamous cell carcinomas (46 cases), in 9.3% of adenocarcinomas (5 cases) and in 5.6% of adenosquamous carcinomas (3 cases).
Regarding the degree of anaplasia, in tumors that tested positive for HPV 18, the prevalence of grade II (moderately differentiated) was 70.8% (17 cases), while 25% (6 cases) of the tumors were classified as grade III (highly differentiated).
For the tumors that tested positive for HPV 16, the prevalence of grade II anaplasia was 66% (33 cases), while 16 cases (32%) were classified as grade III and 1 case (2%) as grade I.
In this sample, only 5 cases were found in which the pelvic lymph nodes were affected by the neoplasia. HPV 18 was detected in 2 of these cases (7.7%), and HPV 16 was detected in another 2 cases (5.6%).
Lymphovascular invasion associated with HPV 18 was observed in 29.4% of the 63 cases for which this information was available in the patients\' charts. With respect to HPV 16, lymphovascular invasion was found in 66.7% of the 66 cases for which this variable was provided.
The majority of recurrences occurred within 30 months after the initial treatment was completed, and the 5-year disease-free survival rate was 91%. The eighth recurrence is not shown on the survival curve because it occurred after 60 months. The 5-year overall survival rate in the study group was 95%. The 5-year disease-free survival rates were 92% and 88% for the patients who tested positive and negative for HPV 16, respectively (*p*\>0.05). The 5-year disease-free survival rates were 83% and 93% for those who tested positive and negative for HPV 18, respectively, and there were no statistically significant differences between the groups.
The overall survival rate among women who tested positive for HPV 16 was 94% compared to 96% among those who tested negative (*p*\>0.05) ([Figure 1A](#f1-cln_68p809){ref-type="fig"}). The women who tested positive for HPV 18 had an overall 5-year survival rate of 91% compared to 96% among those who tested negative (*p*\>0.05) ([Figure 1B](#f1-cln_68p809){ref-type="fig"}). When disease-free survival was analyzed in relation to the other independent variables, none of the factors evaluated were associated with cervical cancer recurrence ([Figure 1](#f1-cln_68p809){ref-type="fig"}). Furthermore, none of the factors evaluated in this study were found to affect overall survival, as shown in [Table 2](#t2-cln_68p809){ref-type="table"}.
DISCUSSION
==========
Of the various prognostic factors for cervical cancer, the clinical staging system proposed by the International Federation of Gynecology and Obstetrics (FIGO) remains the most significant, with a 5-year survival rate of approximately 90% up to clinical stage IB1. Other relevant factors include pelvic lymph node metastases (12-20% at clinical stage IB) and para-aortic lymph node metastases (4-7% at clinical stage I). However, recovery rates may fall considerably when the pelvic lymph nodes are involved, and para-aortic metastases are present principally when the pelvic lymph nodes are also affected. Parametrial invasion and lymphovascular space invasion are also considered relevant findings. As individual factors, age and tumor grade are not as important as other prognostic factors, according to a previous report by Zaino et al. ([@b27-cln_68p809]).
In the present study, HPV 16 and 18 DNA was found in 62.7% and 30.2% of the women, respectively, which is not surprising because the majority of women between 50-59 years of age have been shown to test positive for these HPV types ([@b28-cln_68p809]). In this study, HPV 16 and 18 DNA was found in the majority of the older women between 30-50 years of age, and this finding is also in agreement with the results of other studies ([@b29-cln_68p809],[@b30-cln_68p809]).
Previous work has shown that the presence of HPV 18 may be considered an independent prognostic factor for a poor outcome in early stage cervical cancers ([@b7-cln_68p809],[@b11-cln_68p809],[@b16-cln_68p809]), and the results of the present study are in agreement with this line of reasoning. In fact, the current study included early stage cervical tumors precisely because the literature indicates that the prognosis is otherwise good in these cases.
The current study evaluated the effect of HPV 16 and 18 on the prognosis of women with early stage invasive cervical cancer. The results demonstrated an overall 5-year survival rate of 95%, which may be because only 10.4% of the women in this study sample were staged as IB2 (i.e., tumors larger than 4 cm) according to the staging classification of the FIGO. In other studies, the overall 5-year survival rate for those with early stage tumors has also been high ([@b1-cln_68p809],[@b31-cln_68p809]). Nevertheless, although 5-year survival rates for small-volume tumors are usually as high as 90%, the survival rates for larger tumors may fall to 70% after 5 years ([@b32-cln_68p809]).
The most prevalent histological type was squamous cell carcinoma, which was present in 84.4% of the cases evaluated in this study, followed by adenocarcinoma and adenosquamous cell carcinoma, which were found in 12.4% of the cases. These findings are in agreement with the data published in the literature ([@b7-cln_68p809],[@b11-cln_68p809]). However, it remains controversial as to whether adenosquamous carcinoma is associated with a poor prognosis; although some authors advocate this hypothesis ([@b33-cln_68p809]), others argue that there is no supporting evidence ([@b24-cln_68p809]).
Adenocarcinomas were not associated with a greater likelihood of tumor recurrence or a poorer prognosis in this study group, and only 1 patient experienced tumor recurrence. HPV 18 was only associated with 1 case of adenocarcinoma and 2 cases of adenosquamous cell carcinoma. Furthermore, 20 cases of adenocarcinoma had to be excluded from the study because it was impossible to recover the corresponding paraffin blocks from the anatomopathology department, and it is possible that this exclusion may have affected the final results.
In the present study, HPV type had no effect on the disease-free or overall survival rates and appeared to have no effect on the likelihood of tumor recurrence.
Numerous prognostic factors for cervical cancer have been documented in the literature, including clinical staging, invasion of the lymph nodes by tumor cells, parametrial involvement and invasion of the lymphovascular space ([@b8-cln_68p809],[@b9-cln_68p809],[@b10-cln_68p809],[@b11-cln_68p809]). Nonetheless, various investigators have attempted to clarify the precise role of HPV type in tumor progression ([@b7-cln_68p809],[@b11-cln_68p809]),.
In this study, the number of cases in which the tumor had invaded the pelvic lymph nodes was small (5 cases, 5.5%). Of the 8 patients who experienced recurrences, pelvic lymphatic metastasis was present in only 1 patient. Lymphatic metastasis is considered one of the most important prognostic factors and one that may affect survival. In turn, lymphatic metastasis can be affected by angiolymphatic invasion, an increase in tumor size or the depth of the stromal invasion, as shown in a study conducted by the Gynecology Oncology Group ([@b10-cln_68p809],[@b32-cln_68p809]). The small number of recurrences in the present study may also be attributable to the fact that there were few cases in which the lymph nodes were affected.
When a specific analysis was conducted on patients with early stage tumors (IB and IIA) who had undergone radical hysterectomy and pelvic lymphadenectomy, it was found that women with HPV 18 tended to have a poorer prognosis ([@b7-cln_68p809]). In another study, the presence of HPV 18 in patients with early cervical carcinoma was associated with a significantly poorer prognosis compared to women infected with HPV 16, even after adjusting for other relevant factors, such as clinical stage, lymph node status and histological type ([@b12-cln_68p809]). Nevertheless, in a study conducted in Russian women is not observed in HPV type influences the prognosis of women, which had staged tumors classified as stage I and stage II ([@b19-cln_68p809]). However, in that study, no specific analysis was performed with respect to the type of treatment used, unlike the study mentioned previously ([@b19-cln_68p809]). In the study of van Muyden et al. 1999, HPV was detected in all cases studied, corroborating the hypothesis that HPV-negative cervical cancer does not exist ([@b5-cln_68p809],[@b19-cln_68p809]).
It has also been reported that tumors positive for HPV 16 are more likely to metastasize to the pelvic and parametrial lymph nodes compared to HPV-16-negative tumors ([@b38-cln_68p809]) and that HPV 16 negatively affects the prognosis of patients who receive a radical hysterectomy with pelvic lymphadenectomy ([@b39-cln_68p809]). A study conducted by Pilch et al. ([@b17-cln_68p809]) found a significant association between involvement of the lymph nodes and the lymphovascular space and the presence of HPV types 16 and 18. However, in the present study, the presence of HPV 16, although more prevalent, had no effect on the characteristics mentioned above.
Multiple-type HPV infection has been associated with a poorer response to radiotherapy and a poorer prognosis in patients with local advanced cervical cancer ([@b31-cln_68p809],[@b40-cln_68p809]). Nevertheless, for the patients evaluated in the present study, concomitant infection with HPV 16 and 18 had no detrimental effect on their prognosis.
In recent years, some studies have tried to establish HPV 18 as an indicator of an unfavorable clinical progression. In this respect, the present study sought to select a more homogenous group of women with early cervical cancer (i.e., restricted to clinical stage IB) to evaluate whether HPV 18 had a negative effect on their prognosis. However, it was difficult to obtain an adequate number of cases because an unexpected number of charts (230) could not be located by the Department of Medical Records and Statistics of the Araújo Jorge Hospital. Furthermore, due to a similar situation in the anatomopathology department, it was impossible to recover the paraffin blocks of tumor samples for 68 patients who were eligible for the study, a fact that certainly contributed to the inadequate number of cases for the projected analysis.
Furthermore, it was impossible to identify any factor analyzed that was significantly associated with disease recurrence, which may have been due to the limited size and homogeneity of the sample or the effect of other factors that are still under investigation.
In the present study, the prevalence of HPV 16 in the study group was greater than the prevalence of HPV 18. However, the presence of HPV 16 or 18 was unrelated to the histological type and the degree of anaplasia, vascular invasion or lymph node involvement. Despite the high prevalence of HPV 18 and/or HPV 16, the presence of these HPV types did not affect the prognosis of the study patients with stage I cervical cancer who underwent radical hysterectomy.
ACKNOWLEDGMENTS
===============
This study was conducted within the Postgraduate Program in Tropical Medicine and Public Health at the Institute of Tropical Pathology and Public Health, Federal University of Goiás. The study was supported by the National Council for Scientific and Technological Development (CNPq) and the Minas Gerais State Research Foundation (FAPEMIG).
No potential conflict of interest was reported.
{#f1-cln_68p809}
######
Demographic, clinical and pathological characteristics of the patients with stage I cervical cancer who underwent radical hysterectomy and tested positive for HPV types 16 and 18.
Characteristics HPV 16\* HPV 18\*
------------------------------- ---------- ---------- ------ ------
**Age (years)**
Median 39 39
Range 9.27 8.77
≤30 5 9.3 3 11.5
30--50 39 72.2 19 73.1
\>50 10 18.5 4 15.4
**Clinical stage (FIGO)**
IB1 46 85.2 22 84.6
IB2 8 14.8 4 15.4
**Histological type**
Squamous cell carcinoma 46 85.2 22 84.6
Adenocarcinoma 5 9.3 2 7.7
Adenosquamous cell carcinoma 3 5.6 2 7.7
Others 0 0 0 0
**Grade of differentiation**
Grade I 1 2 1 4.2
Grade II 33 66 17 70.8
Grade III 16 32 6 25
**Metastases to lymph nodes**
Yes 3 5.6 3 12
No 51 94.4 22 88
**Angiolymphatic invasion**
Yes 12 28.6 5 25
No 30 71.4 75 75
**Initial treatment**
Surgery alone 42 77.8 20 76.9
Surgery + radiotherapy 9 16.7 4 15.4
Radiotherapy + surgery 3 5.6 2 7.7
**Recurrence of the disease**
Yes 5 9.8 3 12
No 46 90.2 22 88
**Treatment of recurrence**
Radiotherapy 0 0 0 0
Chemotherapy 1 100 1 100
Radiotherapy + chemotherapy 0 0 0 0
HPV-positive patients.
######
Univariate analysis of the presence or absence of recurrence according to the clinical and pathological characteristics and the HPV type in women with stage IB cervical cancer.
Recurrence
----------------------------- ------------ ------ ---- ------ ------- ----------------------
Age group
≤39 years 5 13.5 32 86.5 1
40-64 years 3 6.5 41 93.5 0.283 2.240 (0.498-0.064)
Clinical staging
IB1 7 9.3 68 90.7 1
IB2 1 12.5 7 87.5 0.572 0.721 (0.077--6.735)
Pregnancies
1-3 pregnancies 2 9.1 20 90.9
\>4 pregnancies 6 10 55 90 0.635 0.900 (0.168--4.832)
Deliveries
1-3 deliveries 1 3.1 31 96.9 1
\>4 deliveries 7 14 43 86 0.141 0.198 (0.023--1.694)
Histological type
Squamous cell carcinoma 7 9.7 65 90.3 1
Non-squamous cell carcinoma 1 9.1 10 90.9 0.947 1.077 (0.120--9.705)
Lymph nodes
None 7 9 71 91
\>1 1 25 3 75 0.342 3.381 (0.309-6.996)
Treatment
Hysterectomy 5 8.1 57 91.9 1
Other treatments 3 14.3 18 85.7 0.604 0.526 (0.114--2.422)
HPV 16
Negative 3 10.7 25 89.3 1
Positive 5 9.8 46 90.2 0.898 1.104 (0.243--5.007)
HPV 18
Negative 5 9.8 46 90.2 1
Positive 3 12 22 88 0.769 0.797 (0.175--3.640)
Fischer\'s exact test.
[^1]: Zampronha RA and Freitas-Junior R contributed to the conception and design of the study, acquisition of the data, analysis and interpretation of data, initial draft of the manuscript and final approval of the submitted version. Murta EF and Michelin MA contributed to the conception and design of the study, acquisition of the data, interpretation of the data, critical review of the manuscript and final approval of the submitted version. Barbaresco AA, Adad SJ and Oliveira AM contributed to the conception of the study, analysis and interpretation of the data and approval of the manuscript final version. Rassi AB contributed to the analysis and interpretation of the data, review of the article and approval of the manuscript final version. Oton GJB contributed to the analysis and interpretation of the data and approval of the manuscript final version.
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Introduction {#s1}
============
Snake venoms are complex mixtures of enzymatic and non enzymatic proteins, together with other components such as carbohydrates, lipids, nucleosides and metals. These function together to immobilize, kill and digest the prey [@pone.0021532-Aird1] `.` Snake venoms have various envenomation effects and can be haemotoxic, myotoxic, neurotoxic and nephrotoxic towards prey and victims [@pone.0021532-Heller1]. Snake venom serine proteases are a major component of venom and have been identified mainly in the venoms of snakes belonging to the viperidae family with a few occurring in members of the elapidae, colubridae and hydrophidae families [@pone.0021532-Serrano1]. Viper venom serine proteases (VVSPs) share similar nucleotide and amino acid sequences (with more than 60% sequence identity) and also three-dimensional structures, but have diverse functions. Generally they have haemotoxic effects, by affecting various stages of the blood coagulation system. They can act either as pro-coagulants via fibrin formation, factor V activation, kininogenolysis or platelet aggregation, or as anti-coagulants via fibrinolysis, plasminogen activation or protein C activation [@pone.0021532-Markland1]. Several VVSP nucleotide sequences have been obtained by screening and sequencing venom gland cDNA libraries. Within these the 5′ untranslated regions (UTRs), N-terminal signal and activation peptide-coding sequences and 3′ UTRs have been found to be more highly conserved than the mature protein coding sequences [@pone.0021532-Siigur1]. Thus it is possible to identify novel VVSPs by screening cDNA libraries or amplifying a cDNA pool using specific primers for the conserved regions. Analysis of the nucleotide and amino acid sequences of these enzymes will help to understand their structure and function and provide insight into their evolution. A knowledge of the diversity of toxins and enzymes present within snake venom will also aid the development of novel treatments for snake bites. In this report, we describe the amplification and sequencing of four serine proteases from the venom gland transcriptome of the Gaboon viper *Bitis gabonica rhinoceros* using specific primers designed for the 5′ signal peptide coding sequence and the 3′ UTR and discuss the possible functions and evolution of these enzymes.
Materials and Methods {#s2}
=====================
Materials used {#s2a}
--------------
Lyophilized venom of *B. g. rhinoceros* was obtained from the Liverpool School of Tropical Medicine, Liverpool, UK. The Illustra mRNA purification system was from GE Healthcare (Amersham, UK). Restriction enzymes, GoTaq® PCR Core System I and Wizard® SV Gel and PCR Clean-Up System were from Promega (Southampton, UK). The ZAP-cDNA synthesis kit was from Stratagene (La Jolla, USA), the TOPO TA Cloning® system and SimplyBlue™ SafeStain were from Invitrogen (Paisley, UK), and the QIAprep Spin Miniprep kit was from Qiagen (West Sussex, UK). TRI Reagent® and all other chemicals used were analytical grade from Sigma Aldrich (Poole, UK).
Ethical statement {#s2b}
-----------------
All activities conducted in the Alistair Reid Venom Research Unit at the Liverpool School of Tropical Medicine are licensed and approved by the UK Home Office (Project Licence 40/3216). Venom extraction from the snakes used in this study is no longer a procedure regulated by the Animals (Scientific Procedures) Act 1986. All efforts were made to minimize the suffering of animals.
Venom gland cDNA synthesis {#s2c}
--------------------------
To enhance the expression of the venom gland genes, venom was extracted from a single specimen of *B. g. rhinoceros* maintained in the Liverpool School of Tropical Medicine three days before the dissection of venom glands. Total RNA was isolated from the venom gland tissues using TRI Reagent® and polyadenylated mRNA was purified using the Illustra mRNA purification system according to the manufacturer\'s protocols. cDNA was synthesized from the purified mRNA using the ZAP-cDNA synthesis kit.
PCR amplification {#s2d}
-----------------
Specific primers were designed for the 5′ signal peptide coding sequence and the 3′ UTR of the known *B. gabonica* serine protease I sequence (NCBI accession number: AAR24534) [@pone.0021532-FrancischettiIMMyPham1] and synthesized by Sigma Aldrich (Poole, UK). The sequences of the primers are: forward primer- 5′TGGTGTTGATCAGAGTGCT3′ and reverse primer- 5′ACAGAAGTACCAATAGAAGAGAAT3′. These primers were used to amplify the serine protease genes present in the venom gland cDNA by PCR (20 cycles) using denaturation at 94°C for 30 seconds, annealing at 52.7°C for 30 seconds, extension at 72°C for 1 minute and a final extension at 72°C for 10 minutes.
Purification, cloning and sequencing of amplified DNA {#s2e}
-----------------------------------------------------
The amplified product was analysed by 1% (w/v) agarose gel electrophoresis and the gel was sliced to purify the amplified DNA using the Wizard® SV Gel and PCR Clean-Up System. Eluted DNA was cloned into a TOPO TA Cloning® system according to the manufacturer\'s protocols. The recombinant colonies were selected and grown in LB broth and the plasmids were purified using a QIAprep Spin Miniprep kit. Restriction digest analysis was used to confirm the presence of inserts and the plasmids were sequenced using M13 forward and reverse primers (as these sites flank the multi cloning site of the TOPO vector) by Cogenics Limited (Essex, UK).
Venom protein separation {#s2f}
------------------------
The venom proteins were separated using a micro rotofor (Bio-Rad, Hemel Hempstead, UK) as described previously [@pone.0021532-Vaiyapuri1]. Briefly, 1 mg of *B. g. rhinoceros* venom was mixed with 3 ml of non-reducing rotofor buffer \[7 M urea, 2 M thiourea, 10% (v/v) glycerol and 2.5% (v/v) amphoyltes (pI 3--10) in Milli-Q water\] and loaded on to the focussing chamber. Isoelectric focussing was performed under cooling setting I (temperature between 4°C and 15°C) with the programmed electric field (150 V/2 W/20 mA for 15 minutes, 200 V/2 W/20 mA for 15 minutes, 300 V/2 W/20 mA for 20 minutes, 350 V/2 W/20 mA for 20 minutes and 400 V/2 W/20 mA for 60 minutes). 0.1 M orthophosphoric acid and 0.1 M sodium hydroxide were used as anode and cathode electrode buffers respectively. Twenty microlitres of separated rotofor fractions were used to analyse the serine protease activity and 10 µl were used for SDS-PAGE to analyse the protein separation patterns.
SDS-PAGE and staining {#s2g}
---------------------
Reducing SDS-PAGE was performed using standard techniques [@pone.0021532-Gibbins1]. The gel was stained with SimplyBlue™ SafeStain and scanned using a Typhoon Trio variable mode imager (GE Healthcare, Amersham, UK) before excising the bands for mass spectrometry.
Serine protease assay {#s2h}
---------------------
Serine protease activity of rotofor separated factions was measured using a fluorescent substrate, Nα-benzoyl-L-arginine 7-amido-4-methylcoumarin.HCl (Arg-AMC) (B7260, Sigma Aldrich) as previously described [@pone.0021532-Bicknell1]. Twenty microlitres of separated rotofor fractions were mixed with Arg-AMC (20 nM) along with trypsin and thrombin as positive controls and incubated at 37°C. The amount of 7-amido-4-methylcoumarin (AMC) released was measured at different time points using a spectrofluorimeter (FLUOstar OPTIMA, BMG Labtech, Offenburg, Germany) at an excitation wavelength of 366 nm and an emission wavelength of 460 nm. All measurements were performed in triplicate.
In-gel digests, liquid chromatography-tandem mass spectrometry {#s2i}
--------------------------------------------------------------
In gel tryptic digestion was performed as described previously [@pone.0021532-Bindschedler1]. Tryptic digests were then reconstituted in 12 µl 0.1% TFA. Four microlitres of sample were loaded for 5 minutes at 30 µl/minute 0.2% TFA on a 10 mm trap column packed with 3.5 µm C~18~ particles (LC Packings/Dionex, Amsterdam, The Netherlands) and eluted in 0.2% formic acid for 10 minutes in a 2 to 15% CAN gradient followed by 80 minutes on a 15--40% gradient and finally 15 minutes on a 40--55% gradient at 250 nl/minute on a 15 cm×75 µm PepMap C~18~ reverse phase analytical column (3.5 µm particles; Packings/Dionex) using an Ultimate™ nLC system (LC Packings/Dionex). The LC system was coupled to a nESI-MS/MS 3D ion trap mass spectrometer (HCT Esquire; Bruker Daltonics, Bremen, Germany) and the nanoESI source was mounted with a 5 cm long stainless steel emitter (Proxeon). The LC system and the ion trap were controlled through HyStar™ and Esquire Control modules in the Compass™ software suite (Bruker Daltonics, Coventry, UK). Mass spectra were acquired from *m/z* 300 to 2,000 using parameters optimised at *m/z* 850 with the trap ion charge control set at 150,000 and a maximum acquisition time of 200 ms averaging three scans per spectrum. The three most abundant ions were selected for MS/MS, the isolation window was 4 *m/z* with a signal threshold of 5,000 and the fragmentation amplitude was 2 V. The selected precursor ions were actively excluded for 45s after two selections. Raw LC-MS/MS data were batch-processed in DataAnalysis 4.0 (Bruker Daltonics, Bremen, Germany). Up to 3,000 2^+^ and 3^+^ compounds (retention time restriction of 10--120 minutes) with a signal-to-noise ratio above 5 were extracted and exported as Mascot Generic Files (MGF).
For protein identification MGF files were submitted to Mascot2.2.2 (Matrix Science) on an in-house server. The Mascot search parameters were the following: 1.2Da error tolerance in MS mode and 0.4Da error tolerance in MS/MS mode, allowance of up to one tryptic missed cleavages and 2^+^,3^+^ and 4^+^ charged ions considered. Cysteine carbamidomethylation was set as a permanent modification and methionine oxidation was included as a variable modification.
Sequence analysis {#s2j}
-----------------
The nucleotide and translated protein sequences were analysed using DNASTAR Lasergene version 7 [@pone.0021532-Burland1]. Multiple sequence alignment and pairwise alignments were performed using ClustalW [@pone.0021532-Larkin1] and EMBOSS [@pone.0021532-Rice1] respectively. The sequence alignment figure was prepared using GeneDoc [@pone.0021532-Nicholas1]. Glycosylation prediction was performed using NetNGlyc [@pone.0021532-Gupta1]. To generate the phylogenetic tree, sequences were aligned using ClustalW within MEGA 4 [@pone.0021532-Kumar1] using a gap opening penalty of 10 and a gap extension penalty of 0.1 for the initial pairwise alignment and gap opening penalty of 3 and gap extension penalty of 1.8 for the multiple alignment and the Gonnet protein weight matrix. The phylogenetic tree was generated within MEGA 4 using the neighbour-joining method and the Jones-Taylor-Thornton substitution model. The bootstrap test was done using 2000 replications. The sequence of bovine α-chymotrypsinogen (NCBI accession number: P00766) was used as an outgroup.
Structural modelling of rhinocerases {#s2k}
------------------------------------
Structural models of rhinocerases 2 to 5 were created using the IntFOLD server [@pone.0021532-Roche1]. Good quality models were obtained for each sequence using the structure of rat trypsin (PDB code: 1co9) as a template. Models were visualised and compared with each other and the structures of bovine α-chymotrpysin (PDB code: 1YPH) and rat trypsin (PDB code: 1CO9) using PyMOL (DeLano Scientific).
Results and Discussion {#s3}
======================
Amplification of venom gland serine protease genes {#s3a}
--------------------------------------------------
In order to amplify the serine protease genes from the venom gland transcriptome of *B. g. rhinoceros*, specific primers were designed for the 5′ signal peptide coding sequence and 3′ UTR of the *B. gabonica* serine protease I sequence [@pone.0021532-FrancischettiIMMyPham1] as these regions are likely to be similar in other venom gland serine protease genes. The amplification resulted in DNA fragments with two different molecular masses, corresponding to around 900 bp and 1200 bp. This suggests the presence of venom gland serine protease genes which have similar 5′ signal peptide coding regions and 3′ UTRs but different lengths. Similar amplified products were obtained previously from the venom gland transcriptome of *Macrovipera lebetina* [@pone.0021532-Siigur1].
Sequence analysis of rhinocerases 2 to 5 {#s3b}
----------------------------------------
Sequencing of the amplified cDNA clones resulted in four distinct serine protease sequences, of lengths 906, 907, 1137 and 1179 bp. Each of these was confirmed by sequencing several clones. The 906 and 907 bp sequences were named rhinocerase 2 and rhinocerase 3, and the 1137 and 1179 bp sequences were named rhinocerase 4 and rhinocerase 5 respectively, following on from the naming of our previously purified serine protease, rhinocerase (now renamed rhinocerase 1) from the venom of *B. g. rhinoceros* [@pone.0021532-Vaiyapuri1]. (These have been deposited in the GenBank database under Accession Numbers FN868645 to FN868648.) Rhinocerases 2 and 3 are very similar to each other (nucleotide sequences 98% identical) and encode similar protein sequences (94% identical) with 259 amino acids ([table 1](#pone-0021532-t001){ref-type="table"}). Similarly rhinocerases 4 and 5 are 90% identical to each other and encode proteins with 257 and 259 amino acids respectively which share 92% sequence identity. However the nucleotide and protein sequences of rhinocerases 2 and 3 are on average only 64% and 69% identical to those of rhinocerases 4 and 5.
10.1371/journal.pone.0021532.t001
###### Features of the nucleotide and protein sequences of rhinocerases 2 to 5.
{#pone-0021532-t001-1}
Sequence Length of cDNA (bp) Predicted coding region (bp) Length of mature protein (aa) Predicted mol. mass (kDa) Predicted isoelectric point No. of predicted N-glycosylation sites
--------------- --------------------- ------------------------------ ------------------------------- --------------------------- ----------------------------- ----------------------------------------
Rhinocerase 2 907 3--779 236 26.28 8.1 1
Rhinocerase 3 906 3--779 236 26.27 8.1 2
Rhinocerase 4 1137 3--773 234 25.65 8.7 3
Rhinocerase 5 1179 3--779 236 26.05 8.9 2
The predicted coding regions, molecular masses and isoelectric points were obtained from DNASTAR Lasergene version 7. The potential N-glycosylation sites were predicted by NetNGlyc.
Rhinocerases 2 to 5 share several common features of viper venom serine proteases: 12 conserved cysteine residues and N-terminal signal (normally 18 amino acids) and activation peptides (normally 6 amino acids). Since the first nucleotide of the start codon was not included in our forward primer, our translated protein sequences show only 17 amino acids in the signal peptide region. However, the signal sequences of the native proteins would be expected to have 18 amino acids, similar to other VVSPs. As is common for VVSPs including rhinocerase 1 [@pone.0021532-Vaiyapuri1] and other venom enzymes [@pone.0021532-Vaiyapuri2], N-glycosylation sites were predicted in all four proteins ([table 1](#pone-0021532-t001){ref-type="table"}) and thus the molecular masses of the native enzymes in the secreted venom may be higher than the predicted molecular masses. The predicted isoelectric points of rhinocerases 2 to 5 were between 8 and 9 which is clearly distinct from rhinocerase 1, the serine protease which we previously purified from the venom of this snake which had an isoelectric point of around 6, although the latter was measured for the glycosylated protein [@pone.0021532-Vaiyapuri1].
Within a gel of rotofor-separated *B. g. rhinoceros* venom we have identified five distinct bands with molecular masses ([figure 1A](#pone-0021532-g001){ref-type="fig"}, bands 1, 7, 8, 11 and 12) and activities ([figure 1B](#pone-0021532-g001){ref-type="fig"}) consistent with serine proteases. The pIs of these proteins together with sequences derived from mass spectrometry analysis of tryptic digests of the gel bands are consistent with rhinocerases 1 to 5 ([figure 1C](#pone-0021532-g001){ref-type="fig"}). This suggests that, in addition to rhinocerase 1, rhinocerases 2 to 5 also exist in the venom of *B. g. rhinoceros*. This is also consistent with a proteomic analysis of *B. g. rhinoceros* venom which identified the N-terminal sequences of five distinct serine proteases [@pone.0021532-Calvete1]. One of these sequences is consistent with rhinocerases 2, 3 and 5, two (identical sequences) are consistent with rhinocerase 4, and a further sequence is consistent with our purified rhinocerase 1 [@pone.0021532-Vaiyapuri1] (underlined in [figure 1C](#pone-0021532-g001){ref-type="fig"}). The fifth sequence identified in the previous proteomic analysis must represent a serine protease distinct from any identified in our research so far. The only complete sequence of a serine protease within *B. gabonica* determined to date is that of serine protease I, which has been found at transcript level only [@pone.0021532-FrancischettiIMMyPham1]. The nucleotide sequence of this serine protease is almost identical to that of rhinocerase 2; there is one substitution in the mature protein-coding region at position 626 and a substitution in the 3′ UTR at position 895. The amino acid sequences of the corresponding proteins are identical, thus the nucleotide substitutions could represent synonymous mutations. Together these data are consistent with rhinocerases 2 to 5 representing novel venom serine proteases present in the venom of *B. g. rhinoceros*.
{ref-type="fig"}) were analysed by mass spectrometry and the corresponding peptide sequences are shown in different colours (grey: band 1; red: band 7; yellow: band 8; blue: band 11; green: band 12) in italics on rhinocerase 1, 2, 4, 3 and 5 respectively. The N-terminal sequences of serine proteases in the venom of *B. g. rhinoceros* identified by proteomic analysis previously are underlined. The symbols ⋆, : and . indicate conserved residues, biochemically related residues and biochemically less related residues respectively.](pone.0021532.g001){#pone-0021532-g001}
Serine proteases in the *B. g. rhinoceros* transcriptome and their homologues {#s3c}
-----------------------------------------------------------------------------
Trypsin-like serine proteases share a catalytic triad which comprises His57, Asp102 and Ser195 (bovine α-chymotrypsinogen numbering). These residues are conserved in rhinocerases 4 and 5 and in the majority of known VVSPs. Thus rhinocerases 4 and 5 would be expected to be catalytically active. However rhinocerases 2 and 3 have His57Arg and Ser195Asn substitutions. Mutations to the catalytic triad residues have previously been observed in a small number of viper venom serine proteases [@pone.0021532-Siigur1], [@pone.0021532-FrancischettiIMMyPham1], [@pone.0021532-Wagstaff1], [@pone.0021532-Wu1]. The majority of these sequences have been identified at transcript level only; prior to this study only one serine protease with catalytic triad substitutions has been identified within the venom of a snake [@pone.0021532-Wu1]. Functional analysis of this protein did not detect any arginine esterolytic, fibrinolytic or proteolytic activity, and such sequences are commonly called serine protease homologues [@pone.0021532-Wu1]. 15 complete amino acid sequences of viper venom serine proteases with mutations to the catalytic triad are available either in the literature or in the NCBI sequence database. These are derived from ten different snakes representing both Crotalinae and Viperinae sub-families of viper snakes. Phylogenetic analysis of the 65 VVSP sequences identified to date in these ten snakes shows that the majority of the serine protease homologues, including rhinocerases 2 and 3 cluster together, although one serine protease with a standard catalytic triad is also in this cluster ([figure 2](#pone-0021532-g002){ref-type="fig"}). The remaining three serine protease homologues occur individually within the phylogenetic tree but Wu *et al.*\'s [@pone.0021532-Wu1] earlier analysis concluded that only one sequence (KNH4 from *Viridovipera stejnegeri*) did not cluster with the main group of serine protease homologues. Our updated analysis suggests that the creation of serine proteases with mutated catalytic triads has occurred several times during the evolution of these sequences.
![Phylogenetic tree showing relationship between serine protease homologues and serine proteases from the same snakes.\
65 amino acid sequences from 10 snakes were included together with bovine α-chymotrypsinogen (NCBI accession number: P00766) which was used as an outgroup. The alignment was generated using ClustalW [@pone.0021532-Larkin1] within MEGA 4 [@pone.0021532-Kumar1] using a gap opening penalty of 10 and a gap extension penalty of 0.1 for the initial pairwise alignment, gap opening penalty of 3 and gap extension penalty of 1.8 for the multiple alignment and the Gonnet protein weight matrix. The phylogenetic tree was generated from this within MEGA 4 using the neighbour-joining method and the Jones-Taylor-Thornton substitution model. The bootstrap test was done using 2000 replications. In the diagram sequences are identified using a code which consists of up to 3 characters representing the snake name, (TG: *Trimeresurus gramineus*; VS: *Viridovipera stejnegeri*; TJ: *Trimeresurus jerdonii;* BJu: *Bothrops jararacussu* ; ML: *Macrovipera lebetina*; EO: *Echis ocellatus*; BG: *Bitis gabonica*; Bja: *Bothrops jararaca*; TF: *Trimeresurus flavoviridis;* BAs: *Bothrops asper*) followed by a dash and then up to 5 characters representing the protein name. Where possible NCBI accession numbers are also included. ML-P3 and ML-P4 sequences were obtained directly from the sequences named VLP3 and VLP4 in [@pone.0021532-Siigur1]. Red circles indicate the sequences with mutations to the catalytic triad.](pone.0021532.g002){#pone-0021532-g002}
[Figure 3](#pone-0021532-g003){ref-type="fig"} shows an alignment of the sequences of the 15 serine protease homologues with all four rhinocerases and bovine α-chymotrypsinogen. The most frequently mutated residue within the catalytic triad is His57, which has been substituted by Arg in 13 sequences (including rhinocerases 2 and 3), by Asn in 2 sequences and by Gln in one sequence. Seven of the sequences have substitutions for the catalytic Ser195; in five proteins including rhinocerases 2 and 3 this has been substituted by Asn, one protein has a Ser195Pro mutation and one has a Ser195Thr mutation. In contrast, Asp102 is absolutely conserved in all the sequences. Structural models of the rhinocerases show their overall similarity, consistent with the sequence similarity of the enzymes ([figure 4A](#pone-0021532-g004){ref-type="fig"}), but differences in their active site regions. Asp102 is at almost identical positions in rhinocerases 2 and 4, and bovine chymotrypsin. The catalytic serine is at a very similar position in rhinocerase 4 and chymotrypsin, and the substituted Asn in rhinocerase 2 is also similarly located. However the substitution of the long arginine side chain instead of histidine at position 57 in rhinocerase 2 has resulted in a significant change to the orientation of the side chain ([figure 4B](#pone-0021532-g004){ref-type="fig"}).
![Amino acid sequence alignment of rhinocerases with other viper venom serine protease homologues.\
The alignment was created using ClustalW [@pone.0021532-Larkin1] and the figure was generated using GeneDoc [@pone.0021532-Nicholas1]. The sequence of bovine α-chymotrypsinogen (NCBI accession number: P00766) (BT-CHY) was included to allow conventional serine protease residue numbering to be assigned. The catalytic triad residues are coloured red, the primary specificity pocket residues are coloured blue and residue 193, involved in the oxyanion hole is coloured green. BG-SP1: AAR24534; BG-RHIN2: CBM40645; BG-RHIN3: CBM40646; EO-SP: ADE45141; ML-P2: Q9PT40; TF-SP2: O13057; TJ-SPH: B0ZT25; TG-SP2A: O13060; VS-KNH7: Q71Q10; VS-SPH1: QAY82; TJ-SP1: Q9DF68; VS-KNH4: Q71QJ4; BJu-SPH: Q7T229; BJa-HP3: Q5W958; ML-P3 and ML-P4 from [@pone.0021532-Siigur1]; Bas-SPL: Q072L6; BG-RHIN4: CBM40647; BG-RHIN5: CBM40648.](pone.0021532.g003){#pone-0021532-g003}
![Structural models of rhinocerases.\
Structural models of rhinocerases 2 to 5 were created using the IntFOLD server [@pone.0021532-Roche1] using the structure of rat trypsin (PDB code: 1co9) as a template. *A.* Schematic diagram showing the overall similarities in structure between rhinocerase 2 (yellow) and rhinocerase 4 (red). The side chain atom positions for the catalytic triad residues are included. *B*. Detailed view of the amino acids corresponding to the catalytic triad residues in rhinocerase 2 (yellow), rhinocerase 4 (red) and chymotrypsin (PDB code: 1yph; cyan). Rhinocerase 2 has substitutions for the serine and histidine residues. *C*. Detailed view of the main constituents of the S1 specificity pocket in rhinocerase 2 (yellow), rhinocerase 4 (red), chymotrypsin (cyan) and trypsin (pdb code: 1co9; green). In chymotrypsin these residues are: S189 at the base of the specificity pocket, with G216 and G226 at the sides. In trypsin D189 is at the base of the pocket, with G216 and G226. All images were generated using PyMOL.](pone.0021532.g004){#pone-0021532-g004}
Proteins with mutations to the catalytic triad are present in many enzyme families; indeed it has been estimated that up to 15% of the members of all encoded enzyme families may have lost their catalytic activity [@pone.0021532-Pils1]. In many cases the inactive homologues are believed to have acquired alternative functions, such as competing with and antagonising the active proteases, or otherwise regulating their function. Within invertebrates, serine protease homologues have been shown to be involved in various defence responses [@pone.0021532-Ross1]. However, it has been suggested that at least some invertebrate serine protease homologues are unlikely to bind peptide substrates by a canonical protease-like mechanism, though other potential protein binding sites have been suggested [@pone.0021532-Fischer1]. Within snake venom, catalytically inactive phospholipase A~2~ such as myotoxins II and IV from the venom of *Bothrops asper* are known to act as toxins and are thought to bind to their target membrane substrates in order to reduce their permeability control and cause subsequent necrosis [@pone.0021532-Angulo1]. Clearly, experiments to determine the function of the serine protease homologues within snake venom need to be performed, but it is possible that they could affect the physiology of victims or prey by binding irreversibly to substrates involved in blood coagulation and preventing their normal function.
There are also differences in the amino acids present in the primary specificity pockets of rhinocerases 2 to 5. The primary specificity pocket of trypsin-like serine proteases normally comprises Asp189, Gly216 and Gly226 (bovine α-chymotrypsinogen numbering) and these confer specificity towards basic residues at the P1 position of potential substrates. Rhinocerases 2 and 3 have Asp189, which might indicate specificity for basic residues but have Glu instead of Gly at position 216 and Ala instead of Gly at position 226. These substitutions are likely to restrict access to the specificity pocket, thus the binding specificity of rhinocerases 2 and 3 is not clear. Rhinocerases 4 and 5 are the only VVSPs represented in the sequence alignment which have substitutions at position 189: they have Gly at this position instead of the negatively charged Asp. This is an unusual substitution, which is shared by the human kallikrein KLK9 whose specificity is unknown [@pone.0021532-LeBeau1]. The substitution might be expected to increase the size of the specificity pocket; *M. lebetina* α and β-fibrinogenases (ML-AF and ML-BF) also have Gly189 [@pone.0021532-Siigur2] and Siigur *et al.* [@pone.0021532-Siigur3] have reported that ML-AF hydrolysed the Tyr16--Leu17, Phe24--Phe25 and Phe25--Tyr26 bonds of the insulin B chain, suggesting that enzymes with Gly189 can cleave substrates with large hydrophobic residues at the P1 position. Unlike ML-AF rhinocerases 4 and 5 also have a Gly to Ala substitution at position 216 which may narrow the specificity pocket slightly. Again, this is similar to human KLK9 which has Gly216 but Ala226. Comparison of the positions and orientations of residues in the S1 specificity pockets of rhinocerases 2 and 4 with those of chymotrypsin and trypsin suggest that, the bottom of the specificity pocket is very similar in rhinocerase 2 and trypsin, while the glycine in rhinocerase 4 is uncharged and protrudes even less into the pocket. At position 226, the glycines and alanines in all four enzymes are very similarly located. In contrast, the substitution of the large negatively charged glutamic acid at position 216 clearly has a significant effect on the S1 pocket in rhinocerase 2 ([figure 4C](#pone-0021532-g004){ref-type="fig"}). However the primary specificity pocket is not the sole determinant of specificity. For serine proteases involved in coagulation, the importance of additional regions of the structure e.g. exosites in recognition of substrates is becoming increasingly recognised [@pone.0021532-Krishnaswamy1].Thus the precise specificity of these enzymes can only be determined experimentally. The potential role of exosites in binding to substrates also strengthens our suggestion above that the serine proteases homologues which lack the catalytic triad may still be capable of interfering with the coagulation cascade through exosite-mediated interactions.
A further interesting feature of these sequences is that Gly193, which is generally highly conserved in serine proteases and is involved in the oxyanion hole and in inhibitor binding, is substituted in 8 sequences: by Val in four sequences including rhinocerases 2 and 3, by Thr in one sequence and by Ala in three sequences. As was found for TSV-PA, a venom plasminogen activator from *Trimeresurus stejnegeri*, which has Phe at position 193 [@pone.0021532-Braud1], bulky residues substituted for Gly193 may reduce the sensitivity of the proteins to inhibitors such as bovine pancreatic trypsin inhibitor as well as reduce their interaction with substrates.
Evolution of rhinocerases 2 to 5 {#s3d}
--------------------------------
The four distinct serine protease genes which we have identified within the venom gland of *B. g. rhinoceros* provide further evidence for the existence of multiple isoforms of toxins within snake venom, yet the processes by which such isoforms have evolved are not yet fully understood. Accelerated evolution is well established as a mechanism for allowing changes at the individual amino acid level, and alternative splicing [@pone.0021532-Siigur1] and accelerated segment switch in exons to alter targeting (ASSET) [@pone.0021532-Doley1], [@pone.0021532-Doley2] have been recently proposed as additional mechanisms used for generating diversity in snake toxin sequences. A closer analysis of the sequences of rhinocerases 2 to 5 suggests that mechanisms such as alternative splicing and ASSET, together with individual amino acid mutations could have played a part in the generation of these isoforms ([figure 5A,B](#pone-0021532-g005){ref-type="fig"}).
![Evolution of rhinocerase enzymes.\
*A*. Amino acid sequence alignment of rhinocerases 2 to 5, *M. lebetina* α-fibrinogenase (ML-AF) and venom serine proteinase-like protein 2 (ML-P2), and *Bothrops atrox* batroxobin (BA-BT). The alignment was created using ClustalW. The amino acids in the batroxobin sequence are coloured according to the exons encoding them: amino acids encoded by exon 2 are coloured green, and residues encoded by exons 3 to 5 are coloured dark red, blue and orange respectively. The catalytic triad residues are coloured red in the *B.s gabonica* and *M. lebetina* sequences. Coloured shading is used to indicate the three surface segments within serine proteases identified by Doley et al. [@pone.0021532-LeBeau1] as undergoing accelerated change (ASSET). Residues in the first surface segment are shaded yellow or red depending on sequence similarity; residues in the second segment are shaded green or turquoise and the residues in the third segment are shaded pink.*B*. Schematic diagram of rhinocerases 2 to 5 indicating the different regions described in the text. The light shading represents the regions of each sequence corresponding to exon 2 and exons 3 to 5. Rhinocerases 2, 3 and 5 have similar N-terminal regions corresponding to exon 2 (pink), while rhinocerase 4 has a different sequence in this region (grey). In the C-terminal regions, corresponding to exons 3 to 5, rhinocerases 2 and 3 are similar (pale green) and rhinocerase 4 and 5 are similar to each other (pale blue). The brighter shading represents the three surface segments as shown in *A*. The first surface segment is identical in rhinocerases 2, 3 and 5 (yellow), and different in rhinocerase 4 (red). The second segment is identical in rhinocerases 3, 4 and 5 (green) and different in rhinocerase 2 (turquoise), and the third segment is very similar in all 3 sequences (pink).](pone.0021532.g005){#pone-0021532-g005}
Although in terms of overall similarity, rhinocerases 2 and 3 are very similar to each other and distinct from rhinocerases 4 and 5, sequence comparisons and BLAST searches of regions of the sequences corresponding to the exons as identified for the *Bothrops atrox* batroxobin gene (the only VVSP whose gene structure has been studied to date [@pone.0021532-Itoh1]) showed that rhinocerases 2, 3 and 5 have very similar N-terminal regions (up to residue 46 of rhinocerase 2; corresponding to exon 2 in batroxobin) which are most similar to the N-terminal region of *M. lebetina* venom serine proteinase-like protein 2 (NCBI accession number Q9PT40; indicated by ML-VLP2 in [figures 3](#pone-0021532-g003){ref-type="fig"} and [5](#pone-0021532-g005){ref-type="fig"}). Rhinocerase 4, on the other hand, has a distinctly different N-terminal region which is only 54% identical in sequence to the other rhinocerases and is most similar (81% sequence identity) to *M. lebetina* α-fibrinogenase (NCBI accession number Q8JH85, ML-AF).
A different pattern is seen within the C-terminal regions (residues 47 onwards of rhinocerase 2; corresponding to exons 3 to 5 in batroxobin). Two distinctly different versions of this region are also observed, however in part of the sequence rhinocerases 2 and 3 are very similar (92%) and rhinocerases 4 and 5 are identical to each other, but only around 60% identical to rhinocerases 2 and 3. The rhinocerase 2 and 3 sequences are most similar to *M. lebetina* venom serine proteinase-like protein 2 (ML-P2; 83%) while rhinocerase 4 and 5 are most similar to *M. lebetina* α-fibrinogenase (ML-AF) in this region (77%). Similar results were obtained when the C-terminal region was divided into separate regions corresponding to exons 3, 4 and 5 of batroxobin.
Thus rhinocerases 2 and 3 as a whole are similar to ML-P2, rhinocerase 4 as a whole is similar to ML-AF, while rhinocerase 5 has an N-terminal region very similar to rhinocerases 2 and 3 and a C-terminal region similar to rhinocerase 4. This could have been generated by splicing together exons corresponding to the N-terminus of rhinocerase 2 or 3 with exons corresponding to the C-terminal region of rhinocerase 4.
Doley *et al.* [@pone.0021532-Doley2] identified three surface segments within serine proteases which seem to be undergoing accelerated change (ASSET). The first of these (residues 19--27) occurs within the N-terminal region corresponding to exon 2 in batroxobin and, consistent with the region as a whole, rhinocerases 2, 3 and 5 have identical sequences to each other within this segment, while the sequence of rhinocerase 4 is distinctly different. However it is interesting to note that His57 is also in the N-terminal region and this is conserved in rhinocerases 4 and 5 but mutated in rhinocerases 2 and 3. Thus this substitution must be an individual mutation occurring independently of any other mechanisms.
The second and third surface segments identified by Doley *et al.* [@pone.0021532-Doley2] within serine proteases are located in the C-terminal region, corresponding to exons 3 to 5 in batroxobin. They found that the third surface segment was the most conserved, and indeed this segment is very similar (75% identical) in all four rhinocerase sequences, even though there are two distinctly different versions of the C-terminal region as a whole. The second surface segment (amino acids 45--52) is identical in rhinocerases 3, 4 and 5, with a different sequence in rhinocerase 2 (3 out of 8 residues matching). Thus this region could be switching independently of the C-terminal region as a whole, in which rhinocerases 2 and 3 are very similar.
Together these results suggest multiple mechanisms at work even in the evolution of this sub-set of *B. g. rhinoceros* serine proteases: mutations of individual amino acids clearly plays a role, but alternative splicing appears to be working alongside, and there are also changes within the surface segments identified by Doley *et al.* [@pone.0021532-Doley2] as undergoing accelerated change within serine proteases. Further as yet unidentified mechanisms may also contribute to the generation of the diversity of toxins present in this and other snakes.
Conclusions {#s3e}
-----------
In this study, we have reported the sequences of four serine proteases (rhinocerases 2 to 5) from the venom gland transcriptome of *B. g. rhinoceros*. These are clearly distinct from the rhinocerase 1 enzyme which we have recently purified from this venom. Mass spectrometry suggests the four enzymes corresponding to these genes are also present in the venom of *B. g. rhinoceros*. All four sequences share several common features of viper venom serine proteases: they have conserved signal and activation peptides, conserved cysteines and are predicted to be N-glycosylated. In addition these sequences have individual characteristics which are likely to affect their catalytic activity, substrate specificity and sensitivity to inhibitors. The variation within these sequences suggests that alternative splicing together with individual amino acid mutations may have been involved in their evolution. Changes within amino acid segments which were previously proposed to undergo accelerated change in venom serine proteases have also been observed. These multiple serine protease isoforms with different substrate specificities may enhance the envenomation effects and help the snake to adapt to new habitats and diets. A better understanding of the diversity of toxin isoforms present in individual snake venom will help in the design of improved therapeutics for treating snake bites.
We would like to thank Mr Paul Rowley for his expertise in the maintenance of snakes and venom glands dissection.
**Competing Interests:**The authors have declared that no competing interests exist.
**Funding:**This study was funded by the Felix Trust, an organization which provides studentships to students from developing countries (<http://www.felixscholarship.org/>). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
[^1]: Conceived and designed the experiments: EGH SV JMG RAH SCW. Performed the experiments: EGH SV. Analyzed the data: EGH SV JMG. Contributed reagents/materials/analysis tools: JMG RAH SCW. Wrote the paper: SV EGH JMG.
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#s1}
============
When a target is presented with distractors in a search array, the distractors are often processed to some extent along with the target, resulting in increased response latencies when the target and distractors indicate different responses compared with when they indicate the same response (e.g., Eriksen and Hoffman, [@B15]; Eriksen and Eriksen, [@B14]; Miller, [@B36]). This response congruency effect has been observed in a variety of paradigms (e.g., Eriksen and St. James, [@B16]; Kramer and Jacobson, [@B26]; Chen and Cave, [@B9]). Even when a search array appears to facilitate target selection via optimal attentional focusing, evidence of distractor processing has still been found (e.g., Gatti and Egeth, [@B19]; Miller, [@B37]), showing that attentional selection is often inefficient. Understanding the mechanisms that modulate the degree of distractor processing is important, because it helps to shed light on the locus of attentional selection and how bottom-up and top-down processes interact in visual information processing. This study focuses on the factors that affect distractor processing in visual search.
Although the response congruency effect is frequently observed in selective attention tasks, it does not always appear. Lavie and Tsal ([@B34]) and Lavie ([@B28]) noted that the magnitude of the effect, which indicates the degree of distractor processing, is closely linked to the perceptual load required of a task, with a larger effect associated with a low perceptual load task and a smaller effect with a high perceptual load task. For example, in one experiment (Lavie, [@B28], Experiment 1), Lavie varied the target-distractor response congruency (congruent, neutral, and incongruent) and the perceptual load involved in selecting the target (low vs. high). Perceptual load was manipulated via adjusting the number of elements in the display. In the low load condition, the target was shown with a single distractor. In the high load condition, it was shown with several neutral stimuli in addition to the distractor. A larger response congruency effect was found in the low compared with the high perceptual load condition. Based on this and other similar results, \[Lavie ([@B28]); see also Lavie and Tsal ([@B34])\] proposed a perceptual load theory, in which perception is an automatic process with a limited pool of resources. To the extent there is spare capacity beyond what is used in processing the target, perception proceeds involuntarily until all the resources are used up. When a task involves a low perceptual load, distractor processing occurs because of the spillover resources. When a task involves a high perceptual load, distractor processing is either reduced or eliminated due to the unavailability of spare resources. Thus, the degree of distractor processing depends on the amount of leftover resources, which, in turn, is determined by the perceptual load of a task. Since its proposal, evidence in support of the perceptual load theory has been reported in many studies (see Lavie, [@B29], for a review).
However, despite this supporting evidence, there is also a growing number of studies that have shown results inconsistent with the perceptual load theory. Whereas the typical perceptual load effect, i.e., a large response congruency effect with low perceptual load, was observed when the low and high perceptual load trials were presented in separate blocks (e.g., Lavie, [@B28]; Lavie and Cox, [@B30]; Lavie and Fox, [@B32]), the effect was reduced or even eliminated when the two types of trials were intermixed within the same block (Murray and Jones, [@B39]; Theeuwes et al., [@B44]). The perceptual load effect was also eliminated, and sometimes even reversed, when the location of the target was precued (Paquet and Craig, [@B41]; Johnson et al., [@B24]), when the target and distractor were placed in separate objects or perceptual groups (Baylis and Driver, [@B1]; Tsal and Benoni, [@B46]; Cosman and Vecera, [@B10]; Yeh and Lin, [@B51]), and when the relevant and irrelevant information belonged to the same object (Chen, [@B8]). Other factors such as the number of locations at which a distractor or a target could appear (Marciano and Yeshurun, [@B35]; Wilson et al., [@B47]) and the relative salience of a target and distractor (Eltiti et al., [@B13]) also influenced the degree of distractor processing in ways inconsistent with the perceptual load theory. Together, these results challenge the perceptual load theory. They suggest that perceptual load, instead of being a determinant in distractor processing as proposed by the perceptual load theory, is one of a number of factors that contribute to the degree of distractor processing.
Recently, several researchers (Benoni and Tsal, [@B4]; Tsal and Benoni, [@B46]; Wilson et al., [@B47]) proposed an alternative account of distractor processing. Tsal and Benoni ([@B46]) noted that evidence supporting the perceptual load theory came largely from experiments that manipulated perceptual load via display set size. Because an increase in display set size entails an increase in the number of neutral stimuli, and previous research on Stroop interference has shown that increasing irrelevant stimuli in a Stroop display dilutes Stroop interference (Kahneman and Chajczyk, [@B25]; Brown et al., [@B6]), this raises the question whether the reduction in distractor processing in a high perceptual load task is caused by the dilution of distractor interference rather than by the unavailability of perceptual resources. To test their hypothesis, Tsal and Benoni measured distractor interference in three types of displays: the typical low and high perceptual load displays that differed in the number of neutral stimuli, and a new dilution display that had the same number of neutral stimuli as that in the high load display but differed from the high load display in that the target and the neutral stimuli were perceptually segregated by color or spatial location. This segregation made it easy for the neutral stimuli to be ignored, so that the dilution display was low in perceptual load but high in display set size. Contrary to the prediction of the perceptual load theory, no response congruency effect was found in the dilution condition.
Based on this and similar results from other experiments, Tsal and Benoni ([@B46]) proposed a dilution account of distractor processing. According to this account, an incongruent distractor causes interference when its representation is sufficiently strong to enter lexical memory and activate the target-opposite response category. When neutral stimuli, regardless of their task relevancy, are present in a display, their features compete with those of the incongruent distractor, degrading the quality of its representation. When the degraded representation of the distractor is not strong enough to enter lexical memory, there can be little distractor interference. In other words, it is the dilution of distractor interference, not the unavailability of spare perceptual resources that eliminates distractor interference in displays with a large set size.
Wilson et al. ([@B47]) proposed a slightly different dilution account to interpret the display set size effect in distractor processing. They proposed a two-stage model, following from Neisser ([@B40]) and Hoffman ([@B21]): there is an initial parallel processing stage, during which the location most likely to contain the target is selected, and a serial processing 2nd stage, during which the selected item is further processed. Because only one item is processed at a time in the 2nd stage, all the other stimuli in the search array are irrelevant in that stage, and this is so regardless of whether a specific stimulus is task relevant or irrelevant in the 1st stage. Dilution occurs during the 2nd stage if there are sufficient spare resources to process the irrelevant stimuli. Increasing the number of neutral stimuli reduces distractor processing, either because of decreased resources to each stimulus or because of increased crosstalk between the distractor and the neutral stimuli. Thus, like Tsal and Benoni ([@B46]), Wilson et al. attribute the display set size effect to the presence of neutral stimuli, which dilute distractor interference regardless of their task relevancy. They manipulated both the display set size and the number of locations at which a target could appear so that the neutral stimuli were relevant on some trials and not on the other trials. The response congruency effect decreased with increasing display set size, and as they predicted, the reduction was comparable regardless of the relevancy of the neutral stimuli to the task. These results are consistent with the notion that the mere presence of neutral stimuli dilutes distractor interference. Experiment 1 below provides an illustration of some of the main aspects of Wilson et al.\'s experiments, and replicates their results.
Wilson et al. ([@B47]) found that the dilution effect was comparable regardless of the cued target locations, but previous research generally shows that the attentional focus modulates the degree of distractor processing. The idea of attentional focus was captured in Eriksen and St. James\' ([@B16]) "zoom lens model," and it was described by Cave et al. ([@B7]) as "attentional zoom," and by Wilson et al. as "attentional breadth." Using a spatial cuing paradigm, Yantis and Johnston ([@B48]) reported that presenting a 100% valid cue before the onset of a target could minimize distractor interference in a search display. Paquet and Lortie ([@B42]) also reported that precuing the target location decreased distractor interference when the target and distractors belonged to the same category. Similar results were shown by LaBerge and his colleagues ([@B27]), who demonstrated that narrowing attention focus so that the distractor appeared outside it decreased distractor interference, and by Eriksen and St. James ([@B16]), who found reduced distractor interference when the number of precued locations decreased. In both cases, an incongruent distractor caused less interference when a task induced a relatively small attentional focus that excluded the distractor. Thus, all else being equal, a larger response congruency effect is more likely to be found when an incongruent distractor is inside rather than outside an observer\'s attentional focus.
Attentional focus has also been shown to mediate the effect of perceptual load on distractor processing. For example, when a 100% valid precue was used to indicate the location of the target, the perceptual load effect was eliminated (Johnson et al., [@B24]). The perceptual load effect was also reduced or eliminated when participants were prevented from varying the extent of attentional focus between the low and high load trials, either by intermixing the two types of trials within the same block (Murray and Jones, [@B39]; Theeuwes et al., [@B44]) or by designing stimuli so that the relevant and irrelevant information pertained to the same object (Chen, [@B8]). Furthermore, intertrial analyses showed that distractor interference on a high perceptual load trial was more likely to occur when it was preceded by a low perceptual load trial rather than by a high perceptual load trial (Theeuwes et al., [@B44]; Biggs and Gibson, [@B5]). As low perceptual load is more likely to induce a relatively broad attentional focus compared with high perceptual load, the observed intertrial contingency, together with the finding that intermixing trials of different perceptual loads within the same block could reduce or eliminate the perceptual load effect, suggests that different response strategies, with variations in attentional focus, may have played a role in the perceptual load effect found in many previous studies.
The results of recent research underscore the importance of understanding the roles of neutral stimuli and attentional focus, and how they interact to influence distractor processing in visual selection. In Wilson et al. ([@B47]), the magnitude of dilution was comparable regardless of the number of cued target locations. Because the extent of attentional focus should correlate with the cue set size, this result indicates that the extent of attention focus did not affect dilution effects. In other words, in Wilson et al.\'s study, whether a stimulus was located inside or outside an observer\'s attentional focus did not influence the degree of processing of that stimulus. As we discussed in the previous section, this finding conflicts with previous research, which shows that a stimulus receives more processing when it is inside rather than outside one\'s attentional focus (e.g., Eriksen and St. James, [@B16]; Yantis and Johnston, [@B48]; LaBerge et al., [@B27]).
In Wilson et al. ([@B47])\'s study, the appearance of the target display was marked by onset transients and the total number of stimuli in the target display varied in accordance with the display set size. Abrupt visual onsets attract attention under most circumstances (Yantis and Jonides, [@B49], [@B50]). Consequently, the use of onset transients in Wilson et al.\'s experiments could undermine the spatial distribution of attention induced by the cue, resulting in a larger attentional focus when the display set size was large rather than when it was small. This could lead to the comparable dilution effects in both the small and large cue set size conditions in Wilson et al.\'s study.
The 4 experiments reported in this study investigated the factors that influence dilution effects. Specifically, we focused on three issues: the role of attentional focus in modulating the effect of display set size on distractor processing, the locus of dilution, and the role of target knowledge in dilution effects. In Experiment 1, we deliberately co-varied display set size with the extent of attentional focus by using luminance increment to signal the appearance of the target display. Our goal was to replicate the findings of Wilson et al. ([@B47]), and we did. The magnitude of the dilution effect was similar regardless of whether 2 or 6 target locations were cued. Experiment 2 used luminance decrement instead of luminance increment so that the stimulus change lowered the contrast rather than raising it, and thus the attentional focus induced by the cue would not be affected very much by the appearance of the target display. A dilution effect was found when the extent of attentional focus was large, but not when it was small. In Experiment 3, we explored the locus of dilution by varying the number of inverted letters in the two display set size conditions. No dilution effects were found, suggesting that dilution occurred beyond a feature level. Finally, in Experiment 4, we tested the effect of preknowledge of the target by making its color predictable for one group of participants but unpredictable for the other group. A dilution effect was found for the latter group, but not for the former one.
Experiment 1
============
Experiment 1 was modeled after Wilson et al. ([@B47]) to replicate their results with our modified experimental paradigm, which differed from Wilson et al.\'s in that the number of stimuli in the target display was held constant via the use of non-letter place-holders. As in Wilson et al., we manipulated cue set size (CueSize) and display set size (DisplaySize) independently (see Figure [1](#F1){ref-type="fig"}). CueSize refers to the number of possible locations at which a target could appear (2 or 6), and DisplaySize refers to the number of letters in the search array (2 or 6, excluding the critical distractor). Luminance increment was used to signal the appearance of the target display. There was always one target letter present in each display, either an H or an S, and the task was to determine as quickly and as accurately as possible which of the two targets was present. Based on Wilson et al.\'s results, we expected our participants to show a dilution effect of similar magnitude in both the 2-cue and 6-cue conditions.
{#F1}
Method
------
### Participants
Nineteen undergraduate students from the University of Canterbury volunteered to participate in the experiment. Each was paid NZ\$10. All reported to have normal or corrected-to-normal vision.
### Apparatus and stimuli
Stimulus displays were shown on a PC with a 16-inch monitor. The participants were tested individually in a dimly lit room. The viewing distance was approximately 60 cm. E-prime 2.0 (Schneider et al., [@B43]) was used to display stimuli and to record responses.
All stimuli were presented against a black background. Each trial consisted of three displays: the fixation, the cue, and the target display. The fixation display consisted of 7 identical gray (RGB = 60, 60, 60) figure-8 stimuli that also served as place-holders in subsequent displays. Each place-holder subtended 0.86° of visual angle in height and 0.57° in width. While six of them were placed at equal distance along the perimeter of an imaginary circle centered on fixation with a radius of 2.48°, the 7th one was always at fixation. The cue display consisted of four frames. Frames 2 and 4 were identical to the fixation display. Frames 1 and 3 differed in that either a pair of place-holders in opposite locations (in the 2-cue condition) or all six place-holders along the imaginary perimeter (in the 6-cue condition) became white (RGB = 255, 255, 255) instead of remaining to be gray. As there was no blank screen between the fixation and the cue display or between any two frames in the cue display, the perception of the cue was that of 2 or 6 place-holders flashing twice.
We are using the DisplaySize label to be consistent with Wilson et al. ([@B47]), but when the DisplaySize was 2 in this experiment, there were 4 figure-8 place-holders added to the display, so that the total number of stimuli with the distractor and the place-holders was always 7. Following Yantis and Jonides ([@B49]), the letters, which were white in color (RGB = 255, 255, 255), were constructed by increasing the luminance of the appropriate line segments of the figure-8 stimuli and deleting the unneeded segments. Thus, the letters were created via luminance increment rather than the onset transients used by Wilson et al. The stimulus at fixation was always the critical distractor. It was white, and was equally likely to be an H or an S. In the 6-letter condition, the search array consisted of a target letter (H or S) and 5 neutral letters (P, E, F, L, and U). In the 2-letter condition, the search array consisted of a target (again an H or an S), a neutral letter selected randomly and with equal probability from the set of neutral letters mentioned above, and 4 place-holders identical to those in the fixation display. On half of the trials (the congruent condition), the target and distractor were identical. On the rest of the trials (the incongruent condition), they were different letters associated with different responses.
### Design and procedure
The experiment used a 2 × 2 × 2 within-participants design. The principal manipulations were CueSize (2-cue vs. 6-cue), DisplaySize (2-letter vs. 6-letter), and target-distractor Congruency (congruent vs. incongruent). The three factors were varied independently. All types of trials were presented randomly within a block.
Each trial started with the presentation of the fixation display. After 500 ms, either 2 or 6 place-holders along the perimeter of the imaginary circle would flash twice, with each flash lasting for 500 ms, with a 500 ms interval after each flash. At the end of the 2nd interval (i.e., the 4th frame of the cue display), the central place-holder would turn into a letter, as would either 2 or 6 of the other place-holders, depending on the DisplaySize condition. The screen went black after 200 ms. The task was to respond, as quickly and as accurately as possible, whether the target was an H or an S. The participants were instructed to maintain fixation at the central place-holder throughout the duration of a trial, and to use the index and middle fingers of their right hand to press one of the two designated keys on a response box (the 4th key if the target letter was an "H," and the 5th key if it was an "S"). They were explicitly informed that the target would only appear at one of the cued locations and that the center letter was always a distractor that they should try to ignore. The entire experiment consisted of 2 blocks of 16 practice trials, followed by 5 blocks of 96 experimental trials with short breaks after each block. It took about 35 min to complete the experiment.
Results
-------
Table [1](#T1){ref-type="table"} shows the mean reaction times (RTs) for correct responses and the error rates, and the graph in Figure [2](#F2){ref-type="fig"} shows the congruency effect across conditions[^1^](#fn0001){ref-type="fn"}. One participant\'s data were not included due to high error rates (greater than 40% in one condition). A 2 × 2 × 2 repeated measures analysis of variance (ANOVA) was conducted on RTs. (See Table [2](#T2){ref-type="table"} for details of the results). All the main effects were significant. The participants were faster in the 2-cue condition (655 ms) than in the 6-cue condition (759 ms), *p* \< 0.001. They were also faster when the display consisted of two letters (654 ms) instead of six letters (760 ms), *p* \< 0.001, and when the target and distractors were congruent (677 ms) rather than incongruent (737 ms), *p* \< 0.001. CueSize interacted with DisplaySize, *p* \< 0.001, suggesting that RT increased more dramatically from the 2-letter condition to the 6-letter condition on the 6-cue trials (an increase of 162 ms) than on the 2-cue trials (an increase of 51 ms). CueSize also interacted with Congruency, *p* \< 0.02. The congruency effect was larger in the 6-cue condition (78 ms) than in the 2-cue condition (42 ms), indicating a positive relationship between the number of target locations and the degree of distractor processing. Furthermore, a dilution effect was found, as evidenced by the significant interaction between DisplaySize and Congruency, *p* \< 0.02, suggesting a larger congruency effect in the 2-letter condition (69 ms) than in the 6-letter condition (51 ms). Finally, there was no significant three-way interaction of CueSize, DisplaySize, and Congurency. The last result indicated that the magnitude of the dilution effect was independent of the cue set size, as can be seen in Figure [2](#F2){ref-type="fig"}.
######
**Experiment 1: mean reaction times and error rates as a function of cue set size, display set size, and target-distractor congruency**.
**Display set size** **Cue set size**
------------------------------- ------------------ ----------- ------------ ------------
**REACTION TIMES (ms)**
2-letter 604 (22) 654 (23) 635 (22) 722 (26)
6-letter 663 (34) 697 (31) 806 (27) 874 (35)
**ERROR RATES (% INCORRECT)**
2-letter 2.2 (0.6) 4.7 (0.9) 3.7 (0.8) 7.0 (1.5)
6-letter 6.7 (1.1) 6.0 (1.1) 13.3 (1.7) 12.4 (1.9)
Standard errors are in the parentheses. C, Congruent; I, Incongruent.
{#F2}
######
**Results of statistical analyses of the reaction times in Experiments 1, 2, and 3**.
**Reaction times**
---------------------------------------------------------------------------------- -------------------------------------------- ------- ------ --------------------------------------------- ------- ------ --------------------------------------------- ------- ------
Cue 34.82[^\*\*\*^](#TN3){ref-type="table-fn"} 0.001 0.67 124.85[^\*\*\*^](#TN3){ref-type="table-fn"} 0.001 0.87 174.00[^\*\*\*^](#TN3){ref-type="table-fn"} 0.001 0.92
Display 73.48[^\*\*\*^](#TN3){ref-type="table-fn"} 0.001 0.81 90.02[^\*\*\*^](#TN3){ref-type="table-fn"} 0.001 0.83 77.63[^\*\*\*^](#TN3){ref-type="table-fn"} 0.001 0.84
Cong 64.77[^\*\*\*^](#TN3){ref-type="table-fn"} 0.001 0.79 58.76[^\*\*\*^](#TN3){ref-type="table-fn"} 0.001 0.76 48.93[^\*\*\*^](#TN3){ref-type="table-fn"} 0.001 0.77
Cue[^\*^](#TN1){ref-type="table-fn"}Display 25.36[^\*\*\*^](#TN3){ref-type="table-fn"} 0.001 0.60 60.74[^\*\*\*^](#TN3){ref-type="table-fn"} 0.001 0.76 54.58[^\*\*\*^](#TN3){ref-type="table-fn"} 0.001 0.78
Cue[^\*^](#TN1){ref-type="table-fn"}Cong 6.87[^\*^](#TN1){ref-type="table-fn"} 0.02 0.29 16.14[^\*\*\*^](#TN3){ref-type="table-fn"} 0.001 0.46 24.70[^\*\*\*^](#TN3){ref-type="table-fn"} 0.001 0.62
Display[^\*^](#TN1){ref-type="table-fn"}Cong 6.94[^\*^](#TN1){ref-type="table-fn"} 0.02 0.29 3.00 0.10 0.14 0.02 0.89 0.01
Cue[^\*^](#TN1){ref-type="table-fn"}Display[^\*^](#TN1){ref-type="table-fn"}Cong 0.03 0.86 0.01 9.44[^\*\*^](#TN2){ref-type="table-fn"} 0.01 0.33 0.06 0.81 0.01
Cue, CueSize; Display, DisplaySize; Cong, Congruency.
p \< 0.05;
p \< 0.01;
p \< 0.001.
A similar ANOVA was conducted on the error rates. (See Table [3](#T3){ref-type="table"} for details of the results). Consistent with the RT results, error rates were lower in the 2-cue condition (4.9%) than in the 6-cue condition (9.1%), *p* \< 0.001, and on the 2-letter trials (4.4%) than on the 6-letter trials (9.6%), *p* \< 0.001. CueSize interacted with DisplaySize, *p* \< 0.02, suggesting a larger increase in error rate from the 2-letter to 6-letter condition on the 6-cue trials (an increase of 7.5%) compared with the 2-cue trials (an increase of 2.9%). Finally, there was a significant interaction between DisplaySize and Congruency, *p* \< 0.02. Whereas a significant congruency effect was found on the 2-letter trials (2.9% error rate), a similar effect was not found on the 6-letter trials (−0.8% error rate). No other effects reached significance. There was no indication of any speed-accuracy tradeoff.
######
**Results of statistical analyses of the error rates in Experiments 1, 2, and 3**.
**Error rates**
---------------------------------------------------------------------------------- -------------------------------------------- ------- ------ -------------------------------------------- ------- ------ -------------------------------------------- ------- ------
Cue 27.09[^\*\*\*^](#TN5){ref-type="table-fn"} 0.001 0.61 52.68[^\*\*\*^](#TN5){ref-type="table-fn"} 0.001 0.73 57.21[^\*\*\*^](#TN5){ref-type="table-fn"} 0.001 0.79
Display 36.96[^\*\*\*^](#TN5){ref-type="table-fn"} 0.001 0.68 69.07[^\*\*\*^](#TN5){ref-type="table-fn"} 0.001 0.78 28.30[^\*\*\*^](#TN5){ref-type="table-fn"} 0.001 0.65
Cong 1.85 0.19 0.10 7.77[^\*^](#TN4){ref-type="table-fn"} 0.02 0.29 6.85[^\*^](#TN4){ref-type="table-fn"} 0.02 0.31
Cue[^\*^](#TN4){ref-type="table-fn"}Display 7.04[^\*^](#TN4){ref-type="table-fn"} 0.02 0.29 79.22[^\*\*\*^](#TN5){ref-type="table-fn"} 0.001 0.81 16.85[^\*\*\*^](#TN5){ref-type="table-fn"} 0.001 0.53
Cue[^\*^](#TN4){ref-type="table-fn"}Cong 0.07 0.80 0.01 2.24 0.15 0.11 2.41 0.14 0.14
Display[^\*^](#TN4){ref-type="table-fn"}Cong 7.99[^\*^](#TN4){ref-type="table-fn"} 0.01 0.32 0.10 0.75 0.01 0.01 0.95 0.01
Cue[^\*^](#TN4){ref-type="table-fn"}Display[^\*^](#TN4){ref-type="table-fn"}Cong 0.18 0.68 0.01 0.12 0.73 0.01 0.04 0.84 0.01
Cue, CueSize; Display, DisplaySize; Cong, Congruency.
p \< 0.05;
p \< 0.001.
Discussion
----------
The results of Experiment 1 were remarkably similar to those of Wilson et al. ([@B47]). In both cases, the congruency effect was substantially larger in the 6-cue condition than in the 2-cue condition. As they pointed out, this result is inconsistent with the perceptual load theory, which predicts a decrease in distractor interference with increasing cue set size, because perceptual load would increase with the number of locations at which a target could appear. Indeed, if RT is a valid indicator of perceptual load, then the longer RT in the 6-cue than the 2-cue condition provides evidence for the higher perceptual load in the former than in the latter. The fact that the perceptual load effect was reversed across the cue conditions is incompatible with the perceptual load theory.
The larger congruency effect in the 6-cue condition was likely caused by the increased RT in that condition compared with the 2-cue condition. As the cue in the 6-cue condition would induce a broader attentional focus than the cue in the 2-cue condition, more irrelevant letters would be within the attentional focus in the former condition, resulting in longer response latencies to the target. Previous research has shown a positive link between the processing time of a target and the magnitude of the congruency effect, and it has been proposed that an increase in the processing time of a target increases the window of opportunity for distractor intrusion, resulting in increased distractor processing (Lavie and De Fockert, [@B31]; Tsal and Benoni, [@B46]; Wilson et al., [@B47]). We agree with this view, and attribute the differential congruency effects in the two cue size conditions to the longer response latencies in the 6-cue condition relative to the 2-cue condition.
As in Wilson et al. ([@B47]), we found that the congruency effect was more diluted when there were more letters in the display, and more importantly, the degree of dilution was comparable in both the 2-cue and 6-cue conditions. However, as we discussed before, the luminance increment that was used to signal the appearance of the target display in the present experiment, which is similar to the onset transient used in Wilson et al.\'s ([@B47]) experiments, could change the extent of attentional focus, raising doubts about the ability to measure the effects of perceptual load and dilution. In Experiment 2, we addressed this issue by using luminance decrement instead of luminance increment to minimize the effect of stimulus appearance on the extent of attentional focus induced by the cue.
Experiment 2
============
In Experiment 2, we replaced luminance increment with luminance decrement so that the target locations in the cue display and the appearance of the letters in the target display were both signaled via luminance decrease instead of luminance increase (see Figure [3](#F3){ref-type="fig"}). Because luminance decrement is less likely to capture attention than luminance increment (Yantis and Jonides, [@B49]), the appearance of the target display should be less likely to affect the extent of attentional focus induced by the cue, allowing the attentional focus to be determined more by the manipulation in CueSize.
{#F3}
As our selective review of the literature in the previous section indicates (e.g., Paquet and Lortie, [@B42]; Paquet and Craig, [@B41]; Johnson et al., [@B24]), there is reason to believe that the effect of neutral stimuli on distractor processing could be strongly affected in this paradigm by their locations relative to the attentional focus. As stimuli are likely to be processed at a greater extent when they are inside rather than outside one\'s attentional focus, we predicted a larger dilution effect in the 6-cue condition compared with the 2-cue condition, for more neutral letters should fall inside the participants\' attentional focus in the former than in the latter.
Method
------
The method of Experiment 2 was the same as that in Experiment 1 except for the following differences. First, the place-holders in the fixation display were white instead of gray. Second, target locations were indicated by luminance decrement instead of luminance increment in the cue display. Frames 2 and 4 were identical to the fixation display, i.e., all the place-holders were white. This ensured that compared with the participants in Experiment 1, those in Experiment 2 were less likely to expand their attentional focus upon the onset of the target display in the 2-cue condition, for the appearance of the target display was signaled by luminance decrement instead of luminance increment. Frames 1 and 3 differed from the fixation display in that the 2 or 6 place-holders in the cued locations were gray. Thus, the perception of the cue was that of 2 or 6 place-holders dimming twice. Third, in the target display, all the stimuli were white regardless of whether they were letters or place-holders. These design features ensured that there was minimal difference in luminance from the last frame of the cue to the target display, or between the target displays in the 2-letter and 6-letter conditions. Twenty new participants took part in the experiment.
Results
-------
Table [4](#T4){ref-type="table"} shows the mean RTs and error rates, and Figure [4](#F4){ref-type="fig"} shows the effects of congruency. Two repeated measures ANOVAs were conducted, one on the RT data (see Table [2](#T2){ref-type="table"}), and the other on the error rates (see Table [3](#T3){ref-type="table"}). As in Experiment 1, all the three main effects were significant. The participants were faster and more accurate in the 2-cue condition (613 ms with 5.7% error rate) than in the 6-cue condition (757 ms with 11.5% error rate), *p* \< 0.001, for both RT and error rates. They were also faster and more accurate in the 2-letter condition (653 ms with 6.3% error rate) than in the 6-letter condition (717 ms with 10.9% error rate), *p* \< 0.001 in both cases. In addition, performance was better on congruent trials (659 ms with 7.2% error rate) than on incongruent trials (711 ms with 10.1% error rate), *p* \< 0.001 for RT; and *p* \< 0.02 for error rates. CueSize interacted with DisplaySize, both in RT, *p* \< 0.001, and in error rates, *p* \< 0.001, suggesting that an increase in display set size impaired performance more when the target could appear at 1 of 6 locations (an increase of 115 ms and 8.8% error rate) rather than at 1 of 2 locations (an increase of 14 ms and 0.5% error rate). In RT, the magnitude of the congruency effect was again affected by CueSize, *p* \< 0.001. The congruency effect was larger in the 6-cue condition (73 ms) than in the 2-cue condition (31 ms). Finally, there was a significant three-way interaction in RT, *p* \< 0.01, which is illustrated in Figure [4](#F4){ref-type="fig"}. No other effects reached significance.
######
**Experiment 2: mean reaction times and error rates as a function of cue set size, display set size, and target-distractor congruency**.
**Display set size** **Cue set size**
------------------------------- ------------------ ----------- ------------ ------------
**REACTION TIMES (ms)**
2-letter 595 (26) 617 (25) 651 (31) 747 (35)
6-letter 600 (25) 640 (26) 789 (41) 839 (38)
**ERROR RATES (% INCORRECT)**
2-letter 4.4 (0.9) 6.6 (1.2) 5.2 (0.8) 9.1 (1.4)
6-letter 5.2 (1.0) 6.7 (1.1) 13.9 (1.6) 17.9 (1.8)
C, Congruent; I, Incongruent. Standard errors are in the parentheses.
{#F4}
To clarify the Three-Way interaction, we conducted two separate ANOVAs, one for the data in the 2-cue condition and the other for the data in the 6-cue condition. In the 6-cue condition, all the effects were significant. RT was longer in the 6-letter condition (814 ms) than in the 2-letter condition (699 ms), *F*~(1,\ 19)~ = 80.65, *MS*~*e*~ = 3238, *p* \< 0.001, η^2^~*p*~ = 0.81, and on incongruent (793 ms) than congruent (720 ms) trials, *F*~(1,\ 19)~ = 53.88, *MS*~*e*~ = 1988, *p* \< 0.001, η^2^~*p*~ = 0.74. DisplaySize interacted with Congruency, *F*~(1,\ 19)~ = 7.58, *MS*~*e*~ = 1404, *p* \< 0.02, η^2^~*p*~ = 0.29. The congruency effect was larger in the 2-letter condition (96 ms) than in the 6-letter condition (50 ms), indicating a significant dilution or perceptual load effect.
The pattern of data differed in the 2-cue condition. The main effects of DisplaySize and Congruency were both significant, with faster RT on the 2-letter trials (606 ms) than on the 6-letter trials (620 ms), *F*~(1,\ 19)~ = 16.21, *MS*~*e*~ = 247, *p* \< 0.001, η^2^~*p*~ = 0.46, and on the congruent trials (598 ms) than on the incongruent trials (629 ms), *F*~(1,\ 19)~ = 20.11, *MS*~*e*~ = 960, *p* \< 0.001, η^2^~*p*~ = 0.51. The interaction between DisplaySize and Congruency was marginally significant, *F*~(1,\ 19)~ = 4.04, *MS*~*e*~ = 360, *p* = 0.06, η^2^~*p*~ = 0.18. Importantly, the direction of the interaction was opposite to what was found in Experiment 1: the congruency effect was larger in the 6-letter condition (40 ms) than in the 2-letter condition (22 ms). Thus, there was no evidence of a dilution effect when the neutral letters were outside the attentional focus in the 2-cue condition.
To confirm statistically that the pattern of data in Experiment 1 differed from that in Experiment 2, we conducted a combined analysis of the RT data across the two experiments, using a mixed ANOVA with Experiment as a between-subjects factor and CueSize, DisplaySize, and Congruency as within-subjects factors. For the sake of brevity, we report only the significant interactions with Experiment, of which there were two. One was a significant interaction between DisplaySize and Experiment, *F*~(1,\ 36)~ = 9.38, *MS*~*e*~ = 3582, *p* \< 0.01, η^2^~*p*~ = 0.21, suggesting that the increase in RT from the 2-letter to 6-letter condition was larger in Experiment 1 (an increase of 106 ms) than in Experiment 2 (an increase of 65 ms). The second was a significant four-way interaction, *F*~(1,\ 36)~ = 4.49, *MS*~*e*~ = 946, *p* \< 0.05, η^2^~*p*~ = 0.11. Subsequent analyses on the 2-cue and 6-cue trials separately indicated that the 4-way interaction arose primarily from the participants in the two experiments behaving differently in the 2-cue condition, where a significant 3-way interaction of DisplaySize, Congruency, and Experiment was found, *F*~(1,\ 36)~ = 5.56, *MS*~*e*~ = 473, *p* \< 0.05, η^2^~*p*~ = 0.13. A similar 3-way interaction was not found in the 6-cue condition, *F*~(1,\ 36)~ = 1.59, *MS*~*e*~ = 1042, *p* = 0.21, η^2^~*p*~ = 0.04. These results confirmed that the pattern of data in Experiments 1 and 2 differed when the cue set size was 2, but not when it was 6.
Discussion
----------
The results of Experiment 2 suggest that the extent of attentional focus modulates the effect of display set size on distractor processing. In the 6-cue condition, the target was equally likely to appear at any location in the search array. To find the target quickly, the best strategy would be to adopt a relatively broad attentional focus that would include the entire target display, including the neutral stimuli. As the neutral stimuli were within the attentional focus, they would compete with the critical distractor for representation. Hence, a dilution effect was found in the 6-cue condition. In contrast, in the 2-cue condition, the participants\' attention was likely to be more narrowly focused, and unlike Experiment 1, there was no abrupt luminance increment to draw attention more widely when the target array appeared. As the letters that appeared at the uncued locations were largely outside the focus of attention, they would not receive the same kind of processing as their counterparts in the 6-cue condition. Whatever processing these letters might have received due to attentional leakage, the level of processing was not sufficient to interfere with the representation of the distractor. As a result, increasing display set size in the 2-cue condition did not lead to a dilution effect.
It is worth noting that the participants in the 2-cue condition of Experiment 2 took longer to respond to the target on the 6-letter trials than on the 2-letter trials despite the fact that the participants knew in advance that the target would never occur at an uncued location. The increased RT in the 6-letter trials indicated that attention could not completely filter out all the irrelevant information. This result is in line with the view that attentional selection is often incomplete, and that some processing can still happen to irrelevant stimuli even with clear spatial separation between a target and irrelevant distractors (Treisman, [@B45]; Miller, [@B37]).
Another interesting aspect of Experiment 2\'s data is the reversed dilution effect in the 2-cue condition. The congruency effect was larger, instead of smaller, when the display consisted of 6 letters rather than 2 letters. It is notable that RT was substantially longer on the 6-letter trials compared with the 2-letter trials. As we discussed in Experiment 1, an increase in response latencies increases the window of opportunity for distractor intrusion. As a result, congruency effect was larger in the 6-letter condition than in the 2-letter condition.
Experiment 3
============
As mentioned earlier, several researchers have proposed a dilution account to interpret the reduction in distractor interference with increasing display set size (Benoni and Tsal, [@B4]; Tsal and Benoni, [@B46]; Wilson et al., [@B47]). Because stimuli of the same category, which share both basic features and response code, were used in these prior studies, the proposed dilution accounts emphasize competition between the features of the added display items and the features of the distractor, which degrades the quality of the distractor representation (e.g., Tsal and Benoni, [@B46]). In other words, they suggest that dilution occurs at a feature level.
Experiment 3 was designed to test this notion empirically. In Experiment 3, both the 2-item and 6-item conditions had 2 upright letters present in the target array, but in the 6-item condition, there were also 4 inverted letters. Because the inverted letters shared basic features but not meaning with the critical distractor, this design allowed us to assess the effect of neutral stimuli on distractor processing at a feature level. If dilution occurs at a feature level, the participants in Experiment 3 should show the same pattern of result as that in Experiment 2. Conversely, if dilution occurs at a level beyond feature processing (e.g., at a categorical, semantic, or response level), no dilution effects should be found in the 6-item condition.
Method
------
The method of Experiment 3 was identical to that of Experiment 2 except for the stimuli in the large display set size condition. Instead of 6 letters, the search array consisted of 2 upright letters (i.e., the target and a neutral letter selected randomly on each trial from the set of neutral stimuli as in Experiment 2) and 4 inverted letters constructed from the original set of neutral letters (i.e., P, F, U, L, E) with a 180 degree rotation. As before, we varied the cue set size (the 2-cue and 6-cue conditions) independently of the display set size (the 2-item and 6-item conditions). Sixteen new participants volunteered for the experiment.
Results
-------
Table [5](#T5){ref-type="table"} shows the response times and error rates, and Figure [5](#F5){ref-type="fig"} shows the congruency effects. As before, we conducted two separate repeated-measures ANOVAs, one on the RT data (see Table [2](#T2){ref-type="table"}), and the other on the error rates (see Table [3](#T3){ref-type="table"}). The participants were again faster and more accurate in the 2-cue condition (615 ms with 4.8% error rate) than in the 6-cue condition (761 ms with 11.5% error rate), *p* \< 0.001 for both RT and accuracy. They were also faster and more accurate when the display set size was 2 (660 ms with 6.0% error rate) rather than 6 (715 ms with 10.2% error rate), *p* \< 0.001 in both cases. In addition, responses were faster and more accurate on congruent trials (664 ms with 7.1% error rate) than on incongruent trials (712 ms with 9.1% error rate), *p* \< 0.001 for RT, and *p* \< 0.02 for accuracy. The interaction between CueSize and DisplaySize was also significant, *p* \< 0.001 for RT and accuracy. This suggests that once again, an increase in display set size impaired performance more in the 6-cue condition (an increase of 105 ms and 8% error rate) compared with the 2-cue condition (an increase of only 6 ms and 0.4% error rate). CueSize interacted with Congruency in RT, *p* \< 0.001, indicating a larger congruency effect in the 6-cue condition (71 ms) than in the 2-cue condition (26 ms). Importantly, neither the two-way interaction between DisplaySize and Congruency nor the 3-way interaction of CueSize, DisplaySize and Congruency was significant, *F*~(1,\ 15)~ \< 1, *ns*. in both cases. These results indicate that the presence of the inverted letters had a negligible effect on the degree of distractor interference regardless of whether the cue set size was 2 or 6. No other effects reached significance, and there was no evidence of a speed-accuracy tradeoff.
######
**Experiment 3: mean reaction times and error rates as a function of cue set size, display set size, and target-distractor congruency**.
**Display set size** **Cue set size**
------------------------------- ------------------ ----------- ------------ ------------
**REACTION TIMES (ms)**
2-item 599 (29) 625 (31) 674 (35) 743 (34)
6-item 605 (31) 630 (33) 777 (33) 849 (38)
**ERROR RATES (% INCORRECT)**
2-item 4.1 (0.7) 5.0 (1.0) 5.8 (1.0) 9.1 (1.5)
6-item 4.4 (0.9) 5.5 (1.5) 14.0 (1.6) 16.9 (2.4)
Standard errors are in the parentheses. C, Congruent; I, Incongruent.
{#F5}
A combined analysis across Experiments 2 and 3 was conducted on the RT data to verify that the pattern of data in the two experiments differed significantly. Again, for the sake of brevity, we report only the significant interactions that involve Experiment. The only significant effect was the four-way interaction of CueSize, DisplaySize, Congruency, and Experiment, *F*~(1,\ 34)~ = 6.12, *MS*~*e*~ = 819, *p* \< 0.05, η^2^~*p*~ = 0.15. Separate analyses on the 2-cue and 6-cue trials confirmed that the four-way interaction in the original analysis arose from the 6-cue condition, where a significant three-way interaction of DisplaySize × Congruency × Experiment was found, *F*~(1,\ 34)~ = 4.95, *MS*~*e*~ = 1086, *p* \< 0.05, η^2^~*p*~ = 0.13. A similar effect was not found in the 2-cue condition, *F*~(1,\ 34)~ = 2.0, *MS*~*e*~ = 356, *p* = 0.17, η^2^~*p*~ = 0.06. These results suggest that the effect of neutral stimuli on distractor processing differed in the 6-cue condition between Experiments 2 and 3.
Discussion
----------
The most important finding of Experiment 3 was the elimination of the dilution effect in the 6-cue condition. Adding inverted letters to the display did not lower the distractor interference in this condition, even though the upright letters added to displays in the same condition of Experiments 1 and 2 lowered the distractor interference in those experiments. This result suggests that the inverted letters in the 6-item condition had a negligible effect on the degree of distractor processing, despite the fact that they increased the overall RT to the target. This RT increase likely reflects the extra difficulty in locating the target due to the increased similarity between the target and the relevant items in the search array. Previous research has shown that an increase in similarity between a target and distractors impairs segmentation, making it hard to distinguish the target from the distractors (Duncan and Humphreys, [@B11], [@B12]). Thus, these items that have been added to the display, which share features with the target and the critical distractor but do not activate responses in the same category, can delay the response to the target but do not necessarily degrade the representation of the distractor.
The absence of a dilution effect in Experiment 3 also suggests that the locus of dilution in Experiments 1 and 2 probably occurred at a semantic level. That said, caution must be taken in generalizing this result to other experimental paradigms. It is quite possible that the locus of dilution depends on participants\' behavioral goals. When a task requires a categorical or semantic level of processing, dilution may occur at these levels. However, when a task requires a feature level of processing, dilution may occur at the feature level. In the present study, although the two target letters could be distinguished on the basis of basic features, they were referred to as individual letters H and S. Naming the letters would likely induce the participants to code them at a semantic level, differentiating them from the inverted letters in terms of task relevancy and avoiding dilution from the inverted letters in the 6-cue condition.
Experiment 4
============
Experiment 3 showed that dilution effects could be eliminated when neutral stimuli did not share the same response code as the target and distractor. In Experiment 4, we investigated whether dilution effects could also be eliminated when participants had preknowledge of the target color. We reasoned that knowing the color of the target in advance would enable participants to use that information to direct their attention to those stimuli that had the task relevant color, thereby excluding the stimuli that had the task irrelevant color from the attention focus. Consequently, if the additional neutral letters in the 6-letter display had a task irrelevant color, they should not affect the degree of distractor processing even when all the locations in the search array were cued in the 6-cue condition. To test this hypothesis, the participants were divided into two groups in Experiment 4. One group (the predictable group) knew in advance the color of the target on each trial. The target was red in one block, and green in a different block. The other group (the unpredictable group) had no preknowledge of the target color on a given trial. The target was equally likely to be red or green. On all trials, 6 locations were cued. If a dilution effect was found in the unpredictable group but not in the predictable group, this would provide additional evidence that the extent of attentional focus modulates dilution effects.
Method
------
The method was similar to that of Experiment 2 except for the following differences. First, as dilution was found only when participants adopted a relatively wide attentional focus, Experiment 4 included only the 6-cue condition. Second, the stimuli in the target display were either red (RGB = 255, 64, 64) or green (RGB = 64, 255, 64). In all conditions, the target had the same color as only one other stimulus: the neutral letter at its opposite location. In other words, the target display consisted of either 2 red and 5 green stimuli, or 2 green and 5 red stimuli. Finally, the participants were randomly and equally divided into two groups. For the predictable group, the color of the target was the same throughout the trials within a block. Half of them completed the red block before the green one, and the order of the blocks was reversed for the other half. For the unpredictable group, the color of the target was unknown on a given trial. The target was equally likely to be red or green within a block. Twenty new participants took part in the experiment.
Results
-------
The data from one participant in the predictable group was excluded from analyses due to high error rates (averaged over 20% across all conditions). Table [6](#T6){ref-type="table"} shows the response times and error rates, and Figure [6](#F6){ref-type="fig"} shows the congruency effects. A mixed ANOVA with DisplaySize and Congruency as within-subjects factors and Group as a between-subjects factor was performed on the RT data (see Table [7](#T7){ref-type="table"}). The results show that RT was faster in the predictable group (581 ms) than in the unpredictable group (701 ms), *p* \< 0.05, indicating that knowing the target color in advance facilitated target responses. As in previous experiments, RT was faster in the 2-letter condition (630 ms) than in the 6-letter condition (651 ms), *p* \< 0.001, and in the congruent condition (630 ms) than in the incongruent condition (651 ms), *p* \< 0.01. DisplaySize interacted with Group, *p* \< 0.01, suggesting a larger set size effect for the unpredictable group (an increase of 33 ms) than for the predictable group (an increase of 8 ms). In addition, there was a significant three-way interaction of DisplaySize, Congruency, and Group, *p* \< 0.05, which is illustrated in Figure [6](#F6){ref-type="fig"}.
######
**Experiment 4: mean reaction times and error rates as a function of the preknowledge of the target color, display set size, and target-distractor congruency**.
**Display set size** **Target color**
------------------------------- ------------------ ----------- ----------- ------------
**REACTION TIMES (ms)**
2-letter 568 (27) 585 (26) 666 (32) 702 (32)
6-letter 574 (29) 595 (25) 713 (38) 721 (32)
**ERROR RATES (% INCORRECT)**
2-letter 3.3 (1.2) 2.9 (0.6) 7.1 (1.1) 6.8 (1.4)
6-letter 3.7 (0.7) 4.1 (0.7) 8.5 (1.2) 10.0 (1.4)
Standard errors are in the parentheses. C, Congruent; I, Incongruent.
{#F6}
######
**Results of statistical analysis of the reaction times and error rates in Experiment 4**.
**Reaction times** **Error rates**
------------------------------------------------------------------------------------ -------------------------------------------- ----------------- ------ ------------------------------------------ ------ ------
Group 7.83[^\*^](#TN6){ref-type="table-fn"} 0.02 0.32 12.30[^\*\*^](#TN7){ref-type="table-fn"} 0.01 0.42
Display 22.72[^\*\*\*^](#TN8){ref-type="table-fn"} 0.001 0.57 8.02[^\*^](#TN6){ref-type="table-fn"} 0.02 0.32
Display[^\*^](#TN6){ref-type="table-fn"}Group 9.17[^\*\*^](#TN7){ref-type="table-fn"} 0.01 0.35 1.80 0.20 0.10
Cong 15.42[^\*\*\*^](#TN8){ref-type="table-fn"} 0.001 0.48 0.34 0.57 0.02
Cong[^\*^](#TN6){ref-type="table-fn"}Group 0.09 0.77 0.01 0.46 0.51 0.03
Display[^\*^](#TN6){ref-type="table-fn"}Cong 4.26 0.06 0.20 1.93 0.18 0.10
Display[^\*^](#TN6){ref-type="table-fn"}Cong[^\*^](#TN6){ref-type="table-fn"}Group 7.86[^\*^](#TN6){ref-type="table-fn"} 0.02 0.32 0.32 0.58 0.02
Display, DisplaySize; Cong, Congruency.
p \< 0.05;
p \< 0.01;
p \< 0.001.
To clarify the three-way interaction, two separate ANOVAs, one for each group, were performed. For the predictable group, while the main effect of congruency was significant, *F*~(1,\ 8)~ = 10.68, *MS*~*e*~ = 298, *p* \< 0.05, η^2^~*p*~ = 0.57, neither the effect of DisplaySize nor its interaction with Congruency reached significant, *p* \> 0.1 in both cases. The predictable group thus showed no evidence of a dilution effect. For the unpredictable group, in addition to the main effects of congruency, *F*~(1,\ 9)~ = 6.88, *MS*~*e*~ = 696, *p* \< 0.05, η^2^~*p*~ = 0.43, and DisplaySize, *F*~(1,\ 9)~ = 22.21, *MS*~*e*~ = 508, *p* \< 0.01, η^2^~*p*~ = 0.71, there was a significant interaction between the two factors, *F*~(1,\ 9)~ = 10.73, *MS*~*e*~ = 175, *p* \< 0.01, η^2^~*p*~ = 0.54, with a larger congruency effect in the 2-letter condition (36 ms) than in the 6-letter condition (8 ms), suggesting dilution.
A mixed ANOVA was also performed on the error rates (see Table [7](#T7){ref-type="table"}). Consistent with the RT results, responses were more accurate in the predictable group (3.5% error rate) than in the unpredictable group (8.1% error rate), *p* \< 0.01, and on the 2-letter trials (5% error rate) than on the 6-letter trials (6.6% error rate), *p* \< 0.05. No other results were significant.
Discussion
----------
The most important finding of Experiment 4 was that preknowledge of the target color could eliminate dilution effects. Whereas a dilution effect was found when participants had no advanced knowledge of the target color, the effect was negligible when the target color was predictable on a given trial. These results are consistent with the notion that attentional focus modulates the effect of display set size on distractor processing. When the color of the target was known in advance, the participants could use this knowledge to deploy attention efficiently. Thus, even though the attentional focus induced by the cue was wide enough to include all the stimuli, the preknowledge of the target color would allow the participants to locate the task relevant color quickly and to adjust their attentional focus accordingly. This means that the neutral letters with the task irrelevant color could be excluded from the attentional focus fairly early in the process, thereby minimizing their effect on distractor processing.
In contrast, the participants in the unpredictable group did not know the target color in advance. For them to use color to guide attention, they would have to first ascertain the task relevant color by determining which color was the minority color and which one was the majority color, which would probably take some time. As attention could not be zoomed in to the target quickly, the irrelevant letters had more opportunity to be processed, resulting in the dilution effect in the unpredictable group.
It is worth noting that although the distractor differed from the target in both color and location, this perceptual segregation did not completely shield the distractor from being processed, as evidenced by the significant congruency effect in both the predictable and unpredictable groups. This distractor interference suggests that the attentional focus included the distractor along with the two cued locations on either side of it[^2^](#fn0002){ref-type="fn"}. A similar result was reported by Harms and Bundesen ([@B20]), who found a significant response congruency effect despite the fact that the target and distractors differed in both color and spatial locations.
Tsal and Benoni ([@B46], Experiment 3) have also investigated the effect of preknowledge of the target color on distractor processing. In two of their experimental conditions most relevant to the present experiment, i.e., the high load and dilution conditions, Tsal and Benoni\'s participants saw multi-stimulus displays that consisted of letters of different colors. Whereas the color of the target was unknown on a given trial in the high load condition, it was known in advance in the dilution condition. Although the average RT was substantially slower in the high load condition than in the dilution condition, no congruency effect was found in either condition. In contrast, a significant congruency effect was found in the low load condition, in which the target display consisted of a single colored target letter and one distractor. Similar results were found by Benoni and Tsal ([@B4], Experiment 2). Once again, no significant congruency effects were found in either the high load or dilution condition, but only in the two low load conditions. (The two conditions differed in that the color of the target was known in one condition but not in the other condition). These results confirmed the researchers\' hypothesis that perceptual load did not influence the degree of distractor processing when the number of neutral items was held constant. Based on their results, Benoni and Tsal also concluded that whereas preknowledge of target location affects both target and distractor processing, preknowledge of target color affects only target processing.
In Experiment 4 of the present study, the pattern of data between the predictable and unpredictable groups differed not only in the overall response latencies to the target (longer in the unpredictable than the predictable group), but also in the effect of display set size on distractor processing. Whereas the magnitude of the congruency effect decreased with an increase in display set size in the unpredictable group, there was no evidence that display set size influenced the degree of distractor processing in the predictable group. These results suggest that preknowledge of the target color affected both target and distractor processing in our paradigm. However, because of the many differences in methodology between the present experiment and the experiments of Benoni and Tsal ([@B4]) and Tsal and Benoni ([@B46]), we do not consider our results contradictory to their claim. Our results simply show that under some conditions, preknowledge of target color can affect participants\' deployment of attention, which in turn can influence the degree of distractor processing.
General discussion
==================
These experiments, and the earlier experiments that they build on, illustrate the complexity of visual processing in multi-element displays with targets and distractors. Attention can select the targets once they are identified, but in many cases it cannot prevent the distractors from being partially processed and interfering with the target. This small bit of processing accorded to the distractor is not guaranteed, however; it can be blocked if extra items are added to the display. The current experiments show that these extra items are themselves subject to changes in attentional allocation triggered by sudden luminance increment and by expectations about target color.
Sorting out these different effects will require an understanding of the different factors governing distractor processing in complex displays. One key question in recent years has been why the interference from an irrelevant distractor diminishes when more items are added to the display. The original perceptual load theory posited that these extra items required processing as part of the task, which took processing resources away from the distractor. However, experiments by Tsal and Benoni ([@B46]) and by Wilson et al. ([@B47]) showed that the extra items can weaken distractor interference even when they are easily identified as irrelevant to the task. They described the effect as dilution, because the mere presence of these items diluted the interfering effects of the distractor, independent of their relevance to the task.
Wilson et al. ([@B47]) explained dilution within a two-stage account in the style of Neisser ([@B40]) and Hoffman ([@B21]), with the dilution occurring in the second stage, after the target has been identified and selected. In this account, any processing resources not used by the target are allocated to the non-target items, but unlike Lavie\'s account, these non-target items are all equal in that their original relevance to the task does not affect their processing. The more non-target items there are, the more interference each item encounters.
Wilson et al.\'s ([@B47]) account predicts that Experiment 2 should show dilution in the 2-cue condition; when extra items are present in the 6-letter display, they should decrease the distractor interference relative to the 2-letter display. Instead, the luminance-decrement items in Experiment 2 do not dilute the effects of the distractor, demonstrating that dilution in this paradigm depends on the attentional effects of the display onsets. All items do not contribute equally to dilution; it depends on whether they benefit from the attentional focus or not. These results are consistent with prior research showing that inducing participants to adopt a small attentional focus so that distractors fall outside it could minimize distractor interference in a search display (e.g., Eriksen and St. James, [@B16]; Yantis and Johnston, [@B48]; LaBerge et al., [@B27]). They are also consistent with the more recent finding that singletons capture attention when they are inside but not outside the attentional focus (Belopolsky et al., [@B3]; Belopolsky and Theeuwes, [@B2]).
The effects of attention are also seen in Experiment 4, in which dilution from the non-targets is eliminated if their color makes it easy to ignore them. The two types of attentional effects on dilution shown in these experiments are consistent with Yeh and Lin\'s ([@B51]) demonstration that dilution is affected by perceptual grouping. One option for explaining both sets of results is to modify the dilution account to allow for attentional effects on all elements in the display at some stage of processing. In other words, the amount of dilution from a particular display item will depend on its location relative to the attentional focus, its grouping with other elements in the display, its features that match the expected features of the target, and other factors that affect attentional allocation. Another option to account for these data is to modify the perceptual load account to include a detailed description of how the different non-targets in the display interact to affect one another\'s processing. As Yeh and Lin have suggested, it may be possible to construct an account somewhere in between the pure perceptual load theory and the pure dilution theory that can explain all of these different experimental results, but it is likely to include a combination of factors that make it more complex than either of those original theories.
While Experiments 2 and 4 show that dilution is affected by the attentional focus, Experiment 3 demonstrates another informative aspect about dilution: that it occurs not because the basic features of the non-targets interfere with processing the features of the distractor, but because the non-target are activating letter representations that compete with the representation for the distractor letter. When the non-targets are inverted so that they do not match any letter representation, the competition is eliminated. The interference that underlies these effects appears to arise at the level of letter representations, and not lower down at the level of simple features.
These results give us a clearer view of how dilution occurs in the processing of multi-element displays, and how it can be prevented. As shown by Tsal and Benoni ([@B46]) and by Wilson et al. ([@B47]), items can contribute to dilution even when their location makes it clear that they are irrelevant to the task, but only if a sudden increment in luminance draws a certain amount of attention to them. Furthermore, the effects of a letter will only be diluted by other letters in the display, and not by items sharing basic features with the letters. Thus, dilution is not as widespread or as uniform as previous accounts predict. These results, like those of Yeh and Lin ([@B51]), suggest that within multi-element displays, there is a complex interaction between the separate elements as they all compete for some level of attention, and that the allocation of attention is shaped by multiple factors working simultaneously.
Conflict of interest statement
------------------------------
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We thank two anonymous reviewers for their helpful comments.
^1^In all experiments, response latencies greater than 2000 ms were excluded. These constituted less than 1% of the total data in each experiment. Only trials that were correct were included in the tables and the statistical analyses.
^2^Although our study was not designed to test the issue of attentional selection over contiguous vs. non-contiguous regions, there is evidence that either type of selection may occur under the right circumstances (see Jans et al., [@B23]; and Cave et al., [@B7], for a review). The fact that we found substantial congruency effects in all our experiments despite the positional certainty of the distractor indicates that attention selected contiguous regions in the present experimental paradigm.
[^1]: Edited by: Tal Makovski, The College of Management Academic Studies, Israel
[^2]: Reviewed by: Chris Blais, Arizona State University, USA; Yaffa Yeshurun, University of Haifa, Israel
[^3]: This article was submitted to Frontiers in Cognition, a specialty of Frontiers in Psychology.
| {
"pile_set_name": "PubMed Central"
} |
Despite dramatic improvements in the hardware resources and computational power available to pharmaceutical researchers over the past few decades, the methods used for assessing the 2D chemical similarity between two molecules hasn\'t changed much since the 1960s. Here we report a novel chemical database search method that allows the exact size of the maximum common edge subgraph (MCES) between a query molecule and molecules in a database to be calculated rapidly. Using a pre-computed index, the 50 nearest neighbors of a query can be determined in a few seconds, even for databases containing millions of compounds. This work builds upon the previous efforts of Wipke and Rogers in the 1980s \[[@B1]\] and of Messmer and Bunke in the 1990s \[[@B2]\], harnessing the advances in high-performance computing and storage technology now available. A graphical depiction of such a \"SmallWorld\" index is shown below.
{#F1}
| {
"pile_set_name": "PubMed Central"
} |
Introduction
============
Obesity has dramatically increased during the past decades and has now reached epidemic proportions in both developed and developing countries. Even in Japan where the self-reported prevalence of obesity has remained consistently low over the last 30 years, obesity is now increasing in middle-aged adults and children \[[@B1],[@B2]\] partly due to a western-style change in diet. The increase in obesity is associated with corresponding increases in type 2 diabetes, hypertension, cardiovascular disease and cancer \[[@B3]\]. Obesity is also associated with an increased incidence of gastrointestinal (GI) disorders \[[@B4]\] suggesting effects on the enteric nervous system (ENS), which controls virtually all gut functions (for review see \[[@B5]\]).
It is generally accepted that obesity is characterized by a low-grade chronic inflammation \[[@B6]\] in which pro-inflammatory cytokines play a pivotal role. The source of the inflammation is regarded as the adipose tissue itself. The adipose tissue of obese individuals, including adolescents, has been shown to produce higher levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6 compared to lean individuals \[[@B7]-[@B10]\]. In animal models of diet-induced and genetic obesity increased production of IL-1, IL-6, TNF-α and Toll-like receptor (TLR) signaling in adipose tissue has also been reported \[[@B11]-[@B13]\]. In this review, the cause of the inflammation has been reevaluated and the GI tract as a potential source of inflammation and the role of gut microbiota are explored.
A high fat (HF) diet not only modulates the release of inflammatory mediators from adipocytes but also has a major impact upon the gut microbiota \[[@B14]\], the trillions of bacteria that normally reside within the human GI tract and upon fermentation of non-digestible carbohydrates generate short-chain fatty acids \[[@B15]\] and promote their absorption and storage as fat \[[@B16],[@B17]\]. HF feeding in mice induced a low-grade inflammatory tone that was associated with changes in gut microbiota towards a decreased number of bifidobacteria \[[@B18]-[@B20]\], a group of bacteria, which has been shown to reduce lipopolysaccharide (LPS) levels in mice and to improve mucosal barrier function \[[@B21]-[@B23]\]. Interestingly, feeding obese mice with prebiotics and changing gut microbiota in favor of the *Bifidobacterium*spp. led to a significant improvement of gut permeability that correlated with lower portal plasma LPS levels and inflammatory tone (i.e., decreased circulating cytokines) \[[@B18],[@B21]\]. The leakage of gut microbiota-derived LPS into the portal blood is a well-established mechanism of metabolic endotoxemia that triggers liver inflammation and oxidative stress \[[@B17],[@B24]\]. The gut microbiota differs in composition between lean and obese individuals \[[@B14],[@B25]\]. Moreover, alterations in gut microbiota seen in morbidly obese subjects are modulated by weight loss due to calorie restriction (CR) \[[@B26]\] or gastric bypass surgery and are correlated with a reduction in inflammatory state \[[@B27],[@B28]\].
This article will discuss recent advances in understanding the role of gut microbiota and inflammation in the pathogenesis of obesity and obesity-related GI dysfunction. Since the silent information regulator (SIR) genes (sirtuins) are protective against obesity-induced inflammation and mediate, at least in part, the beneficial effects of CR (for review see \[[@B29],[@B30]\]), the potential contribution of sirtuin signaling in the bowel under inflamed states is also considered.
Gut microbiota
--------------
The human GI tract is dominated by anaerobic bacteria belonging to three bacterial phyla: Firmicutes, Bacteroidetes, and Actinobacteria \[[@B31]\]. Greater than 90% of the microbiota in a normal distal gut is represented by the Bacteroidetes and Firmicutes phyla \[[@B32]\]. The Firmicutes, which is the largest bacterial phylum, comprises over 200 genera of predominantly Gram-positive bacteria, including *Lactobacillus*, *Mycoplasma*, *Bacillus*, and *Clostridium*species. The Bacteroidetes phylum is composed of three large classes of Gram-negative bacteria: Cytophaga, Flavobacterium, and Bacteroidales. Members of the Bacteroidetes and Firmicutes phyla have been shown to be influenced by HF feeding and obesity \[[@B32]-[@B34]\]. The Actinobacterium phylum consists of Gram-positive bacteria and includes the genus *Bifidobacterium*, which is increased upon consumption of prebiotics, indigestible carbohydrates, which stimulate the growth of particular species of the microflora (for review see \[[@B35]\]).
Initial observations to suggest that the gut microbiota contribute to obesity were prompted by the observations of Gordon and colleagues \[[@B16],[@B36]\]. Studies in leptin-deficient *ob/ob*mice showed a different proportion of the two dominating divisions, Bacteroidetes and Firmicutes compared to lean wild-type (WT) mice. Compared with lean littermates fed the same polysaccharide-rich diet, obesity was associated with a 50% reduction in Bacteriodetes and a proportional division-wide increase in Firmicutes in obese mice. To definitively demonstrate that gut microbiota composition is a cause and not a consequence of obesity, cecal microbiota from lean and obese mice were transplanted into the gut of germ-free (GF) mice. GF mice, raised from birth in sterile conditions, are significantly thinner than microbially colonized mice, despite eating the same amounts of food. Furthermore, after 2 weeks, conventionalization of GF mice with the cecal microbiota from normal mice produced a 57% increase in total body fat, a 2.3-fold increase in hepatic triglycerides, and much higher levels of leptin production and insulin resistance which was not dependent on an increase in chow consumption or changes in energy expenditure \[[@B37],[@B38]\]. Evidence was obtained to suggest that an increased energy harvest from the diet contributed to obesity in host GF mice. This might be due to an increase in energy extraction due to bacterial fermentation of polysaccharides and also to the ability of the gut microbiota to upregulate fasting-induced adipose factor, a circulating lipoprotein lipase, which increases cellular uptake of fatty acids and triglyceride accumulation in adipocytes. Shotgun metagenomic analysis of the gut microbiome in obese and lean mice revealed an enrichment of genes involved in energy harvest in obese mice \[[@B34],[@B38]\]. These included genes involved in sensing and degrading dietary polysaccharides, transporters of the resulting mono- and oligosaccharides, and genes involved in their intracellular metabolism. Thus, the microbiome of obese mice had increased fermentation capabilities resulting in increased levels of short chain fatty acids (SCFAs) in the cecum \[[@B38]\]. Similarly, the gut microbiome of obese individuals has increased fermentation capacity resulting in elevated SCFA production \[[@B38]\]. Interestingly, mice deficient in either of the SCFA receptors are leaner than their wild-type counterparts \[[@B39]\], further implicating SCFAs in the development of obesity. Currently, there is no consensus as to whether the gut microbiota plays a causative role in obesity or is modulated in response to the obese state itself or the diet in obesity. Further studies, especially on the regulatory role of SCFA in human energy homeostasis are needed to clarify the physiological consequences of an \"obese style\" microbiota and any putative dietary modulation of associated disease risk.
Data from human studies were generally consistent with the results from animal models. The first study describing qualitative changes of the gut microbiota in human obese subjects was published a few years ago \[[@B40]\]. In this study, the analysis of fecal samples of obese versus matched lean individuals showed a shift in bacterial phyla (lower Bacteroidetes and more Firmicutes). Interestingly the authors observed that after weight loss (following a fat restricted or a carbohydrate restricted low calorie diet) the ratio of Bacteroidetes to Firmicutes approached a lean type profile after 52 weeks \[[@B40]\]. In addition, humans who have undergone gastric bypass surgery for morbid obesity, have a microbial composition that is different from both obese and slim individuals \[[@B41]\]. A metagenomic study which included monozygotic and dizygotic twins concordant for leanness or obesity and their mothers, also showed that obesity was associated with a markedly reduced bacterial diversity, a relative depletion of Bacteroidetes, and a higher proportion of Actinobacteria \[[@B42]\].
The hypothesis of more specific modulation of the gut microbiota community in obesity (instead of those obtained at the wide *phylum*levels) is supported by several studies. Children with developmentally unusual gut microbiota appear to have predispositions to obesity \[[@B43]\] and another study found that the response of overweight adolescents to a diet and exercise weight-loss program was dependent on the initial gut microbiota prior to the treatment \[[@B26]\]. Moreover, differences in fecal microbiota were shown to predict overweight in children during the first year in life \[[@B43]\]. In a prospective study, *Bifidobacteria*spp. number was higher in children who exhibited a normal weight at 7 years than in children developing overweight. Moreover, they observed that the *Staphylococcus aureus*were lower in children who maintain a normal weight than in children becoming overweight several years later. The authors proposed that *S. aureus*may act as a trigger of low-grade inflammation, contributing to the development of obesity \[[@B43]\]. In agreement with these last findings, significant differences have been observed in gut microbiota composition according to the body weight gain during pregnancy \[[@B44]\]. They also found that the *Bifidobacterium*genus was present in higher numbers in normal-weight than in overweight women and also in women with lower weight gain during pregnancy \[[@B44]\]. The *Bifidobacterium*genus was also poorly represented in the fecal samples of diabetic patients compared with healthy individuals \[[@B45]\].
Similar results linking gut microbiota to obesity have been described in models of diet-induced obesity. In mice, ingestion of a HF diet resulted in an increase of Firmicutes and this was transmissible to lean GF recipient mice \[[@B34]\]. In addition, an increase in Bacteroidales and Clostridiales was found in rats fed a HF diet regardless of whether they exhibit either an obesity-prone or obesity-resistant phenotype. However, an increase in Enterobacteriales was seen in the microbiota of obesity-prone rats only \[[@B17]\]. Gut inflammation has been shown to promote the growth of Enterobacteriaceae \[[@B46]\]; therefore, an increase in this family may be a consequence of gut inflammation in the HF obesity prone mice rather than a cause. Taken together, these data demonstrate that it is the consumption of a HF diet rather than obesity that accounts for the change in the gut microbiota, but it is the development of inflammation that is associated with the appearance of hyperphagia and an obese phenotype.
Although humans are interested in manipulating microbiota to aid in weight loss, the food industry has been engaged for decades in manipulating microbiota to increase in weight gain through the use of low-dose antibiotics, usually called antibiotic growth promoters (AGPs) as feed additives \[[@B47]\]. Evidence from the food industry has shown that antibiotics, such as avoparcin (a vancomycin analogue) and Firmicute probiotics (e.g., Lactobacillus and Enterococcus) that modify the microbiota can act as growth promoters increasing the size and weight of farm animals \[[@B48]\]. Notably, a recent human clinical study showed significant weight gain can occur in humans after a six-week intravenous treatment of vancomycin plus gentamycin for infective endocarditis with a risk of obesity. Lactobacillus sp, a microorganism intrinsically resistant to vancomycin was found at higher concentration in the feces of obese patients \[[@B49]\]. In contrast, the absence of specific microbiota or its almost complete reduction with broad-spectrum antibiotics prevents or reverses HF-induced obesity \[[@B36],[@B50]\]. Treatment with rifaximin (Xifaxan^®^, Salix Pharmaceuticals, Morrisville, NC, USA), a nonsystemic rifamycin-derived antibiotic that exhibits low gastrointestinal absorption while retaining potent antibacterial activity \[[@B51]\] for two weeks has recently been shown to provide significant relief of symptoms associated with IBS, such as bloating, abdominal pain, and loose or watery stools \[[@B52]\], which are also observed in obese individuals. Modulation of the gut microbiota with antibiotic therapy has been reported in obese mice. In addition, antibiotics reversed insulin resistance improving glycemic control \[[@B50]\].
Gut inflammation and barrier function
-------------------------------------
It is now well established that obesity is an inflammatory condition and that \"low grade chronic\" inflammation, associated with insulin and leptin resistance exists in obese individuals \[[@B6]\]. The source of the inflammation is commonly regarded as the adipose tissue itself, which is known to produce inflammatory mediators \[[@B53]\]. However, the gut microbiota could also be a potential source of inflammation.
A HF diet is associated with the expression of two inflammatory biomarkers in the intestine, TNF-α and nuclear factor kappa B (NF-κB). The presence of gut bacteria is required for the induction of TNF-α and NF-κB since GF mice given a similar diet did not exhibit up-regulation of these pro-inflammatory markers \[[@B54]\]. Moreover, the observation that increases in intestinal TNF-α precede yet significantly correlate with body weight gain, body fat, and subsequent development of insulin resistance, supports a potential role of gut-derived TNF-α in the development of HF-induced obesity and obesity-related disease \[[@B54]\].
Recent work has shown that gut microbiota can initiate the inflammatory state of obesity through the activity of LPS, part of the outer membrane of Gram-negative bacteria that is released into the gut lumen when bacteria die. Cani et al. \[[@B18]\] reported that mice fed a HF diet present a chronic increase in circulating LPS, which they called \"metabolic endotoxemia\". The level of serum LPS is increased by about twice in obese, diabetic, or high-fat fed individuals, by processes involving an increase in chylomicron formation, a decrease in gut barrier integrity, and a decrease in alkaline phosphatase activity, which is the enzyme responsible for the cleavage of the LPS in the intestine \[[@B55],[@B56],[@B21]\].
LPS can trigger the inflammatory process by binding to the CD14 toll-like receptor-4 (TLR4) complex in the gut wall. When metabolic endotoxemia was reproduced by subcutaneous infusion of LPS, animals developed the same metabolic abnormalities induced by a HF diet, while LPS knock out (CD14*-/-*) mice were resistant to the effects of both HF diet and LPS infusion \[[@B57]\]. Moreover, chronic (4 week) administration of LPS in mice causes hyperphagia and an increase in adiposity and metabolic changes seen with ingestion of HF diet \[[@B21],[@B57]\]. In a subsequent experiment, changes in gut microbiota composition induced by antibiotic treatment reduced the cecal content of LPS and improved measures of inflammation, such as macrophage infiltration of adipose tissue, closely correlating with an improvement in the obese phenotype in both HF fed and *ob/ob*mice \[[@B19]\].
Activation of TLR4 causes the secretion of IL-6 and TNF-α, supporting the role of LPS in triggering the downstream inflammatory processes associated with obesity, such as metabolic disease \[[@B20],[@B57]\]. Ingestion of a HF diet induced a significant postprandial elevation of LPS, accompanied by an increased mononuclear cell expression of TLR-4, NF-ΚB and suppressor of cytokine signaling-3 (SOCS-3), an adipokine involved in insulin resistance \[[@B58]\].
Interestingly, mice genetically deficient in TLR-5 have an altered gut microbiota composition that correlates with obesity and several features of the metabolic syndrome including hyperlipidemia, hypertension, and insulin resistance, which could at least in part be attributed to increased food consumption \[[@B59]\]. In contrast to the *ob*/*ob*mouse model of obesity, which is characterized by a phylum-level shift in Bacteroidetes and Firmicutes, the TLR5-deficient mice exhibit altered abundance in over one-hundred specific bacterial phylotypes. A direct causal relationship between the altered gut microbiota and obesity was demonstrated by gut microbiota transplants where GF mice receiving the gut microbiota from TLR-5-deficient mice gained significantly more weight compared to mice that received gut microbiota from wild-type mice \[[@B59]\]. These results support the emerging view that the gut microbiota contributes to obesity and suggest that malfunction of the innate immune system may promote the development of obesity-related disorders such as metabolic syndrome.
Mucosal barrier function is maintained by several interrelated systems, including mucous secretion, chloride and water secretion, and binding together of epithelial cells at their apical junctions by tight junction (TJ) proteins. The disruption of the TJ complex leads to leakage of water and proteins into the lumen, as described in relapsing diarrhea, and to the translocation of intraluminal solutes, such as bacterial endotoxins (LPS), into the system circulation \[[@B60]\]. Activation of TLR4 has previously been shown to alter the TJ complex and increase intestinal permeability \[[@B61]\]. Modulation of gut bacteria following a HF diet strongly increases intestinal permeability, by reducing the expression of genes coding for two intestinal TJ proteins, ZO-1 and occluding \[[@B19]\]. Alteration in occludin distribution has also been reported *in vitro*on epithelial cells stimulated with pro-inflammatory cytokines; thus, occludin was chosen as a marker of TJ disruption \[[@B24],[@B62]\].
Since aberrant gut microbiota and a \"leaky\" mucosal barrier are found in obesity they offer potential targets for intervention that would include modulation of the intestinal microbiota to correct an imbalance, as well as tightening of interepithelial junctions. Enhancement of barrier function by probiotic bacteria has been observed both in *in vitro*models and *in vivo*in the whole animal \[[@B63]\]. Probiotics are live microorganisms that have a beneficial effect on the intestinal mucosa via several proposed mechanisms that include inhibition of the mucosal adhesion of pathogens, improvement of the barrier function of the epithelium, and alteration of the immune activity of the host. They may also regulate intraluminal fermentation and stabilize the intestinal microbiota \[[@B64]\]. Probiotic bacteria are *Lactobacilli spp*., certain types of *Streptococcus*, and *Bifidobacteria spp*., but also other non-pathogenic bacilli such as *E. coli*-Nisle 1917 and yeasts such as *Saccharamyces boulardii*. They secrete short chain fatty acids, an action that results in decreased luminal pH and production of bactericidal proteins. Butyric acid, a byproduct of bacterial fermentation of fiber, has been shown to nourish colonic enterocytes, enhancing mucosal integrity \[[@B65]\].
Researchers have demonstrated the utility of probiotics for obesity in HF fed mice \[[@B57],[@B66]\], which is associated with a decrease in the number of *Bifidobacteria*\[[@B21]\]. An increase in *Bifidobacteria*in *ob/ob*mice was associated with a significant improvement of gut permeability measured *in vivo*; this improvement was linked to an increase in TJ mRNA expression and protein distribution \[[@B21]\]. In addition, the rise in *Bifidobacteria*was correlated with a decrease in plasma LPS concentrations and therefore, a significant reduction in markers of oxidative and inflammatory stress \[[@B21]\]. Potential beneficial effects of probiotics on gut motility via a direct action on the ENS or epithelial cells have also been demonstrated \[[@B67]\]. In experimental studies, *Lactobacillus*inhibited post-infective intestinal hypercontractility through an unidenfied, heat-labile fermentation-product and by blocking calcium-dependent potassium channels \[[@B68],[@B68],[@B69]\]. Supernatant of *Escherichia coli*Nissle 1917 enhanced human colonic motility *in vitro*and acute exposure of colonic mucosa to *Lactobacillus rhamnosus*GG significantly reduced the acetylcholine-stimulated human colonic contractions in a dose- and time-dependent manners \[[@B70]\]. Administration of *L. reuteri*altered the motility of *ex vivo*colonic segment of rat; it decreased the amplitudes of contractions and increased intraluminal fluid filling pressure thresholds for evoking pressure pulses \[[@B71]\]. Overall, probiotics will likely have an emerging therapeutic role in preventing and treating obesity and obesity-related inflammation.
Gut dysfunction and obesity
---------------------------
Many obese individuals report symptoms suggestive of gut dysfunction including bloating, abdominal pain and diarrhea \[[@B4],[@B72],[@B73]\]. Bloating and upper abdominal pain increased in frequency with increasing body mass index (BMI). There was also a significant positive relationship between BMI and diarrhea. In contrast, no significant relationship was observed between BMI and constipation, even though it was more frequent in obese patients \[[@B4]\]. Potential mechanisms to explain the increased bowel frequency would be rapid gastric emptying, which has been reported in some groups of obese patients \[[@B74]\], and increased colonic motility, although the latter has not yet been demonstrated.
Obesity is associated with gastroesophageal reflux disease (GERD), nonalcoholic fatty liver disease (NAFLD), and increased occurrence of cholelithiasis \[[@B75],[@B76]\]. GERD has been shown to be more common in obese patients than in those with a BMI within normal range, and an increase in the BMI above the 95 percentile for age and gender is a significant risk factor for GERD \[[@B77],[@B78]\]. Also, a higher BMI is associated with more frequent and more severe heartburn and regurgitation in patients with GERD and increasing BMI is a strong predictor of heartburn during sleep \[[@B79],[@B80]\].
In patients with irritable bowel syndrome (IBS), heartburn was more likely to be present in subjects with obesity, and epigastric pain and nausea, were also more common in overweight patients with IBS. However, in an adjusted log linear model, no significant interaction was found between BMI and any other studied symptom and heartburn was found to be independent of IBS \[[@B81]\].
An important and well described correlation also exists between obesity and colorectal cancer \[[@B82]\]. Epidemiologic data have shown that obesity independently increases colorectal cancer risk, particularly in males, but the mechanisms are poorly understood \[[@B83]\]. Serum leptin level in colon cancer patients who were overweight or obese were significantly higher compared to patients with normal weight \[[@B84]\]. mRNA levels of the novel inflammatory factors lipocalin-2, chitinase-3 like-1 and osteopontin are increased in human visceral adipose tissue of individuals with colon cancer \[[@B85]\]. Leptin upregulates pro-inflammatory cytokines in discrete cells within the mouse colon \[[@B86]\]. IL6, IL1β and CXCL1 were upregulated by leptin and localized to discrete cells in gut epithelium, lamina propria, muscularis and at the peritoneal serosal surface.
Diet-induced weight loss in obese individuals reduces colorectal inflammation and greatly modulates inflammatory and cancer-related gene pathways \[[@B87]\]. After weight-loss, rectosigmoid biopsies showed a 25-57% reduction in TNF-α, IL-1β, and MCP-1 concentrations. Gene arrays showed dramatic down-regulation of pro-inflammatory cytokine and chemokine pathways, prostaglandin metabolism, oxidative stress pathways and the transcription factor CREB. These data imply that obesity is accompanied by inflammation in the colorectal mucosa and that weight loss reduces this inflammatory state and may thereby lower colorectal cancer risk \[[@B87]\].
Obesity predicts persistence of abdominal pain in children with functional gastrointestinal disorders \[[@B88]\]. Obese children (mean age 13 years) were more likely to have abdominal pain, higher intensity of pain, higher frequency of pain, school absenteeism and disruption of daily activities than non-obese children \[[@B88]\]. Obesity is more common in children with celiac disease, a T cell-mediated chronic autoimmune enteropathy occurring in genetically susceptible individuals, and manifested by a permanent intolerance of gluten-containing products \[[@B89]\]. The most common presenting symptoms among obese patients were abdominal pain, diabetes, and diarrhea. Symptoms improved in all patients on a gluten-free diet.
Role of the ENS
---------------
Under both physiological and pathological conditions, the ENS, the intrinsic innervation of the bowel, regulates intestinal mucosal function and coordinates the activity of the GI tract. The ENS is a component of the autonomic nervous system with the unique ability to function independently from the CNS (for review, see \[[@B90]\]). Enteric ganglia are organized into two major ganglionated plexuses, namely the myenteric (Auerbach\'s) and submucosal (Meissner\'s) plexus, and contain a variety of functionally distinct neurons, including primary afferent neurons, interneurons, and motor neurons, synaptically linked to each other in microcircuits. While the myenteric plexus mainly regulates intestinal motility, the submucosal plexus together with nerve fibers in the lamina propria are involved in regulating epithelial transport. These nerves form networks within the lamina propria of both crypts and villi with the terminal axons in close contact with the basal lamina, an ideal position not only to affect epithelial cell functions but also to detect absorbed nutrients and antigens. Substances released from epithelial cells may act on nerve terminals to change the properties of enteric neurons and cause peripheral sensitization. Consequently, permanent or even transient structural alterations in the ENS disrupt normal GI function.
The ENS is increasingly recognized as a regulatory housekeeper of the epithelial barrier integrity, in addition to its ascribed immunomodulatory potential (for review see \[[@B5]\]). Inflammation affects both epithelial integrity and barrier function and, in turn, loss of barrier function perpetuates the inflammatory condition. Several studies have demonstrated structural changes within enteric ganglia in gut inflammation (see \[[@B91],[@B92]\] for review). For example, damage to axons has been observed in the inflamed human intestine in episodes of inflammatory bowel disease (IBD) \[[@B93]\]. Other changes that occur in the ENS during inflammation include altered neurotransmitter synthesis, content, and release, changes in glial and myenteric cell numbers and a myenteric ganglionitis associated with infiltrates of lymphocytes, plasma cells and mast cells \[[@B94]-[@B96]\]. In fact, experimental data show that gut inflammation, even if mild, could lead to persistent changes in GI nerve and smooth muscle function, resulting in dysmotility, hypersensitivity, and dysfunction (for review see \[[@B91],[@B92]\]). Furthermore, alterations in gut function were observed even after the resolution of an acute intestinal inflammation \[[@B97]-[@B99]\]. Thus, the breakdown of mucosal barrier function as occurs in obesity could cause alterations in the patterns of gut motility, abnormal secretion, and changes in visceral sensation that contributes to symptom generation. In a rodent model of diet-induced obesity the secretomotor function of submucosal neurons was compromised, which may lead to an altered host defense with a resultant change in intestinal flora contributing to the maintenance of obesity \[[@B100]\]. The breakdown of mucosal barrier function may at least partially explain the link between obesity and gut dysfunction. Whether the persistent alterations in GI motility observed in many obese patients are due to inflammation-related changes in the properties of enteric nerves is not known. However, probiotic lactic acid producing-bacteria have been shown to prevent and alleviate GI disturbances and to normalize the cytokine profile which might be of an advantage for patients suffering from obesity \[[@B101]\].
Role of sirtuins
----------------
In the past decade, a novel class of regulators, the silent regulator 2 (SIR2), has been linked to metabolic regulation and aging and shown to mediate CR-induced longevity in yeast and possibly other organisms (for review see \[[@B29]\]). Mammalian sirtuins are conserved with seven genes (SIRT1-7) homologous to the yeast *Sir2*gene. Like their yeast homologs, the mammalian sirtuins are class III histone deacetylases and require NAD(+) as a cofactor to deacetylate substrates ranging from histones to transcriptional regulators. The nuclear sirtuins (SIRT1, SIRT6, and SIRT7), the mitochondrial sirtuins (SIRT3, SIRT4, and SIRT5), and the cytosolic sirtuin (SIRT2) regulate diverse metabolic functions. For example, SIRT6 functions in genomic stability and transcriptional control of glucose metabolism and its deficiency (SIRT6-/-) causes a lethal hypoglycemia \[[@B102],[@B103]\]. SIRT6 is highly expressed in the CNS and mice overexpressing SIRT6 are protected against diet-induced obesity \[[@B104]\]. In contrast, neural-specific SIRT6 knockout mice become obese during adult life \[[@B105]\], further highlighting the importance of SIRT6 in the context of nutrient metabolism. SIRT3 is an integral regulator of mitochondrial function and its depletion results in hyperacetylation of critical mitochondrial proteins that protect against hepatic lipotoxicity under conditions of nutrient excess. Livers of mice fed on a HF diet had reduced SIRT3 activity \[[@B106]\].
The beneficial effect of CR on aging and various metabolic disorders is dependent on the activation of SIRT1 and can be mimicked by resveratrol, a product present in grape skin and red wine, which activates the SIRT1 enzyme \[[@B107],[@B108]\]. Mild to moderate red wine consumption has anti-inflammatory properties, and can reduce the risk of cardiovascular disease and cancer. The resveratrol content in red wine is often cited to account for this \"French paradox.\" Evidence for a role of sirtuins in obesity comes from emerging understanding of the regulatory role sirtuins play in metabolic pathways and adaptations linked with obesity and aspects of metabolic syndrome. These include the expression of adipocyte cytokines (adipokines), the maturation of fat cells, insulin secretion, modulation of plasma glucose levels, cholesterol and lipid homeostasis and mitochondrial energy capacity \[[@B29]\].
SIRT1, for example, is involved in regulating the expression of adipokines such as adiponectin and TNF-α, has been linked to hypothalamic control of energy balance, plays a role in adipogenesis, and is involved in the regulation of lipolysis and fatty acid mobilization in response to fasting.\[[@B29]\] Evidence from animal studies in which sirtuins are under- or over-expressed, and from limited human evidence, also suggest a role for sirtuins in obesity. Existing evidence on resveratrol suggests that this compound may have sirtuin-mediated anti-obesity effects \[[@B109]\].
Fasting leads to the up-regulation of SIRT1 in adipose tissue of mice, pigs and humans whereas decreased SIRT1 expression is associated with obesity \[[@B29]\]. In both *db/db*and obese HF fed mice SIRT1 expression is low in adipose tissue \[[@B110]\]. Circumstances that result in SIRT1 under-expression in adipose tissue enhanced adipogenesis, while circumstances that promote fat SIRT1 over-expression were characterized by attenuated adipogenesis and increased lipolysis \[[@B111]\]. Consistent with this idea, lean women had more than twofold higher SIRT1 expression compared to obese women \[[@B112]\]. Benefits of SIRT1 over-expression also included less inflammation, better glucose tolerance, and almost complete protection against hepatic steatosis, suggesting that SIRT1 plays an important role in obesity-associated metabolic adverse effects. Consequently, if activation of SIRT1 results in loss of body fat without decreasing caloric intake, this could open the door for novel treatment and prevention strategies for obesity and related diseases.
Genetic variation in SIRT1 is related to BMI and risk of obesity in humans \[[@B113],[@B114]\]. In a recent Belgian case/control study of 1,068 obese patients (BMI ≥ 30 kg/m^2^) and 313 normal weight control subjects, a SIRT1 single nucleotide polymorphism (SNP) associated with visceral obesity parameters in obese men but not women \[[@B113]\]. In two large and independent Dutch Caucasian populations, two common variants in *SIRT1*were associated with lower BMI. Carriers of these two common genetic variants had 9-11% decreased risk of being overweight and 13-18% decreased risk of being obese compared with noncarriers \[[@B114]\].
SIRT1 has recently been implicated in the regulation of obesity-related inflammation. Zhu et al. \[[@B115]\] demonstrated that resveratrol, a SIRT1 activator, decreased TNF-α-induced MCP-1 secretion in 3T3-L1 adipocytes. Pfluger et al. \[[@B116]\] showed over-expression of SIRT1 in mice resulted in a lower level of IL-6 and TNF-α in the serum of transgenic mice fed a HF diet and an attenuated response to TNF-α-induced NF-κB activation in transgenic mouse embryonic fibroblasts. Resveratrol regulates human adipocyte number and down-regulates the expression and secretion of IL-6 and IL-8 from mammalian adipocytes in a SIRT1-dependent manner \[[@B117]\]. Furthermore, mice fed a diet supplemented with 0.4% resveratrol for 10 weeks showed significantly lower body weight and visceral fat-pad weights than HF diet fed mice. Resveratrol significantly attenuated the HF diet-induced up-regulation of a number of pro-inflammatory cytokines such as TNF-α and IL-6, and their upstream molecules, including TLR4 and NF-κB in epididymal adipose tissues of mice \[[@B12]\]. Thus, increased SIRT1 activity appears to be anti-inflammatory in mice and resveratrol may improve obesity-induced inflammation and add to the potential of this dietary polyphenol in the control of obesity. In contrast, inhibition of SIRT1 appears to be pro-inflammatory. Studies using small interfering RNA (siRNA) to knock down SIRT1 reported an increase in TNF-α-induced MCP-1 and other pro-inflammatory genes in 3T3-L1 adipocytes \[[@B118]\]. Taken together, these data demonstrate that a decrease in SIRT1 activity increases activation of NF-κB and transcription of pro-inflammatory mediators. These results have important clinical implications and may thus provide a valuable new strategy for treatment of obesity and its related diseases.
In addition to adipose tissue, SIRT1 is highly expressed in the hypothalamus where it appears to be involved in regulating energy homeostasis, food intake and body weight \[[@B108],[@B119]\]. Fasting up-regulates hypothalamic SIRT1 expression, which is associated with a fasting-induced increase in hunger, and presumably part of the complex adaptations against CR-induced weight loss \[[@B119]\]. Conversely, pharmacological inhibition of hypothalamic SIRT1 decreases food intake and body weight gain in rodents \[[@B120]\], suggesting that hypothalamic SIRT1 decreases food intake and body weight gain in rodents. Lack of SIRT1 in pro-opiomelanocortin (POMC) neurons causes hypersensitivity to HF obesity \[[@B121]\].
SIRT1 is also expressed in the gut where it exerts anti-inflammatory effects in acute intestinal inflammation and suppresses intestinal tumorigenesis and colon cancer associated with colitis \[[@B122]-[@B124]\]. Rats fed with 1 mg of resveratrol/kg/day (a human equivalent dose) for 25 days, and in the last five days, 5% DSS to induce colitis, displayed increased lactobacilli and bifidobacteria as well as a reduced increase in enterobacteria upon DSS treatment. In addition, resveratrol significantly protected the colonic mucosa architecture, reduced body weight loss, diminished the induced anemia and reduced systemic inflammatory markers possibly via the down-regulation of NF-κB \[[@B124]\]. Also, resveratrol up-regulated SIRT1 expression in the mucosa and mitigated the increase in the number of mucosal CD4+ T cells suggesting that resveratrol may exert its anti-inflammatory effects by modulating activated immune cells \[[@B123]\]. Thus, activation of SIRT1 maintains gut barrier function, which is compromised in obesity \[[@B125]\] and through its regulation of gut inflammation controls colitis and colon cancer, which are also more prevalent in obese individuals \[[@B126]\].
The ENS also contains sirtuins. We have shown for the first time that neurons in the murine colon display SIRT1 immunoreactivity (Figure [1](#F1){ref-type="fig"}). The cellular localization of SIRT1 is predominantly nuclear and displayed by neurons in both the submucosal and myenteric plexus consistent with areas strongly compromised by aging \[[@B127]\]. These findings propose a role for SIRT1 in gut motility and secretion and suggest a previously unrecognized role of enteric SIRT1 in regulating energy homeostasis. Moreover, activation of enteric sirtuin pathways could offer a therapeutic approach to treat obesity-related gut dysfunction. Clearly, further research is required to explore the role of sirtuin proteins in enteric neurobiology during normal and inflamed states.
![**Immunohistochemical localization of the class III histone deacetylase SIRT1 (Sir2) in the murine enteric nervous system**. **A**. Confocal image of a whole mount preparation of colon stained with a goat antibody to the neuronal marker human neuronal protein (HuD; 1:100; Santa Cruz; sc-5977; green). HuD immunoreactivity is displayed by neurons in a myenteric ganglion. **B**. Double label confocal image of the same area depicted in **A**stained with an antibody to HuD and a SIRT1-specific antibody made in rabbit (1:500; Abcam Inc. Cambridge MA; ab 16640). Myenteric neurons display both HuD (green) and nuclear SIRT1 immunoreactivity (red). For whole-mount preparations, segments of colon were cut along the mesenteric border and the resulting sheet of gut was pinned flat, mucosal side up, in a silicone elastomer (Sylgard, Dow Corning, Midland, MI)-coated dish. The tissue was fixed for 3 hours with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). After fixation, the preparations were washed in phosphate-buffered saline (PBS) for 1 hour and whole-mount preparations of longitudinal muscle with adherent myenteric plexus (LMMP) were generated as previously described \[[@B128]\]. Non-specific binding was blocked by incubating the preparations with 6% (v/v) normal horse serum, with Triton X-100 (0.5%), in PBS for 60 minutes. The preparations were then exposed for 24 h to primary antibodies at 4°C. After washing with PBS, sites of bound primary antibodies were detected by incubation with donkey anti-rabbit or donkey anti-goat secondary antibodies coupled to DyLight™ 549 (1:400; Jackson ImmunoResearch Labs.West Grove, PA) or DyLight™ 488 (1:400; Jackson ImmunoResearch Labs.) for 3 hours. Confocal images were obtained using an Olympus FluoView FV300 confocal microscope. Scale bar, 30 μm.](1479-5876-9-202-1){#F1}
Conclusions
===========
Obesity is considered a major public health concern globally as it predisposes to a number of chronic human diseases. Recent studies report an aberrant gut microbiota in obese individuals and that gut microbial metabolic activities, especially fermentation can impact on a number of mammalian physiological functions linked to obesity. Those data suggest that specific changes in the gut microbiota characterize the obese state and associated metabolic diseases, including diabetes. Gut microbiota, which affect barrier function also modulate the activity of the ENS, a key player in gut dysfunction. Since aberrant gut microbiota and a \"leaky\" mucosal barrier are found in obesity they offer potential targets for intervention that would include modulation of the intestinal microbiota to correct an imbalance, as well as tightening of interepithelial junctions. However, we may not conclude, from the papers published until now, that targeting one specific bacterial target is sufficient to get an improvement of a complex disease such as obesity. Future studies using newly developed techniques to evaluate the gut microbiota in obese patients and/or animal models of obesity are certainly needed. In addition, future research should focus on the role of sirtuin proteins in the gut and their function in obesity and gut dysfunction. Studies suggest that resveratrol acts as an anti-inflammatory agent in the gut by targeting the SIRT1 gene. Thus, resveratrol may be a therapeutic target against obesity-related inflammation and gut dysfunction.
List of abbreviations
=====================
Body mass index: BMI; calorie restriction: CR; enteric nervous system: ENS; gastroesophageal reflux disease: GERD; gastrointestinal: GI; germ-free: GF; high-fat diet: HF; inflammatory bowel disease: IBD; irritable bowel syndrome: IBS; interleukin: IL; interferon: IFN; lipopolysaccharide: LPS; nonalcoholic fatty liver disease: NAFLD; nuclear factor kappa B: NF-κB; pro-opiomelanocortin: POMC; short chain fatty acid: SCFA; silent information regulator: SIR; silent information regulator genes: sirtuins; small interfering RNA: siRNA; suppressor of cytokine signaling-3: SOCS-3; tight junction: TJ; Toll-like receptor: TLR; tumor necrosis factor: TNF; wild type: WT.
Competing interests
===================
The authors declare that they have no competing interests.
Authors\' contributions
=======================
All authors participated in the preparation of the manuscript, and read and approved the final manuscript.
Acknowledgements
================
This development of this work was supported by the Global Neuroscience Initiative Foundation (GNIF). The authors wish to extend special thanks to GNIF research assistants/associates Violeta Osegueda, Nirali Shah, and Magdalena Hofer for their suggestions and editing support.
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#Sec1}
============
Disability is a public health priority \[[@CR1]\]. The level of disability is inversely associated with socioeconomic position \[[@CR2]\]. Musculoskeletal (MSK) diseases are the most common cause of disability worldwide \[[@CR3]\] and their disabling effects are more severe in developing countries \[[@CR4]\].
Arthritis is a common term used to group different chronic MSK diseases of the joints, mainly osteoarthritis (OA) and rheumatoid arthritis (RA) \[[@CR5], [@CR6]\]. Arthritis is considered a leading cause of disability \[[@CR5]\]. One out of nine persons with arthritis experience disease-related limitations in fulfilling their life roles \[[@CR7]\].
The level of disability associated with arthritis is higher for people living in low socioeconomic conditions \[[@CR7], [@CR8]\]. For example, the functional status of people living with knee and/or hip OA in middle- and low-income countries is lower than those living in high-income countries \[[@CR9]\]. Consequently, the negative effects of arthritis are likely to be associated with social determinants linked with increased disability within low-socioeconomic areas in developing countries.
Considered together, OA and RA are the leading MSK diseases in the Mexican Southern state of Yucatán \[[@CR10]\]. This state includes several indigenous rural communities from the Maya-Yucateco culture. Mexican indigenous communities experience low socioeconomic living conditions and limited access to appropriate health care services \[[@CR11]\]. As a result, these communities are more vulnerable to the disabling effects of arthritis, underscoring the need to develop rehabilitation interventions aimed at preventing and decreasing these effects.
Developing rehabilitation interventions for arthritis requires an understanding of its impact on physical function and identifying modifiable factors associated with the manifestation of this condition. This will help in designing actions to decrease the prevalence and prevent the disabling effects of arthritis at the community level. This study was part of a project designed to develop a community-based rehabilitation program to ameliorate the disabling effects provoked by MSK diseases in a rural, lower-socioeconomic, and underserved Maya-Yucateco community in Yucatán \[[@CR12]\].
The objectives of this study were to evaluate the impact of arthritis on the physical function of people living in the Mayan municipality of Chankom, Yucatán, México, and to evaluate the association between modifiable factors and the prevalence of overall arthritis and its main types (OA and RA) in this community.
Materials and methods {#Sec2}
=====================
Study design {#Sec3}
------------
This was an observational, cross-sectional, community-based study undertaken in three stages: (a) survey, (b) home-based assessment, and (c) confirmatory assessment (see Fig. [1](#Fig1){ref-type="fig"}). These stages were based on the Community Oriented Program for the Control of Rheumatic Diseases (COPCORD) phase one methodology, as described elsewhere \[[@CR13]\] ([www.copcord.org](http://www.copcord.org/)). The survey stage consisted of a census conducted in the adult population (≥18 years) of the municipality of Chankom, Yucatán. Trained local personnel applied a cross-culturally validated questionnaire \[[@CR14]\] designed to detect MSK symptoms and quantify relevant clinical and socioeconomic factors. Two family physicians, trained in rheumatologic evaluation and assisted by local translators, assessed all people who reported MSK symptoms in their homes (home-based assessment stage) within the same week in which the surveys had been applied. During the confirmatory assessment stage, a physiatrist and a rheumatologist evaluated all possible OA or RA cases, respectively, within 1 month of the initial contact and with the help of local Mayan translators.Fig. 1Study stages and participants' flowchart
This study was approved by the Hamilton Health Sciences/McMaster University Research Ethics Board (12--544), the Ethics Committee of the Faculty of Medicine, Universidad Anáhuac-Mayab, and the Ethics Committee of the "Hospital General de México, Dr. Eduardo Liceaga" (DI/11/4044B/3/123). All participants signed an informed consent before participating in the study.
Setting {#Sec4}
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This study was conducted in the rural municipality of Chankom, which is located at the Southeast of Yucatán, Mexico, and comprised of 11 small rural villages inhabited entirely by people of the Maya-Yucateco ethnicity. Chankom is situated on a flat rocky land of tropical forest, 27 m above the sea level with a predominantly warm humid climate. The municipality of Chankom has a population of 4464 habitants of whom 80 % are considered to be living in poverty \[[@CR15]\]. The three stages were conducted between June and December 2012. The confirmatory assessments were performed at various locations, including people's homes, villages' public facilities, and a municipal evaluation center. People identified as having possible OA received a radiographic evaluation at the nearest public hospital.
Participants {#Sec5}
------------
Osteoarthritis diagnosis in participants were confirmed by the physiatrist following the American College of Rheumatology (ACR) clinical criteria for hand OA \[[@CR16]\] and radiological and clinical criteria for hip and knee OA \[[@CR17], [@CR18]\]. A rheumatologist certified by the Mexican Board of Rheumatology and with ≥15 years of clinical practice evaluated all participants detected with inflammatory arthritis and confirmed all cases of RA. Cases with clear signs of arthritis but which did not fulfill OA or RA criteria were classified as "unspecific arthritis."
Measurements {#Sec6}
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### Physical function {#Sec7}
Physical function was evaluated through the health assessment questionnaire disability index (HAQ-DI) applied during the survey stage. The HAQ-DI has shown good psychometric properties when used in OA \[[@CR19]\] and RA \[[@CR20]\] populations. This instrument was cross-culturally translated, adapted, and validated for use in the Maya-Yucateco population \[[@CR14]\] and demonstrated good test-retest reliability (ICC = 0.69) when applied to a sample of 30 individuals living with MSK diseases in Chankom \[[@CR21]\]. The HAQ-DI was scored following standard procedures \[[@CR22]\] and then transformed to a dichotomous variable to indicate the presence or absence of disability using a cutoff point score of 0.25, as reported in a population-based study \[[@CR23]\].
### Modifiable risk factors {#Sec8}
Arthritis prevalence has been associated with several modifiable social, physical, and behavioral factors \[[@CR24], [@CR25]\]. On the one hand, OA prevalence has been associated with socioeconomic factors (i.e., gender, race, and nutrition) that provoke joint vulnerability, and with factors that increase joint loading, such as body mass index (BMI) and the execution of repetitive movements, which augments the mechanical stress within the joints \[[@CR26]\]. On the other hand, RA prevalence has been associated with low level of wealth, high BMI, and smoking \[[@CR25], [@CR27]\].
The participants' level of wealth, or property that can be sold and converted to cash for the benefit of the owner, was assessed as a socioeconomic status variable during the survey stage. Property owned by participants was registered, selected, and classified by local staff according to the type of properties that better differentiate between people's level of wealth in the community. Property related to entertainment, electro-domestic appliances, communication, and transportation were organized into a hierarchical format and combined to derive a "level of wealth" variable ranging from 0 "no properties owned" to 14 "ownership of the highest-valued properties."
Body mass index was registered as the Quetelet's index (weight/height^2^). Weight and height were measured during the survey stage following Lohman's technique \[[@CR28]\]. Weight was measured with a portable digital scale (Tanita Model 804), and height was measured with a portable ultrasonic digital stadiometer (ADE, Ultraschall/Messstab/MZ10020, Germany).
Finally, the following behavioral variables were assessed during the survey: self-reported smoking status, as a yes/no question, and the regular performance of repetitive movements, by asking participants to define their lifetime's principal occupation and whether this occupation involved frequent repetitive movements such as: jolting hands, lifting or pushing ≥20 kg, climbing, standing, kneeling for longer than 30 min, and constant shifting from sit-to-stand positions. Two variables were then created to reflect the cumulative mechanical stress within the joints, one for static stress formed by standing and kneeling longer than 30 min (0 to 2) and one for dynamic stress formed by jolting hands, lifting, and pushing ≥20 kg, climbing stairs or slopes, and constant shifting from sit-to-stand positions (0--5).
### Confounders {#Sec9}
Factors known to be associated with physical function such as age, gender, and number of comorbidities were evaluated at the survey stage through self-report. These factors were considered to be confounders for determining the association between arthritis and disability after no interaction effects were found between these and the association of interest \[[@CR29]\]. The number of comorbidities was determined by adding the number of reported diseases by participant from a list including: diabetes, hypertension, cardiovascular disease, dyslipidemia, gastritis, anxiety and depression, resulting in a continuous variable ranging from 0 to 7.
Statistical analyses {#Sec10}
--------------------
Descriptive statistics were generated through the calculation of means and frequencies. *T* tests adjusting for unequal variances (Satterthewaite's approximation) and chi^2^ tests were used to compare means and frequencies between those participants assessed or not for diagnostic confirmation and between participants with and without arthritis. A logistic regression model was used to evaluate the relationship between the presence of overall arthritis (independent variable) and the presence of disability (dependent variable), adjusting for previously described confounders.
Modifiable risk factor analyses were conducted separately for overall arthritis, OA, and RA using logistic regression models considering the presence of disease as the dependent variable and all the factors evaluated as the independent variables. The OA analysis was adjusted for age and gender. Gender-based subgroup analyses were conducted for hand and lower extremity (hip and/or knee) OA, adjusting by age. Penalized maximum likelihood estimation (Firth's method) was used to estimate logistic regression parameters and profile penalized likelihood confidence intervals in the RA, hand and lower extremity OA analyses, accounting for the observed phenomenon of complete separation in the data \[[@CR30]\].
All regression models were constructed following a complete-case analysis strategy. Assumptions and the models' goodness of fit were confirmed using likelihood ratio tests, hat^2^ tests, Hosmer and Lemenshow tests, and the area under the curve. Hypothesis testing was deemed statistically significant at *α* = 0.05. Two statistical analysis packages (STATA 12.1. and R 3.1.1) were used.
Results {#Sec11}
=======
Fifteen hundred and twenty three adults answered the questionnaire during the survey stage and 823 (54 %) reported MSK symptoms. Forty-four participants (5 %) who reported MSK symptoms could not be seen by a specialist during the confirmatory assessment stage because either they refused or were not found at their homes after five visits and were excluded from the analysis. Proportionally, more men (70 vs. 39 %, Chi^2^~\[1\]~ = 17.78, *p* \< 0.0001) and more smokers (21 vs. 9 %, Chi^2^~\[1\]~ = 5.95, *p* = 0.01) were not assessed for diagnostic confirmation. Arthritis was confirmed in 169 cases (22 %): 144 with OA, 17 with RA, and 8 with unspecific arthritis (Fig. [1](#Fig1){ref-type="fig"}).
Table [1](#Tab1){ref-type="table"} shows the general characteristics of the population. The mean age of the whole population was 45 years, while for the group of OA and RA, the means were 63 and 55 years, respectively. In total, 61 % of the population was female, whereas 74 % of the hand OA and 76 % of the RA groups were women. The OA population reported twice the average number of comorbidities than the rest of the population. Mean BMI was higher in the arthritis group than in the rest of the population. The smoking prevalence in the whole population was 9 %, while 0 % of the RA population reported this behavior. On average, level of wealth was significantly higher in the non-arthritis group (6 vs. 5, *T* = 3, *n* = 1478, *p* = 0.002).Table 1Participants' general characteristicsTotal^a^\
(*n* = 1479)Arthritis^b^Hand OA^c^\
(*n* = 35)Hip or knee OA^c^\
(*n* = 126)RA\
(*n* = 17)Mean years of age (SD)45 (18)63 (12)63 (13)55 (13)Female (%)904 (61)26 (74)68 (54)13 (76)Mean comorbidities (SD)1 (1.2)2 (1)2 (1)1 (0.4)Mean BMI (SD)28 (5)\
\[missing: 22\]29 (5)29 (5)29 (4)\
\[missing:1\]Smokers (%)139 (9)2 (6)10 (8)0 (0)Mean level of wealth (SD)6 (3)\
\[missing 1\]5 (3)5 (3)5 (2)Disability (%)251 (17)23 (63)60 (48)10 (59)*SD* standard deviation, *BMI* body mass index, *missing* number of participants with missing data, *OA* osteoarthritis, *RA* rheumatoid arthritis^a^Data from participants not assessed for diagnostic confirmation are not included^b^Data from participants diagnosed with non-specific arthritis are not described^c^Cases with combined hand and hip or hand and knee OA were considered in the estimation of both the hand and the hip or knee osteoarthritis parameters
Disability was present in 165/1310 participants without arthritis (13 %) and 86/169 participants with arthritis (51 %). Disability frequencies per arthritis subgroup are shown in Table [1](#Tab1){ref-type="table"}. Having arthritis was significantly associated with having disability after adjusting for gender, age, and number of comorbidities \[prevalence odds ratio (POR) 3.8 (95 % confidence interval (CI) 2.6--5.6); Wald test 5.1, *p* \< 0.0001\].
Table [2](#Tab2){ref-type="table"} shows the frequency of participants' performance of regular repetitive movements during their main occupations. All men with hand OA reported doing repetitive jolting-hand and sit-to-stand movements. Performance of repetitive activities such as lifting, pushing, climbing, standing, kneeling, walking, and sitting-to-standing were reported less often in the RA group than in the non-arthritis group.Table 2Frequency of regular repetitive movements performance during principal occupationArthritisNo arthritis^b^Hand OA^a^Hip or knee OA^a^RAWomen\
(*n* = 26)Men\
(*n* = 9)Women\
(*n* = 68)Men\
(*n* = 58)(*n* = 17)(*n* = 1310)Jolting hands (%)21 (81)9 (100)54 (79)47 (81)14 (82)981 (76)\
\[missing 11\]Lifting 20 kg (%)10 (38)5 (56)30 (44)46 (79)4 (24)777 (60)\
\[missing 11\]Pushing 20 kg (%)11 (42)6 (67)34 (50)45 (78)5 (29)846 (65)\
\[missing 12\]Climbing (%)12 (46)4 (44)36 (53)24 (41)4 (24)635 (49)\
\[missing 12\]Standing \>30 min (%)19 (73)8 (89)60 (88)46 (79)5 (29)1169 (90)\
\[missing 12\]Kneeling \>30 min (%)14 (54)6 (67)42 (61)39 (67)7 (41)786 (61)\
\[missing 12\]Sit-to-stand (%)19 (73)9 (100)57 (83)50 (86)11 (65)1080 (83)\
\[missing 12\]*OA* osteoarthritis, *RA* rheumatoid arthritis, *missing* number of participants with missing data^a^Cases with combined hand and hip or hand and knee OA were considered for both hand and hip or knee OA parameters' estimation^b^ Includes data from participants without musculoskeletal symptoms and participants diagnosed with other than arthritis during the confirmatory stage
Prevalence odds ratios expressing the associations between modifiable risk factors and the presence of arthritis, OA, and RA are presented in Table [3](#Tab3){ref-type="table"}. Body mass index was directly associated with higher arthritis prevalence, while levels of wealth and static cumulative mechanical joint stress were associated with lower arthritis prevalence. Only BMI was significantly associated with a higher prevalence of OA, and only static cumulative mechanical joint stress was significantly associated with a lower prevalence of RA.Table 3Prevalence odds ratios for the associations between selected risk factors and arthritis, OA and RARisk factorsArthritis (95 % CI)OA (95 % CI)^a^RA (95 % CI)^b^BMI1.1 (1.03, 1.1)\*1.01 (1.06, 1.15)\*1.0 (0.9, 1.1)Wealth0.9 (0.8, 0.9)\*1.1 (0.9, 1.1)0.9 (0.7, 1.1)Static mechanical stress0.7 (0.5, 0.9)\*0.1 (0.7, 1.3)0.3 (0.1, 0.6)\*Dynamic mechanical stress1.0 (0.9, 1.2)1.1 (0.9, 1.3)1.0 (0.7, 1.4)Smoking0.7 (0.4, 1.3)0.1 (0.5, 1.9)0.4 (0, 3.0)*BMI* Body mass index, *OA* osteoarthritis, *RA* rheumatoid arthritis^a^Adjusted by age and gender^b^Firth logistic regression and profile penalized likelihood confidence intervals\*Significant at *α* = 0.05
Adjusted by age prevalence odds ratios estimated during the OA subgroup analyses are presented in Table [4](#Tab4){ref-type="table"}. Body mass index was significantly associated with a higher prevalence of lower extremity OA in women and men. Repetitive lifting of ≥20 kg was significantly associated with a lower prevalence of hand OA in men. Finally, repetitive standing for longer than 30 min was significantly associated with a lower prevalence of lower extremity OA in men.Table 4Adjusted prevalence odds ratios for the associations between selected risk factors and hand OA and hip or knee OA in women and menRisk factorsHand OA (95 % CI)^a^Hip or knee OA (95 % CI)^a^WomenMenWomenMenWealth1.0 (0.9, 1.2)0.9 (0.7, 1.3)0.9 (0.9, 1.0)1.0 (0.9, 1.1)BMI1.1 (0.9, 1.1)1.0 (0.8, 1.2)1.1 (1.1, 1.2)\*1.1 (1.1, 1.2)\*Smoking0.70 (0.0, 11.4)1.4 (0.2, 5.7)2.4 (0.2, 16.6)0.9 (0.4, 1.9)Jolting hands1.8 (0.7, 5.7)4.9 (0.6, 609)1.4 (0.7, 2.8)1.3 (0.6, 2.9)Lifting ≥20 kg1.2 (0.3, 5.7)0.1 (0.0, 0.8)\*1.1 (0.4, 2.7)0.8 (0.2, 4.8)Pushing ≥20 kg0.9 (0.2, 3.5)7.5 (0.4, 129)1.0 (0.4, 2.4)1.5 (0.3, 8.5)Climbing1.7 (0.6, 4.7)1.2 (0.3, 5.4)1.5 (0.8, 2.9)1.0 (0.5, 2.0)Standing \>30 min0.4 (0.1, 1.2)1.5 (0.1, 20.2)1.3 (0.5, 3.6)0.3 (0.1,0.7)\*Kneeling \>30 min1.0 (0.4, 2.7)0.6 (0.1, 3.5)0.9 (0.5, 1.9)1.4 (0.6, 3.2)Sit-to-stand0.5 (0.2, 1.4)3.2 (0.3, 447)0.9 (0.4, 2.0)1.0 (0.4, 2.6)Adjusted by age*OA* osteoarthritis, *BMI* Body mass index, *CI* confidence interval^a^Firth logistic regression and profile penalized likelihood confidence intervals\*Significant at α = 0.05
Discussion {#Sec12}
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Principal findings {#Sec13}
------------------
Overall, the presence of arthritis is common in the municipality of Chankom, which aligns with what has been reported in other Mexican \[[@CR31]\] and international reports \[[@CR6]\]. The disability prevalence ratio between the arthritis and non-arthritis populations is 2.8:1, as calculated by Zhang's method \[[@CR32]\]. This means that people living with arthritis in this community are 2.8 times as likely to have disability as the people living without arthritis after controlling for age, gender, and number of comorbidities. Consequently, this group of chronic conditions have important disabling effects in this community, as has been observed in other populations \[[@CR7]\].
The results from the evaluation of associations between modifiable risk factors and the overall prevalence of arthritis and its main types show that this group of chronic diseases are linked with factors that either increase the vulnerability or increase the loading of the joint, as has been previously suggested \[[@CR26]\]. On the one hand, social factors, such as low level of wealth, may have increased joints' vulnerability to be affected by degenerative and/or inflammatory processes. On the other hand, physical and behavioral factors, such as BMI or doing repetitive movements, may have increased the loading within the joints facilitating the manifestation of joint damage.
Being wealthier was associated with less probability of presenting overall arthritis in this community, similar to what has been reported in a population-based study conducted in Brisbane, Australia \[[@CR24]\]. Chankom is a Mexican indigenous community, where the people face health inequities \[[@CR11]\]. These inequities impede the delivery of timely and appropriate care for solving initial MSK problems for all community members, increasing vulnerability to develop arthritis. Having less wealth in Chankom could also be associated with inadequate nutritional intake, which may foster the progression of joint degeneration and/or inflammation.
Factors that increase joint loading, such as BMI, were significantly associated with a higher prevalence of overall arthritis in this community. This association only held for the prevalence of lower extremity OA, which has been consistently reported in other epidemiologic studies \[[@CR33]\]. A person with a BMI of 29 was 1.5 times more likely to present with lower extremity OA than a person with a BMI of 24.
The lack of a significant association observed between BMI and hand OA prevalence does not support the suggested systemic effects of obesity in OA \[[@CR34]\]. Consequently, it is possible that in our analysis BMI acted only as a joint loading factor and not as a systemic factor that increased joint vulnerability through serologic inflammatory markers, as has been proposed in the literature \[[@CR35]\].
Results related to cumulative mechanical joint stress, the other joint-load increasing factor addressed in this study, were inconsistent and conflict with what has been reported in the literature. On the one hand, static cumulative joint mechanical stress was associated with a lower prevalence of overall arthritis. This association only held for the prevalence of RA, and persons in the RA group reported doing regular repetitive climbing, standing, kneeling, and sitting-to-standing less often than people without RA (Table [3](#Tab3){ref-type="table"}). A recent cross-sectional study conducted in Colombia found that people with RA usually performed low levels of physical impact work \[[@CR36]\]. This implies that our findings could be related to a lower engagement in physically demanding activities by the RA group, which can be considered a case of "inversed causality" \[[@CR37]\].
On the other hand, the OA subgroup analyses showed that for men, repetitive hand jolting and sit-to-stand movements approached a significant association with hand OA prevalence. In fact, all men with hand OA reported doing these repetitive activities, supporting the notion that dynamic cumulative mechanical joint stresses were linked to the manifestation of this condition. However, repetitive lifting was significantly associated with a lower prevalence of hand OA and repetitive standing for longer than 30 min was significantly associated with a lower prevalence of low-extremity OA in men; while the latter has been linked with a higher prevalence of hip and knee OA \[[@CR38]\].
The inconsistent findings observed in the cumulative joint mechanical stress analyses could be related to the high frequency with which participants reported doing repetitive movements during their main occupation. More than 60 % of the population reported doing ≥2 static and dynamic cumulative repetitive movements (data not shown). This indicates some homogeneity among the occupations performed by these community inhabitants; usually, men do the same type of agricultural work while women do similar housework activities. This homogeneity makes it difficult to explore differences between groups. Consequently, we cannot conclude anything solid about the role that joint mechanical stress has on the manifestation of arthritis in this population.
Interestingly, smoking behavior was not significantly associated with RA prevalence in this study, which contradicts several reports in the literature \[[@CR25], [@CR27]\]. It has been suggested that only heavy smoking and therefore the dosage, and not just the presence of smoking, is associated with the "seropositive" type of RA \[[@CR39]\]. We could not determine whether serologic markers were present in participants with RA. However, we are sure that none of the participants with confirmed RA in Chankom, where smoking is uncommon, reported this behavior. Considering that the prevalence of RA observed in this community (1 %) aligns with the prevalence reported worldwide \[[@CR27]\], we could argue against the existence of a real association between smoking and the manifestation of RA.
Strengths and limitations {#Sec14}
-------------------------
The main strengths of this study are related to the methods used for screening and defining arthritis cases and the use of locally grounded measurement instruments. The census strategy, involving the majority of adults living in Chankom, allowed us to conduct a comprehensive analysis of the OA and RA problems in this community. The COPCORD methodology we followed has been validated and used with success in detecting MSK diseases, including OA and RA at the community level in Mexico \[[@CR40]\]. This methodology involved a duplicate assessment of cases, including diagnostic confirmation by specialists, which increases our confidence in the validity of the prevalence estimates observed. Finally, the use of a cross-culturally validated instrument, which involved the participation of local people in its development, increased confidence about the local relevance of observed results. For example, people who lived in Chankom decided the properties on which to differentiate levels of wealth among community members, increasing the cultural relevance of the measurements "level of wealth."
The main limitations of this work are related to the cross-sectional design, the measurement of disability, and the measurement of regular repetitive movements performed in the main occupation. The cross-sectional nature of this study precludes us from establishing causal associations between known risk factors and arthritis incidence. For instance, the accuracy of the counterintuitive associations observed between mechanical joint stress and a lower prevalence of hand and low extremity OA in men can only be established through longitudinal data. The cross-sectional design also prevents the further assessment of "non-specific arthritis" cases, limiting the possibility of observing how these cases progress over time and with which type of arthritis (inflammatory or degenerative) they will ultimately be diagnosed. Responding to this design-related limitation, we initiated a longitudinal surveillance of this population and results will be available in the future.
The measurement of disability is complex and it has been suggested that considering only one dimension of physical function, such as what people think they can do from a pre-defined list of activities contained in a questionnaire is not enough to understand the whole disabling effects of an illness \[[@CR41]\]. Therefore, we may not have detected the entire disabling effects of arthritis for people living in Chankom. This is a common limitation in epidemiologic studies of arthritis-related disability. Finally, we did not incorporate a measurement of the actual time (hours, days, months or years) spent on doing the repetitive movements explored in the main occupation. The lack of this temporal component limits our analysis and interpretations about the role that cumulative joint mechanical stress plays in the presentation of arthritis in this community.
Implications for practice and policy {#Sec15}
------------------------------------
Overall, arthritis produces high rates of disability in this indigenous population. From a "social determinants of health" perspective, it seems that the conditions of social disadvantage faced by this rural community result in health inequities that condition the manifestation of arthritis. This social disparity has also been observed in a large Mexican multilevel epidemiological study where the prevalence of OA was clearly associated with higher social underdevelopment \[[@CR42]\] and in a large Latin American study where the rheumatoid arthritis disabling effects were higher in people of low socioeconomic status \[[@CR43]\].
Consequently, there is a need to develop a culturally appropriate community-based rehabilitation intervention directed to prevent the manifestation of arthritis and decrease the associated disabling effects. This intervention should include the early detection and management of RA, as this will potentially improve the functioning outcomes for this population. Appropriate local and regional health policy analyses and strategies need to be undertaken to increase community access to proper health care for people living with chronic MSK diseases in Chankom and other indigenous rural communities. These strategies should also target a reduction of BMI of the adult population as a way to decrease the prevalence of OA in these communities. Finally, our findings support the call for implementing rheumatologic disease prevention and control strategies in Latin American countries \[[@CR44]\].
Implications for research {#Sec16}
-------------------------
The disabling effects of arthritis need to be further assessed by incorporating measurements of other dimensions of physical function such as the execution of standardized tasks or the limitations conditioned by the disease on the performance of real life activities. In addition, it is important to evaluate the presence of modifiable factors linked with the progression of arthritis in this community and how these relate with its disabling effects. The association between social factors, such as the level of wealth, and the prevalence of arthritis in Mayan rural communities, should be further explored using quantitative and qualitative methods. Finally, there is a need for longitudinal studies that explore possible causal associations between the significant factors detected in this study and the prevalence of the various arthritis types, especially for those unexpected and counterintuitive associations (i.e., cumulative mechanical joint stress analyses).
Conclusions {#Sec17}
===========
Overall, arthritis is a common chronic condition in Chankom and an important source of disability. Higher level of wealth was associated with lower arthritis prevalence, while higher body mass index was associated with higher OA prevalence. Action is required to decrease the prevalence and disabling effects of these chronic diseases in this community.
We like to thank Mr. Diego Yeh Cen and Mr. Carlos Castillo Kuyoc for their valuable contributions to the analysis and data acquisition in this study. We also thank research assistants Maria Lizbeth Escudero and Beatriz Quintero, for their great work capturing data and maintaining the database. Finally, we thank Dr. Rudy Coronado Bastarrachea and Radiographer Gabriel Ivan de León Ojeda for supporting the conduction of radiographic studies. This project was funded by the Consejo Nacional de Ciencia y Tecnología \[CONACYT\] (grant \#162154). ALS received funding from CONACYT's doctoral scholarship for studying abroad (scholarship \#209621) and from the Doctoral Vanier Canada Scholarships (\# 268078).
Authors' contributions {#FPar1}
======================
ALS participated in the conception, design, data acquisition, analysis, and drafting of this manuscript. JR and IPB participated in the conception, design, analysis, and drafting of this manuscript. JAN, JL, MW, and SW participated in the design and drafting of this manuscript. All authors read and approved the final version of this manuscript.
| {
"pile_set_name": "PubMed Central"
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All relevant data are within the paper and its Supporting Information files.
Introduction {#sec001}
============
With the rapid aging of the global population, the prevalence of chronic diseases such as diabetes and cardiovascular disease is increasing every year \[[@pone.0151336.ref001]--[@pone.0151336.ref003]\] and has become a worldwide public health problem \[[@pone.0151336.ref002]\]. In 2010, 284.8 million patients suffered with diabetes worldwide in 2010 and this number is predicted to increase to 438.7 million by 2030 \[[@pone.0151336.ref004]\], with the burden of disease being especially heavy in developing countries \[[@pone.0151336.ref005]\]. Additionally, the risk of cardiovascular disease greatly increases with diabetes progression \[[@pone.0151336.ref006]\], patients with diabetes are more likely to present with dyslipidemia than those without \[[@pone.0151336.ref007]\], and approximately 75 to 80% of individuals with diabetes die from cardiovascular disease \[[@pone.0151336.ref008]\]. Although considerable advances have been recently attained in diabetes research, the specific mechanism underlying diabetes has yet to elucidated.
Apolipoprotein E (*APOE*) is a protein that is rich in arginine and originates from very low density lipoprotein (VLDL) in normal individuals \[[@pone.0151336.ref009]\]. *APOE* is related to proteins such as the LDL receptor and VLDL receptor ligands, and is primarily synthesized in the liver and the brain. *APOE* regulates plasma lipoprotein metabolism by modifying the storage and distribution of cholesterol and lipids, and is closely related to lipid metabolism and atherosclerosis \[[@pone.0151336.ref010], [@pone.0151336.ref011]\]. Many studies have demonstrated a correlation between *APOE* genotypes and coronary heart disease and Alzheimer's Disease (AD), especially with the *APOE* ε4 genotype; however, the relationship between *APOE* genotypes and diabetes or blood glucose levels has not been confirmed. Although one study reported that no such association was found \[[@pone.0151336.ref012]\], others have found that *APOE*4 or *APOE*2 are associated with blood glucose level \[[@pone.0151336.ref013]--[@pone.0151336.ref015]\]. Notably, the correlation between the *APOE* gene and cognitive function is influenced by age \[[@pone.0151336.ref016]\]; for example cognitive functioning in young people carrying the *APOE* ε4 alleles is better than that of non-carriers \[[@pone.0151336.ref017]\], but gradually weakens after age 50 \[[@pone.0151336.ref016]\], and after age 65 *APOE* ε4 becomes a risk factor for AD. Age also influences the association between *APOE* genotypes and lipid metabolism \[[@pone.0151336.ref018]\]. Therefore, we supposed that the correlation between APOE genotypes and blood glucose might have been difficult to determine because previous researchers did not consider the effect of age.
To address this issue, we based this study on a sample comprising community dwelling participants aged 60 and above of Han descent and determined the correlation between *APOE* genotypes and fasting plasma glucose and blood lipids in this homogenous cohort.
Materials and Methods {#sec002}
=====================
Study design and participants {#sec003}
-----------------------------
The study was conducted in two neighborhoods in Shanghai, China (one in the Beixinjing area of the Chang Ning District and the other from the Xiangjing area of the Pudong District) from June to September, 2012. Overall, 810 individuals aged 60 and above resided in the Beixinjing neighborhood whereas 1033 lived in the Xiangjing neighborhood, for a total of 1843 elders. Using a random number chart, 660 residents were selected from the Beixinjing site and 758 from the Xiangjing site for a total of 1418 potential participants. Within this group there were 513 residents who had either moved, passed away, or were unreachable, and 348 residents declined to participate. The remaining 555 residents provided informed consent and were included in the original assessment. Within this group, 280 refused and 275 consented to have blood drawn and tested. However, from the latter group, 32 of the blood samples were not suitable for blood glucose and *APOE* tests. Thus, a total of 243 elderly participants completed blood and *APOE* genetic testing and were included in this study ([Fig 1](#pone.0151336.g001){ref-type="fig"}). This study was approved by the ethics committee of the Shanghai Mental Health Center. All participants provided written informed consent.
{#pone.0151336.g001}
The 243 final participants were of Han Chinese descent, and consisted of 96 men (39.5%) and 147 women (60.5%) with ages ranging from 60 to 95 years \[mean age 71.67 (± 8.331)\] and years of education ranging from 0 to 20 \[mean 8.36 (± 4. 693)\]. Of the 312 individuals that did not enter the study, 135 were men (43.3%) and 177 were women (56.7%), with ages ranging from 60 to 97 years \[mean age 72.54 (± 7.983)\], and their years of education ranged from 0 to 21 years \[mean 9.18 (± 4.721)\]. No statistically significant difference (*P* \> 0.05) existed between those that entered the study and those that did not enter the study in gender and age; however, the difference in years of education did reach statistical significance (*P* \< 0.05).
All general participant information was recorded including gender, age, years of education, height, weight, and body mass index (BMI) was calculated. Included participants received cognitive assessment, including a Chinese version of the Mini Mental State Examination (MMSE) \[[@pone.0151336.ref019]\], and battery cognitive assessment \[[@pone.0151336.ref020]\]. Participants with intact activities of daily life and an MMSE score \> 24 were considered as having normal cognitive function. AD and vascular dementia (VD) were diagnosed by two senior psychogeriatrists according to the criteria of the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV) (1994) and The National Institute of Neurological and Communicative Disorders and the Stroke-Alzheimer\'s Disease and Related Disorders Association \[[@pone.0151336.ref021]\]. In addition, the majority of participants with cognitive impairment provided the results of computed tomography or magnetic resonance imaging scans that had previously been performed. Furthermore, comorbidities were recorded through participant report and carefully checked by the senior psychogeriatrists. The diagnosis of diabetes (type 2 diabetes mellitus) was previously administered by endocrinologists according to the criteria from the World Health Organization (WHO 1999) for diabetes diagnosis \[[@pone.0151336.ref022]\]. High blood pressure, coronary heart disease, hyperlipidemia, and other comorbidities were also recorded as diagnosed by previous clinical specialists.
Blood glucose and lipid measurements {#sec004}
------------------------------------
To determine the levels of blood glucose and lipids, all participants fasted for 12 h and the next morning 4 mL of blood was taken intravenously and stored at room temperature for 30 min before being centrifuged at 1710 × *g* for 15 min to extract the plasma. All participants were tested for plasma glucose, triglycerides, cholesterol, and high- and low-density lipoprotein. Plasma glucose \> 6.1 mmol/L was defined as hyperglycemia according to WHO criteria \[[@pone.0151336.ref022]\].
*APOE* genotyping by real time polymerase chain reaction (PCR) {#sec005}
--------------------------------------------------------------
We have developed an *APOE* genotyping method based on allele-specific PCR methodology adapted to real time PCR using a TaqMan probe \[[@pone.0151336.ref023]\]. Briefly, the *APOE* genotyping assay includes three reactions that detect the alleles of *APOE* ε2, ε3, and ε4, respectively. Each PCR reaction mixture (20 μL) contained the following reagents: 1×AccuPower Plus DualStar TM qPCR PreMix (Bioneer, Daejeon, Korea, K-6603), *APOE* primers, and an *APOE* TaqMan probe (FAM labeled), 20% glycerol, and 20 ng genomic DNA. Positive control DNA template (ε2, ε3, and ε4, plasmid DNA) and negative controls (DNA/RNA-free water) were included in each genotyping panel. The PCR amplification protocol was as follows: initial pre-denaturation at 95°C for 5 min, followed by 40 cycles with denaturation at 95°C for 10 s and annealing/extension at 58°C for 1 min. The fluorescence signals were collected during the annealing/extension step, with FAM signal indicating the *APOE* alleles. The amplification was performed using the Applied Biosystems 7500 Fast Real-Time PCR System. All samples were repeated at least twice and the assays were performed by two investigators.
Data analysis {#sec006}
-------------
The Statistical Package for Social Science (SPSS) version 19.0 (SPSS IBM, Inc., Chicago, IL, USA) was used for analysis and processing of the data. *APOE* allele frequencies were calculated using a Hardy-Weinberg equilibrium *χ*^*2*^ test for goodness of fit. Continuous data (age, years of education, fasting plasma glucose, triglycerides, cholesterol, high-density lipoprotein, low-density lipoprotein, and BMI) are showed as the means ± standard deviation, Categorical data (gender, AD diagnosis, comorbidity with diabetes, high blood pressure, coronary heart disease, hyperlipidemia, and fasting plasma glucose \>6.1 mmol/L) are expressed as percentages. The *APOE* genotypes were divided into three groups for comparison: the *APOE* ε2 group (ε2/ε2 and ε2/ε3), *APOE* ε3 group (ε3/ε3); and *APOE* ε4 group (ε2/ε4, ε3/ε4, and ε4/ε4). Continuous data was compared among the three group by one-way analysis of variance and multicomparison of fasting plasma glucose was performed using Tamhane's T2 method, whereas categorical data was compared using a *χ*^*2*^ test. *APOE2*, *APOE3*, and *APOE4* carrier and non-carrier status was converted into a dichotomous variable and then their correlation with plasma glucose, lipid, and BMI was tested using Spearman's correlation analysis. The significance level was set at *P* \< 0.05.
Results {#sec007}
=======
*APOE* genotype distribution among community-dwelling elderly {#sec008}
-------------------------------------------------------------
We identified a total of 6 *APOE* genotypes, among which 1 subject (0.4%) carried ε2/ε2, 32 (13.2%) were ε2/ε3, 2 (0.8%) were ε2/ε4, 171 (70.4%) carried ε3/ε3, 35 (14.4%) were ε3/ε4, and 2 (0.8%) carried ε4/ε4 ([Fig 2](#pone.0151336.g002){ref-type="fig"}). A Hardy Weinberg goodness of fit test, *χ*^*2*^ = 0.59, *df* = 3, *P* \> 0.05 showed that this cohort exhibited Hardy Weinberg equilibrium. The allele frequencies were as follows: ε2, 36 (7.4%), ε3, 409 (84.2%), and ε4, 41 (8.4%).
{#pone.0151336.g002}
Comparison of *APOE* genotypes with subject characteristics {#sec009}
-----------------------------------------------------------
The difference between fasting plasma glucose in subjects with different *APOE* genotypes was statistically significant (*P* \< 0.05), and the fasting glucose level over 6.1 mmol/L almost reached statistical significance (*P* = 0.068). No statistically significant difference was identified between the three groups in gender, age, years of education, presence of AD, high blood pressure, coronary heart disease, diabetes, triglycerides, cholesterol, high-density lipoprotein, low density lipoprotein, or BMI ([Table 1](#pone.0151336.t001){ref-type="table"}).
10.1371/journal.pone.0151336.t001
###### Comparison of *APOE* types with patient characteristics.
{#pone.0151336.t001g}
Item *APOE* ε2 (n = 33) *APOE* ε3 (n = 171) *APOE* ε4 (n = 39) *x*^*2*^*/F* *P*-value
----------------------------------- -------------------- --------------------- -------------------- -------------- -----------
Male (%) 11 (33.3%) 69 (40.4%) 16 (41.0%) 0.62 0.735
Mean age (±SD) 71.91 ± 8.10 71.99 ± 8.60 70.03 ± 7.27 0.90 0.407
Mean years of education (±SD) 7.67 ± 4.81 8.20 ± 4.79 9.67 ± 3.98 1.99 0.139
Alzheimer's disease (%) 1 (3.0%) 12 (7.0%) 4 (10.3%) 1.33 0.528
High blood pressure (%) 20 (60.6%) 89 (52.0%) 22 (56.4%) 0.93 0.627
Coronary heart disease (%) 4 (12.1%) 21 (12.3%) 4 (10.3%) 0.12 1.000
Hyperlipidemia (%) 11 (33.3%) 43 (25.1%) 10 (25.6%) 0.97 0.617
Diabetes (%) 4 (12.1%) 35 (20.5%) 4 (10.3%) 3.09 0.213
Plasma glucose (mmol/L) 5.18 ± 1.10 5.86 ± 2.23 5.17 ± 1.12 3.09 0.047
Plasma glucose \>6.1 mmol/L (%) 6 (15.2%) 53 (31.0%) 8 (17.9%) 5.38 0.068
Triglycerides (mmol/L) 1.89 ± 0.98 1.82 ± 1.30 1.90 ± 1.89 0.07 0.932
Cholesterol (mmol/L) 4.89 ± 1.10 4.90 ± 1.15 4.96±0.95 0.06 0.945
High-density lipoprotein (mmol/L) 1.22 ± 0.34 1.18 ± 0.28 1.12±0.23 1.11 0.330
Low-density lipoprotein (mmol/L) 2.72 ± 0.85 2.95 ± 0.94 3.07±0.73 1.44 0.240
BMI (Kg/m^2^) (±SD) 24.58 ± 3.41 24.11 ± 3.36 23.77±3.08 0.53 0.587
*APOE* genotype groups consist of *APOE* ε2 (ε2/ε2 and ε2/ε3); *APOE* ε3 (ε3/ε3); and *APOE* ε4 (ε2/ε4, ε3/ε4, and ε4/ε4). SD = standard deviation; BMI = body mass index.
Comparison of plasma glucose among different *APOE* carriers {#sec010}
------------------------------------------------------------
In comparison with study participants carrying *APOE* ε4 and *APOE* ε2 genotypes, the fasting glucose levels in those with *APOE* ε3 were significantly higher (*P* \< 0.05), whereas no significant difference was identified between *APOE* ε2 compared to *APOE* ε4 participants (*P* \> 0.05) ([Fig 3A](#pone.0151336.g003){ref-type="fig"}). Fasting plasma glucose levels ≤ 6.1 mmol/L are considered normal, whereas higher levels are considered abnormal. Comparisons between genotype groups showed that *APOE* ε3 carriers exhibited abnormal fasting plasma glucose compared to *APOE* ε2 carriers to a degree approaching statistical significance (*P* = 0.065);. differences between the other two groups were not statistically significant (*P* \> 0.05) ([Fig 3B](#pone.0151336.g003){ref-type="fig"}).
{#pone.0151336.g003}
Correlation between *APOE* alleles and subject characteristics {#sec011}
--------------------------------------------------------------
Carriers of the ε2 allele were defined as *APOE*2 carriers with a variable of 1; non-carriers were given a variable of 0. Those having the ε3/ε3 genotype were defined as *APOE*3 carriers with a variable of 1, those carrying other genotypes were defined as non-*APO*E3 carriers with a variable of 0. Carriers of the ε4 allele were defined as *APOE*4 carriers with a variable of 1, non-carriers were assigned a variable of 0. These three types of carriers and non-carriers were converted into a dichotomous variable and Spearman correlational analysis was performed for plasma glucose, blood lipid, and BMI ([Table 2](#pone.0151336.t002){ref-type="table"}). The presence of *APOE*3 was significantly correlated with abnormal plasma glucose levels (*P* \< 0.05) and nearly reached statistical significance (*P* = 0.082) as related to a history of diabetes. In addition, *APOE*2 had a close association with lower levels of low-density lipoprotein (*P* = 0.052). However, *APOE* carrier type was not associated with any other type of lipid level or with a diagnosis of AD (*P* \> 0.05).
10.1371/journal.pone.0151336.t002
###### Correlation of Elderly Community-dwelling Han Ethnicity *APOE* Carrier Status with Subject Characteristics (*r*).
{#pone.0151336.t002g}
Item *APOE*2 *APOE*3 *APOE*4
-------------------------- --------- ---------- ---------
Fasting plasma glucose
***r*** −0.077 0.128 −0.101
***P*** 0.235 0.047 0.115
Glucose \>6.1 mmol/L
***r*** −0.115 0.148 −0.087
***P*** 0.072 0.021 0.177
Presence of diabetes
***r*** −0.067 0.112 −0.085
***P*** 0.296 0.082 0.185
Presence of AD
***R*** −0.067 0.001 0.056
***P*** 0.301 0.984 0.386
Triglycerides
***r*** 0.074 −0.045 −0.014
***P*** 0.249 0.488 0.831
Cholesterol
***r*** −0.039 −0.019 0.035
***P*** 0.542 0.768 0.591
High-density lipoprotein
***r*** 0.019 0.025 −0.072
***P*** 0.768 0.699 0.263
Low-density lipoprotein
***r*** −0.125 0.009 0.085
***P*** 0.052 0.890 0.189
BMI
***r*** 0.060 \- 0.011 −0.037
***P*** 0.353 0.863 0.563
AD = Alzheimer's disease; BMI = body mass index.
Discussion {#sec012}
==========
There are 6 kinds of common human *APOE* genotypes made up of the 3 *APOE* alleles (ε2, ε3, ε4): ε2/ε2, ε2/ε3, ε3/ε3, ε2/ε4, ε3/ε4, and ε4/ε4. Of these, ε3/ε3 is the most common with a greater than 60% frequency, followed by ε2/ε3 and ε3/ε4; all of these contain the ε3 allele \[[@pone.0151336.ref024]\], which accordingly exhibits the highest frequency distribution \[[@pone.0151336.ref025]\]. However, the frequency distributions of the *APOE* alleles differ among various groups and races \[[@pone.0151336.ref026]\], with the frequency of the ε4 allele in Asians being lower (7.4% in both China and Japan) and in European higher (18.6% in both Finnish and Hungarian) \[[@pone.0151336.ref024]\]. The two areas of Asia from which the research samples in this study were chosen cover the east and west areas of downtown Shanghai, representing the newer and older districts of the city, respectively. Our research shows that the ε3/ε3 frequency was highest among the *APOE* genotypes detected in this Shanghai-based Han population and the frequency of the ε3 allele was also the highest, similar to that seen in other Asian groups. The *APOE* genotype and allele frequency distributions were found to be in accordance with Hardy-Weinberg equilibrium after examination using the *χ*^2^ test, which indicated the general representativeness of the sample.
Previous research has shown that people with diabetes are more likely to present with dyslipidemia than those without diabetes \[[@pone.0151336.ref007]\], and that the *APOE* gene is associated with lipid metabolism and heart disease \[[@pone.0151336.ref010], [@pone.0151336.ref011], [@pone.0151336.ref027]\]. Therefore, it was speculated that the *APOE* gene might also exhibit a certain relevance to diabetes. However, no agreement has been reached regarding the relationship between *APOE* gene variation and blood glucose level. Research focusing on the relationship between the ε4 allele and plasma glucose \[[@pone.0151336.ref013]\] has suggested that this allele was related to diabetes with or without the presence of coronary heart disease; however, other studies have found no correlation between the *APOE* gene and blood glucose \[[@pone.0151336.ref012]\]. Our research first proposed that fasting plasma glucose was higher in the elderly population carrying the *APOE* ε4 allele and that the incidence of diabetes by subjective report in this group was also higher. Notably, our study sample constituted community dwelling elders aged 60 and above; however, previous studies did not include participants in this age group, with an average age of about 50 years old. This difference in age, which has been shown to impact the influence of the *APOE* gene on cognitive function and lipid metabolism \[[@pone.0151336.ref016], [@pone.0151336.ref018]\], might therefore be one of the reasons explaining the difference between our results and those of other studies. In addition, Scuteri et al. \[[@pone.0151336.ref028]\] conducted long-term follow-up research and discovered that fasting plasma glucose increased with the increase of age for elders carrying *APOE*4+. At baseline, the blood glucose levels were higher in the *APOE*4+ carriers than in the APOE4− carriers, which is not consistent with our results. This inconsistency might be related to race, as the cohort studied by Scuteri consisted primarily of Caucasians, wherein the frequency of the ε4 allele reached 25.5%. In contrast, our subjects were all of Han ethnicity from Asia with an ε4 allele frequency of only 8.4%. On the other hand, one recently published review of Asian populations showed that the ε3 allele was likely related to coronary heart disease \[[@pone.0151336.ref029]\], indirectly demonstrating that *APOE* ε3/ε3 was a probable risk genotype for glycolipid metabolic disorder in Asian populations. Furthermore, Sapkota et al. \[[@pone.0151336.ref030]\] investigated an Asian sample and showed that ε2 and ε4-containing genotypes had protective OR of 0.64 in diabetes when compared to the ε3 genotype, which was also consistent with our results. Findings with respect to ε2 have, however, been controversial, as other research has suggested that the ε2 allele is associated with blood glucose and that this allele might instead increase the risk of diabetes \[[@pone.0151336.ref014], [@pone.0151336.ref015]\], which is not consistent with our findings. These differences might also be related to the age or race of the groups used in these studies. Our results support the assertion that *APOE*2 and *APOE*4 are protective factors for diabetes in Asian populations, in concordance with Sapkota's results; however further research with larger sample sizes is needed to confirm this hypothesis.
Our correlation analyses also showed that ε2 carriers had relatively lower levels of LDLs. APOE is one of the main apolipoproteins in the blood and is related to lipid metabolism abnormalities \[[@pone.0151336.ref031]\]. Other research has also suggested that ε2 allele could reduce the levels of low density lipoprotein \[[@pone.0151336.ref032]\] and that ε4 could increase these levels \[[@pone.0151336.ref033]\]. Notably, lower levels of low density lipoprotein are associated with a lower incidence of coronary heart disease \[[@pone.0151336.ref034]\]. Although our research showed no difference among the carriers of these three alleles with respect to the incidence of coronary heart disease, it is important to keep in mind that the diagnosis of coronary heart disease in these cases was based on subjective report.
It is known that *APOE* ε4 is a risk factor for the development of AD \[[@pone.0151336.ref035]\] whereas *APOE* ε2 has a preventive and protective function \[[@pone.0151336.ref036]\]. Consistent with this, in our study, the results showed that subjects carrying *APOE* ε4 had the highest incidence of AD and that those carrying *APOE* ε2 had the lowest. However, an important note is that the diagnosis of AD was based on clinical interview data obtained from participants by psychiatrists. No further diagnosis was made, so it is therefore possible that other types of dementia such as front temporal lobe dementia and Lewy Body dementia were not fully identified.
This study has several limitations: First, the sample was consisted only of participants of Han ethnicity from the Shanghai area and is not representative of all Han Chinese. Furthermore, the disease history was based on the self-report of community dwelling elderly participants, and therefore included a certain amount of bias. In addition, we lacked other adult samples that could be compared with this elderly sample. These factors need to be improved in future research in order to clarify the impact of *APOE* genotypes on different groups of aged individuals.
Conclusions {#sec013}
===========
In conclusion, our research is the first study to report that elders of Han ethnicity with the ε3/ε3 genotype are more likely to suffer from diabetes, and that the *APOE* ε2 allele is a possible protective factor for diabetes and blood lipids. This might serve as a new approach to studying the effect of *APOE* on glycolipid metabolism in persons of Han ethnicity and possibly other Asian ethnicities as well.
Supporting Information {#sec014}
======================
######
(SAV)
######
Click here for additional data file.
The authors wish to thank Sheng-yu Zhang, Jing Dai, and Yan Cheng for their help with the psychological assessment, and Mr. Tong Lian for his excellent coordination work. The authors are also grateful to Mr. Andrew Fralick for his valuable comments and language editing.
[^1]: **Competing Interests:**The authors have declared that no competing interests exist.
[^2]: Conceived and designed the experiments: XL SFX. Performed the experiments: LZ TW MJZ JHW ZLZ ZW NS YYL YCS. Analyzed the data: CXB XL. Contributed reagents/materials/analysis tools: LZ ZLZ ZW SFX XL. Wrote the paper: CXB LZ XL.
[^3]: ‡ These authors are co-first authors on this work.
| {
"pile_set_name": "PubMed Central"
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According to the WHO report *Global atlas on cardiovascular disease prevention and control*, cardiovascular diseases are the leading causes of death and disability in the world. Heart failure is a very common condition that is costly, disabling, and potentially deadly. In developed countries, around 2% of adults suffer from heart failure, but in those over the age of 65, this increases to 6--10% (Dickstein et al., [@B15]). Conditions that damage or overwork the heart muscle can cause heart failure. The most common causes of heart failure are coronary heart disease (CHD), high blood pressure, obesity, and diabetes (He et al., [@B17]). Treating these problems can prevent or improve heart failure. Drugs acting on the renin--angiotensin system (RAS), such as angiotensin converting enzyme (ACE) inhibitors or angiotensin receptor blockers are first-line therapy for all heart failure patients. Since the RAS has both endocrine and local tissue components, RAS drugs have been developed to attain increased tissue penetrability and volume of distribution and consequently an efficient inhibition/blockage of both RAS components. Of the tissue systems, the brain RAS is of particular interest for us. Accumulating evidence indicates that angiotensins produced locally in various brain nuclei involved in homeostasis control mainly in the hypothalamus and brain stem interact with several neurotransmitter systems to regulate cardiovascular and fluid--electrolyte homeostasis, their biology and mechanisms of action representing an active area of actual research interests (Baltatu et al., [@B6]; Diz et al., [@B16]). The brain RAS is physically separated from the endocrine one by the presence of the blood--brain barrier which hampers the penetration of angiotensin II (Ang II) from blood into the brain. Exceptions are some areas lacking the blood--brain barrier through which circulating Ang II can transmit its effects inside the brain. The brain RAS is involved in the modulation of cardiovascular and fluid--electrolyte homeostasis, complementing the classical roles of the endocrine RAS. Several lines of evidence demonstrate that chronic over activation of the brain RAS is responsible for the development and maintenance of hypertension in several animal models of disease (Baltatu et al., [@B6]; Diz et al., [@B16]).
To elucidate a contributory role of the brain RAS and its significance in diverse pathophysiological processes we developed a transgenic rat \[TGR(ASrAOGEN)\] to inhibit the production of angiotensinogen (AOGEN) specifically in the brain (Schinke et al., [@B22]). The brain levels of AOGEN in TGR(ASrAOGEN) rats are 90% low due to an antisense RNA expressed against AOGEN, induced by means of the astrocyte-specific glial fibrillary acidic protein promoter. As a consequence, these rats have low blood pressure and a diabetes insipidus-like syndrome with altered central vasopressinergic system (Campos et al., [@B8]).
The TGR(ASrAOGEN) rats were investigated in experimental conditions of hypertension and heart pathology. We demonstrated that the brain RAS significantly contributes to the development of hypertension in a transgenic model with overactive tissue RAS by crossbreeding the TGR(ASrAOGEN) rats with the hypertensive TGR(mREN2)27 strain (Schinke et al., [@B22]). We were further interested to investigate whether the brain RAS is participating in the development of hypertensive pathology in experimental reno-vascular hypertension. Ang II when infused at doses of 50--250 ng/kg/min subcutaneously, which do not produce direct vasoconstriction are described as "subpressor" or "slow-pressor" and can induce a gradual increase of blood pressure in days to weeks. It represents a model of reno-vascular hypertension with low plasma renin activity (PRA). The subpressor Ang II-induced hypertension at TGR(ASrAOGEN) rats was significantly attenuated supporting the importance of the brain RAS in this process (Baltatu et al., [@B5]). Furthermore, the Ang II-induced cardiac hypertrophy and fibrosis was considerably attenuated as well. This attenuation of the cardiac phenotype in this model of experimental hypertension might not only be due to the reduction of blood pressure but also because of a disturbed autonomic nervous system.
Several authors presented findings indicating that Ang II interacts with the autonomic nervous system at several levels, namely at postganglionic nerve terminals, sympathetic ganglia, and within the central nervous system (reviewed in Bader et al., [@B1]). Ang II signaling in the brain modulates reflex control of heart rate and sympathetic outflow (Dampney et al., [@B14]; Carlson and Wyss, [@B12]). Studies from our group provide evidence that a permanent inhibition of brain RAS may cause a decreased basal sympathetic outflow, as it has been observed in TGR(ASrAOGEN) rats (Campos et al., [@B9]; Parrish et al., [@B21]). Power spectral analysis of heart rate and blood pressure indicates an altered autonomic nervous system activity with a dominance of vagal tone over sympathetic tone and exaggerated spontaneous baroreflex sensitivity (Baltatu et al., [@B4]). As a consequence to a diminished basal activity of sympathetic nervous system, the hearts of TGR(ASrAOGEN) exhibit an increased sensitivity to β-receptor agonists (Campos et al., [@B9]; Parrish et al., [@B21]).
The brain RAS -- sympathetic nervous system connection is important in the development of left ventricular dysfunction after myocardial infarction (Huang and Leenen, [@B18]). Sympathetic activity surges in parallel with the progress of damage in cardiac performance, contributing to the progression of the heart failure (Packer, [@B20]). TGR(ASrAOGEN) rats exhibit marked attenuation of both sympathetic hyperactivity and of left ventricular remodeling and dysfunction after myocardial infarction (Wang et al., [@B23]). As a consequence of a lower post-infarct sympathetic activation in the TGR(ASrAOGEN) rats, there was a blunted increase in tyrosine hydroxylase (TH; the rate-limiting enzyme in norepinephrine synthesis; Parrish et al., [@B21]). Lower cardiac TH levels may represent a mechanism contributing to the lower pathological elevation of norepinephrine.
Not only does peripheral Ang II interact with the brain RAS to activate the sympathetic nervous system in chronically overactive conditions, but also the brain RAS appear to influence renal renin release. The sympathetic nervous system, which is involved in the fight-or-flight reaction, is one of the main stimulators of renal renin release. During the stress reaction, there is an increase in blood levels of renin in addition to epinephrine. We provided evidence that brain RAS is an important player involved in the stress-induced renin release. The TGR(ASrAOGEN) rats with low brain AOGEN exposed to stress have elevations of only 50% on PRA levels (marker of both renal renin release and of the reactivity to different types of stressors) from those of the stressed control rats (Baltatu et al., [@B3]). Therefore, inhibition of the brain RAS may represent a protective strategy against chronic and generalized activation of both endocrine and local systems in disease.
Brain RAS modulates the cardiovascular and fluid--electrolyte homeostasis not only by interacting with the autonomic nervous system but also by modulating hypothalamic--pituitary axis and vasopressin release (Baltatu et al., [@B3]). Since the angiotensinogen-deficient TGR(ASrAOGEN) rats have lower plasma levels of vasopressin and an altered central vasopressinergic system (Schinke et al., [@B22]; Campos et al., [@B8]), we considered that this might be also one mechanism contributing to the attenuated Ang II-induced hypertension and cardiac hypertrophy. Therefore, we studied the Ang II-induced hypertension in vasopressin-deficient Brattleboro rats, as well. Basal systolic arterial pressure (SAP) monitored by telemetry was significantly lower in Brattleboro rats as in TGR(ASrAOGEN) in comparison with the respective parent strain Long--Evans (LE) rats (119.4 ± 1.1 vs. 129.4 ± 1.4 mmHg, *p* \< 0.001, respectively) or Sprague-Dawley (SD; 122.5 ± 1.5 vs. 128.9 ± 1.9 mmHg, *p* \< 0.05, respectively). The increase in SAP induced by 7 days of chronic Ang II infusion (100 ng/kg/min) in Brattleboro rats was similar to the LE rats (Figure [1](#F1){ref-type="fig"}; delta SAP: 25.1 ± 4.8 vs. 18.2 ± 7.7 mmHg, *p* \> 0.05, respectively), in contrast to the significantly attenuated hypertension in TGR(ASrAOGEN; delta SAP: 29.8 ± 4.2 vs. 46.3 ± 2.5 mmHg, *p* \< 0.005 vs. control SD rats). Heart hypertrophy measured as heart weight per body weight was evident neither in LE nor in Brattleboro rats, probably due to the mild hypertension levels. These results together with our previous results (Baltatu et al., [@B5]) indicate that the brain RAS but not vasopressin system plays an important role in mediating the hypertension induced by slow-pressor doses of Ang II.
![**(A)** Effect of Ang II infusion (100 ng/kg/min s.c.) on systolic arterial pressure (SAP). Data are extracted from telemetry recording and data acquisition as day mean. The start of Ang II infusion is at the end of day 0. LE indicates Long--Evans rats (*n* = 6); Brattleboro rats (*n* = 6). **(B)**. Increase of systolic BP after Ang II infusion calculated by subtracting the 2-day mean basal values from the mean values of the last 2 days at the end of Ang II infusion \[mean day (5, 6) -- mean day (−1, 0)\] (Long--Evans rats, *n* = 6; Brattleboro rats, *n* = 6). Data are expressed as mean ± SE.](fphys-02-00115-g001){#F1}
Circadian variability of blood pressure may constitute another mechanism through which brain RAS may participate to the hypertensive target organ injury and cardiovascular events. According to the Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure (JNC7), ambulatory blood pressure monitoring is warranted for evaluation of "white-coat" hypertension in the absence of target organ injury (Chobanian et al., [@B13]). It is also helpful to assess patients with apparent drug resistance, hypotensive symptoms with antihypertensive medications, episodic hypertension, and autonomic dysfunction. In most individuals, BP decreases by 10--20% during the night; those in whom such reductions are not present are at higher risk for cardiovascular events. A "non-dipping" blood pressure (reversed nighttime BP dipping pattern) was associated with a 2.2-fold higher incidence of heart failure (Ingelsson et al., [@B19]). Our group was the first to determine that slow-pressor Ang II infusion may induce a shift in BP circadian rhythm, making it a convenient and reproducible experimental model of Ang II-induced non-dipping hypertension (Baltatu et al., [@B4]). These alterations in BP circadian rhythm are not synchronized with alterations of heart rate or locomotor activity, contributing to the concept that the circadian variability of blood pressure and heart rate are differentially regulated (Baltatu et al., [@B4]; Campos et al., [@B10]). The brain RAS appears to be also an important modulator of the circadian BP rhythm (Baltatu et al., [@B4]; Campos et al., [@B10]). Ang II not only induces alterations in the circadian rhythm of BP but also its local production in the brain modulates the synthesis of both melatonin and vasopressin, which are important hormones regulating circadian rhythms (Baltatu et al., [@B2]; Campos et al., [@B7], [@B8]). Furthermore, melatonin itself may alter the short-term variability of cardiovascular parameters, including the baroreflex (Campos-Santos et al., [@B11]). With these studies we demonstrated that the brain RAS is an important regulator of circadian rhythm of blood pressure. The experimental model of Ang II-induced hypertension might be useful to further dissect the pathophysiology and molecular biology of non-dipping hypertension.
In summary, several lines of evidence indicate that the brain RAS is involved in cardiovascular diseases including heart failure contributing to pathophysiological alterations (Figure [2](#F2){ref-type="fig"}). Chronically overactive RAS is thus recruiting tissue RASs through a positive biofeedback in order to maintain a state of alert diseased conditions. A circadian pattern becomes quite obvious in the occurrence of acute cardiovascular diseases, such as ischemia, infarction, stroke, and sudden death, and new chronotherapeutic approaches in antihypertensive therapy are trying to exploit the knowledge of circadian rhythms in order to reduce these events. Therefore, targeting brain RAS with drugs such as renin or ACE inhibitors or receptor blockers having increased brain penetrability could be of benefit. These RAS-targeting drugs are first-line therapy for all heart failure patients. Since the RAS has both endocrine and local tissue components, RAS drugs are being developed to attain increased tissue penetrability and volume of distribution and consequently an efficient inhibition of both RAS components.
{#F2}
Conflict of Interest Statement
==============================
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The work of the authors was supported by the Deutsche Forschun gsgemeinschaft (BA1374/13-2, BA1374/16-1, and BA1374/20-1).
[^1]: Edited by: Jacqueline Kathleen Phillips, Macquarie University, Australia
[^2]: Reviewed by: Robert A. Augustyniak, Oakland University William Beaumont School of Medicine, USA; Michael McKinley, Florey Neuroscience Institutes, Australia
[^3]: This article was submitted to Frontiers in Integrative Physiology, a specialty of Frontiers in Physiology.
| {
"pile_set_name": "PubMed Central"
} |
Introduction
============
Alzheimer disease (AD) is a late onset neurodegenerative disorder characterized by progressive neuronal loss, especially in the cortex and the hippocampus ([@b30]). The vast majority of AD is sporadic (SAD), but mutations in the amyloid precursor protein (APP) and in presenilin-1 (PS1) and -2 (PS2), which are components of the γ-secretase complex that processes APP to produce amyloid-β (Aβ), have been identified in the familial form (FAD), which is similar to SAD but has an earlier age of onset. To date, there is no unifying hypothesis that can explain the diverse and apparently unrelated morphological and biochemical abnormalities---extracellular plaques containing Aβ fibrils ([@b71]), intracellular tangles containing hyperphosphorylated forms of the microtubule-associated protein tau ([@b71]), elevated serum cholesterol ([@b86]), altered phospholipid metabolism ([@b103]; [@b70]), aberrant calcium homeostasis ([@b5]), and mitochondrial dysfunction ([@b102])---detected in tissues and in cultured cells from AD patients ([@b71]), with no apparent direct link among them.
In the case of FAD, investigations into a common source of these apparently unrelated features have been complicated by uncertainty regarding the subcellular distribution of the presenilins. PS1 and PS2 have been located to numerous subcellular compartments, including endoplasmic reticulum (ER) ([@b2]), Golgi ([@b2]), plasma membrane (PM) ([@b57]), nuclear envelope ([@b46]), endosomes ([@b99]), lysosomes ([@b68]), and mitochondria ([@b1]). On the other hand, it is generally accepted that APP ([@b48]), presenilins, Aβ, and γ-secretase activity are enriched in lipid rafts (LRs) ([@b94]; [@b98]), which are specialized domains rich in cholesterol and sphingolipids that form detergent-insoluble aggregates in cell membranes (i.e., detergent-resistant membranes, or DRMs) ([@b84]). These regions have a liquid-ordered structure with unique biophysical characteristics that differs from the rest of the cell\'s liquid-disordered membranes ([@b84]). Traditionally, LRs have been considered to be present only in the PM ([@b84]). However, γ-secretase activity is negligible in this compartment, forming the basis of what has been called 'the spatial paradox\' ([@b18]). Recent evidence, however, has in fact indicated the existence of intracellular LRs/DRMs that are different in protein composition from those in the PM ([@b6]).
We recently found that PS1, PS2, APP, and γ-secretase activity are not homogeneously distributed in the ER, but rather are enriched in mitochondria-associated ER membranes (ER-MAMs or MAMs) ([@b3]). MAM is a dynamic subcompartment of the ER connected physically and biochemically to mitochondria that is involved in a number of key metabolic functions ([@b36]), including cholesterol metabolism ([@b78]), the synthesis and transfer of phospholipids between the ER and mitochondria ([@b96]), and calcium homeostasis ([@b17]).
We now report that MAM is a complex elaboration of the ER with the characteristics of an LR. Moreover, using a number of relevant tissues and cell lines ([Supplementary Table S1](#S1){ref-type="supplementary-material"}), we show that mutations in presenilins perturb MAM function significantly, and that these perturbations are also present in cells from FAD patients with mutations in PS1, PS2, and APP, and in SAD patients with no known genetic aetiology. These findings may shed new light on the biology of presenilins and on our understanding of some of the features associated with the pathogenesis of AD.
Results
=======
*MAM displays the characteristics of an intracellular* LR
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MAM is a dynamic domain of the ER responsible for the integration of several cellular functions, including Ca^2+^ signalling, lipid transport, energy metabolism, and cellular survival. For this reason, we speculated that MAM might have the characteristics of an LR/DRM in order for it to recruit and orientate the different signalling proteins needed for cellular homeostasis and for the effective cross-talk between mitochondria and ER ([@b35]; [@b109]; [@b26]). In addition, the fact that γ-secretase activity is present in LRs ([@b98]) and is also enriched in MAM ([@b3]) provided indirect support for the idea that MAM could be an LR/DRM.
We therefore incubated purified MAM from mouse tissues ([@b3]; [Supplementary Figure S1](#S1){ref-type="supplementary-material"}) with and without Triton X-100 (TX100), and loaded both samples onto a Percoll gradient under the same conditions used for its initial isolation. The TX100-treated MAM sample was fundamentally intact and migrated to the identical position in the gradient as did the untreated sample, consistent with the behaviour of a DRM ([Figure 1A](#f1){ref-type="fig"}). To separate LR from other cell contents, we loaded TX100-treated and untreated control MAM from mouse brain onto a sucrose gradient, and analysed fractions for the known MAM markers Pemt (phosphatidylethanolamine *N*-methyltransferase; [@b95]), Vdac1 (voltage-dependent anion channel 1; [@b36]), and Ps1 ([@b3]; [Figure 1B](#f1){ref-type="fig"}). The proteins migrated at similar positions in the lower density fractions, and were unaffected by detergent treatment ([Figure 1B](#f1){ref-type="fig"}), consistent with the behaviour of MAM as a DRM. By contrast, purified mitochondria and bulk ER from the bottom of the gradient behaved like detergent-soluble fractions ([Supplementary Figure S2](#S1){ref-type="supplementary-material"}), indicating the absence of DRMs in these organelles, as expected ([@b108]). MAM was not contaminated with PM rafts, as Src, a marker for PM rafts ([@b62]), was observable in sucrose gradient fractions from purified PM, but not from the crude mitochondrial fraction from which the MAM fraction was derived ([Figure 1C](#f1){ref-type="fig"}). Moreover, the cholesterol content of MAM was higher than that found in the cytoplasm, mitochondria, bulk ER, and total PM, and was comparable to that of LR from PM ([@b84]; [Figure 2A](#f2){ref-type="fig"}). These results are consistent with the numerous reports showing that presenilins and γ-secretase activity reside in LRs and suggest that MAM is an intracellular LR-like domain that may recruit and orient various signalling proteins needed to regulate cross-talk between ER and mitochondria.
Elevated cholesteryl ester synthesis in PS-mutant cells and AD patient fibroblasts
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Given that MAM is an LR/DRM, the regulation of cholesterol metabolism should be an important determinant of its structure and function. Acyl-CoA:cholesterol acyltransferase (ACAT), which catalyses the conversion of free cholesterol to cholesteryl esters (CE) and controls the equilibrium between membrane-bound free cholesterol and CE stored in cytoplasmic lipid droplets ([@b75]), is enriched in MAM ([@b78]). We confirmed that MAM is indeed the predominant locus for CE synthesis, as ACAT protein was not only more abundant in MAM as compared to bulk ER and mitochondria ([Figure 2B](#f2){ref-type="fig"}, inset), but also had an ∼3-fold higher enzymatic activity ([Figure 2B](#f2){ref-type="fig"}).
Using CE synthesis as a probe of MAM function, we determined the role of presenilins in this process by analysing mouse embryonic fibroblasts (MEFs) lacking Ps1 (Ps1-KO), Ps2 (Ps2-KO), or both proteins (DKO). Compared to wild-type (WT) MEFs, the mutant lines showed increased steady-state levels of total ([@b32]) and free cholesterol ([Figure 2C](#f2){ref-type="fig"}), but more importantly, the relative differences in CE content were even greater, with up to ∼5-fold more CE in DKO than in WT MEFs ([Figure 2C](#f2){ref-type="fig"}). Upon labelling the cells with ^3^H-cholesterol to monitor its conversion by ACAT into ^3^H-cholesteryl esters, we found significantly higher ACAT activity in the Ps-mutant MEFs (up to ∼2.5-fold over controls) ([Figure 2D](#f2){ref-type="fig"}). Both presenilin isoforms contributed to CE synthesis, as the average increase in CE content was far more pronounced in the Ps1+Ps2 DKO MEFs (145% greater than WT) than in either individual knockout alone (96 and 63% greater than WT in Ps1- and Ps2-KO, respectively) ([Figure 2D](#f2){ref-type="fig"}). In a control experiment, we determined that this upregulation was not due to an increase in *Acat1* expression ([Supplementary Figure S3](#S1){ref-type="supplementary-material"}). Because CE synthesis in the Ps1-KO cells was higher than in the Ps2-KOs, and because PS1 plays a more significant role in FAD than does PS2 ([@b44]), we also examined CE synthesis in immortalized mouse MEFs in which Ps1 expression had been knocked down (Ps1-KD) ([Supplementary Figure S4](#S1){ref-type="supplementary-material"}). There was an ∼3-fold increase in the kinetics of CE formation in Ps1-KD cells versus control ([Figure 2E](#f2){ref-type="fig"}). Notably, we detected significant increases in CE synthesis in PS-mutant FAD cells (∼1.8-fold higher than controls), and equally strikingly, in SAD cells as well (∼1.7-fold higher) ([Figure 2F](#f2){ref-type="fig"}).
Consistent with the increase in CE, we observed numerous structures that appeared to be lipid droplets in electron microscopic images of DKO, but not control, MEFs (asterisks in [Figure 3A](#f3){ref-type="fig"}). We therefore looked for the presence of lipid droplets after staining cells with HCS LipidTox Green^TM^ ([Figure 3B](#f3){ref-type="fig"}) and Oil Red O ([Supplementary Figure S5](#S1){ref-type="supplementary-material"}). Whereas the LipidTox stain in WT MEFs was diffuse, the PS-mutant MEFs contained numerous discrete LipidTox-positive droplets (∼5-fold greater signal) ([Figure 3B](#f3){ref-type="fig"}; see also [Supplementary Figure S6](#S1){ref-type="supplementary-material"}). Similar results were also obtained with the Ps1-KD cells ([Figure 3C](#f3){ref-type="fig"}); importantly, the increase in lipid droplets in Ps1-KD cells (∼5-fold over control) was rescued by overexpression of human WT PS1, but not of human PS1 harbouring the A246E mutation found in many FAD patients ([Figure 3C](#f3){ref-type="fig"}). Notably, we detected significantly more lipid droplets in fibroblasts from FAD patients (with mutations in PS1, PS2, and APP) and in SAD fibroblasts (an average of ∼20--30% of the cells was LipidTox positive) versus controls (∼3% positive) ([Figure 3D](#f3){ref-type="fig"}; [Supplementary Figures S5 and S6](#S1){ref-type="supplementary-material"}). These observations may help explain the elevated numbers of lipids droplets found in fibroblasts ([@b66]) and neurons ([@b31]) of SAD patients.
Taken together, the data indicate that MAM activity, as measured by CE synthesis, is altered in AD, and that presenilins and APP, and perhaps also γ-secretase activity, may play a key role in regulating MAM function.
Elevated phospholipid synthesis in PS-mutant cells and AD patient fibroblasts
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It is well known that ER--mitochondrial communication via MAM is necessary for phospholipid synthesis ([@b100]). Phosphatidylserine (PtdSer) is synthesized in the MAM ([@b36]); it then translocates to mitochondria, where it is converted to phosphatidylethanolamine (PtdEtn); PtdEtn then translocates back to the MAM, where it is methylated (by PEMT) ([@b97]) to generate phosphatidylcholine (PtdCho). The trafficking of these phospholipids between ER and mitochondria is a recognized measure of the communication between both organelles and of the regulation of MAM function ([@b100]). Therefore, to test directly the role of presenilins in ER--mitochondrial communication, we incubated Ps-mutant MEFs in medium containing ^3^H-serine (^3^H-Ser) and measured the incorporation of the label into newly synthesized ^3^H-PtdSer and ^3^H-PtdEtn. The levels of both labelled species increased \>3-fold in the DKO MEFs as compared to WT ([Figure 4A](#f4){ref-type="fig"}), suggesting an upregulation of MAM function and of ER--mitochondrial cross-talk in these cells. In a control experiment, we determined that this upregulation was not due to an increase in the expression of *Ptdss1*, *Ptdss2*, or *Pisd*, three key genes involved in transport of phospholipids between ER and mitochondria ([Supplementary Figure S3](#S1){ref-type="supplementary-material"}).
To determine the kinetics of this upregulation, we performed pulse-chase analysis by incubating the MEFs with ^3^H-Ser for 1 h, followed by a chase with cold serine for various time periods ([Figure 4B](#f4){ref-type="fig"}). The incorporation of label into ^3^H-PtdSer during the pulse (time 0 in [Figure 4B](#f4){ref-type="fig"}) was significantly higher in the Ps1-KO and DKO MEFs than in control. During the chase, the amount of ^3^H-PtdSer decreased and that of ^3^H-PtdEtn increased, consistent with the conversion of the former into the latter, with higher rates in the Ps1-KO and DKO MEFs (up to three-fold higher). While the increase in lipid synthesis in the pulse-chase was not altered significantly in the Ps2-KO MEFs, Ps2 clearly contributes to phospholipid metabolism and MAM function, as lipid synthesis in the Ps1+Ps2 double knockout was much more pronounced than in the Ps1-knockout alone ([Figure 4B](#f4){ref-type="fig"}). These results were confirmed in isolated MEF crude mitochondrial fractions (containing essentially only ER, MAM, and mitochondria; [@b3]) ([Figure 4C](#f4){ref-type="fig"}). The rate of phospholipid synthesis was also increased in Ps1-KD cells ([Figure 4D](#f4){ref-type="fig"}) and, importantly, in FAD and SAD fibroblasts (by ∼1.5- to 2-fold over controls) ([Figure 4E](#f4){ref-type="fig"}).
Since some of the PtdEtn synthesized is exported to the inner leaflet of the PM ([@b97]), we hypothesized that it would be elevated in the PM of mutant cells. Accordingly, we treated cells with two highly related antibiotics, cinnamycin (Cin; also called Ro09-0198) ([@b16]) and duramycin (Dura) ([@b58]), both of which are 19-aa cyclic peptides that form a complex specifically with PtdEtn to induce pore formation in the PM in a PtdEtn concentration-dependent manner, followed by rapid cell death ([@b54]) (see example in [Figure 5A](#f5){ref-type="fig"}, left and middle panels). Ps1-KO and DKO MEFs were ∼3.5-fold more Cin sensitive than were controls ([Figure 5A](#f5){ref-type="fig"}, right panel). Similarly, Ps1-KD cells were ∼3-fold more Cin sensitive than were controls; as before, this sensitivity could be rescued by overexpression of human WT, but not A246E mutant, PS1 ([Figure 5B](#f5){ref-type="fig"}, left panel). Notably, FAD and SAD cells were significantly (∼3- to 5-fold) more Cin sensitive than were controls ([Figure 5B](#f5){ref-type="fig"}, right panel). We also were able to visualize the presence of PtdEtn on the cell surface by staining cells with FL-SA-Ro ([Figure 5C](#f5){ref-type="fig"}), a fluorescent-conjugated form of cinnamycin that binds to PtdEtn on the PM but does not initiate cell death ([@b23]). In agreement with the Cin-sensitivity results, ∼4 times as many FAD and SAD cells were stained with FL-SA-Ro as compared to controls ([Figure 5C](#f5){ref-type="fig"}; [Supplementary Figure S7](#S1){ref-type="supplementary-material"}).
Together with the CE data, these results point to an upregulation of MAM function in AD, either by mutations in presenilins or APP or, in the case of SAD, by unknown causes.
*Increased ER*--*mitochondrial contacts in PS-mutant cells and AD patient fibroblasts*
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The increased biochemical activity of MAM in PS-mutant cells prompted us to see if ER--mitochondrial contacts were physically altered. We therefore transfected cells with DsRed-Mito to detect mitochondria (in red) and with GFP-Sec61β to detect ER (in green), and used confocal microscopy and Image J analysis to detect and quantitate regions where the two signals were in close apposition (see example in [Figure 6A](#f6){ref-type="fig"}). Using this method, we found that the degree of ER--mitochondrial apposition was significantly higher in Ps1-KO (∼34±10% of the total signal), Ps2-KO (34±6%), and DKO (56±6%) MEFS than in WT MEFS (12±4%) ([Figure 6B](#f6){ref-type="fig"}, left). Moreover, the degree of apposition was significantly higher in fibroblasts from both FAD (26±4%) and SAD (24±6%) patients than in those from controls (11±2%) ([Figure 6B](#f6){ref-type="fig"}, right).
In order to observe ER--mitochondrial apposition at higher resolution, we imaged MEFs and patient cells by electron microscopy (EM). We observed a significant increase in the length of mitochondrial-ER contacts (i.e., MAM) in DKO as compared to WT MEFs ([Figure 7](#f7){ref-type="fig"}). Specifically, there were significantly more numerous 'long\' (50--200 nm; [Figure 7C](#f7){ref-type="fig"}) and 'very long\' (\>200 nm; [Figure 7E](#f7){ref-type="fig"}) contacts in DKO MEFs than in WT MEFs (∼5-fold and \>10-fold, respectively; [Figure 7F](#f7){ref-type="fig"}, left), whereas connections in WT MEFs were much shorter and more 'punctate\' (\<50 nm; [Figure 7B](#f7){ref-type="fig"}). We found a similar increase in 'long\' and 'very long\' contacts in AD patients ([Figure 7F](#f7){ref-type="fig"}, right; [Supplementary Figure S8](#S1){ref-type="supplementary-material"}). Thus, the increased biochemical activity of MAM in PS-mutant and in AD cells correlated with an increased area of physical association between the two organelles.
*Analysis of MAM function in cells deficient in ER*--*mitochondrial communication*
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If upregulated MAM function in PS-mutant and AD cells is related to increased ER--mitochondrial communication, then decreased contacts between the two organelles should have an opposite effect. We therefore assayed cholesterol ester and phospholipid synthesis in MEFs lacking mitofusin-2 (Mfn2), a protein that is required for MAM-mediated ER--mitochondrial interactions ([@b19]). In agreement with our hypothesis, we found an ∼2-fold lower rate of CE synthesis in Mfn2-KO MEFs ([Figure 8A](#f8){ref-type="fig"}), and an ∼25% decrease in phospholipid synthesis and transport, as compared to WT MEFs ([Figure 8B](#f8){ref-type="fig"}). Finally, we asked whether the deficient ER--mitochondrial communication present in Mfn2-KO cells affected γ-secretase activity. We measured the levels of APP and its C-terminal cleavage products C99 and AICD (APP intracellular domain) ([@b3]; [@b24]) (see scheme in [Figure 8C](#f8){ref-type="fig"}). There was an ∼2-fold reduction in the amount of AICD in Mfn2-KO cells as compared to WT, and a concomitant increase in C99 ([Figure 8C](#f8){ref-type="fig"}). As a control, we also measured these three proteins in WT and DKO MEFs. As expected, no AICD was produced in the DKO cells ([@b3]), while C99 accumulated accordingly ([Figure 8C](#f8){ref-type="fig"}). Importantly, the reduction in γ-secretase activity in Mfn2-KO cells was not due to a relocalization or mislocalization of presenilins in these cells, as they were still highly enriched in the MAM compartment ([Figure 8D](#f8){ref-type="fig"}). Thus, the deficiency in γ-secretase activity in Mfn2-KO cells supports the view that ER--mitochondrial communication is required for this functionality.
Given that mutations in presenilins increase both MAM function and ER--mitochondrial connectivity, whereas mutations in Mfn2 decrease the latter ([@b19]), we asked if one could complement the other. Accordingly, we knocked down *Ps1* and *Ps2* transcripts simultaneously by ∼80% in WT and Mfn2-KO MEFs ([Supplementary Figure S9A](#S1){ref-type="supplementary-material"}), and measured phospholipid synthesis. We found that the decreased production of ^3^H-PtdSer and ^3^H-PtdEtn in Mfn2-KO MEFs was reversed when *Ps1/2* expression was knocked down ([Figure 9A](#f9){ref-type="fig"}, left panel). Conversely, when we knocked down *Mfn2* transcripts in WT and Ps1/2-DKO MEFs by ∼80% ([Supplementary Figure S9B](#S1){ref-type="supplementary-material"}), we obtained the opposite result, as the increased phospholipid synthesis in the DKO MEFs was significantly reduced upon knocking down *Mfn2* expression ([Figure 9A](#f9){ref-type="fig"}, right panel); notably, the phospholipid transport data correlated with the degree of ER--mitochondrial colocalization in these cells ([Figure 9B](#f9){ref-type="fig"}). Analysis of the cells for duramycin sensitivity confirmed the biochemical findings, especially in the Ps1/2-DKO cells, where knockdown of *Mfn2* blunted the increase in Dura sensitivity relative to that in WT MEFs ([Figure 9C](#f9){ref-type="fig"}). Finally, we measured CE synthesis (as assayed by LipidTox staining) and found that the increase in lipid droplet formation seen in Ps1/2-DKO MEFs was reversed by *Mfn2* silencing ([Figure 9D](#f9){ref-type="fig"}).
Taken together, these results imply that presenilins and Mfn2 may affect the same pathway to regulate MAM activity and the apposition of ER to mitochondria.
Analysis of MAM function in cells deficient in γ-secretase activity
-------------------------------------------------------------------
Our data clearly show that mutations in presenilins affect MAM function and ER--mitochondrial connectivity, but it was unclear whether the enzymatic activity of γ-secretase plays a direct role in these processes. Accordingly, we examined HeLa cells in which PS1 expression had been knocked down by shRNA ([Supplementary Figure S9C](#S1){ref-type="supplementary-material"}) and asked if we could rescue MAM function by overexpressing either PS1-WT or a 'catalytically dead\' construct encoding a D385A mutation in PS1 ([@b105]). Whereas adding back the WT version of PS1 rescued the knockdown phenotype, as expected, cells transfected with the D385A mutant showed an increase in ER--mitochondrial communication and MAM activity (as measured by phospholipid synthesis and lipid droplet formation; [Supplementary Figure S10](#S1){ref-type="supplementary-material"}), similar to the increase observed in the PS1 knockdown MEFs ([Figure 3C](#f3){ref-type="fig"}). The failure of 'catalytically dead\' PS1 to rescue the knockdown phenotypes would suggest that γ-secretase activity itself is responsible for the regulation of MAM activity. However, it has been shown that full-length PS1 containing the D385A mutation cannot be cleaved into the N- and C-terminal fragments required for γ-secretase activity ([@b105]; [@b45]); this cleavage defect may also affect the proper subcellular localization of the protein ([@b13]). Moreover, PS1-D385A has been shown to depress the activity of WT PS1, in a dose-dependent manner ([@b45]), as well as to compete with WT PS1 for limiting factors required for γ-secretase activity ([@b91]). For these reasons, we believe that overexpressing the D385A mutant basically mimics the KO/KD phenotype ([@b15]), and therefore does not really answer the question as to whether γ-secretase activity itself regulates ER--mitochondrial communication and/or MAM function.
We therefore took a more direct biochemical approach. We treated various cell types with DAPT ((*N*-\[*N*-(3,5-difluorophenacetyl)-[L]{.smallcaps}-alanyl\]-S-phenylglycine t-butyl ester)), a highly specific γ-secretase inhibitor ([@b61]), for 24 h ([Supplementary Figure S9D](#S1){ref-type="supplementary-material"}), and then assayed them for MAM function. DAPT had a profound effect on CE synthesis, as measured by the detection of lipid droplets, with an increase in the % of LipidTox-positive cells ranging from ∼2-fold in MEFs to ∼7-fold in 3T3 and HeLa cells ([Figure 10A](#f10){ref-type="fig"}). Surprisingly, however, the increase in phospholipid transport was much lower (only on the order of ∼20%; [Figure 10B](#f10){ref-type="fig"}), a value far lower than the robust increases in phospholipid transport that were detected in cells (including MEFs) harbouring presenilin mutations (see [Figure 4](#f4){ref-type="fig"}). We therefore quantitated the amount of ER--mitochondrial colocalization (performed as in [Figure 6](#f6){ref-type="fig"}) in these DAPT-treated cells, and found that the drug had essentially no effect on the degree of ER--mitochondrial apposition ([Figure 10C](#f10){ref-type="fig"}).
Taken together, these data imply that the catalytic activity of γ-secretase can have significant effects on MAM function (at least with respect to cholesterol ester synthesis) but appears to have little effect on establishing ER--mitochondrial connectivity. They also point to the possibility that there may be two separate classes of functions at the ER--mitochondrial interface: those required for 'horizontal\' activity (i.e., intraorganellar enzymatic functions residing within the MAM compartment itself, as measured, for example, by CE synthesis) and those required to maintain 'vertical\' MAM activity (i.e., interorganellar communication between ER and mitochondria, as measured, for example, by phospholipid transport).
Discussion
==========
We previously showed that MAM is the predominant subcellular locus for presenilins and for γ-secretase activity ([@b3]). We now show that mutations in PS1, PS2, and APP can upregulate MAM function and increase ER--mitochondrial connectivity significantly, implying that presenilins are negative regulators of these behaviours. Moreover, we found that cells from patients with SAD, in which PS1, PS2, and APP structure (but perhaps not expression; [@b60]; [@b80]) are normal, also display the same hallmarks of upregulated MAM function and ER--mitochondrial communication as do those with FAD.
Because MAM dysfunction is the common denominator underlying the phenotypes seen in both FAD and SAD, we therefore propose that increased MAM activity and ER--mitochondrial communication lies at the heart of AD pathogenesis ([Figure 11](#f11){ref-type="fig"}). In support of this view, we note that many of the biochemical and morphological phenotypes associated with AD---altered cholesterol, phospholipid, glucose, and calcium metabolism, aberrant mitochondrial behaviour and function, and notably, altered Aβ production---are the very functions that are associated with MAM and with connections between ER and mitochondria.
The first clue that MAM might be involved in AD came from the observation that presenilins and γ-secretase activity are enriched in MAM ([@b3]). This speculation was reinforced by the finding that MAM is an LR-like domain ([Figure 1](#f1){ref-type="fig"}; [@b35]; [@b109]; [@b26]), which is consistent with previous data showing that APP processing to produce Aβ depends on LRs ([@b22]; [@b94]; [@b98]; [@b48]; [@b65]). Apart from the lack of appropriate markers to detect MAM, one of the reasons that MAM was overlooked as a detergent-resistant membrane, and as the region in which PS\'s, APP, and γ-secretase activity are enriched, may be that most methods used to isolate rafts do not separate the PM from intracellular membranes ([@b53]). Thus, intracellular DRMs like MAM will be co-isolated with PM rafts during the subfractionation process, obscuring the localization of PS\'s and γ-secretase activity at ER--mitochondria connections. This ambiguity might help explain earlier observations indicating that, along with the presenilins, the other components of the γ-secretase complex---nicastrin, APH-1, and PEN-2---are not only present in the MAM ([@b3]) but are also enriched in DRMs ([@b98]).
Furthermore, the fact that MAM is a DRM may also help explain the lack of consensus regarding the subcellular localization of presenilins and γ-secretase activity, as detecting proteins embedded in DRMs can be technically challenging ([@b81]). It may also explain why MAM markers such as VDAC ([@b90]) and calnexin ([@b63]) were found in some studies of PM rafts ([@b25]; [@b108]) and why Aβ was reported to be present in mitochondria ([@b52]; [@b55]). Likewise, crude mitochondrial preparations contain MAM that can lead one into thinking that mitochondria contain *bona fide* LRs ([@b85]) when in fact they do not ([@b108]). It has long been known that presenilins, APP, Aβ, and γ-secretase activity are enriched in LRs/DRMs ([@b94]; [@b48]), but it had always been assumed that these rafts were located at the PM ([@b49]). Our data are consistent with an alternate interpretation, namely, that APP processing to produce Aβ occurs not at LRs in the PM, but intracellularly in the MAM ([@b35]). We note that several groups have suggested the existence of intracellular rafts in the ER or mitochondria ([@b6]); we believe that these LRs/DRMs are, in fact, MAMs ([@b35]; [@b109]; [@b26]).
As a locus for the integration of key cellular functions and signalling ([@b36]), MAM apparently requires a more rigid liquid-ordered phase to recruit, orient, regulate, and limit the lateral mobility of the membrane proteins within its boundaries, so as to promote the cross-talk of mitochondria and ER. If MAM is indeed an LR, then alterations in its structure and composition may affect profoundly the cellular functions localized within it. It is possible that the loss or alteration of γ-secretase activity (via, e.g., mutations in presenilins and APP in the case of FAD) affects the structure and lipid composition of cellular membranes in general and of MAM in particular, especially in AD ([@b10]). Conversely, alterations in MAM via, for example, disrupting Mfn2 expression, can also affect γ-secretase activity, with the physical distance separating ER from mitochondria playing a critical role in the regulation of MAM function ([@b17]). Thus, we believe that perturbations in ER--mitochondrial connectivity are the key element underlying AD pathogenesis, and perhaps other neurodegenerative diseases as well ([@b82]).
As noted above, many of the apparently unrelated cellular functions that are misregulated in AD can be ascribed to increased MAM function and ER--mitochondrial connectivity. First, it has long been known that AD patients have elevated ACAT levels ([@b66]), elevated cholesterol and/or CEs ([@b86]; [@b66]), and deposition of lipid droplets in peripheral cells ([@b67]; [@b56]) and in neurons ([@b31]). In addition, there is compelling evidence that MAM plays a role in lipid droplet formation ([@b101]) and that both PtdEtn synthesis ([@b65]) and ACAT activity ([@b75], [@b74]) are required for Aβ production. The connection of MAM-localized ACAT1 to AD is intriguing: the enzyme is absolutely required for the generation of Aβ, by modulating the equilibrium between free and esterified cholesterol ([@b75], [@b76], [@b74]; [@b72]), presumably as a result of its affects on APP processing ([@b42], [@b43]; [@b7]). While it is unclear how mutated presenilins affect Aβ production, it has been shown that pathogenic mutations in PS1 affect the conformation of the γ-secretase active site ([@b11]). Thus, one possibility is that altered cholesterol and lipid composition within the MAM membrane changes the orientation of APP and its cleavage by γ-secretase, and hence, the generation of total Aβ and an alteration in the ratio of Aβ~42~:Aβ~40~ ([@b93]; [@b40]). In support of this view, we note that the membrane thickness of LRs is greater than that of the surrounding bilayer ([@b77]; [@b51]), and that the thickness of model membranes affects the cleavage specificity of γ-secretase as well as the relative amounts of Aβ~40~ and Aβ~42~ ([@b104]). A connection between MAM and Aβ production is also supported by the recent finding that extracellular plaques have an intracellular origin, and that this process is mediated by sphingomyelin and GM1 gangliosides ([@b106]), both of which are components of MAM and which play a significant role in its regulation ([@b79]; [@b33], [@b34]). In addition, the finding of increased phospholipid synthesis in PS-mutant cells, indicative of increased cross-talk between ER and mitochondria ([@b100]), is consistent with the reported aberrations in phospholipid profiles in AD patients ([@b70]; [@b10]).
Alterations in MAM likely affect other cellular functions as well. One of the most critical of these is calcium homeostasis ([@b17]; [@b28]), as MAM is highly enriched in the sarco-ER calcium-ATPase ([@b20]), the sigma-1 receptor ([@b35]), ryanodine receptors (RyRs) ([@b27]), and inositol-1,4,5-trisphosphate receptors (IP3Rs) ([@b36]). Thus, increased ER--mitochondrial communication in AD could explain the altered intracellular Ca^2+^ trafficking via RyRs ([@b88]) and IP3Rs ([@b50]; [@b107]), leading to the aberrant calcium homeostasis found in AD patients ([@b5]). Surprisingly, [@b107] concluded that PS2, but not PS1, modulates ER--mitochondria interactions and calcium cross-talk, a finding that differs with the data reported here showing that *both* presenilin isoforms mediate MAM function and ER--mitochondrial communication; it also differs with the observation that mutations in PS1 enhance IP3R-mediated calcium trafficking ([@b14], [@b15]; [@b59]), which is clearly an MAM-mediated function ([@b36]). Their findings would also be difficult to reconcile with the fact that mutations in PS1 are more prevalent in FAD than are those in PS2, and result in a more 'severe\' clinical course ([@b44]), and would also imply that the mechanisms by which these two highly related proteins cause FAD are radically different, even though both proteins are components of the γ-secretase complex. However, we note that [@b107] relied on experiments in which the presenilin and multiple reporter constructs were overexpressed transiently, which may have introduced a degree of ambiguity in the interpretation of their data ([@b87]). In addition, these transfections were performed in cells expressing endogenous WT chromosomal PS1 and PS2 alleles, which could have resulted in potential feedback of the introduced constructs on their regulation, a potential problem that was circumvented in our experimental design, which relied instead on knockdown and knockout constructs and on AD patient cells expressing endogenous mutated alleles. We also note that, contrary to what we observed here, [@b107] found reductions in *Mfn2* expression upon knockdown of the presenilins; such reductions may have inadvertently masked any effect on MAM function due to reduced presenilin expression. Taken together, we believe that the conclusion of [@b107] that only PS2, and not PS1, affects MAM behaviour is open to question.
Mitochondrial dysfunction in AD has been reported extensively ([@b89]), but there is no clear evidence as to whether it is cause or effect. Altered connections between ER and mitochondria would almost certainly affect mitochondrial dynamics (e.g., shape, distribution, and movement; [@b47]) and function (e.g., oxidative energy metabolism, calcium buffering capacity, and free radical production; [@b69]; [@b38]; [@b29]; [@b8]), and thus may be the underlying cause of the reported aberrant mitochondrial phenotypes seen in AD ([@b89]).
Our assays of MAM function upon inhibition of γ-secretase activity yielded the surprising result that whereas the catalytic activity of the enzyme is required for at least some aspects of MAM function, it is apparently not required to maintain ER--mitochondrial connectivity. Together with the Mfn2 rescue experiments, this result, which was obtained in cells expressing endogenous WT presenilins, implies that the presenilin protein itself (and not just its proteolytic activity) is acting as a regulator of the distance between ER and mitochondria ([@b17]). A corollary of this finding is that the presenilins might play a role in ER--mitochondrial communication independent of their enzymatic function. These results also underscore the fact that MAM function and ER--mitochondrial communication are two separate aspects of this single cellular subdomain: on the one hand, MAM is a true compartment of the ER, with its own suite of biochemical activities, whereas on the other, it is a bridge that connects two organelles, namely the ER and the mitochondria. We note, however, that all of our data indicate that in AD, both aspects---MAM function and interorganellar distance---are affected, but whether both aspects must be affected consistently in order to cause disease remains to be determined.
Finally, our findings have implications for understanding the genetics of AD ([@b39]; [@b64]). Most presenilin mutations causing FAD are dominant and are presumed to cause a gain of function ([@b21]; [@b15]). However, the increased MAM function in PS-KD and -KO cells reported here imply that the effects of many (but probably not all; [@b12]) pathogenic PS1 mutations are more likely to be due to loss of function or to a gain of negative function ([@b60]; [@b4]; [@b83]; [@b37]; [@b44]; [@b9]).
We believe that the increased MAM function and ER--mitochondrial cross-talk found in all PS mutant cells analysed could help explain some of the features and early events in AD, such as elevated serum cholesterol levels, mitochondrial dysfunction, oxidative stress, and calcium deregulation ([@b81]), which are all upstream of the appearance of plaques and tangles ([@b73]) and which are detected routinely in AD patients. As such, upregulated MAM function could play a hitherto unrecognized and critical role in the pathogenesis of AD ([@b81]) that may help us to understand better this devastating disease.
Materials and methods
=====================
Cells and reagents
------------------
WT, Ps1-KO, Ps2-KO, and DKO mouse MEFs were gifts of Dr Bart De Strooper (University of Leuven). WT and Mfn2-KO MEFs were gifts of David Chan (California Institute of Technology). PS1- and APP-mutant FAD cells were kind gifts of Gary E Gibson (Cornell University) and Richard Cowburn (Karolinska Institute). Other AD and control cell lines were obtained from the Coriell Institute for Medical Research (Camden, NJ) (see [Supplementary Table S1](#S1){ref-type="supplementary-material"}). We used antibodies to PS1 (aa 1--65 (Calbiochem 529591) and aa 303--316 (Calbiochem PC267)), PEMT (a gift of Joan Vance, University of Alberta), VDAC1 (Abcam ab15895), ACAT1 (Abcam ab39327), Na^+^/K^+^-ATPase (Abcam ab7671), SRC tyrosine kinase (Abcam ab32102), NDUFA9 (monoclonal; Molecular Probes A21344), MFN2 (Abcam ab50838), and SSRα (a gift of Howard Worman and Martin Wiedemann, Columbia University). Thin layer chromatography (TLC) silica plates were from EMD Biosciences (5748-7). Phosphatidylserine (P7769), phosphatidylethanolamine (60648), cholesteryl palmitate (C6072), cholesteryl oleate (C9253), lipid markers for TLC (P3817), cinnamycin (C5241), duramycin (D3168), and DAPT (D5942) were from Sigma. Radiolabelled ^3^H-serine and ^3^H-cholesterol were from Perkin-Elmer; ^3^H-oleoyl-CoA was from Moravek Biochemicals (MT1649); and fatty acid-free bovine serum albumin (FAF-BSA) was from MP Biomedical (820472).
Subcellular fractionation and western blotting
----------------------------------------------
Purification of ER, MAM, and mitochondria was performed and analysed as described ([@b3]).
Isolation of LRs
----------------
To identify detergent-resistant domains, samples were resuspended in 400 μl of isolation buffer (IB: 250 mM mannitol, 5 mM HEPES pH 7.4, and 0.5 mM EGTA) containing 1% Triton X-100 and incubated at 4°C with rotation for 1 h. Samples were adjusted to 80% sucrose, placed at the bottom of a 5--30% sucrose gradient, and centrifuged at 250 000 *g* for 18 h. After fractionation, equal volumes of each fraction were loaded on an SDS--PAGE gel and analysed by western blot.
Measurement of cholesterol species
----------------------------------
Quantification of total cholesterol and CEs was performed using the Cholesterol/Cholesteryl Ester Quantitation kit from Calbiochem (428901).
Lipid droplet staining
----------------------
Staining of lipid droplets was performed using HCS LipidTox™ Deep Green neutral lipid stain (Invitrogen H34475) according to manufacturer\'s instructions. Lipid droplet staining was quantified using ImageJ. When reported as intensities, the values in the text represent the product of the intensity and the area covered by the fluorescent signal above background in every cell examined. For each cell type and/or condition, we took between 15 and 20 images for analysis. The images were first taken at lower magnification (× 20) in order to view 50--100 cells per field. The numbers reported in the text represent the averages derived from the quantification of staining in 500--600 cells.
For lipid droplet staining by Oil Red O, we first prepared a stock solution (35% Oil Red O in isopropanol), stirring the mix for 12 h and filtering it before use. Cultured cells were fixed in 10% formalin in PBS for at least 1 h and washed twice in ddH~2~O. The plates were incubated in 60% isopropanol for 5 min at RT, the alcohol was discarded, and the cells were allowed to dry completely at RT. One microlitre of a filtered Oil Red O working solution (60% stock solution in H~2~O) was added to the cells at RT for 10 min. The Oil Red O solution was removed and the samples were immediately washed in ddH~2~O before acquiring images under the microscope.
Analysis of phospholipid synthesis in cultured cells
----------------------------------------------------
Cells were incubated for 2 h with serum-free medium to ensure removal of exogenous lipids. The medium was then replaced with MEM containing 2.5 μCi/ml of ^3^H-serine for the indicated periods of time. The cells were washed and collected in DPBS, pelleted at 2500 *g* for 5 min at 4°C, and resuspended in 0.5 ml water, removing a small aliquot for protein quantification. Lipid extraction was done by the Folch method. Briefly, three volumes of chloroform/methanol 2:1 were added to the samples and vortexed. After centrifugation at 8000 *g* for 5 min, the organic phase was washed twice with two volumes of methanol/water 1:1, and the organic phase was blown to dryness under nitrogen. Dried lipids were resuspended in 60 μl of chloroform/methanol 2:1 and applied to a TLC plate. Phospholipids were separated using two solvents, composed of petroleum ether/diethyl ether/acetic acid 84:15:1 v/v and chloroform/methanol/acetic acid/water 60:50:1:4 v/v. Development was performed by exposure of the plate to iodine vapour. The spots corresponding to the relevant phospholipids (identified using co-migrating standards) were scraped and counted in a scintillation counter (Packard Tri-Carb 2900TR).
Analysis of phospholipid synthesis in subcellular fractions
-----------------------------------------------------------
Crude mitochondrial fractions were isolated from MEFs as described ([@b3]). Two hundred micrograms were incubated in a final volume of 200 μl of phospholipid synthesis buffer (10 mM CaCl~2~, 25 mM HEPES pH 7.4, and 3 μCi/ml ^3^H-Ser) for 30 min at 37°C. The reaction was stopped by addition of three volumes of chloroform/methanol 2:1. Lipid extraction and TLC analysis were performed as described above.
Assay of ACAT activity
----------------------
To measure ACAT activity *in vivo*, whole cells were incubated in serum-free medium for 2 h to remove all exogenous lipids. After that, 2.5 μCi/ml of ^3^H-cholesterol was added to FBS-free DMEM containing 2% FAF-BSA, allowed to equilibrate for at least 30 min at 37°C, and the radiolabelled medium was added to the cells for the indicated periods of time. Cells were then washed and collected in DPBS, removing a small aliquot for protein quantification. Lipids were extracted as described above and samples were analysed by TLC along with an unlabelled CE standard. A mixture of chloroform/methanol/acetic acid 190:9:1 was used as solvent. Iodine stains corresponding to CE bands were scraped and counted.
Cinnamycin and duramycin assays
-------------------------------
Cinnamycin and duramycin were diluted in FBS-free DMEM to the appropriate concentration and added to the cells for the indicated time periods at 37°C. After incubation, cells were washed in PBS twice before testing cell viability by Live/Dead assay (Invitrogen \#L3224) according to manufacturer\'s instructions. Samples were then analysed under the microscope and cell death was quantified by counting live (green) and dead (red) cells manually. To detect fluorescent cinnamycin with FL-SA-Ro, cells were washed three times with Hanks\' buffered saline containing 0.5% BSA (0.5% BSA--HBS) and incubated with 50 μg/ml Cy3-conjugated cinnamycin (FL-SA-Ro) for 30 min at 37°C. Cells were then washed with 0.5% BSA--HBS and analysed under the microscope. To visualize cell morphology, cells were then counterstained with 5 mM calcein (AnaSpec \#89203) in PBS for 20 min at room temperature, fixed in 4% paraformaldehyde, mounted, and analysed under the microscope.
Analysis of ER--mitochondrial apposition
----------------------------------------
Cells under were co-transfected with GFP-Sec61-β (Addgene plasmid \#15108) and DsRed-Mito (Clontech, \#632421) at a 1:1 ratio, using Lipofectamine 2000 (Invitrogen, \#11668-027) in serum-free DMEM. Twelve hours post transfection, cells were analysed in a single-plane with a Zeiss LSM510 microscope. Interactions between mitochondria and ER were calculated using Image J software (<http://rsbweb.nih.gov/ij/>), determining the area occupied by one organelle and using its signal as a mask for the other one. The various 'colocalization\' data sets were compared using Mander\'s coefficient.
Transmission EM
---------------
Cells were fixed with 2.5% glutaraldehyde in 0.1 M Sorenson\'s phosphate buffer (pH 7.2) for at least 1 h. Cells were then postfixed for 1 h with 1% OsO~4~ in Sorenson\'s buffer. Staining was performed using 1% tannic acid. After dehydration, cells were embedded in a mixture of Lx-112 (Ladd Research Industries) and Embed-812 (EMS, Fort Washington, PA). Thin sections, cut on an MT-7000 ultramicrotome, were stained with uranyl acetate and lead citrate, and examined in a JEOL JEM-1200 EXII electron microscope. Pictures were taken on an ORCA-HR digital camera (Hamamatsu) and recorded with an AMT Image Capture Engine.
Transcriptional silencing
-------------------------
To knockdown mouse *Ps1*, small hairpin (sh) RNA oligonucleotide M2 @ nt 179--197 in mouse *Psen1* (Genbank NM_008943: gacaggtggtggaacaaga) and mismatch control shRNA M3 (gacagg**a**gg**a**ggaacaaga; mismatches in bold) were inserted into pSUPER-Retro-Puro vector pSR (OligoEngine). We also replaced the puromycin-resistance cassette with a blasticidine-resistance cassette inserted at the *Nhe*I-*Dra*III sites in the vector, generating pSR-Blast, to allow for 'double transduction\' using two different selection markers to increase shRNA expression. Viral supernatants (3 ml) from plasmid-transfected Amphotrophic Phoenix ΦNX-A packaging cells supplemented with polybrene were added to immortalized mouse MEFs (derived from primary MEFs of mixed C57Bl/6-129 mice, using a 3T3 subculture schedule; [@b92]), seeded 1 day prior to infection at 100 000/well in 6-well culture plates, followed by infection for 24 h. Cells were selected in medium containing puromycin, blasticidine, or both antibiotics, for 14 days. Using this protocol, PS1 expression was reduced by \>75% ([Supplementary Figure S3](#S1){ref-type="supplementary-material"}). For complementation experiments, human WT PS1 and mutated PS1 (A246E) cloned in pSuper-Retro-Neo and viral supernatants were prepared as described above. Transduced cells were selected using G418.
To knockdown presenilin expression, shRNAs against mouse *Psen1* (Sigma SASI_Mm01_00048853) and *Psen2* (Sigma SASI_Mm02_00310708) and against human *PSEN1* (Sigma SASI_Hs01_00043630) and *PSEN2* (Sigma SASI_Hs01_00033516) were transfected transiently together into relevant mouse and human cells, respectively, according to manufacturer\'s recommendations. Briefly, cells plated at low confluence were transfected with each shRNA to a final concentration of 30 nM, using Lipofectamine 2000 (Invitrogen, 11668-027) at a 1:1 ratio in serum-free DMEM. After 5 h, the medium was changed to 2% FBS DMEM and the cells were incubated for 12 more hours. Successful silencing of the targeted proteins was checked by western blot. To knockdown mouse *Mfn2* in WT and Ps1/2-DKO MEFs, shRNA against *Mfn2* (Sigma SASI_Mm01_00027313) was transfected and analysed in a similar fashion.
Where indicated, cells were transfected with pcDNA3-PS1WT or pcDNA3-PS1D385A mutant (a kind gift of Dr Tae-Wan Kim, Columbia University) at a 1:1 ratio, using Lipofectamine 2000 (Invitrogen \#11668-027) in serum-free DMEM. After 5 h, the medium was changed to DMEM containing 2% FBS. Cells were analysed 12 h post transfection.
Inhibition of γ-secretase activity
----------------------------------
Cells were treated with 2--5 μM DAPT in 2% FBS DMEM, a highly specific γ-secretase inhibitor ([@b61]), for 24 h. Following treatment, the cells were collected in PBS and then analysed, as described.
Quantitative RT--PCR (qRT--PCR)
-------------------------------
Total RNA was extracted from MEFs using TRIzol® Reagent (Invitrogen 15596-018) according to manufacturer\'s instructions, and was quantified by NanoDrop2000 (Thermo Scientific). One microgram of total RNA was used to obtain cDNA by RT--PCR using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems; PN 4368813, 4374966). Real-time PCR was performed in triplicate in a StepOnePlus™ Real-Time PCR System (Applied Biosystems; 4376600). The expression of each gene under study was analysed using specific predesigned TaqMan Probes and normalizing against *Gapdh* expression (Applied Biosystems, 4352339E) as an internal standard.
Supplementary Material {#S1}
======================
###### Supplementary Information
###### Review Process File
We thank Kristy Brown for assistance with the electron microscopy; Bart de Strooper for providing the Ps-mutant MEFs; David Chan for providing the Mfn2-mutant MEFS; Mariska te Lindert and Bé Wieringa for assisting in establishing the Ps1-KD MEFs; Tae-Wan Kim for providing PS1 constructs; Karen Duff, Ellen Steinbart, Gary Gibson, and Richard Cowburn for providing patient cells; and Salvatore DiMauro, Robert W Gilkerson, Michio Hirano, Larry Honig, and Serge Przedborski for comments. This work was supported by grants from the Ara Parseghian Medical Research Foundation (to SLS), the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to MU and JI), the Japan Science and Technology Agency (to JI), the National Institute of Aging (AG005136) and Veterans Affairs Research Funds (to TDB), the John Douglas French Alzheimer Foundation (to EAG), and the American Health Assistance Foundation, the Ellison Medical Foundation, the Alzheimer Drug Discovery Foundation, the US Department of Defense (W911NF-12-1-0159), and the Marriott Mitochondrial Disorder Clinical Research Fund (to EAS).
*Author contributions*: EA-G conceived and designed experiments; acquired, analysed, and interpreted data; wrote the paper. MCLC, MDT, CG-L, AJCG, and MM acquired, analysed, and interpreted data. JI, MU, and TDB provided critical reagents and helped acquire data. SLS provided critical experimental expertise and technology and helped acquire data. EAS conceived and designed experiments; analysed and interpreted data; wrote the paper.
The authors declare that they have no conflict of interest.
{#f1}
{ref-type="supplementary-material"}), the data point represents an average of the assays. Boxes with centred lines denote averages±s.d.; asterisks denote significant difference versus WT (*P*\<0.05).](emboj2012202f2){#f2}
{ref-type="supplementary-material"}. Other notation as in [Figure 2](#f2){ref-type="fig"}.](emboj2012202f3){#f3}
{ref-type="fig"}.](emboj2012202f4){#f4}
{ref-type="supplementary-material"}. Other notations as in [Figures 2](#f2){ref-type="fig"} and [3](#f3){ref-type="fig"}.](emboj2012202f5){#f5}
{ref-type="fig"} and [3](#f3){ref-type="fig"}.](emboj2012202f6){#f6}
{ref-type="supplementary-material"}.](emboj2012202f7){#f7}
{ref-type="fig"}) in Mfn2-KO MEFs (note decreased slope versus control). (**B**) Phospholipid synthesis after 6 h (as in [Figure 4A](#f4){ref-type="fig"}) in Mfn2-KO cells (right) (*n*=3). (**C**) Western blot to detect APP and its C-terminal cleavage products C99 and AICD (cleavage scheme at top) in Ps1/2-DKO and Mfn2-KO MEFs (image at the left is a composite of two non-adjacent lanes from the same gel; vertical dashed line indicates where the two lanes were apposed). Note the absence of AICD in DKO MEFs, and the shift in the ratio of C99:AICD in Mfn2-KO versus WT MEFs. (**D**) Subcellular distribution of presenilins in Mfn2-KO versus WT MEFs. Note that the loss of Mfn2 does not alter the predominant localization of presenilins in MAM. Other notations as in [Figure 2](#f2){ref-type="fig"}.](emboj2012202f8){#f8}
{ref-type="fig"}), and quantitation (average of 20 cells analysed ±s.e.), of ER--mitochondrial contacts in WT, Ps1+Ps2-DKO, and DKO cells 'rescued\' by knockdown of *Mfn2*. (**C**) Effect of presenilins and Mfn2 on duramycin sensitivity. Note that Dura sensitivity correlates with the change in PtdEtn synthesis shown in (**A**). (**D**) Quantitation of lipid droplets in *Ps1+Ps2* DKO MEFs upon knockdown of *Mfn2* (average of five images ±s.e.). Other notation as in [Figure 2](#f2){ref-type="fig"}.](emboj2012202f9){#f9}
{ref-type="fig"}. Note the large increase in LipidTox staining, ranging from ∼200 to ∼700% over control. (**B**) Phospholipid transport in the indicated cells (as in [Figure 4](#f4){ref-type="fig"}). Note the modest increase in PtdSer and PtdEtn synthesis, of only ∼20%. (**C**) Colocalization of ER and mitochondria (as in [Figure 6](#f6){ref-type="fig"}) in HeLa cells. Note that DAPT had no effect on the degree of colocalization. Other notation as in [Figure 2](#f2){ref-type="fig"}.](emboj2012202f10){#f10}
{#f11}
[^1]: Present address: Merck/Intervet International bv, Wim de Körverstraat 35, PO Box 31, 5830 AA Boxmeer, The Netherlands
| {
"pile_set_name": "PubMed Central"
} |
(J Am Heart Assoc. 2016;5:e003846 doi: [10.1161/JAHA.116.003846](10.1161/JAHA.116.003846))
Introduction {#jah31902-sec-0004}
============
A large proportion of myocardial infarctions (MIs) are unrecognized,[1](#jah31902-bib-0001){ref-type="ref"}, [2](#jah31902-bib-0002){ref-type="ref"}, [3](#jah31902-bib-0003){ref-type="ref"}, [4](#jah31902-bib-0004){ref-type="ref"}, [5](#jah31902-bib-0005){ref-type="ref"} often because they are accompanied by few or no symptoms. Unrecognized MI is associated with a similar risk of death and recurrent MI as recognized MI.[3](#jah31902-bib-0003){ref-type="ref"}, [6](#jah31902-bib-0006){ref-type="ref"}, [7](#jah31902-bib-0007){ref-type="ref"}, [8](#jah31902-bib-0008){ref-type="ref"}, [9](#jah31902-bib-0009){ref-type="ref"}, [10](#jah31902-bib-0010){ref-type="ref"} Persons with unrecognized MI may be identified by presence of Q waves on ECG.
It is unknown why some persons experience unrecognized MI. One possible explanation for the absence of chest pain is attenuated pain sensitivity. Small sampled experimental studies have suggested a relationship between attenuated pain sensitivity and silent ischemia.[11](#jah31902-bib-0011){ref-type="ref"}, [12](#jah31902-bib-0012){ref-type="ref"}, [13](#jah31902-bib-0013){ref-type="ref"}, [14](#jah31902-bib-0014){ref-type="ref"} To our knowledge, no previous study has examined the relationship between pain sensitivity and recognition of MI.
We examined the cross‐sectional relationship between cold pressor pain tolerance and recognized and unrecognized MI in the Tromsø Study, a large population‐based health study in Tromsø, Norway. We also investigated sex differences in the association between infarct recognition and pain sensitivity. This is of interest because a larger proportion of MIs are unrecognized in women than in men.[8](#jah31902-bib-0008){ref-type="ref"}, [9](#jah31902-bib-0009){ref-type="ref"}, [15](#jah31902-bib-0015){ref-type="ref"}
Methods {#jah31902-sec-0005}
=======
Study Population {#jah31902-sec-0006}
----------------
The Tromsø Study is a population‐based cohort study conducted in the municipality of Tromsø, Norway, and was initiated in 1974. The population consists of predominantly white Caucasians. The design of the study includes repeated cross‐sectional health surveys. The sixth survey (Tromsø 6) took place in 2007--2008 and consisted of 2 visits. Total birth cohorts and random samples of birth cohorts were invited to the first visit, and 12 981 attended (attendance rate 66%).[16](#jah31902-bib-0016){ref-type="ref"} Those eligible for the second visit were first‐visit participants in the age groups 50 to 62 years and 75 to 84 years, a 20% random sample in the age group 63 to 74 years, and those who had attended the second visit of the fourth survey (Tromsø 4) if aged older than 75 years in 1994.[16](#jah31902-bib-0016){ref-type="ref"} A total of 7306 (64%) patients attended the second visit. The first visit included testing of pain tolerance with the cold pressor test.[17](#jah31902-bib-0017){ref-type="ref"} The second visit included a standard 12‐lead ECG. A total of 4899 participants were examined with ECG and the cold pressor test. We excluded 50 participants: 18 ECGs had pathologic noninfarct Q waves due to altered conduction (eg, left bundle branch block and Wolff‐Parkinson‐White syndrome) or ventricular enlargement; 14 ECGs were uncodable (eg, pacemaker rhythm or missing leads); and 18 ECGs were not available for manual review (ECG files were missing). The final sample consisted of 4849 participants who had undergone the cold pressor test and had valid ECGs. Figure [1](#jah31902-fig-0001){ref-type="fig"} shows a flow diagram of the participants in the study.
{#jah31902-fig-0001}
All participants gave informed, written consent to research and agreed to linkage with public records of disease and death. The Tromsø Study and projects based on this have approval from the regional ethical committee.
Data Collection {#jah31902-sec-0007}
---------------
Baseline information on potential confounding variables and use of medication was obtained by self‐reported questionnaires and physical examinations. Data on previous MI are retrospectively registered for all first‐time participants of each survey of the Tromsø Study by linkage to the electronic patient records of the University Hospital of North Norway. Admissions to other hospitals are unlikely as the nearest hospital is more than 200 km from Tromsø. Each event was reviewed and validated by persons with medical expertise based on local hospital records and records from other hospitals.[16](#jah31902-bib-0016){ref-type="ref"}
Cold Pressor Pain {#jah31902-sec-0008}
-----------------
The cold pressor test is a common pain assay that has been used in experimental pain research for several decades.[18](#jah31902-bib-0018){ref-type="ref"} The stimulus consists of submerging the hand or foot in circulating cold water and elicits a deep aching pain thought to originate from activation of venous nociceptors.[19](#jah31902-bib-0019){ref-type="ref"} It was historically used as an aid in the diagnosis of angina. Participants had the testing procedures verbally explained and were placed in a comfortable chair. They were asked to insert their dominant hand and wrist into a container with circulating cold water at 3°C and a flow rate of 22 L/min, and sustain the cold immersion for as long as they could endure, up to a maximum of 106 seconds. Cold pressor tolerance was defined as time to withdrawal of the hand from the water.
Electrocardiography {#jah31902-sec-0009}
-------------------
A 12‐lead resting ECG was recorded using a computer‐based electrocardiograph (Cardiovit AT‐104 PC, Schiller AG, Baar, Switzerland). We used a computer‐based algorithm to extract all ECGs with a Q wave of amplitude ≤−0.1 mV and duration ≥0.02 seconds in any lead. Two authors (A.M.Ø. and H.L.) independently assessed the extracted ECGs. Disagreement was resolved after discussion with an expert cardiologist (H.S.). We used the third universal definition of myocardial infarction[20](#jah31902-bib-0020){ref-type="ref"} to define prior MI on the ECG as (1) any Q wave in leads V2 to V3 ≥0.02 seconds or QS complex in leads V2 and V3; (2) Q wave ≥0.03 seconds or QS complex in any 2 leads of a contiguous lead grouping (I, aVL; V1--V6; II, III, aVF); or (3) R wave ≥0.04 seconds in V1 to V2 and R/S ≥1 with a concordant positive T wave in absence of conduction defect. We defined a Q wave as a negative deflection on the ECG with amplitude ≤−0.1 mV without any initial positive QRS deflection. We defined a QS wave as a negative deflection on the ECG with amplitude ≤−0.1 mV without any positive deflection in the QRS complex.
Myocardial Infarction {#jah31902-sec-0010}
---------------------
We used the ECGs and the end point registry of hospital admissions for MI to categorize the patients into 3 groups: (1) no MI, (2) unrecognized MI, and (3) recognized MI. We defined participants with unrecognized MI as those with findings of MI on the ECG in Tromsø 6 without any clinical event in the end point registry or a registered silent MI in the end point registry up to the date of examination (diagnosis of MI based on echocardiography, ECG, or radionuclide angiogram). We defined participants with recognized MI as those with a clinical event of definite or probable MI, defined as typical or atypical symptoms with either ECG findings of acute MI or elevated cardiac biomarkers.
Selection of Potential Confounding Variables {#jah31902-sec-0011}
--------------------------------------------
We selected age, diabetes mellitus, sex, hypertension, depression, anxiety, physical activity, and smoking as potential confounding variables. Age, diabetes mellitus, and female sex have previously been reported to be associated with increased risk for unrecognized MI[6](#jah31902-bib-0006){ref-type="ref"}, [7](#jah31902-bib-0007){ref-type="ref"}, [10](#jah31902-bib-0010){ref-type="ref"}, [21](#jah31902-bib-0021){ref-type="ref"}, [22](#jah31902-bib-0022){ref-type="ref"} and are also associated with pain sensitivity.[23](#jah31902-bib-0023){ref-type="ref"}, [24](#jah31902-bib-0024){ref-type="ref"}, [25](#jah31902-bib-0025){ref-type="ref"} Diabetes mellitus was defined as glycated hemoglobin \>6.5 or use of antidiabetic medication. Hypertension is a risk factor for unrecognized MI,[21](#jah31902-bib-0021){ref-type="ref"} and an association between increasing blood pressure and hypoalgesia has been demonstrated.[26](#jah31902-bib-0026){ref-type="ref"}, [27](#jah31902-bib-0027){ref-type="ref"}, [28](#jah31902-bib-0028){ref-type="ref"} We modeled systolic blood pressure as a continuous variable and also included current use of blood pressure--lowering medication. We modeled hypertension (defined as systolic blood pressure \>140 mm Hg, diastolic blood pressure \>90 mm Hg or use of antihypertensive medication) as a dichotomous variable for interaction analyses. Depression and anxiety has been reported to be differently associated with unrecognized and recognized MI,[29](#jah31902-bib-0029){ref-type="ref"} and is associated with increased risk for pain disorders.[30](#jah31902-bib-0030){ref-type="ref"}, [31](#jah31902-bib-0031){ref-type="ref"} Depression/anxiety was measured by Hopkin symptom checklist 10‐item version, and modeled as a dichotomous variable (cutoff ≤1.85). Physical activity is considered protective of coronary heart disease[32](#jah31902-bib-0032){ref-type="ref"}, [33](#jah31902-bib-0033){ref-type="ref"} and was reported to relate to pain sensitivity.[34](#jah31902-bib-0034){ref-type="ref"} Physical activity was self‐reported and divided into 3 levels based on the participants' answer to the question of whether their average physical activity in leisure time was limited to "reading, watching TV, or other sedentary activity," "walking, cycling, or other forms of exercise at least 4 hours a week" (eg, walking or cycling to place of work, Sunday walking)," or "participation in recreational sports, heavy gardening, etc (note: duration of activity at least 4 hours a week)." It was modeled as a categorical variable. Smoking is an established cardiovascular risk factor that is also linked to pain sensitivity.[35](#jah31902-bib-0035){ref-type="ref"} Smoking was self‐reported and defined as "current daily smoker," "former daily smoker," or "never daily smoker" and modeled as a categorical variable.
Statistical Analyses and Data Management {#jah31902-sec-0012}
----------------------------------------
We calculated descriptive statistics for 3 groups: participants with recognized MI, participants with unrecognized MI, and participants without MI. We used Pearson\'s chi‐square test to compare categorical variables and *t* test to compare continuous variables between unrecognized and recognized MI.
We used the Cox proportional hazard model to compare cold pressor tolerance between unrecognized and recognized MI. Since we could only study the association between cold pressor pain and MI and not causality, and because time to withdrawal of the hand is right‐censored data, we used time to withdrawal as the time to event in the Cox model. Data were right‐censored if the participant endured the cold pressor test to the maximum 106 seconds. Participants with no prior MI were excluded from the main analyses. MI was included as a binary variable (prior recognized MI, prior unrecognized MI). We used participants with recognized MI as the reference group. Hazard ratios (HRs) of aborting the cold pressor test were calculated with 95% CIs. Compared with the reference group, HRs \<1 indicated higher tolerance, whereas HRs \>1 indicated lower tolerance for pain. Potential confounding factors (listed in Table [1](#jah31902-tbl-0001){ref-type="table-wrap"}) were included in multivariable models. We examined interactions by adding cross‐product terms of MI group and each of the potential confounding variables to the model. Evaluation of Schoenfeld residuals and inspection of log‐log survival plots did not indicate that the proportional hazards assumption was violated. We performed additional analyses including participants without MI, using this group as a reference group. This was done to describe the relationship of pain sensitivity in the general population without MI with that of persons with unrecognized and recognized MI. All analyses were preplanned and performed in STATA (version 12.0, Stata Corp, College Station, TX).
######
Characteristics of the Study Population by MI Status----The Tromsø Study, 2007--2008
No Prior MI (n=4235) Unrecognized MI (n=387) Recognized MI (n=227) *P* Value[a](#jah31902-note-0003){ref-type="fn"}
--------------------------------------------------------------------- ---------------------- ------------------------- ----------------------- --------------------------------------------------
Age, y 62±9 64±8 68±8 \<0.001
Women 2482 (59) 145 (37) 49 (22) \<0.001
Systolic blood pressure, mm Hg 139±22 144±23 139±23 0.01
Current use of blood pressure medication 1032 (24) 125 (32) 127 (56) \<0.001
Hypertension[b](#jah31902-note-0004){ref-type="fn"} 2380 (56) 255 (66) 168 (74) 0.036
Smoking habits 0.002
Current daily smoker 789 (19) 63 (17) 38 (17)
Former daily smoker 1935 (46) 195 (52) 142 (64)
Never daily smoker 1511 (36) 129 (33) 47 (21)
Diabetes mellitus[c](#jah31902-note-0005){ref-type="fn"} 279 (7) 35 (9) 28 (12) 0.20
Physical activity 0.65
Sedentary lifestyle (reading, watching TV) 698 (18) 72 (21) 41 (21)
Walking, cycling, or other forms of exercise \>4 h/wk 2544 (66) 218 (62) 123 (63)
Participation in recreational sports, heavy gardening, etc \>4 h/wk 651 (16) 59 (17) 30 (16)
Psychological distress (HSCL‐10 score \>1.85) 483 (10) 32 (8) 25 (11) 0.26
Cold pressor tolerance \<106 s 1338 (32) 95 (25) 76 (33) 0.02
Medication use
Antiplatelet drugs 399 (9) 73 (19) 188 (83) \<0.01
Anticoagulants 81 (2) 13 (3) 32 (14) \<0.01
Statins 498 (12) 82 (21) 200 (88) \<0.01
β‐Blockers 418 (10) 65 (17) 172 (76) \<0.01
ACEIs 582 (14) 79 (20) 84 (37) \<0.01
Weekly use of painkillers (with or without prescription) 697 (16) 42 (11) 23 (10) 0.78
Values are expressed as mean±SD or number (percentage). ACEIs indicates angiotensin‐converting enzyme inhibitors; HSCL‐10, Hopkin symptom checklist 10‐item version; MI, myocardial infarction.
*t* or chi‐square tests comparing unrecognized and recognized MI.
Defined as systolic blood pressure \>140 mm Hg, diastolic blood pressure \>90 mm Hg, or use of antihypertensive medication.
Defined as glycated hemoglobin \>6.5 or use of antidiabetic medication.
We also calculated descriptive statistics for participants included (had undergone cold pressor test and had valid ECG) and excluded (not undergone cold pressor test or no valid ECG) from our analyses.
Results {#jah31902-sec-0013}
=======
Women had fewer MIs than men (7% versus 19%, *P*\<0.001), but a larger proportion of MIs were unrecognized in women than in men (75% versus 58%, *P*\<0.001). Unrecognized MI was present in 387 (8%) and recognized MI in 227 (4.7%) of the 4849 included participants. Baseline characteristics, by MI, are shown in Table [1](#jah31902-tbl-0001){ref-type="table-wrap"}.
Pain Tolerance and Presentation of MI {#jah31902-sec-0014}
-------------------------------------
A total of 1509 participants (31%) aborted the cold pressor test before the maximum time of 106 seconds. Fewer participants with unrecognized MI aborted the cold pressor test compared with those with recognized MI (25% versus 33%, *P*\<0.02). Figure [2](#jah31902-fig-0002){ref-type="fig"} shows Kaplan--Meier curves for time to aborting the cold pressor test, by MI and sex. HRs for aborting the cold pressor test, by MI and sex, are shown in Table [2](#jah31902-tbl-0002){ref-type="table-wrap"}. Participants with unrecognized MI endured the cold pressor test significantly longer than participants with recognized MI (HR for aborting the cold pressor test, 0.64; CI, 0.47--0.88). After adjustment for additional potential confounding factors (mean systolic blood pressure, use of blood pressure--lowering drugs, diabetes mellitus, daily smoking, psychological distress, and physical activity), the association was attenuated and nonsignificant, but the direction of the effect was unaltered.
{#jah31902-fig-0002}
######
HRs for Cold Pressor Tolerance (Aborted Cold Pressor Test), by MI Status and Sex----The Tromsø Study, 2007--2008
Adjusted for Age and Sex[a](#jah31902-note-0006){ref-type="fn"} Multivariable Adjusted[b](#jah31902-note-0007){ref-type="fn"}
------------------------------- ----------------------------------------------------------------- --------------------------------------------------------------- ------ ------------
Women and men
Prior recognized MI (n=227) 1.00 --- 1.00 ---
Prior unrecognized MI (n=387) 0.64 0.47--0.88 0.68 0.46--1.00
Women only
Prior recognized MI (n=49) 1.00 --- 1.00 ---
Prior unrecognized MI (n=145) 0.52 0.33--0.84 0.54 0.28--1.03
Men only
Prior recognized MI (n=178) 1.00 --- 1.00 ---
Prior unrecognized MI (n=242) 0.75 0.49--1.12 0.81 0.50--1.31
HRs indicates hazard ratios; MI, myocardial infarction.
The analyses of both women and men were adjusted for sex.
Adjusted for sex, age, mean systolic blood pressure, use of blood pressure--lowering drugs, diabetes mellitus, daily smoking, psychological distress, and physical activity.
Sex Differences {#jah31902-sec-0015}
---------------
More women aborted the cold pressor test compared with men (38% versus 23%, *P*\<0.0001); however, the association between pain tolerance and infarct recognition was not significantly different in men and women (*P* for interaction=0.14). We also investigated whether the association between infarct recognition and pain tolerance varied with the potential confounders differently in men and women, and none of these 3‐way interactions were significant (results not shown).
Interaction Analyses {#jah31902-sec-0016}
--------------------
We did not find any statistically significant interaction between groups of MI and systolic blood pressure (*P*=0.77), hypertension (yes/no) (*P*=0.32), use of blood pressure--lowering drugs (*P*=0.10), diabetes mellitus (*P*=0.66), daily smoking (*P*=0.32), psychological distress (*P*=0.48), or physical activity (*P*=0.58) with regards to pain tolerance.
Additional Analyses {#jah31902-sec-0017}
-------------------
Table S1 shows additional analyses by adding participants without MI and using this group as the reference group. Participants with unrecognized MI did not endure the cold pressor test statistically significantly longer than participants without MI (HR for aborting the cold pressor test, 0.84; 95% CI, 0.68--1.03). Participants with recognized MI endured the cold pressor test significantly shorter than participants without MI (HR for aborting the cold pressor test, 1.30; 95% CI, 1.02--1.65).
Table S2 shows descriptive statistics for participants excluded and included in our analyses. The excluded participants were older (62±9 versus 66±10 years), more often women (55% versus 60%), hypertensive (58% versus 64%), and above the cutoff of 1.85 for the HSCL score for psychological distress (11% versus 14%).
Discussion {#jah31902-sec-0018}
==========
The main finding of the present study is that participants with unrecognized MI had higher pain tolerance compared with participants with recognized MI. Our results suggest that differences in pain sensitivity affect the perception of MI.
To our knowledge, no previous study has examined pain tolerance in persons with recognized and unrecognized MI. However, 2 studies have examined pain sensitivity in patients hospitalized in the acute phase of MI. One study of 92 MI patients found that patients with painful acute MI had increased sensitivity to heat pain compared with patients with painless acute MI.[36](#jah31902-bib-0036){ref-type="ref"} Another study of 67 persons with ST‐elevation MI found an association between conditioned pain modulation (a test of pain inhibition) and increased patient delay in seeking treatment, suggesting that more efficient pain inhibition reduced pain presentation in acute MI.[37](#jah31902-bib-0037){ref-type="ref"} Both studies examined hospitalized patients in the acute phase of MI and are not directly comparable to our study. However, they imply that increased pain tolerance is associated with less symptomatic MI.
Our findings propose that pain tolerance is associated with recognition of MI. It is plausible that ischemic myocardial pain is modulated through similar central processes as other pain modalities, such as cold pressor pain. This can partly explain our results. Experimental studies have shown that asymptomatic ischemia is associated with attenuated response to pain.[38](#jah31902-bib-0038){ref-type="ref"}, [39](#jah31902-bib-0039){ref-type="ref"}, [40](#jah31902-bib-0040){ref-type="ref"} Differences in central modulation of pain have been proposed as part of the explanation for absence of pain in silent ischemia. Activation of the thalamus is seen in both angina and silent ischemia, but activation of the frontal cortex seems to be necessary for the conscious sensation of myocardial pain.[13](#jah31902-bib-0013){ref-type="ref"}, [41](#jah31902-bib-0041){ref-type="ref"} Comparison of peripheral nerve conduction has shown similar conduction in patients with angina and silent myocardial ischemia, signifying that peripheral nerve transmission is not altered.[40](#jah31902-bib-0040){ref-type="ref"}
It is possible that the size of MI and differences in pathophysiology also influence symptom severity. A recent study reported that coronary microvascular dysfunction was related to silent positive exercise testing in persons with normal coronary arteries,[42](#jah31902-bib-0042){ref-type="ref"} suggesting a different pathophysiology between silent and symptomatic myocardial ischemia. Studies also indicate that unrecognized MIs are smaller[43](#jah31902-bib-0043){ref-type="ref"}, [44](#jah31902-bib-0044){ref-type="ref"} and manifest less regional wall‐motion abnormalities[45](#jah31902-bib-0045){ref-type="ref"}, [46](#jah31902-bib-0046){ref-type="ref"} than recognized MIs. However, other studies show no association between pain ratings and the ischemic area at risk or MI size,[37](#jah31902-bib-0037){ref-type="ref"}, [47](#jah31902-bib-0047){ref-type="ref"}, [48](#jah31902-bib-0048){ref-type="ref"} suggesting that MI size cannot be reliably assessed by the patient\'s symptoms. In the present study, we did not have the opportunity to study these potential influencers on infarct recognition.
Additional analyses shown in Table S1 demonstrate that participants with unrecognized MI do not have a statistically significantly lower pain tolerance than those with no MI. It is, however, possible that persons with no MI have a pain tolerance intermediate to unrecognized and recognized MI, as Figure [2](#jah31902-fig-0002){ref-type="fig"} indicates. However, we cannot exclude the possibility that it is patients with recognized MI who have an increased pain sensitivity compared with those without MI and with unrecognized MI. However, the main purpose of this article was to study differences between recognized and unrecognized MI.
Sex Differences {#jah31902-sec-0019}
---------------
Women are more likely to present without chest pain or to have atypical symptoms in the setting of an acute MI[2](#jah31902-bib-0002){ref-type="ref"}, [49](#jah31902-bib-0049){ref-type="ref"} and the proportion of unrecognized MI is larger in women compared with men.[8](#jah31902-bib-0008){ref-type="ref"}, [15](#jah31902-bib-0015){ref-type="ref"}, [50](#jah31902-bib-0050){ref-type="ref"} Most studies of experimental pain show significant sex differences, although sometimes relatively minor and affected by numerous confounding variables.[51](#jah31902-bib-0051){ref-type="ref"}, [52](#jah31902-bib-0052){ref-type="ref"} When differences are observed, they consistently show that women have higher sensitivity and lower tolerance to pain than men.[53](#jah31902-bib-0053){ref-type="ref"}, [54](#jah31902-bib-0054){ref-type="ref"} It therefore seems contradictory that women have more unrecognized MIs.
As in previous studies, we found a higher proportion of unrecognized MIs in women and that women were less tolerant to pain than men. We found that the association between unrecognized MI and lower pain tolerance was stronger in women than in men, and statistically significant in women only, but the sex difference was not statistically significant (*P* for interaction=0.14). It is possible that the larger proportion of unrecognized MIs in women is explained by the fact that they are more likely to have coronary disease misdiagnosed or dismissed because of deficient knowledge and more difficult diagnostics, and not because they do not experience symptoms of the MI. In addition, it might also be that women recognized as having an MI are those most sensitive to pain, presenting with the most severe symptoms, and therefore are more likely to receive a diagnosis. This can potentially explain the stronger association in women, as we have investigated pain sensitivity in those with unrecognized MI relative to those with recognized MI.
Pain Sensitivity and Clinical Implications {#jah31902-sec-0020}
------------------------------------------
This study contributes to increased awareness of unrecognized MI. More specifically, it contributes to the knowledge that pain tolerance affects the presentation of MI. Absence of chest pain should not lower alertness of doctors towards ischemic heart disease. Questions on pain sensitivity or factors that affect this might be important in the assessment of patients at risk for cardiovascular disease. ECG is a low‐cost and widely available investigative method and should be considered in persons with high cardiovascular risk despite no history of chest pain. Pain is a common symptom when seeking medical assistance and is a crucial factor in how health workers recognize and assess the severity of a disease. An increasing body of evidence suggests that pain sensitivity modulates the clinical expression of disease.[55](#jah31902-bib-0055){ref-type="ref"}, [56](#jah31902-bib-0056){ref-type="ref"} The present study adds to this by showing that pain sensitivity may be of importance in the recognition of MI and contributes with an important aspect to the further research of underlying reasons for unrecognized MI. We encourage researchers of future studies in this field to consider including variables associated with pain sensitivity.
Strengths and Limitations {#jah31902-sec-0021}
-------------------------
Strengths of the present study include the population‐based design, large sample size, rigorous validation of previous recognized MI, and that the technicians were blinded to MI status. There are also some limitations. First, as Q‐wave criteria were used to identify those with unrecognized MI, there is probably a larger proportion of non--Q‐wave MIs among participants with recognized compared with unrecognized MI. It is unknown whether this may have affected the results. Second, we do not know whether participants with unrecognized MI experienced symptoms or whether the MIs were truly silent. Third, more women with increased risk factor levels were excluded from the analyses because they had not undergone the cold pressor test or had no valid ECG. Our main results indicate that the ability of this study to detect differences in pain tolerance between persons with unrecognized and recognized MI, and possibly sex differences, were attenuated because of these differences. Fourth, the cross‐sectional design precludes causal inference. Last, the external validity refers to Caucasian middle‐aged and elderly adults and may not be generalizable to other groups.
Conclusions {#jah31902-sec-0022}
===========
Our findings suggest that persons who experience unrecognized MI have reduced pain sensitivity compared with persons who experience recognized MI, adjusting for age and sex. This may partially explain the lack of symptoms associated with unrecognized MI.
Sources of Funding {#jah31902-sec-0024}
==================
This work was funded by the Norwegian National Advisory Unit on Women\'s Health, Oslo University Hospital, Oslo, Norway.
Disclosures {#jah31902-sec-0025}
===========
None.
Supporting information
======================
######
**Table S1.** Hazard Ratios (HRs) for Cold Pressor Tolerance (Aborted Cold Pressor Test) With No Prior Myocardial Infarction (MI) as Reference, by MI Status and Sex----The Tromsø Study, 2007--2008
**Table S2.** Characteristics of Participants Included and Excluded From Analyses----The Tromsø Study, 2007--2008
######
Click here for additional data file.
The authors would like to thank all of the participants and technicians of the Tromsø Study.
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#Sec1}
============
Talus fractures are rare yet serious fractures that usually necessitate operative intervention with a relatively high risk of complications \[[@CR1]\].
Peroneal tendon dislocation is a commonly missed soft tissue injury which may have a significant impact on the outcomes. Available literature suggests that operative management offers better functional outcomes for such injuries \[[@CR2]\]. Complications, including persistent pain and swelling, have been reported. It has been associated with other hindfoot injuries such as calcaneal fractures as well as talus fractures \[[@CR3], [@CR4]\].
The Fleck sign is a cortical avulsion fracture of the distal tip of the lateral malleolus, also known as rim fracture or fibular sleeve avlusion fracture, van Dijk et al. \[[@CR5]\] which is best visualized in internal rotation views. It has been reported to be associated with peroneal tendon dislocation in talus fractures \[[@CR4]\].
Aim {#Sec2}
---
This study aims to determine the prevalence of peroneal tendon dislocation in talar fractures as well as the diagnostic value of the fleck sign.
Materials and methods {#Sec3}
=====================
After obtaining approval from the Institutional Review Board (IRB), the authors retrospectively reviewed 93 consecutive talus fractures in the period between January 1, 2011, to January 1, 2018, at a tertiary care center with a well-established foot and ankle service.
Inclusion criteria were:
The patient underwent open reduction and internal fixation, had a pre-operative CT scan that is available for review, had three views of ankle plain radiographs, and the operative report is available on EMR.
Three independent reviewers (AA, KM, and TT) studied the CT scans for peroneal tendon dislocations, plain radiographs and CT scans for the fleck sign (Fig. [1](#Fig1){ref-type="fig"}), and fracture classification, if any. Peroneal tendon dislocation was assessed from the axial and coronal CT images according to the criteria of Ho et al. \[[@CR6]\]. In the axial image, the peroneal tendon should lie in a triangle formed by the posterolateral margin of the distal fibula, the superior peroneal retinaculum, and the calcaneofibular ligament. In the coronal image, the peroneal tendons should lie within the fibular groove. When dislocated, the tendon lies lateral and anterior to the posterolateral margin of the distal fibula. Figure [2](#Fig2){ref-type="fig"} Subluxations and dislocations were both considered dislocations for the feasibility of the study and statistical analysis. Hawkins classification was used for talus neck fractures. Any dispute was resolved by the senior author (MM). The three reviewers had an almost perfect inter-observer agreement. Patient records were also reviewed for laterality, age, sex, mode of injury, associated injuries and operative interventions, and intra-op findingsFig. 1Axial CT cut (Fig. [2a](#Fig2){ref-type="fig"}) and internal rotation ankle plain X-ray (Fig. [2b](#Fig2){ref-type="fig"}) showing talar neck fracture and fleck sign (circled)Fig. 2Axial cut from CT scan of a left ankle showing anterior dislocation of peroneal tendons (circled)
A total of 50 talus fractures matched the inclusion criteria. Almost all the patients were males. Mean age was 32.5 years (SD = 9.5 years). The predominant mode of injury was falling from a height followed by road traffic accidents. Most of the fractures were closed while open fractures accounted for 16% of the fractures (Table [1](#Tab1){ref-type="table"}).Table 1Patient Demographics. Abbreviations: FFH= Fall from height, HO= Heavy object, RTA= road traffic accident, M= Male, F=FemaleMean age (years) (SD)32.5 (9.5)Gender M4998% F12%Mechanism of injuryNumberPercentageFFH2142.0HO48.0RTA1122.0Unknown1428.0Open fracture?NumberPercentageClosed4284.0Open816.0
Talus fractures were isolated in 23 ankles (46%). Others were associated with other foot and ankle injuries. These injuries were further classified anatomically into ankle malleolar fractures, calcaneal fractures, and midfoot fractures (Table [2](#Tab2){ref-type="table"}).Table 2Associated foot and ankle injuriesAssociated injuriesAnkle (lat mal/med mal/bimalleolar)15Calcaneus7Midfoot6None22
Talus fractures were classified according to fracture location into neck, body, and posterior processes. Neck fractures were further classified according to Hawkins classification. Interestingly, a majority of the fractures (72%) were neck fractures, mostly Hawkins type 1.
Results {#Sec4}
=======
Peroneal tendon dislocation was found in ten ankles out of 50 (20%). Almost all dislocations occurred in talar neck fractures, and up to 50% of Hawkins type III and IV talar neck fractures were associated with peroneal tendon dislocation. Risk of dislocation increased with the severity of the fracture (Table [3](#Tab3){ref-type="table"}).Table 3PT dislocation with talus fracture locationFracture locationNumberPT dislocationNeck368/36 (22.2%)H1201/20 (5%)H273/7 (42.8%)H352/5 (40%)H442/4 (50%)Body72/7 (28.5%)Post process60Total50
In terms of concomitant injuries with peroneal tendon dislocation cases, there were three cases of ankle fractures, while one patient had calcaneum fracture. However, the association between those fractures in peroneal tendon dislocation did not reach statistical significance.
The Fleck sign was present in five out of ten PT dislocations both on plain radiographs and CT scans. There was a statistically significant correlation between the fleck sign and PT dislocation (*p* = 0.005).
Table [4](#Tab4){ref-type="table"} summarizes the clinical details of those who had peroneal tendon dislocation associated with talus fracture.Table 4Summary of peroneal tendon dislocation casesCaseAge (years)SexR/LMech of injuryTalus fractureClassificationAssociated injuriesFleck signSurgery for talusSurgery for PTDiagnosed by radiologistCase 126MRRTAClosedNeckHawkins IIOCDYORIFNoneNCase 225MRFFHOpenNeckHawkins IVBimalleolarNEx fix + ORIFNoneNCase 334MRHOOpenNeckHawkins IVLat mal, heel pad avulsion, navicular, med cuneiformNORIFanchor repairYCase 425MLFFHClosedNeckHawkins IIInoneNORIFanchor repair, Revision (lat mal pain)NCase 529MLFFHClosedBodycalcaneumYORIFnoneNCase 637MLFFHOpenNeckHawkins IIIMedial MalYORIFNoneNCase 728MLFFHClosedNeckHawkins IBimalleolarYORIFNoneNCase 838MRUnknownClosedNeckHawkins IICuboidYORIFNoneNCase 924MRUnknownClosedNeckHawkins IINoneNORIFNoneNCase 1041MLRTAClosedBodyNoneNORIFAnchor repairY
Discussion {#Sec5}
==========
Anatomy of the peroneal tendons is crucial to understanding. Peroneal tendons are contained within a common synovial sheath that splits at the level of the peroneal tubercle. The sheath runs in the retromalleolar sulcus on the fibula that is deepened by a fibrocartilaginous rim. They are covered by superior peroneal retinaculum (SPR) which originates from the posterolateral ridge of the fibula and inserts onto the lateral calcaneus at the peroneal tubercle. The inferior aspect of the SPR blends with the inferior peroneal retinaculum. SPR is thought to be the primary restraint of the peroneal tendons within the retromalleolar sulcus \[[@CR7]\]. It is suggested that the axial loading force associated with falling from height leads to disruption of lateral ankle structures including the SPR which consequently leads to peroneal tendon dislocation or instability. Moreover, the presence of the fleck sign has been reported by multiple studies on calcaneal fractures to be predictive of rim fractures \[[@CR8], [@CR9]\]. Reviewing the axial and coronal CT images using Ho et al. criteria is suggested. It is also worth mentioning that CT scans may overestimate the prevalence of peroneal tendon dislocation; hence, intraoperative assessment of peroneal tendon stability is paramount for proper management \[[@CR10], [@CR11]\].
We have demonstrated that peroneal tendon dislocation is associated with talus fractures and that its incidence is higher in patients with a fleck sign. This finding should compel surgeons to have a high index of suspicion when treating such injuries. The incidence is higher in neck fractures and increases with the severity of the fracture on Hawkins classification \[[@CR12]\].
Unfortunately, most of the dislocations (7 out of 10) were missed and no additional procedures were done to address such an injury. Functional outcomes of those patients are not documented on the EMR. The other three patients required additional anchor fixation and, out of those three patients, one patient required revision of peroneal tendon fixation at a later stage. Only two dislocations were diagnosed by the musculoskeletal radiologist, an unfortunate yet common occurrence. Literature reports 75 to 90% of injuries being missed by the radiologists \[[@CR11], [@CR13], [@CR14]\].
Approximately one-quarter of the patients who underwent internal fixation for a fracture of the talus had evidence of peroneal tendon dislocation. This figure is comparable with that of dislocation associated with calcaneal fractures \[[@CR15]\]. However, associated injuries have not affected the incidence of peroneal tendon dislocation in our study and the association was statistically insignificant.
Peroneal tendon dislocation occurred almost exclusively in talar neck fractures which had a statistically significant correlation. It also occurred more frequently in patients with more severe fractures (Hawkins types III and IV) than in patients with less severe fractures (Hawkins types I and II), although the difference did not reach statistical significance. Our findings are consistent with previous studies of talus fractures, as well as calcaneus fractures \[[@CR4], [@CR9], [@CR11], [@CR15], [@CR16]\].
The importance of recognition of peroneal tendon dislocation is that those injuries left untreated are associated with significant morbidity including lateral ankle instability, pain, and clicking. Chronic dislocation is associated with tear of the peroneus brevis due to attrition against the posterolateral ridge of the fibula. This adds to the morbidity of talus fractures and may lead to inferior outcomes of surgical treatment (17--19). Another implication of the presence of peroneal tendon dislocation is that it may affect approach selection for open reduction and internal fixation to allow for examination and treatment of the peroneal dislocation while maintaining maximum exposure. The surgeon may choose to use a posterolateral approach just lateral to the Achilles tendon utilizing the interval between the peronii and flexor halucis muscles in order to tackle the talus fracture as well as peroneal tendon dislocation. The recommended examination technique by Chen et al. is to introduce a Freer elevator within the peroneal tendon sheath followed by application of anterior and lateral forces. Any advancement over the anterior border of the fibula is diagnostic \[[@CR10]\]. An anterolateral approach may also be utilized by making an incision in line with the fourth ray between the tibia and fibula, just lateral to the extensor digitorum longus; however, this approach is not ideal for tackling the peroneal tendons. The choice of the lateral approach needs to be carefully considered if a combined anteromedial approach is also indicated to ensure sufficient skin bridge to avoid wound healing complication \[[@CR20]\]. Variables such as the surgical approach, age, and gender were found to influence the microcirculation of the lateral hindfoot and those need to be considered in the decision-making process as well \[[@CR7]\].
There is a number of limitations to this study. First, we only included patients whose talar fracture underwent operative fixation. Minimally displaced talus fractures that were treated conservatively may not necessarily have the same incidence of peroneal tendon dislocation.
Secondly, CT scans may not always show peroneal dislocation; however, we reviewed the operative reports for peroneal tendon dislocation and two dislocations have been identified intra-op despite inconclusive CT scans. Although MRI evaluation of peroneal tendons might be superior to CT scans, it is not readily available for all cases and needs prior arrangement. Moreover, it leads to additional costs and delays in surgical management. Routine MRI for all talus fractures is not feasible; hence, we relied on CT scans that are usually requested for displaced talar fractures at our institute, are more cost effective, and can be done in the presence of an MRI-incompatible external fixator. Another limitation is that functional outcomes of those cases with peroneal tendon dislocation were not uniformly recorded, which precludes comparison to those with talus fractures with no concurrent peroneal tendon dislocation. However, morbidity associated with isolated peroneal tendon dislocation has been described in literature and touched upon earlier in this discussion \[[@CR17]--[@CR19]\].
Lastly, the authors acknowledge the retrospective nature of the study as well as the number of subjects included. We believe that more patients could have been included whose data were lost when migrating from paper-based to EMR a few years back, not to mention the rarity of talus fracture. However, this study has the most significant number of subjects reported in the literature so far \[[@CR21]--[@CR23]\].
Conclusion {#Sec6}
==========
Peroneal tendon dislocation is associated with as high as 20% of talus fractures. The authors recommend carefully reviewing CT scans by surgeons and radiologists alike to avoid missing such injury and allow for appropriate surgical approach utilization.
The Fleck sign is a highly specific radiographic sign that has a statistically significant correlation with PT dislocation; hence, we recommend intra-operative assessment of peroneal tendons in patients with a fleck sign.
**Publisher's note**
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Open Access funding provided by the Qatar National Library.
| {
"pile_set_name": "PubMed Central"
} |
Introduction
============
Lung cancer is a major cause of morbidity and mortality worldwide and the most common cause of cancer-related death ([@b1-or-34-06-2871]). Non-small cell lung cancer (NSCLC) accounts for \~85% of all lung cancers. Although surgical and chemotherapeutic treatments have made great contributions in lung cancer, these methods may induce serious long-term adverse effects. Various natural herbal products have gained increasing attention due to their potential anticancer effects against NSCLC ([@b2-or-34-06-2871],[@b3-or-34-06-2871]).
Phloretin (Ph) (2′,4′,6′-trihydroxy-3-(4-hydroxyphenyl)-propiophenone) is a natural polyphenolic compound existing in apples, pears and other plants of the rosaceae family and has been found to have anti-inflammatory and immunosuppressive effects on both lymphoid- and myeloid-derived cell lines ([@b4-or-34-06-2871]). Ph has also been shown to have antitumor activities by inducing apoptosis in human leukemia cells, bladder cancer and human colon cancer cells ([@b5-or-34-06-2871]--[@b7-or-34-06-2871]), and inhibiting the growth, invasiveness and migration of human liver cancer cells ([@b8-or-34-06-2871]). However, little is known about its effects on human lung cancer cells.
In the present study, we investigated the possible anticancer effects of Ph on A549 lung adenocarcinoma cells *in vitro* and *in vivo*, and discussed the underlying molecular mechanisms. We demonstrated that Ph could inhibit A549 cell proliferation by inducing apoptosis, and that upregulation of JNK, ERK, Bax and P38 MAPK by Ph was associated with the downregulation of Bcl-2 and NF-κB, and the activation of caspase-3 and -9, and P53, suggesting that Ph may be a useful plant product for the treatment of lung cancer.
Materials and methods
=====================
Chemicals and reagents
----------------------
Ph, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethylsulfoxide (DMSO) were purchased from Sigma Chemical Co. Dulbecco\'s modified Eagle\'s medium (DMEM) and fetal bovine serum (FBS) were obtained from Life Technologies. FITC-Annexin V/PI apoptosis detection kit was purchased from BD Biosciences. Ph stock solution was prepared into 50, 100 and 200 *µ*M concentrations in DMSO and stored at -20°C. The final concentration of DMSO for all treatments was consistently \<0.1%. Specific inhibitors for JNK1/2 (SP600125), ERK1/2 (U0126) or P38 (SB202190) were purchased from Calbiochem. The following antibodies were used: JNK1, p-JNK1/2 (Cell Signaling), P53, cleaved caspase-3, caspase-9, NF-κB, MMP-9 (Santa Cruz), P38, p-P38, ERK1/2, p-ERK1/2, Bcl-2, Bax and PARP (Bioworld Technology), and GAPDH (Sigma).
Cell culture
------------
Human NSCLC A549, H1299 and bronchial epithelial cells (Beas-2b) were from the Institute of Biochemistry and Cell Biology (Shanghai Institutes for Biological Sciences, CAS). Cells were maintained in DMEM supplemented with 10% FBS in a humidified incubator under 5% CO~2~ at 37°C.
In vitro cytotoxicity assay
---------------------------
MTT was performed as previously described ([@b9-or-34-06-2871]). Cells were cultured into a 96-well plate (1×10^4^/well), stimulated with different concentrations (0, 25, 50, 100 and 200 *µ*M) of Ph in culture medium when the cells were 80--90% confluent. After 48 h Ph of stimulation, the medium was removed and 100 *µ*l MTT was added to each well (0.5 mg/ml final concentration) for further incubation for 4 h. Then the medium was removed, and 100 *µ*l DMSO was added to dissolve the solid formazan for 15 min. The absorbance of each well was read at 570 nm using a microplate reader (Thermo Fisher).
Fluorescence observation of cell death
--------------------------------------
A549 cells (6×10^3^ cells/well) on 96-well plates were incubated with phosphate-buffered saline (PBS) control and Ph (50, 100 and 200 *µ*M) for 24 h, then treated with Hoechst 33342 (10 mg/ml) for another 1 h, stained with propidium iodide (PI; 100 mg/ml) for 15 min ([@b10-or-34-06-2871]), washed with PBS three times and observed on Operetta high content analysis system (Perkin-Elmer).
Annexin V/PI double staining
----------------------------
To detect apoptosis in A549 cells after exposure to Ph, the FITC-Annexin V apoptosis detection kit was used to quantify the number of cells in different stages of cell death. Briefly, A549 cells seeded into 6-well plates, and treated with PBS control and Ph (50, 100 and 200 *µ*M) for 48 h. Then, 1×10^5^ cells were re-suspended in 100 *µ*l 1X binding buffer. After addition of FITC-Annexin V and PI (5 *µ*l each), the cell suspension was gently vortexed and incubated for 20 min at room temperature in the dark. After addition of 400 *µ*l 1X binding buffer to each tube, cells were analyzed by flow cytometry (BD Calibur).
Cell cycle analysis
-------------------
To determine the effect of Ph on the cell cycle, A549 cells were seeded into 6-well plates, treated with PBS control and Ph (50, 100 and 200 *µ*M) for 48 h, fixed with 70% ethanol at 4°C for 30 min, and then incubated for another 30 min in the dark, at room temperature with PI buffer \[50 mg/ml containing ribonuclease A (50 ng/ml)\]. Cell cycle distribution was analyzed for 10,000 collected cells with Aria II flow cytometer (BD Biosciences).
Transwell migration assay
-------------------------
The effect of Ph on migration of A549 cells was further analyzed using Transwell chambers with 8-mm porous membrane (Corning, Corning, NY, USA). Cells were treated with PBS control and different concentrations (10, 20 and 40 *µ*M) of Ph for 24 h, and then loaded into the migration chamber at 1×10^5^. Medium containing 10% FBS was placed in the lower chambers. After allowing cell migration for 6 h, cells were removed from the upper side of the membrane; migratory cells on the lower side of the membrane were fixed with 4% paraformaldehyde for 20 min, and then washed with PBS three times before being stained with crystal violet for another 10 min ([@b11-or-34-06-2871]). The number of migratory cells was counted by fluorescence microscopy (magnification, ×100).
Western blotting
----------------
A549 cells were seeded into 6-well plates and incubated with PBS control and different concentrations (50, 100 and 200 *µ*M) of Ph for 48 h, lysed in RIPA buffer (50 mM Tris-HCl, pH 7.2, 150 mM NaCl, 1% NP40, 0.1% SDS, 0.5% DOC, 1 mM PMSF, 25 mM MgCl~2~, supplemented with a phosphatase inhibitor cocktail) and finally subjected to immunoblotting analysis with indicated antibodies. GAPDH were diluted to 1:2,000, P53, cleaved caspase-3 and -9, NF-κB, MMP-9 were diluted to 1:200; and JNK1, p-JNK1/2, P38, p-P38, ERK1/2, p-ERK1/2, Bcl-2, Bax and PARP were diluted to 1:1,000.
In vivo antitumor effect
------------------------
Female nude mice (BK Biotech) aged 5 weeks were used. A549 cells (5×10^6^) were suspended in Matrigel (BD Biosciences) and injected subcutaneously (s.c.) into the mice. All animal procedures were performed following the protocol approved by the Institutional Animal Care Committee of Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). Mice bearing evident tumors were randomly divided into PBS control group, low-dose (10 mg/kg) Ph group, and high-dose (20 mg/kg) Ph group. Ph was dissolved in PBS for intraperitoneal (i.p.) administration to the mice every two days for three weeks. Animals were euthanized with carbon dioxide. Tumor masses were isolated and tumor weight was measured as previously described ([@b12-or-34-06-2871]).
Statistical analysis
--------------------
Results are expressed as mean ± SD (range) or percentage. The difference between two groups was analyzed using the Student\'s t-test. Statistical analyses were performed using the one-way analysis of variance (ANOVA) followed by Tukey\'s post hoc test when more than three groups were analyzed. A P-value \<0.05 was considered to indicate a statistically significant result. The differentiation of amount of protein expressions were calculated using Image Lab version 4.0 software (Bio-Rad Laboratories, Inc.). All calculations were performed using GraphPad Prism software (GraphPad Software, San Diego, CA, USA).
Results
=======
Cytotoxic effects of Ph-treated A549 and H1299 cells
----------------------------------------------------
The chemical structure of Ph is shown in [Fig. 1A](#f1-or-34-06-2871){ref-type="fig"} ([@b13-or-34-06-2871]). The cytotoxicity comparison of Ph on A549, H1299 and Beas-2b cells was evaluated. After cells were treated with different concentrations (0, 25, 50, 100 and 200 *µ*M) of Ph for 48 h, Ph exhibited a moderate effect on normal human Beas-2b cells, and Ph had more cytotoxic effects on A549 than H1299 cells ([Fig. 1B](#f1-or-34-06-2871){ref-type="fig"}). Furthermore, A549 cancer cells were dose- and time-dependently observed when the cells were treated with Ph for 6, 24, 36 and 48 h ([Fig. 1C](#f1-or-34-06-2871){ref-type="fig"}).
Ph-induced cell death in A549 cells
-----------------------------------
To further compare the cytotoxicity between different concentrations of Ph on A549 cells, cell death assay was performed on Operetta high content analysis system. A549 cells were incubated with PBS control and different concentrations (50, 100 and 200 *µ*M) of Ph for 24 h, and then double stained with Hoechst 33342 (blue indicates the nucleus) and PI (red indicates dead cells). As shown in [Fig. 2](#f2-or-34-06-2871){ref-type="fig"}, Ph significantly increased the cell death rate in a dose-dependent manner, and A549 cells became more curved and thinner with the concentration of Ph increasing.
Ph-induced cell apoptosis in A549 cells
---------------------------------------
To determine whether the inhibitory effect of Ph on cell viability was associated with the induction of cell apoptosis, A549 cells were treated with different concentrations (0, 50, 100 and 200 *µ*M) of Ph for 24 h. As shown in [Fig. 3A](#f3-or-34-06-2871){ref-type="fig"}, in PBS control group, the percentage of cells in the sub-G1 fraction was low (0.43%), and a significant proportion of cells (20.45%) went into sub-G1 phase when treated with Ph at 200 *µ*M. Apoptotic cells with a lower DNA content should fall into similar sub-G1 region in cell cycle on flow cytometric analysis ([@b10-or-34-06-2871]). Cell cycle analysis by flow cytometry showed a dose-dependent increased accumulation of cell population in sub-G1 phase. Annexin V and PI double staining displayed an increased percentage of apoptotic cells and dead cells after Ph treatment for 24 h ([Fig. 3B and C](#f3-or-34-06-2871){ref-type="fig"}). These results suggest that the strong effect of Ph on A549 cells may be due to induction of more apoptosis with the increased concentration.
Effect of Ph on A549 cell migration
-----------------------------------
The potential function of Ph on A549 tumor cell migration was characterized by Transwell migration assay. Cells were treated with indicated concentrations of Ph for 24 h, and loaded into the migration chamber at 1×10^5^. The results showed that Ph treatment slowed down the migration of A549 cells in a concentration-dependent manner. As shown in [Fig. 4](#f4-or-34-06-2871){ref-type="fig"}, 40 *µ*M Ph markedly inhibited the migration of A549 cells.
Ph induces activation of caspase-3 and -9 in A549 cells
-------------------------------------------------------
To further investigate whether caspase activation was involved in Ph-induced apoptosis, activation of caspase-3 and -9 and PARP was detected. As shown in [Fig. 5A](#f5-or-34-06-2871){ref-type="fig"}, exposure of A549 cells to Ph (0, 50, 100 and 200 *µ*M) for 24 h increased the number of cleaved fragments of caspase-3 and -9 in a dose-dependent manner. It is known that PARP is a characteristic marker of apoptosis ([@b14-or-34-06-2871]), the abundance of the cleaved form of PARP was increased compared to the control. Ph treatment at 200 *µ*M for 24 h increased the expression level of cleaved caspase-3 and -9 and cleavage form of PARP by 2.6-, 1.3- and 1.5-fold compared to the control, respectively. Ph decreased Bcl-2 and NF-κB, and increased the expression level of P53 and Bax in a dose-dependent manner. As shown in [Fig. 5B](#f5-or-34-06-2871){ref-type="fig"}, compared to the control, Ph treatment at 200 *µ*M for 24 h significantly increased the expression level of P53 and Bax by 1.8- and 2.3-fold, respectively, and decreased the expression level of Bcl-2 and NF-κB by 34 and 32%, respectively. These data suggest that caspase-3, and -9, PARP, Bcl-2, Bax, NF-κB and P53 were involved in Ph-induced apoptosis. MMPs can degrade the basement membrane and play main roles in promotion of cancer invasion and metastasis ([@b15-or-34-06-2871]). As anticipated, we also found that the expression level of MMP-9 was decreased in Ph-treated A549 cells by 22%, which is consistent with previous data from the migration assay ([Fig. 4A](#f4-or-34-06-2871){ref-type="fig"}).
Ph-induced apoptosis is involved in the regulation of P38 MAPK and JNK signaling pathways in A549 cells
-------------------------------------------------------------------------------------------------------
MAPK signaling pathway plays an important role in the action of chemotherapeutic drugs in the regulation of apoptosis ([@b16-or-34-06-2871]--[@b18-or-34-06-2871]). To see whether MAPKs were involved in Ph-induced apoptosis, we first examined the activation status of JNK, ERK and P38 by western blotting with antibodies specific to the phosphorylated forms of these kinases. As shown in [Fig. 6](#f6-or-34-06-2871){ref-type="fig"}, treatment of cells with (0, 50, 100 and 200 *µ*M) Ph increased the phosphorylated form of JNK, ERK and P38 in a dose-dependent manner, with the total protein levels remaining steady, indicating the activation of JNK, ERK and P38 in A549 cells. In contrast, A549 cells were pretreated with 25 *µ*M SP600125 (a JNK inhibitor), U0126 (an ERK inhibitor) or SB202190 (a P38 inhibitor) for 45 min, treated with 200 *µ*M Ph for another 24 h, and then cleaved caspase-3 and -9 were analyzed by western blotting. SP600125 and SB202190 treatment significantly attenuated Ph-induced caspase-3 and -9 activation, as shown in [Fig. 7](#f7-or-34-06-2871){ref-type="fig"}. These findings suggest that activation of JNK1/2 and P38 MAPK may play a crucial upstream role in Ph-mediated caspase activation in A549 cells.
Antitumor activity of Ph on A549 lung tumor xenografts
------------------------------------------------------
To further evaluate the tumor-suppressing effect of Ph *in vivo*, a model for tumorigenicity of A549 cancer cells in nude mice was established. A549 cells (5×10^6^ cells) were injected s.c. into the female nude mice aged 5 weeks and weighing \~20 g. After three days, 15 mice bearing visible tumors were equally randomized to a PBS control, a low-dose (10 mg/kg) Ph group, and a high-dose (20 mg/kg) Ph group. Ph was dispersed in PBS and administered i.p. every two days for three weeks. After three weeks, mice were sacrificed and tumors were dissected and weighed. Tumor images and mean tumor weight in each group are shown in [Fig. 8](#f8-or-34-06-2871){ref-type="fig"}. As anticipated, the tumor size was decreased significantly in both Ph groups, compared to that in the control group. The mean tumor mass in low- and high-dose Ph groups was \~61 and 38% of that in the control group respectively, indicating that Ph had an inhibitory effect on lung carcinoma xenograft growth in mice.
Discussion
==========
Lung cancer is the most commonly diagnosed cancer and one of the leading causes of cancer death in males, and was the 4th most commonly diagnosed cancer and the 2nd leading cause of cancer-related death in females in 2008 worldwide. Lung cancer accounted for 13% (1.6 million) of the total cases and 18% (1.4 million) of cancer deaths in 2008 ([@b19-or-34-06-2871],[@b20-or-34-06-2871]). How to enhance antitumor function and expand survival in lung cancer patients has been an open question for decades. Apoptosis (programmed cell death), is not only essential to the development and maintenance of homeostasis during cell growth but plays an important role in the prevention of tumor development ([@b21-or-34-06-2871],[@b22-or-34-06-2871]). Natural herbal products are currently studied for their antitumor activities including apoptosis induction and antiproliferative activities ([@b23-or-34-06-2871]--[@b25-or-34-06-2871]). However, their active components and molecular mechanisms of action are not well understood. Ph is a natural phenol existing in apples and a variety of vegetables ([@b26-or-34-06-2871],[@b27-or-34-06-2871]). Ph has been previously reported with anticancer effects on breast and hepatocellular cancer and colon cancer cell lines ([@b5-or-34-06-2871],[@b12-or-34-06-2871],[@b28-or-34-06-2871]). The present study for the first time demonstrated that Ph induced apoptosis and inhibit migration of NSCLC A549 cells.
During the apoptotic process, pro-apoptotic Bcl-2 members such as BAX redistribute from the cytosol to mitochondria, resulting in increased membrane permeability. Induction of BAX results in a downstream program of mitochondrial dysfunction and activation of caspases. Due to this event, the released mitochondrial cytochrome participates in this process, leading to caspase-9 activation and subsequent activation of caspase-3 ([@b29-or-34-06-2871]), thus increasing the cleavage form of PARP and inducing A549 cell apoptosis. It was found in the present study that the expression of BAX and fractured PARP protein was increased, the expression of Bcl-2 was decreased, and caspase-3 and -9 were activated in a dose-dependent manner after Ph treatment. In addition, protein MMP-9 was inhibited after Ph treatment, particularly in the 200 *µ*M group. These findings are consistent with the results of cell apoptosis assay and migration assay in the previous experiments. These results proved that Ph not only induced mitochondrial activation-mediated apoptotic cell death but inhibited migration of A549 cells.
Previous studies have suggested that MAPKs can be induced by various compounds and are involved in cell death in NSCLC A549 cells ([@b30-or-34-06-2871],[@b31-or-34-06-2871]). The MAPK family includes three kinase members, including c-Jun NH2-terminal protein kinase/stress activated protein kinases (JNK/SAPKs), P38 MAPK, and extracellular signal-regulated kinase (ERK). Previous results tempted us to ask whether the tumor-suppressing effect of Ph relied on the presence of the P38 MAPK signaling system in A549 cells. To answer this question, we further investigated activation of the MAPK family proteins in Ph-treated A549 cells. The results showed that the phosphorylation of ERK1/2, JNK1/2 and P38 MAPK was increased in Ph-treatment A549 cells in a dose-dependent manner with the total protein level remaining steady. However, treatment with JNK1/2 specific inhibitor (SP600125) or the P38 MAPK specific inhibitor (SB202190) effectively inhibited activation of caspase-3 and caspase-9 induced by Ph, whereas U0126 (an ERK1/2 inhibitor) showed no effect on Ph-induced caspase activation. These findings suggest that activation of JNK1/2 and P38 MAPK plays a critical role in Ph-induced apoptosis in NSCLC A549 cells.
This study was supported by the Key Program of the Shanghai Committee of Science and Technology (no. 12JC1410901) and the National Natural Science Funds of China (no. 81402449).
{#f1-or-34-06-2871}
{#f2-or-34-06-2871}
{#f3-or-34-06-2871}
{#f4-or-34-06-2871}
{#f5-or-34-06-2871}
{#f6-or-34-06-2871}
{#f7-or-34-06-2871}
{#f8-or-34-06-2871}
[^1]: Contributed equally
| {
"pile_set_name": "PubMed Central"
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Erratum to: J Orthopaed Traumatol DOI 10.1007/s10195-014-0292-0 {#Sec1}
===============================================================
The author would like to correct the following error in the publication of the original article:
In the discussion section, eighth sentence of the first paragraph should read as:
"In our study, though there were no significant differences in clinical outcomes, we identified a significant reduction in final VT, UV, and change in VT".
The online version of the original article can be found under doi:10.1007/s10195-014-0292-0.
This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.
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"pile_set_name": "PubMed Central"
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Executive Editors
=================
*Senior Editors*: K.R.Fox, *Southampton, UK*; foxnar\@soton.ac.uk B. Stoddard, *Seattle, WA, USA*; stoddnar\@fhcrc.org
M.Madan Babu, *Cambridge, UK*D.Corey, *Dallas, TX, USA*M.Wegner, *Erlangen, Germany*<[email protected]><[email protected]><[email protected]>J.M.Bujnicki, *Warsaw, Poland*W.Dynan, *Atlanta, GA, USA*E.Westhof, *Strasbourg, France*<[email protected]><[email protected]><[email protected]>M.Churchill, *Aurora, CO, USA*A.D.Sharrocks, *Manchester, UK*J.A.Wise, *Cleveland, OH, USA*<[email protected]><[email protected]><[email protected]>
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S.H.Wilson, *Research Triangle Park, NC, USA*
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1. Introduction {#sec1}
===============
Ulcerative colitis (UC) is a chronic, relapsing, and remitting inflammation of the large intestine \[[@B1]\]. It often starts at young age and lasts throughout life. Debilitating symptoms, such as increased frequency of bloody stools, pain, fever, and lack of effective treatment, lead to the need for surgical removal of the whole large intestine in one-third of the patients \[[@B2]\]. For these reasons it is of great interest to understand the molecular mechanism behind the disease in order to identify new targets that can be modified as therapy. The aetiology and pathogenesis of UC are unclear \[[@B3]\] but are known to include the intestinal microflora, the intestinal barrier function, and the immune system and modifications of these by genetic polymorphisms \[[@B4]\].
Galectins, a family of soluble carbohydrate-binding proteins (lectins), have emerged as one possible therapeutic target in inflammatory bowel disease (IBD). This is based mainly on experiments in animals and cell culture \[[@B5]\] pointing to potential pathophysiology roles in IBD, the fact that galectins tend to be well tolerable, and the therapeutic effects of galectin inhibitors in inflammatory disease of other tissues. Galectins are defined by a conserved carbohydrate recognition domain (CRD) with affinity for *β*-galactosides as found in glycoproteins and glycolipids \[[@B6]\] and occur in different types as shown in [Figure 1](#fig1){ref-type="fig"}. Galectins are synthesized in the cytosol and may have functions there and in the nucleus \[[@B7]\].
An important emerging mechanism of action involves their transfer, by nonclassical secretion, into vesicles or extracellularly, where they encounter *β*-galactoside containing glycoproteins, which they may cross-link. This enables galectins to direct subcellular trafficking, organize membrane architecture, affect cell adhesion, and/or induce cell signals in the same or other cells \[[@B7]--[@B9]\]. This in turn is manifested at the organism level as rate-limiting effects on inflammation, immunity, and cell growth \[[@B10]--[@B12]\]. In the large intestine, additional potential galectin mediated effects exist including modification of barrier function and interaction with microbes \[[@B13]\]. For this reason, galectins represent targets for therapeutic intervention of disease \[[@B14]--[@B16]\].
Various aspects for the role of galectins in IBD pathogenesis have been studied, most of these in mouse colitis models \[[@B5]\]. In these models, both pro- and anti-inflammatory properties of different galectins have been identified \[[@B17]--[@B23]\] and related to concomitant changes in glycan structures \[[@B21], [@B22]\]. Galectin-1 is mainly anti-inflammatory, by induction of apoptosis in T-cells \[[@B5]\]. Galectin-2 has been studied much less but has been found mainly to be anti-inflammatory and supporting wound healing in the intestine \[[@B18]\]. Galectin-3 has been studied extensively and it is required for polarized targeting of some glycoproteins in intestinal epithelial cells \[[@B7], [@B23]\]. It is also highly expressed in macrophages and is mainly proinflammatory but also protects against tissue damage. Recent studies suggest rate-limiting roles of galectin-3 in the fibrosis accompanying some chronic inflammation \[[@B24]\]. The role of these different effects in intestinal inflammation remains unclear \[[@B5]\]. Galectin-4 is highly and specifically expressed in intestinal epithelial cells and may direct polarized trafficking \[[@B25]\] and formation of super rafts \[[@B26]\] as well as being bactericidal \[[@B27]\]. In IBD, galectin-4 has the most distinct pathogenic role as a specific activator of intestinal, but not other, CD4-positive T-cells \[[@B28]\].
Galectins have been studied in human IBD both as serum biomarkers \[[@B29]\] and in tissue samples, but there are restrictions in the clinical interpretation of data obtained since patient classification in terms of disease severity, intestinal inflammatory grade, and pharmacological treatment is limited \[[@B30]--[@B32]\]. Most studies were performed using tissue biopsies obtained by endoscopy that only allow evaluation of a limited amount of superficial mucosal tissue from selected areas of the intestinal wall. Considering the complexity of UC, the aim of this study was to explore the galectin expression in whole intestinal wall surgical specimens, not earlier performed, as well as correlate epithelial galectin-1, galectin-2, galectin-3, and galectin-4 expression with degree of intestinal inflammation.
2. Material and Methods {#sec2}
=======================
2.1. Patients {#sec2.1}
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All patients with IBD undergoing acute or elective colectomy, proctocolectomy, or rectal resection at the Colorectal Unit, Sahlgrenska University Hospital/Östra Campus, from 2008 to 2011 were, prior to surgery, asked to participate in a multidisciplinary IBD research project approved by the Regional Ethical Review Board, University of Gothenburg, Sweden (<http://www.epn.se>). Among 78 included IBD patients, 22 consecutive patients with UC disease were included in this study and patient data is listed in [Table 1](#tab1){ref-type="table"}. Excluded patients were those with UC operated with extirpation of ileal pouch-anal anastomosis (IPAA) and those with Crohn\'s disease. In addition, some patients had to be excluded due to logistic reasons such as lack of laboratory staff and out-of-office-hour surgery. Prior to surgery, all patients were examined and categorized using a modified Mayo-score \[[@B33]\] to measure disease severity with a scale range of 0--12, 12 being most severe disease. Variables measured were frequency of bowel movements (range 0--3), blood in stool (range 0--3), quality of life (range 0--3), and endoscopic evaluation (range 0--3). Only two patients undergoing acute resection were included in this study (cases 3 and 28). Ongoing and previous medications, in particular the use of steroids, 5-ASA, and immunomodulators such as TNF inhibitors, were listed. Median age of included patients was 38 (20--65). Median laboratory tests were haemoglobin 136 (101--156) g/L, C-reactive protein (CRP) 5 (1--160) mg/L, and albumin 38 (20--46) g/L. Symptom duration was 8 (2--25) years. Total modified Mayo-score was median 8 (5--11) and there was no correlation between the Mayo-score and the histopathological inflammatory grade. Indications for surgery were chronic colitis in 19 patients and dysplasia in one patient and two patients underwent surgery due to acute colitis.
The patients excluded due to logistic reasons did not differ concerning age, blood tests, or disease severity. Of 12 excluded patients, 7 were males. Median age was 42 (21--67). Median laboratory tests at surgery were haemoglobin 132 (107--155) g/L, C-reactive protein (CRP) 13 (1--190) mg/L, and albumin 30 (26--40) g/L. Symptom duration was 15 (3--30) years. Total Mayo-score was median 7 (4--11). Eight patients in the excluded group were on oral steroids and one was on locally administrated steroids. Six had oral 5-ASA and one had 5-ASA locally. One patient was on immunomodulating therapy.
Control tissues were obtained from patients undergoing elective resection of the sigmoid colon and right-sided hemicolectomy due to repeated sigmoid volvulus (*n* = 2) or colonic cancer (*n* = 8), respectively. Collection of control tissues was sampled as far as possible (minimum 10 cm) from the tumor.
2.2. Tissue Specimens {#sec2.2}
---------------------
Resected colonic tissue was immediately embedded in a plastic bag in the operating theatre, covered with crushed ice, and transported within 45 minutes to the Department of Pathology. Tissue specimens were collected according to a standardized protocol from each colonic and rectal area; from each patient, 5-6 (rectal resection) to 15--20 separate (colectomy) samples were analysed. In addition, specimens from selected areas were collected according to the pathologist judgment. The specimens were fixed in formalin and embedded in paraffin.
Paraffin sections of full wall colon tissue samples were obtained and stained with haematoxylin-eosin. The inflammatory activity for each patient was graded, according to suggested criteria for grading disease activity in UC \[[@B34]\], as mild (cryptitis), moderate (crypt abscesses), or severe (ulcerations) illustrated in [Figure 2](#fig2){ref-type="fig"}. All specimens were coded and analysed in a blinded fashion by two investigators (Johan Mölne and Mattias Block). The clinical course of each patient as well as the final histopathological evaluation based on all biopsies from each specimen was unknown to the investigators when they were evaluating the inflammatory grade and galectin expression in each tissue slide.
2.3. Anti-Galectin Antibodies {#sec2.3}
-----------------------------
Polyclonal antisera were raised in rabbits and characterized as described for anti-rat galectin-1 (diluted 1 : 800) \[[@B35]--[@B37]\], anti-human galectin-2 (1 : 600) \[[@B38]\], and anti-rat galectin-4 (1 : 50000) \[[@B39], [@B40]\]. A commercially available rat monoclonal anti-mouse galectin-3 (anti-Mac-2, clone 3/38) (1 : 500) \[[@B41]\] has been used extensively by us \[[@B39]\] and others.
2.4. Immunohistochemistry {#sec2.4}
-------------------------
The EnVision Flex High pH (Link) detection kit (Dako K8000, Copenhagen, Denmark) was used. The most important steps were as follows. Consecutive series of paraffin sections were produced at a 4 *μ*m constant thickness setting. Antigen retrieval was done in tris/EDTA buffer, pH 9 (Dako K8004), by microwave oven heating and endogenous peroxidase activity was blocked by immersion in peroxidase-blocking solution (Dako K8000) for 5 minutes at RT. Immunostaining was performed in a computer-assisted Autostainer Plus processor (Dako). Incubation time for primary antibodies was 30 minutes at RT, terminated by repeated washing, followed by incubation with a dextran polymer conjugated with secondary antibodies and horseradish peroxidase (HRP) for another 30 minutes. Slides were transferred to fresh hydrogen peroxide plus 3-3-diaminobenzidine tetrahydrochloride (DAB) solutions for 4 minutes. Finally, slides were stained with Mayer\'s haematoxylin and permanently mounted under cover slips. Omitting or replacing the primary antibodies with irrelevant antibodies produced negative controls. Optimal primary antibody dilutions were defined by staining normal colon using serial dilutions of each antibody.
2.5. Grading of Inflammation and Immunohistochemical Labelling {#sec2.5}
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Intestinal tissue specimens from each patient, 5-6 (rectal resection) to 15--20 separate (colectomy), were studied. Galectin-1, galectin-2, galectin-3, and galectin-4 expressions were examined in 3 representative tissue blocks from each patient. The epithelial cell galectin staining intensity was recorded in epithelia with cryptitis and crypt abscess and in areas adjacent to ulcerations as well as in noninflamed areas as illustrated in [Figure 2](#fig2){ref-type="fig"}. The intensity of immunoperoxidase (IP) staining was recorded on a 4-level scale: negative: 0, trace amounts: 1, weakly positive: 2, and strongly positive: 3. If galectin expression showed a focal pattern this was assigned as (f). Representative tissue sections illustrating galectin-2--galectin-4 expression in noninflamed colon tissue and IBD tissue with abscesses and ulcerations are shown in [Figure 3](#fig3){ref-type="fig"}. Leukocytes were identified by morphology (neutrophil and eosinophil granulocytes) or identified by monoclonal antibodies using CD3 (Dako, N1617) for T-cells, CD68 (Dako, N1576) for macrophages, and CD138 (Dako, IR642) for plasma cells.
3. Results {#sec3}
==========
3.1. Epithelial Galectin Expression in Control Colon Tissue {#sec3.1}
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Galectin-1 in normal colon epithelial cells was negative except in a few cases showing a focal, minimal staining. This is in accordance with earlier studies on normal human colon \[[@B42]\] and stomach \[[@B43]\]. In nonepithelial cells (fibroblasts, endothelium, and smooth muscle), galectin-1 was weakly expressed, as described before \[[@B44]\].
Galectin-2, galectin-3, and galectin-4 were all strongly positive as exemplified in Figures [2(d)](#fig2){ref-type="fig"}, [2(g)](#fig2){ref-type="fig"}, and [2(j)](#fig2){ref-type="fig"}, respectively, and did not show any individual variation ([Table 2](#tab2){ref-type="table"}).
Galectin-2 staining was localised to the entire cytoplasm of the cells but mucus droplets were negative ([Figure 2(d)](#fig2){ref-type="fig"}). An increased staining was seen on the epithelial cell apical membrane ([Figure 2(d)](#fig2){ref-type="fig"}, insert).
Galectin-3 staining was localised to the entire cytoplasm of the cell but mucus droplets were negative ([Figure 2(g)](#fig2){ref-type="fig"}). There was a gradient of galectin-3 staining intensity with strong surface epithelial expression and diminishing expression in crypts.
Galectin-4 in the control group showed, like galectin-2 and galectin-3, a strong expression pattern in all cases. However, compared to galectin-2 and galectin-3, labelling was different with a localised supranuclear distribution (Figures [2(j)](#fig2){ref-type="fig"}, insert, and [2(k)](#fig2){ref-type="fig"}) and no staining was present in the remaining cytosol or plasma membrane.
3.2. Epithelial Galectin Expression in UC Colon (Study Group) {#sec3.2}
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*Galectin-1*. Most of the UC patients, as well as the control individuals, lacked galectin-1 expression in the epithelial cells (not shown). Only a few cases showed a weak focal epithelial cell staining.
*Galectin-2*. In patients with mild inflammatory activity, the epithelial cell galectin-2 expression was identical to that seen in control individuals ([Table 2](#tab2){ref-type="table"}). In those with moderate activity, most patients had a strong galectin-2 expression but some patients with severe activity showed a reduced-to-minimal epithelial expression in all 3 slides (Figures [2(e)](#fig2){ref-type="fig"} and [2(f)](#fig2){ref-type="fig"} and [Table 2](#tab2){ref-type="table"}). Due to a reduced amount of mucus the entire cytoplasm was galectin-2 positive in areas of inflammation (Figures [2(e)](#fig2){ref-type="fig"} and [2(f)](#fig2){ref-type="fig"}).
*Galectin-3*. The colon epithelium showed the same gradient of galectin-3 staining intensity with strong surface expression and diminishing expression in crypts, as in the control group ([Figure 2(g)](#fig2){ref-type="fig"}). In inflamed areas, staining was seen in the whole cytoplasm, as for galectin-2 (Figures [2(h)](#fig2){ref-type="fig"} and [2(i)](#fig2){ref-type="fig"}). A reduced expression of galectin-3 in some individuals with increased inflammatory activity was observed but the majority of patients expressed normal levels in severely inflamed specimens ([Table 2](#tab2){ref-type="table"}).
*Galectin-4*. A decreased galectin-4 expression in several patients with severe inflammatory activity (Figures [2(k)](#fig2){ref-type="fig"} and [2(l)](#fig2){ref-type="fig"} and [Table 2](#tab2){ref-type="table"}) was observed but there was a great interindividual variation. The supranuclear distribution was seen also in inflamed epithelium ([Figure 2(k)](#fig2){ref-type="fig"} insert).
3.3. Individual Changes in Epithelial Cell Galectin Expression {#sec3.3}
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In addition to [Table 2](#tab2){ref-type="table"}, the epithelial galectin expression in areas with cryptitis, for the different individuals grouped according to the inflammatory grade (mild, moderate, and severe), is summarized in [Figure 3](#fig3){ref-type="fig"}. This shows that the galectin expression in the epithelial cells is decreased related to the severity of inflammation. However, when comparing individual patients, changes in epithelial cell galectin expression varied considerably between individual patients reflecting an individual pattern rather than a general reduction in galectin expression ([Table 2](#tab2){ref-type="table"}). In the two cases with mild inflammation there was no change in galectin expression related to the inflammatory grade. Of the 4 cases with moderate inflammatory grade, 2 cases (No 48 and No 50) showed a galectin expression in the normal epithelial cells that was identical to that of the healthy controls (all positive) and no change in their expression was found irrespectively of inflammatory grade. Patients 1 and 14 had very small changes in galectin expression. The 16 patients with severe inflammation showed different pattern of changes in their epithelial cell galectin-2, galectin-3, and galectin-4 expression. These heterogenic patterns can be explained by the focality of UC disease and also individual factors among the patients as shown in [Figure 1](#fig1){ref-type="fig"}, where a patient with severe inflammation showed cryptitis, abscess, and ulceration in the same specimen.
3.4. Galectin Expression in Inflammatory Cells {#sec3.4}
----------------------------------------------
In control colon tissue, inflammatory cells were seen in the lamina propria. Individual cells showed a labelling intensity comparable to inflammatory cells in the study. As expected, the number of inflammatory cells in the control tissue was considerably lower than in the study group.
The majority of inflammatory cells found in the intestinal wall in the study group were macrophages, lymphocytes, and plasma cells identified by morphology and specific CD markers. As expected, neutrophilic granulocytes were seen in areas of cryptitis, crypt abscesses, and ulcerations but were otherwise sparse. In the mucosa, approximately 50% of the leukocytes were plasma cells, 25% macrophages, and 25% T-cells. In the submucosa, the dominating cell type was macrophages, while only occasional and focal infiltrates of lymphocytes and plasma cells were seen.
Galectin-1 and galectin-4 were not present in any inflammatory cells.
Galectin-2 was strongly expressed in inflammatory cells in the majority of the patients irrespectively of the intestinal inflammatory grade. The labelling intensity was comparable to the galectin-2 expression in epithelial cells in very few inflammatory cells (\<2%), while about 50% of the cells showed a weak expression (Figures [2(e)](#fig2){ref-type="fig"} and [2(f)](#fig2){ref-type="fig"}). Almost all of the strongly galectin-2 positive cells were identified as macrophages. However, in total, only a minority (10--20%) of the macrophages were positive. The majority of plasma cells (75--100%) were weakly positive as well as few (\<5%) of the T-cells.
Galectin-3 expression in the inflammatory cell infiltrates varied between individual patients. Both negative and positive as well as trace amount of staining were noted for patients irrespectively of the intestinal inflammatory grade. For patients expressing galectin-3 in inflammatory cells, the expression was weaker compared to galectin-2, with an overall strong expression in \<1% of the inflammatory cells and a weak expression in 10--15%. The majority of the positive inflammatory cells were macrophages and 20--30% of macrophages were positive for galectin-3. About 10% of the plasma cells and \<1% of T-cells were positive for galectin-3 ([Figure 2(h)](#fig2){ref-type="fig"}).
4. Discussion {#sec4}
=============
The UC tissues analysed in this study were obtained after bowel resection. This permits evaluation of the entire intestinal wall as well as several different intestinal regions compared to a limited number of small biopsies collected during endoscopy \[[@B31], [@B32]\]. Another difference compared to previous studies is that the surgical specimens were obtained from patients being at the end of the road regarding medical treatment of their disease and therefore representing, on average, a later state of the disease. However, there was still considerable heterogeneity regarding the degree of inflammation within a single intestine (exemplified in [Figure 1](#fig1){ref-type="fig"}) and also between individual patients. The control tissue in this study was from both the sigmoid area (*n* = 2) and ascending area of colon (*n* = 8) and no difference in galectin pattern was seen irrespective of which anatomical part the samples were collected from, nor was there any difference in galectin pattern depending on the anatomical localisation found in the UC study patients ([Table 1](#tab1){ref-type="table"}). Therefore, it is not likely that the differences in galectin expression depend on the anatomical localisation of the tissue sections.
The galectin expression did not differ considerably between the colon epithelium in the control group and that of epithelial cells having a normal histological appearance in the UC study group. When the galectin expression in areas with cryptitis was summarized for each inflammatory grade ([Figure 3](#fig3){ref-type="fig"}) it seems that a decrease in epithelial galectin expression is correlated to inflammation. However, since the expression pattern for each individual patient was very complex ([Table 2](#tab2){ref-type="table"}) we argue that there was no clear-cut correlation between the expressions of galectin-2, galectin-3, and galectin-4 in the colonic epithelium and the inflammatory activity in our study group of consecutive patients. This is in contrast to some reports that suggest decreased galectin expression in conjunction with intestinal inflammation \[[@B17], [@B19], [@B31]\]. There may be several explanations for this discrepancy. The evaluation of a certain antigen(s) in tissues/cells involves many technical aspects of the methodologies used that affect the quality of analysis. Immunohistochemistry has a well-known variation depending on the techniques for tissue handling and antibody reagents used. Molecular analysis strategies using homogenized biopsies also have limitations due to sampling errors. For example, galectin-3 has been quantified using mRNA reverse transcription technique \[[@B19]\]. However, there is usually a patchy appearance of the intestinal inflammatory process both macroscopically and, especially, at the microscopic level, with differences in intensity between different cryptitis, abscess, and ulceration areas (Figures [1](#fig1){ref-type="fig"} and [2](#fig2){ref-type="fig"}). Therefore, quantification of whole biopsies collected by endoscopy has to be carefully evaluated regarding representativeness of tissue specimens analysed. Furthermore, changes in galectin expression in biopsies will be due to either loss of epithelial cells, reduced expression in individual cells, the amount of infiltrating inflammatory cells, or a combination of these factors. Many of the individual patients express a stable amount of galectins in their epithelial cells independently of the degree of inflammation ([Table 2](#tab2){ref-type="table"}). Therefore, results from previous studies indicating that a reduced galectin expression correlates with inflammation \[[@B19], [@B31]\] may be due to lack of epithelial cells due to tissue damage as well as sampling error of small biopsies and not to a specific downregulation of epithelial galectin biosynthesis. Furthermore, to increase the number of individual cases analysed in this investigation will most likely not result in a statistical significant correlation between galectin expression and inflammation due to the great heterogeneity of epithelial cell galectin expression both within and between individual cases as shown in [Table 2](#tab2){ref-type="table"}. Even if a statistical significant correlation should be obtained, the biological significance of this must be highly questioned.
The tissue localisation of galectin-1--galectin-4 found here agreed in general with previous immunohistochemical studies of human and mouse intestines. In addition, we also found some features not described before. Galectin-1, one of the most studied galectins, has typically not been found in human epithelial cells \[[@B42], [@B43]\], and this was also the case here. This is in contrast to mouse where galectin-1 is expressed in the intestinal epithelial cells \[[@B45], [@B46]\] but also shows a strain variation \[[@B46]\]. Variable expression of galectin-1 has been found in human nonepithelial tissue like muscle, lymphocytes, and fibroblasts \[[@B42]\] but here no or low expression was found. However, this does not rule out a role in colitis, as galectin-1 can bind both epithelial cells and lymphocyte glycans, resulting in apoptosis and other cell regulations \[[@B47]\].
Galectin-2 is the closest compared to galectin-1 but much less studied in mammals. Similar to galectin-1, it is a noncovalent dimer, but unlike galectin-1 it has characteristic localisation to the gastrointestinal tract and it has a different carbohydrate-binding specificity, seemingly adapted to intestinal glycans \[[@B17], [@B18]\]. Thus, it can be regarded as an intestinal paralogue of galectin-1. Here we found high expression of galectin-2 in colon epithelial cells, as reported before, but also the novel observation of high expression in some submucosal macrophages, suggesting an immunoregulatory role. Added galectin-2 can support epithelial wound healing and suppress lamina propria T-cells to ameliorate experimental colitis in mice \[[@B17]\].
Galectin-3, the other most studied galectin, was abundant in epithelial cells and also in some macrophages, in agreement with previous studies \[[@B19], [@B23]\]. Galectin-3 behaves mainly as a proinflammatory protein, and studies using null mutant mice support a rate-limiting role in chronic inflammation with fibrosis in many tissues. In a mice colitis experimental model, intraperitoneally administrated galectin-3 reduced inflammation \[[@B48]\].
In contrast to galectin-2 and galectin-3, which were distributed in the entire cytoplasm, galectin-4 had specific supranuclear localisation presumed to be in the Golgi network. To our knowledge, this has not been reported before. Galectin-4 is expressed only in the digestive tract and restricted to epithelial cells. Previous studies have showed that there is no significant difference in the expression of galectin-4 in epithelial cells from inflamed colon versus controls \[[@B17]\] which is confirmed by this study.
Information regarding galectin expression in the infiltrating inflammatory cells in IBD intestine is very limited. In the macrophages, we found a strong expression of galectin-2 and a weak expression of galectin-3 with a significant interindividual variability, while staining for galectin-1 and galectin-4 was completely negative. There was no increase in the expression of galectin-2 and galectin-3 in the inflammatory infiltrates in the UC study group compared to the expression in inflammatory cells in controls. Reports on galectin expression in intestinal inflammatory cells have, to our knowledge, only been reported for galectin-3. A study of patients with ileal pouch-anal anastomosis \[[@B49]\] revealed a significant decrease in the galectin-3 staining index in subepithelial macrophages in patients with chronic or recurrent acute pouchitis compared to the noninflamed controls. A reduced expression of galectin-3 has been found in intestinal macrophages of patients with Crohn\'s disease but not in UC patients \[[@B50]\]. The observation of a high specific expression of galectin-2 in submucosal macrophages has not been reported earlier. Galectin-2 is evolutionary and structurally most related to galectin-1, but it has a tissue expression and carbohydrate-binding specificity more adapted to glycan structures found in intestinal epithelial cells (galactosides with blood group determinants) \[[@B47]\] and does not bind sialylated glycans as found in serum glycoproteins bound by galectin-1 \[[@B51]\].
5. Conclusions {#sec5}
==============
The findings that several UC patients did not show any changes in colon epithelial cell galectin expression while others showed an individual specific change not correlated to the inflammatory grade indicate that the variation in epithelial galectin expression may not be related primarily to the inflammatory grade but rather to the focal presentation of the disease as well as individual factors not defined at present.
The Ihre Foundation, Sahlgrenska University Hospital, and Gothenburg Medical Society supported this study.
Conflict of Interests
=====================
The authors declare that they have no competing interests.
Authors\' Contribution
======================
Mattias Block, Hakon Leffler, Lars Börjesson, and Michael E. Breimer conceived and designed the experiments. Mattias Block, Johan Mölne, Lars Börjesson, and Michael E. Breimer analysed the data. Mattias Block, Johan Mölne, Hakon Leffler, and Michael E. Breimer wrote the paper. All the authors read and approved the final paper.
![Illustration of the histological features for inflammatory grading of UC colon mucosa according to \[[@B34]\]. Top panel (a) shows a haematoxylin-eosin stained colon section from a patient with severe inflammatory activity ([Table 1](#tab1){ref-type="table"}, patient 11, left colon). Below are shown magnifications of three selected areas illustrating cryptitis (panel (b), marked with black arrows), abscess (c), and an ulceration (d) all present in close proximity to each other. Bar in (a) = 2 mm, (b) = 50 *μ*m, (c) = 100 *μ*m, and (d) = 1 mm. Note that the sizes of biopsies obtained by endoscopy usually are 3-4 mm.](BMRI2016-5989128.001){#fig1}
{ref-type="table"}. The noninflamed epithelium (a) contains cells filled with mucous and the crypts have a normal architecture. Immunohistochemical staining of serial sections from the same specimen for galectin-2, galectin-3, and galectin-4 is shown in rows 2 to 4, respectively. Galectins are predominantly expressed in the colonic epithelium. The inserts illustrate that galectin-2 and galectin-3 are homogeneously distributed in the cytoplasm while galectin-4 has a distinct perinuclear distribution most easily seen in (k) where mucus droplets are less abundant. In addition, a low number of inflammatory cells express a strong staining for galectin-2 and galectin-3, but not galectin-4, and this is illustrated for galectin-2 and galectin-3 by arrowheads in panels (e), (f), and (h). Bar = 200 *μ*m.](BMRI2016-5989128.002){#fig2}
{ref-type="table"}.](BMRI2016-5989128.003){#fig3}
######
Clinical data of patients with UC included in the study. Study patients are grouped according to tissue inflammation degree as mild (cryptitis, *n* = 2), moderate (crypt abscesses, *n* = 4), and severe (ulcerations, *n* = 16) as described in [Section 2](#sec2){ref-type="sec"}. In addition, control colon tissues (*n* = 10) obtained from patients with volvulus and colon cancer were studied.
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No^1^ Gender/age^2^ Performed procedure^3^ Disease duration^4^ Medical therapy\ Mayo-score^6^ CRP\ Infl-grade^8^
SS/SL/AS/AL/IM^5^ mg/L^7^
------- --------------- ------------------------ --------------------- ------------------- --------------- --------- ---------------
18 M/49 C 19 −/−/+/−/− 6 20 Mild
19 F/49 RR 21 −/−/−/−/− 10 5 Mild
1 M/29 C 3 +/−/+/−/+ 7 5 Moderate
14 M/65 RR 10 −/−/+/−/− 10 5 Moderate
48 F/36 C 4 −/−/+/−/+ 10 4 Moderate
50 F/38 C 6 −/−/+/−/+ 5 9 Moderate
2 F/49 C 21 +/+/−/−/− 5 28 Severe
3^9^ F/22 C 7 +/+/−/−/− 10 5 Severe
4 F/53 C 2 +/+/+/−/− 9 5 Severe
5 F/28 RR 6 −/−/−/−/− 9 5 Severe
8 F/52 RR 25 −/−/−/−/− 5 Severe
11 F/38 C 21 +/−/+/−/+ 8 44 Severe
13 F/27 RR 5 +/−/−/−/− 10 5 Severe
15 F/62 PC 8 +/−/+/+/− 7 5 Severe
17 F/33 C 7 +/+/+/+/− 8 160 Severe
22 M/42 C 14 +/−/−/+/− 11 5 Severe
26 F/39 RR 3 −/−/−/−/− 8 5 Severe
28^9^ M/24 C 9 +/−/+/−/− 11 22 Severe
29 M/20 C 7 +/+/+/−/+ 6 18 Severe
40 M/35 RR 6 +/+/−/−/+ 6 1 Severe
52 M/41 PC 5 −/−/−/−/+ 11 2 Severe
53 F/27 C 17 −/−/−/−/+ 10 10 Severe
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^1^No = patient study number in IBD biobank.
^2^M = male. F = female. Age = years.
^3^C = colectomy. PC = proctocolectomy. RR = rectum resection.
^4^Disease duration = years.
^5^SS = steroids systemic. SL = steroids local. AS = azathioprine systemic. AL = azathioprine local. IM = immunomodulation.
^6^Mayo-score = clinical evaluation of the severity of the patient\'s disease.
^7^CRP = C-reactive protein.
^8^Infl-grade = total histopathological evaluation of the patients disease.
^9^Acute surgery.
######
Galectin expression in colon epithelial cells in patients with UC. Study patients are grouped according to degree of tissue inflammation (see [Table 1](#tab1){ref-type="table"}) and normal controls (*n* = 10). Epithelial cell staining intensity for individual anti-galectin antibodies was graded as 0 to 3. Galectin expression is shown for the epithelial cells present in the uninflamed, cryptitis, abscess, and ulceration areas. Areas not applicable (i.e., ulceration in mild inflammation cases) are marked with ---. Staining for galectin-1 was negative in the epithelial cells of both controls and UC cases and is not listed.
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Patient number Inflammatory grade^*∗*^ Epithelial cell galectin expression^\#^
---------------- ------------------------- ----------------------------------------- --- --- ----- ------- ----- -------- ------- ----- ------- ------- -------
18 Mild 3^†^ 3 3 3 3 3 ---^¶^ --- --- --- --- ---
19 Mild 3 3 3 3 3 3 --- --- --- --- --- ---
1 Moderate 2 3 1 2 3 1 2 3 1 --- --- ---
14 Moderate 3 3 3 2 3 3 2 3 3 --- --- ---
48 Moderate 3 3 3 3 3 3 3 3 3 --- --- ---
50 Moderate 3 3 3 3 3 3 3 3 3 --- --- ---
2 Severe 1 3 2 3 1 1 3 1 0 3 3 0
3 Severe 3 3 2 2 1 1 1 1 1 1 3 2
4 Severe 3 3 3 1 1 3 1 3 2 2 2 3
5 Severe 2 (f)^‡^ 3 3 1 1 3 1 3 3 1 2 3
8 Severe 3 3 3 2 3 2 --- --- --- 1 1 1
11 Severe 3 3 3 2 3 2 2 3 2 2 3 2
13 Severe 2 3 2 --- --- --- --- --- --- 1 1 2
15 Severe 3 3 3 3 3 2 3 3 2 3 3 3 (f)
17 Severe 3 3 3 2 3 3 2 3 3 3 3 3
22 Severe 3 3 3 3 3 3 3 3 3 2 3 2
26 Severe 3 3 3 1 3 3 1 3 3 3 (f) 3 2
28 Severe 1 3 1 3 3 (f) 3 3 (f) 3 (f) 3 3 (f) 3 (f) 3
29 Severe 3 3 3 3 2 3 3 2 3 2 2 2
40 Severe 3 3 2 --- --- --- 2 2 3 3 3 3
52 Severe 3 3 1 3 3 1 --- --- --- 3 3 1
53 Severe 3 3 3 3 3 3 3 3 3 3 3 3
Controls\ Normal 3 3 3 --- --- --- --- --- --- --- --- ---
*n* = 10
---------------------------------------------------------------------------------------------------------------------------------------------------------------
^*∗*^Histopathological classification of inflammatory activity.
^\#^Galectin-2, galectin-3, and galectin-4 expression in epithelial cells present in the area.
^†^Galectin expression is graded according to a 4-level scale as follows: 0 = negative, 1 = trace, 2 = weak expression, and 3 = strong expression.
^¶^Empty fields (not marked with 0--3) mean no presence of cryptitis, abscess, or ulceration.
^‡^Focal distribution in some cells in the area.
[^1]: Academic Editor: Atsushi Sakuraba
| {
"pile_set_name": "PubMed Central"
} |
INTRODUCTION
============
The anterior clinoid process (ACP) is the apex portion of the lesser wing of the sphenoid bone. ACP\'s anatomical location is important for its relation with neighboring structures including optic nerves, internal carotid artery (ICA), and other neurovascular structures.[@B7] Anterior clinoidectomy (AC) is an essential technique that is of utmost importance for clipping aneurysms involving the paraclinoid region. Dolenc introduced the extradural AC (EAC) and opening of the optic sheath in 1985.[@B2] We can retract the brain minimally to remove the whole ACP since the dura provides a natural barrier to preserve the neurovascular structures from drilling, and separates the subarachnoid space from the bone dusts. We Reports the current techniques for EAC with clipping of paraclinoid aneurysm.
MATERIALS AND METHODS
=====================
Two patients with paraclinoid aneurysm (1 ruptured, 1 unruptured) were treated, and it was found that pterional craniotomy and EAC are more suitable than coil embolization for the clipping of aneurysm. The patients were females aged 42 and 46. One patient was diagnosed using magnetic resonance imaging (MRI) due to a visual field defect, and the other patient was diagnosed using computed tomography (CT) due to the presence of intracerebral hemorrhage with ruptured aneurysm.
SURGICAL TECHNIQUE
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Pterional approach with interfacial muscle dissection
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The ipsilateral pterional approach is often used for treating paraclinoid aneurysms. The patient\'s head is elevated 10-30 degrees and rotated 30-45 degrees to the contralateral side and fixed onto the Mayfield head fixator. The ipsilateral neck is draped so that the carotid artery can be compressed manually in an emergency. The cervical carotid artery is usually not exposed to allow temporary occlusion.
A curvilinear skin incision starts at the zygomatic arch 1 cm anterior to the tragus and curves to the midline, just behind the hairline. Interfacial temporalis muscle dissection is often done for more temporal-base exposure, and the temporal branch of the facial nerve is preserved.
Frontotemporal craniotomy is done using 2 or more burr holes. The craniotomy follows the temporalis incision posteriorly then arcs anteromedially to the supraorbital norch, and inferiorly to the bottom of the orbital and temporal base. The drill is used to take off the lesser wing of the sphenoid medially to the superior orbital fissure, with a flat surface over the orbit connecting the anterior and middle cranial fossae ([Fig. 1](#F1){ref-type="fig"}, blue area).
Extradural removal of the ACP: Periosteal dural dissection
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The extradural removal of the ACP is accomplished using a microscope after the outer two-thirds of sphenoid ridge is drilled. The dura on the orbital roof is softly detached with a dissector. This stage yields mobility for the neurovascular structures of the superior orbital fissure (SOF) and exposes the dural fold at the top of the SOF, where the meningo-orbital band (MOB) is located. The SOF is an inset of the external dural layer of the temporal fossa, which runs through the superior orbital fissure and conjoins the periorbita at the level of the sphenoparietal sinus ([Fig. 2A](#F2){ref-type="fig"}). The ACP is drilled partially, and the MOB that runs the bone contour SOF and attaches to the periorbita opened. The dura mater of the frontal and temporal fossa is divided to expose the MOB superomedially and the SOF inferolaterally ([Fig. 2B](#F2){ref-type="fig"}). The MOB and the junction of periorbital dura mater are opened at the lateral wall of the cavernous sinus. The MOB is mutilated using a pair of sharp scissors or an 11 blade. The inner and outer dura layer of the lateral cavernous sinus is carefully split off to expose all the dimensions of the ACP.
Extradural removal of the ACP: Drilling of the lesser wing of sphenoid bone
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The posterior half of the roof and lateral wall of the orbit and sphenoid ridge covering the SOF are resected until the orbital portion of the optic nerve is clearly identified. The ACP drilling is done with a 3 mm or less diamond drill under continuous irrigation to prevent thermal injury to the neurovascular structures. The drilling starts from the lateral side of the ACP to medial part ([Fig. 1](#F1){ref-type="fig"}, red area). The oculomotor nerve passes the inferolateral side of the ACP. Attention is required while drilling the inferolateral side of the ACP.
Removal of the roof of the optic canal and optic strut
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The apex of the ACP is removed using a diamond burr with constant irrigation inside the cancellous bone to lateral cortical bone. The cortical bone of the ACP is remained like a thinned onion shell on the periosteal layer in the clinoid space. The last step involves centrally hollowing out the dense cortical bone in the center of the ACP ([Fig. 3A](#F3){ref-type="fig"}). During this procedure, the surgeon must be aware of the relative positions of the optic nerve, the carotid artery, and the oculomotor nerve with reference to the ACP. It is withdrawn using a microdissector to expose the clinoid space ([Fig. 1](#F1){ref-type="fig"}, green area). The optic canal is broken using a diamond drill. Last, the optic strut remains between the opened clinoid space and the optic canal. The optic strut is dissected from the optic sheath and cleared away using a micropunch ([Fig. 3B](#F3){ref-type="fig"}). Bleeding is easily controlled with bone wax and hemostatic material such as gellfoam.
Dural opening and intradural procedure
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The dura is opened with a semicircular incision extending from the floor of the temporal base at the posterior-inferior aspect of the exposure to the floor of the frontal base at the anterior-inferior aspect exposure. The dura covering the anterior clinoid can be excised to extend the view. We do not need an extended dural incision along the ACP. Wide splitting of the sylvian fissure allows easier retraction of the brain, but this is not done routinely. We can dissect the aneurysm from the carotid artery and optic nerve with a blunt dissector and micro scissors. The clipping techniques of the aneurysms differ depending on the anatomical situation, the shape, and size of the aneurysm.
Closure
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At the end of the clipping, the intradural space is filled with warm saline. The dural incision is closed with prolene 4-0 continuous sutures. The dural around the anterior clinoid leaves covering the medial sphenoid wing are then closed primarily, followed by a watertight closure of the more superficial dural opening. The bone flap is fixed with titanium miniplates and screws. The bone flap should be fixed tightly to achieve optimal cosmetic results. Biopore™ burr hole covers are thus used on the burr hole site or skull defect area. After the final meticulous hemostasis, the muscle and subcutaneous layers are closed layer by layer.
Case
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### Illustrative case 1
A 46-year-old female presented with an incidental right paraclinoid aneurysm. The 8.66×4.81-mm aneurysm was located at the ophthalmic segment of the ICA, anteriorly and superiorly directed. The preoperative axial T2 MRI scans showed a signal void lesion near the right optic nerve. The images obtained from the digital subtraction angiography of the right carotid artery showed that the saccular aneurysm was directed medial-superiorly and compressed the right optic nerve superolateraly ([Fig. 4A](#F4){ref-type="fig"}). The patient experienced dizziness and diplopia. Her visual-field examination showed right inferior quadrantanopia. A decision was thus made to clip the aneurysm through decompression of the optic nerve instead of coil embolization. The 2-dimensional (2D) CT angiography showed that the ACP was an obstacle to clipping the aneurysm ([Fig. 4B](#F4){ref-type="fig"}). The aneurysm clipping was finished without complications. The postoperative 2D CT angiography scan shows that the entire ACP was removed, and the aneurysm was clipped with 2 15-mm straight clips ([Fig. 4C](#F4){ref-type="fig"}). Her visual field was much improved at an 8 months postoperative exam.
### Illustrative case 2
A 42-year-old female presented with sudden-onset severe headache. The CT scan showed right-frontal-base hemorrhage. The 2D angiography showed that the aneurysm was situated in the site of the hemorrhage. The ACP was an obstacle to clipping the aneurysm ([Fig. 5A](#F5){ref-type="fig"}). The 3D CT angiography showed the paraclinoid internal carotid artery aneurysm directed to superolateral side ([Fig. 5B](#F5){ref-type="fig"}). A decision was made to clip the aneurysm through hematoma evacuation. The postoperative CT scan showed that the entire ACP was removed, and the aneurysm was clipped with 2 15-mm curved clips ([Fig. 5C](#F5){ref-type="fig"}).
DISCUSSION
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The lesser wing of the sphenoid presents a sharp posterior border, the sphenoid ridge, which overhangs the middle cranial fossa and projects into the lateral sulcus of the cerebral hemisphere. It ends medially in the ACP, which attaches to a fold of the dura called tentorium cerebelli,[@B4] the medial side of the tentorium with the periclinoid folds, the inter-clinoid folds and the falciform ligament connecting from the ACP to the planum sphenoidale wrapping up the optic canal roof.[@B5] The ICA entering the cavernous sinus forms the anterior loop between the proximal and distal rings. The ICA, the proximal dural ring at the height of the ACP, becomes the clinoid segment. The ophthalmic segment extends above the distal dural ring. It is continuous to the falciform ligament, to the segment of the posterior communicating artery. The clipping of this paraclinoid segment aneurysm does not have sufficient space for the clipping of the aneurysm neck and proximal control, and that is all essential to remove the apex of the ACP and to open the falciform ligament lateral to the clinoid process. In addition, for clipping large paraclinoid aneurysms, for which it is necessary to open the external dural ring and to mobilize the clinoidal segment of the ICA, total AC is generally required.
The removal of the ACP provides a better field of the structures in and around the optic nerve, the ICA. AC and optic-sheath opening significantly increases the exposure and mobilization of the optic nerve and ICA as well as 3 to 4 fold expansion of the opticocarotid triangle width.[@B3]
Yasargil et al. described the pterional approach and intradural AC for the clipping of carotid-ophthalmic aneurysms.[@B10] The pterional approach offers a familiar visualization for most surgeons operating in circle of Willis. Complete splitting of the sylvian fissure makes it easier to identify the proper anatomy of the lesion that may be an obstacle in the initial approach. At that point intradural AC is done. The benefit of intradural AC is the early exposure of the lesion and tailored ACP resection. If an unintended event occurs during surgery, such as the rupture of an aneurysm, most vascular surgeons will feel more able to control that event.
Dolenc introduced the EAC procedure as part of an extensive transcavernous approach with the opening of the optic sheath in 1985.[@B2] The advantage of EAC is minimal brain retraction. EAC will leave the dura intact when it is frequently opened later in the operation. Thus, the dura mater provides a natural barrier protecting the neurovascular drill burr, and protects the subarachnoid space to the bone ducts. The disadvantage is the risk of injuring the neurovascular structures passing through the SOF during dissecting.[@B12]
We favor extradural anterior clinoidectomy for the clipping of paraclinoid aneurysm due to this procedure is done by the second operator. The first operator focuses on the intradural procedure with clipping of the aneurysm.
AC-shaped dural incision is enough to clip of a paraclinoid aneurysm. An additional dural incision parallel to the sylvian fissure is needs to open the distal dural ring for proximal control. We do not need this incision only for proximal control, but we think opening of the distal dural ring for proximal control of the ruptured aneurysm is so risky. In that case we propose to open cervical ICA.
The described major complications related to AC in the modern series include postoperative cerebrospinal fluid leakage, damage to the optic nerve in the form of visual-field deficits due to direct neuronal damage or ischemic injury, oculomotor palsy, and intraoperative aneurysm rupture.[@B1][@B6][@B9][@B11] Aneurysm rupture during AC is rarely reported.[@B8]
The illustrative cases of paraclinoid aneurysm demonstrate the lack of complications. With understanding of the bony anatomy and the dura space around the ACP, EAC can be performed easily.
CONCLUSION
==========
The authors\' experience reviewed in the present literature demonstrates that AC is practicable for clipping paraclinoid aneurysms. The patients were treated with pterional craniotomy and EAC with clipping of paraclinoid aneurysm, and good outcomes were achieved. EAC is also recommended for clipping paraclinoid aneurysms.
{#F1}
{#F2}
{ref-type="fig"}). The outer two-thirds of sphenoid ridge (Asterisk) and the entire lesser wing (Open arrow) are drilled. The temporalis muscle (Dark circle) is retracted red inferiorly. (A) The apex of the ACP is removed using a diamond burr and constant irrigation in an inside cancellous bone to lateral cortical bone. The cortical bone of the ACP (Closed arrow) is remained like a thinned onion shells on the periosteal layer in the clinoid space. The diamond drill is aimed so that the walls can be easily fractured and circumferentially dissected free of the surrounding dural-fold-canal drilling. (B) Intraoperative photography: The optic canal (Open arrow) is broken using a diamond drill. The optic strut (Arrowhead) is remained between the opened clinoid space and the optic canal. The optic strut is dissected from the optic sheath and cleared away using a micropunch.](jcen-15-260-g003){#F3}
{#F4}
{#F5}
| {
"pile_set_name": "PubMed Central"
} |
Nanoparticles are of advantages of unique optical, electrical and chemical properties, and have been used for protein and nucleic acid analysis, biosensors, biochips and nanocatalytic analysis[@b1][@b2][@b3][@b4][@b5][@b6][@b7][@b8]. Noble metal nanoparticles have high electron density, good biocompatibility, good catalysis and good stability, easy preparation, so it has attracted people's attentions. Haruta found that nanogold was a good catalyst which load on the transition metal oxides[@b9], not only has high catalytic activity for CO oxidation at low temperature, but also have the advantages of good water resistance, stability and the enhanced effect of humidity[@b10]. It has broken the traditional ideas that nanogold has no catalytic activity. In the analysis of trace contaminants, nanocatalysis provides opportunities to establish a high sensitive and selective analysis method to amplify analytical signal, and improve selectivity that combine with immunoreaction and nucleic acid aptamer reaction[@b11][@b12][@b13][@b14][@b15][@b16]. Xu *et al*.[@b15] have reported a new light scattering method for determination of nucleic acid using immunonanogold catalytic amplification, with a detection limit of 10 fmol/L. Our group developed two new technologies including immunonanocatalyis and aptamer-modified nanoparticle catalysis, that have been used for detecting 7.2 pg/mL urine albumin and 0.09 ng/mL IgG[@b14][@b17][@b18]. These demonstrate that exploring new highly sensitive nanocatalysis analytical reaction is very significant. Among the nanoparticles, nanogold in solution has best stability and strong catalysis. H~2~O~2~ not only has no effect for subsequent SPR research but also is colorless, accessible and with non-toxic product. As far as we know, there are no reports about H~2~O~2~-HAuCl~4~-nanogold catalytic analytical reaction and used for the SPR-S analysis platform.
The SPR-S techniques included the RRS and SERS, which the former is elastic and the later is inelastic scattering that both were based on the nanoparticle scattering. RRS is simple, sensitive spectral analysis method and has been used for protein, nucleic acid and metal ions analysis[@b19][@b20][@b21][@b22][@b23]. Lead is a harmful heavy metal, which has been listed as key detection project in food, drugs, environmental pollutants and supervision inspection. Based on the reaction of Pb^2+^ reacting with excessive I^-^ to form \[PbI~4~\]^2−^, and further associated with rhodamine 6 G (Rh6G) to produce particles with a strong RRS peak, Luo *et al*.[@b24] developed a RRS method for detection of Pb^2+^ as low as 0.04 μg/mL. Luo *et al*.[@b25] reported a RRS method for detection Pb^2+^ as low as 1.0 nmol/L, based on the binding of Pb(II) with thrombin and aptamer. Pb^4+^ was reduced to PbH~4~ gas by NaBH~4~ and the gas trapped by Au^3+^ to form nanogold that exhibited a RRS effect at 286 nm. This principle was used to detect Pb^2+^ as low as 7.0 × 10^−8 ^mol/L[@b26]. Based on the dsDNA cracked by Pb(II) to release a short single-stranded DNA that conjugated gold nanoparticles (AuNPs) to form a stable AuNPs-ssDNA complex, and its nanocatalysis of HAuCl~4~-vitamin C particle reaction, a sensitive RRS method was developed for detection of Pb(II)[@b27]. However, there are no reports about the HAuCl~4~-H~2~O~2~ nanogold catalysis SPR-RS analytical platform being utilized to detect trace Pb^2+^, combing with the DNA enzymes cracked reaction. SERS is a sensitive and selective molecular spectrometry, based on the molecular probes adsorbed on rough surface of nanoparticles[@b28][@b29][@b30][@b31][@b32][@b33]. Although there are many SERS detection techniques, a few SERS quantitative methods have been reported, with good accuracy, simplicity and practicality. Liu *et al*.[@b34] proposed a SERS biosensor to detect lead ion, combining the DNAzyme cracking and nanocatalytic reaction. Zhang *et al*.[@b35] used the prepared tree-shape nanogold-DNA as a signal amplifier to fabricate a SERS biosensor for detection of 100 pmol/L Pb^2+^. A label-free rhodamine 6G SERS probe was reported for detection of trace Pb(II) in Au~core~Ag~shell~ nanosol substrate, based on the Pb(II) cracking the DNAmyze[@b36]. However, there are no reports about aptamer combining with the nanocatalysis of H~2~O~2~-HAuCl~4~ in SERS quantitative analysis of Pb^2+^. In this paper, we have considered the new nanocatalytic reaction of AuNP-HAuCl~4~-H~2~O~2~, and two new SPR methods were developed for detection of Pb(II), combining the analysis platform with the DNAzyme cracking.
Results
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RRS spectra
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The RRS signals of small particle size gold, silver, platinum and palladium nanosol are very weak. Different concentrations of AgNO~3~ were added to preparation of AuNR~1~, AuNR~2~ and AuNR~3~, with the diameter of 32 nm, 37 nm and 43 nm respectively that RRS values gradually reduced ([Fig. S1](#S1){ref-type="supplementary-material"}). With the increase of AuNR concentration, the RRS peak linear increased at 370 nm ([Fig. S1D](#S1){ref-type="supplementary-material"}). Nanoparticles can catalytic hydrogen peroxide reduction of HAuCl~4~ under the condition of 0.67 mmol/L HCl, and with the increase of nanogold solution concentration, the RRS intensity of system linear increase at 370 nm ([Fig. 1](#f1){ref-type="fig"}, [Fig. S2](#S1){ref-type="supplementary-material"}). The catalytic activity of AuNP~B~ was better than that of AuNP~c~ because the particle size of AuNP~B~ was smaller than AuNPc, which lead to the surface energy more larger and the surface of AuNP~B~ nanoparticles can absorb more HAuCl~4~. Different particle size of AuNR was added as catalyst, with the AuNR concentration increased , the RRS peak linear increased at 370 nm ([Figs S3--S5](#S1){ref-type="supplementary-material"}). When AgNPs, PdNPs, PtNPs nanosol solution was used as catalyst, with the increase of nanoparticles concentration, the RRS peak linear increased at 370 nm ([Fig. S6](#S1){ref-type="supplementary-material"}). It can be used to quantitative of HAuCl~4~ and H~2~O~2~ through this catalytic system, with the increase of HAuCl~4~ concentration, the RRS peak of system linear increased and color from colorless gradually became red ([Fig. S7](#S1){ref-type="supplementary-material"}), with the increase of H~2~O~2~ concentration, the RRS peak of system linear increased ([Fig. S8](#S1){ref-type="supplementary-material"}). When AuNPc-ssDNA solution was used as catalyst, AuNP~c~ modified by aptamer catalytic activity is stronger than AuNP~c~ solution, with the increase of AuNPc-ssDNA concentration, the RRS peak linear increased at 370 nm ([Fig. S9](#S1){ref-type="supplementary-material"}).
DNAzyme catalytic strand hybridize with substrate strands to form double-stranded DNA (dsDNA). In pH 8.0 Tris-HCl buffer solution and 6.7 mmol/L NaCl, AuNP~c~ were aggregated to the AuNP~c~ aggregations which exhibited a strong RRS peak at 370 nm. Upon addition of Pb^2+^, the substrate chain of dsDNA could be cracked catalytically by Pb^2+^ to produce a short single-stranded DNA (ssDNA) that adsorbed on the AuNPc surface to form stable AuNPc -ssDNA conjugate to prevent aggregation by NaCl, With the increase of Pb^2+^ concentration, the RRS peak linear decreased at 370 nm ([Fig. 2](#f2){ref-type="fig"}). The AuNPc-ssDNA probe of the apt-AuNP~c~-Pb^2+^ system reaction solution has strong catalytic effect on the slow reaction between H~2~O~2~ and HAuCl~4~, the products gold nanoparticles had a stronger RRS peak at 370 nm, with the increase of Pb^2+^ concentration, the RRS peak linear increased at 370 nm ([Fig. 3](#f3){ref-type="fig"}).
SERS spectra
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Au^3+^ was reduced to Au and growing around the surface of nano-gold under the action of reducing agent H~2~O~2~, and the irregular shape, big particle size of nanoparticles was obtained. Upon addition of Rhs, it was adopted on the surface of gold nanoparticles which exhibited SERS peaks at 618 cm^−1^, 732 cm^−1^, 1199 cm^−1^, 1277 cm^−1^, 1356 cm^−1^, 1507 cm^−1^, 1527 cm^−1^ and 1645 cm^−1^. Among them, the SERS peak at 1645 cm^−1^ is the biggest, and the SERS peak intensity linearly increased with the concentration of AuNP~B~ increasing ([Fig. S10](#S1){ref-type="supplementary-material"}). Upon addition of VBB, VBB molecular probes exhibited SERS peaks at 795 cm^−1^, 1167 cm^−1^, 1200 cm^−1^, 1364 cm^−1^, 1394 cm^−1^ and 1612 cm^−1^. Among them, the SERS peak at 1612 cm^−1^ is the biggest, and the SERS peak intensity linearly increased with the concentration of AuNP~B~ increasing ([Fig. 4](#f4){ref-type="fig"}). Upon addition of Tibetan red T, Tibetan red T molecular probes exhibited SERS peaks at 349 cm^−1^, 612 cm^−1^, 1240 cm^−1^, 1372 cm^−1^, 1551 cm^−1^ and 1639 cm^−1^. Among them, the SERS peak at 1372 cm^−1^ is the biggest, and the SERS peak intensity linearly increased with the concentration of AuNP~B~ increasing ([Fig. S11](#S1){ref-type="supplementary-material"}), and we can know that the SERS signal strength of Tibetan red T is weaker than that of RhS and VBB. When Rh6G was used as SERS probe, the SERS signal is very weak. When PdNPs solution was used as catalyst and VBB was used as SERS probe, with the increase of PdNPs concentration, the SERS peak linearly increased at 1612 cm^−1^ ([Fig. S12](#S1){ref-type="supplementary-material"}). For the apt-nanogold-Pb^2+^ catalytic system, VBB and RhS was used as SERS probe respectively, the SERS peak intensity linearly increased with the concentration of AuNP~c~ increasing ([Fig. 5](#f5){ref-type="fig"}, [Fig. S13](#S1){ref-type="supplementary-material"}).
Scanning electron microscopy(SEM)
---------------------------------
According to the procedure to get the aptamer reaction solution, a 1.0 mL the solution was taken into a 1.5 mL centrifuge tube, and centrifuged in 15000 r/min for 20 min before abandoned the supernatant. A 1.0 mL water was added into the centrifuge tube and dispersed by ultrasonic 30 min, and centrifuged again. The operation was repeated, and the dispersed sample solution was dropped onto a silicon wafers and dried naturally, then the scanning electron microscope (SEM) was recorded. The size of gold nanoparticles and silver nanoparticles are uniform and small ([Fig. S14a,b](#S1){ref-type="supplementary-material"}). Different concentration of AgNO~3~ was added to preparation of AuNR~1~, AuNR~2~, AuNR~3~, the diameter of them was 32 nm, 37 nm, 43 nm respectively ([Fig. S14c--e](#S1){ref-type="supplementary-material"}). For AuNP~B~-HAuCl~4~-H~2~O~2~ system, in the absence of AuNP~B~, the reaction of H~2~O~2~ and HAuCl~4~ is slow under the condition of 0.67 mmol/L HCl medium and 60 °C, and the products of gold nanoparticles is less ([Fig. 6a](#f6){ref-type="fig"}). Upon addition of the AuNP~B~, the reaction was accelerated by nano-catalyst of small gold nanoparticles (AuNP~B~), and it would reacted to form a large number of gold nanoparticles which was irregular shape, big particle size. With the increase of AuNP~B~ concentration, the amount of gold nanoparticles increased which had high SERS and RRS singals ([Fig. 6b,c](#f6){ref-type="fig"}). When AuNPc-ssDNA was used as catalyst, the products gold nanoparticles were gathered into small clusters ([Fig. 6d](#f6){ref-type="fig"}). Compared with the same concentration of nangold, the catalytic activity of nanogold modified by aptamer was better due to its size was smaller. For the apt-AuNPc-Pb^2+^ system, with the increase of Pb^2+^ concentration, the amount of reaction product gold nanoparticles increased ([Fig. 6e,f](#f6){ref-type="fig"}).
Research of gold nanoparticle-HAuCl~4~-H~2~O~2~ reaction
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The effect of HCl concentration was examined. It was found that the influence of hydrochloric acid concentration has a great influence on the formation of gold nanoparticles. The results showed that the Δ*I* value reached its maximum when the concentration was 0.5 mmol/L ([Fig. S15](#S1){ref-type="supplementary-material"}). But under this condition, the color of the blank was pink and the RRS value was 3506 which indicated that the blank had formed a large number of gold nanoparticles. Thus, the 0.67 mmol/L was chosen for use which RRS value was 506 and colorless. The effect of HAuCl~4~ and H~2~O~2~ concentration was studied. The results showed that the Δ*I* value reached its maximum when the concentration was 4.48 μmol/L and 3.33 mmol/L H~2~O~2~ respectively ([Figs S16 and S17](#S1){ref-type="supplementary-material"}). Thus a 4.48 μmol/L of HAuCl~4~ and 3.33 mmol/L H~2~O~2~ solutions were chosen for use. The effect of the incubation temperature was considered, when the temperature was greater than 60 °C, with the increase of temperature, the RRS value and color of blank increased gradually. When the incubation temperature was 60 °C, the blank RRS value was 745 and the color was colorless, meanwhile the catalytic reaction was very slow below 60 °C. Therefore the best temperature was 60 °C ([Fig. S18](#S1){ref-type="supplementary-material"}). The effect of incubation time on the catalytic reaction was considered, a fixed reaction time of 15 min was chosen for use, giving a good compromise between high sensitivity, short analytical time and low blank ([Fig. S19](#S1){ref-type="supplementary-material"}). After quenching the catalytic reaction, cooling with ice water to quench the reaction, the scattering intensity was constant within 90 min ([Fig. S20](#S1){ref-type="supplementary-material"}). The effect of Raman probe RhS, VBB and Tibetan red T concentration were examined, and the results showed that the Δ*I* value reached its maximum when their concentration were 7 μmol/L, 13.2 μmol/L, 6.7 μmol/L respectively ([Figs S21--S23](#S1){ref-type="supplementary-material"}).
The gold nanoparticle reaction of HAuCl~4~-H~2~O~2~ was slow in diluted HCl solution at 60 °C and was accelerated by nano-catalyst of small gold nanoparticles (AuNP~B~, AuNP~c~, AgNPs, PdNPs and PtNPs). Under the optimal conditions, the RRS intensity for different AuNP concentrations (C) was recorded and the working curves were drawn according the relationship between C and their corresponding Δ*I* values. We have investigated the influence of different kinds of AuNP on the working curve ([Fig. 7](#f7){ref-type="fig"}, [Fig. S24](#S1){ref-type="supplementary-material"}). [Table 1](#t1){ref-type="table"} showed that the AuNP~B~ system was the best, with the most wide linear range and lowest detection limit. We have investigated the influence of different size of AuNR on the working curve ([Fig. S25](#S1){ref-type="supplementary-material"}), [Table 1](#t1){ref-type="table"} showed that with the increase of AuNR particle size, the catalytic activity was weaker. As for AuNP~B~-HAuCl~4~-H~2~O~2~ system, under the optimal conditions, RhS, VBB, and Tibetan red T was added as SERS probe respectively, the increased SERS intensity responded linearly with the concentration of AuNP~B~ over 3.8--456, 19--285, 4--190 ng/mL respectively, with a linear regression equation of Δ*I*~*1645 cm−*1~ = 2.28 *C* + 72.77, Δ*I*~*1612 cm−*1~ = 5.94 *C* + 86, Δ*I*~*1372 cm−*1~ = 1.47 *C*−9.1 respectively ([Fig. 8](#f8){ref-type="fig"}). Results showed that when VBB used as SERS probe was the most sensitive. When PdNPs was used as catalyst and VBB was used as SERS probe, the increased SERS intensity at 1612 cm^−1^ responded linearly with the concentration of PdNPs over 500--5950 ng/mL, with a linear regression equation of Δ*I*~*1612 cm−*1~ = 14.71 *C* + 2.06 ([Fig. S26](#S1){ref-type="supplementary-material"}). We have investigated the influence of different AuNP on the working curve, and the results ([Table 1](#t1){ref-type="table"}) showed that the AuNP~B~ system was the best, with the lowest detection limit.
The gold nanoparticle reaction of HAuCl~4~-H~2~O~2~ was slow in diluted HCl solution at 60 °C. Upon addition of the nanoparticles, HAuCl~4~ would be adsorbed on the surface of nanoparticles catalyst. The surface energy was higher when the nanoparticles particle size was very small, and it can absorb a large number of HAuCl~4~ in the surface of the nanoparticles. When Au^3+^ was reduced to Au and growing around the nano-gold surface under the action of reducing agent H~2~O~2~, irregular shape, big particle size of nanoparticles were obtained. The products had a strong RRS signal because of the particle size was very large. When VBB, RhS and Tibetan red T were added as SERS probe respectively, the products had strong SERS signal because of the shape of gold nanoparticles was random. From [Table 1](#t1){ref-type="table"}, we can know that the AuNP~B~ RRS system was the best.
Optimization of aptamer detection of Pb^2+^ system analysis conditions
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The effect of Tris-HCl concentration and pH were examined, the results showed that the Δ*I* value reached its maximum when the concentration was 4 mmol/L and pH was 8.0 ([Figs S27](#S1){ref-type="supplementary-material"} and [28](#S1){ref-type="supplementary-material"}). The effect of AuNP~c~ and NaCl concentration were examined, the results showed that the Δ*I* value reached its maximum when the concentration were 9.55 μg/mL and 10 mmol/L respectively ([Figs S29](#S1){ref-type="supplementary-material"} and [30](#S1){ref-type="supplementary-material"}), thus, 9.55 μg/mL of AuNP~c~ and 10 mmol/L of NaCl solution were chosen for use.
Effect of foreign substances
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According to the procedure, the effect of foreign substances on the determination of 0.167 μmol/L Pb^2+^ was tested, with a relative error within ±10%. Results ([Table S1](#S1){ref-type="supplementary-material"}) showed that common ions did not interfere with the determination, which indicated that this method had good selectivity.
Working Curve
-------------
Under the optimal conditions, the RRS intensity for different Pb^2+^ concentrations (C) were recorded and the working curves were drawn according the relationship between C and their corresponding Δ*I* values. With the increase of Pb^2+^ concentration, the RRS peak increased at 370 nm and the decreased RRS intensity responded linearly with the concentration of Pb^2+^ over 125--425 nmol/L with a linear regression equation of Δ*I*~*370 nm*~ = 1.26C−20.56, coefficient R^2^ of 0.9836. For the apt-nanogold-Pb^2+^ catalytic system, The increased RRS intensity at 370 nm responded linearly with the concentration of Pb^2+^ over 16.7 -- 666.7 nmol/L, the linear regression equation is Δ*I*~*370 nm*~ = 9.85 C + 470, coefficient R^2^ of 0.9856 ([Fig. S31](#S1){ref-type="supplementary-material"}). VBB and RhS were added as SERS probe, the SERS intensity *I*~*1612 cm−*1~ and *I*~*1645 cm−*1~ responded linearly with the concentration of Pb^2+^ over 17--250, 17--167 nmol/L respectively ([Fig. S32](#S1){ref-type="supplementary-material"}).
Sample analysis
---------------
Three natural water samples were filtered to obtain water sample solutions, and were analyzed according to the procedures. Results ([Table S2](#S1){ref-type="supplementary-material"}) showed that two of them had been detected out of Pb^2+^. A known amount of Pb^2+^ was added into the water sample to obtain the recovery. The relative standard deviation was in the range of 4.4--5.5%, and the recovery was in the range of 98.0--102%.
Discussion
==========
Analytical principle
--------------------
Nanocatalytic reaction is an important way to amplify the signal of analysis method, so explore a new method to use nanocatalytic reaction is great significance. It was found that, the gold nanoparticle reaction of HAuCl~4~-H~2~O~2~ is slow in diluted HCl solution at 60 °C, upon addition of nanoparticles such as AuNP~B~, AuNP~c~, AuNR, AgNPs, PdNPs and PtNPs, HAuCl~4~ would be adsorbed on the surface of nanoparticles catalyst. The specific surface area is larger because of the nanoparticles particle size is very small, therefore it can absorb a large number of HAuCl~4~ in the surface of the nanoparticles, owing to the fact that the small nanoparticles have a high surface energy. When Au^3+^ was reduced to Au and growing around the nano-gold surface under the action of reducing agent H~2~O~2~, it can obtain irregular shape, big particle size of nanoparticles ([Fig. 9](#f9){ref-type="fig"}), which have highly SERS signals and RRS signals. Thus the nanogold catalytic reaction can be used to build SPR-S analysis platform.
The DNAzyme catalytic strand hybridized with substrate strands to form double-stranded DNA (dsDNA) which couldn't protect AuNP~c~ in pH 8.0 Tris-HCl buffer solution containing 6.7 mmol/L NaCl, and were aggregated to AuNP~c~ aggregations with a strong RRS peak at 370 nm. Upon addition of Pb^2+^, the substrate chain of dsDNA could be cracked catalytically by Pb^2+^ to produce a short single-stranded DNA (ssDNA) that adsorbed on the AuNPc surface to form stable AuNPc-ssDNA conjugate to prevent aggregation by NaCl. Combining the nanocatalytic SPR-RRS analytical platform and the DNAzyme cracking reaction, the AuNPc-ssDNA conjugates have strong catalytic activity to HAuCl~4~-H~2~O~2~ particles reaction, and its product gold nanoparticles had a stronger RRS peak at 370 nm. With the increase of Pb^2+^ concentration, the concentration of AuNPc--ssDNA probe increase and lead to the catalytic activity stronger ([Fig. 10](#f10){ref-type="fig"}). Based on this, the new sensitive RRS and SERS quantitative analysis methods were developed for detection of Pb^2+^.
Conclusion
==========
In 0.67 mmol/L HCl medium at 60 °C, HAuCl~4~ adsorbed on the surface of nanoparticles catalyst, Au^3+^ was reduce to Au and growing around the nano-gold surface, the products have highly SERS signals and RRS signal, thus the AuNP-HAuCl~4~-H~2~O~2~ nanogold catalytic reaction RSS and SERS analysis platform were built. The AuNPc-ssDNA probe of the apt-AuNP~c~-Pb^2+^ system reaction solution has strong catalytic effect on the slow reaction between H~2~O~2~ and HAuCl~4~. Combing the nanocatalysis and the DNAmyze reaction, a new nanocatalysis analysis platform was developed for the detection of Pb^2+^ by the RRS and SERS, with advantages of high sensitivity, good selectivity, simplicity and rapidity. Compared with the reported methods, the methods are easier to operate and more sensitive. Further more, it is a rapid RRS and SERS quantitative method for Pb^2+^ ([Table S3](#S1){ref-type="supplementary-material"}).
Methods
=======
Apparatus
---------
A model of DXR smart Raman spectrometer (Thermo companies in the United States) with a laser wavelength of 633 nm and power of 2.5 mW, a model of the F-7000 Hitachi Fluorescence spectrometer (Hitachi Company, Japan), a model of the TU-1901 double-beam UV-Vis spectrophotometer (Beijing Purkinje General Instrument Co., Ltd., China), a model of FEI 200 FEG field emission scanning electron microscope (Dutch philips), and a model of C-MAG HS7 incubation magnetic stirrer (Germany IKA company) were used.
Reagents
--------
A 1.0 μmol/L DNAzyme catalytic strand with sequence of 5′-(T)10 CAT CTC TTC TCC GAG CCG GTC GAA ATA GTG AGT-3′, 1.0% HAuCl~4~, 1.0% sodium citrate, 10 mmol/L sodium borohydride, 0.2 mol/L cetyltrimethyl ammonium bromide (CTAB), 4.0 mmol/L AgNO~3~, 77.8 mmol/L vitamin C (VC), 0.01 mol/L HCl, 0.3% H~2~O~2~ (0.1 mol/L), 50 mmol/L pH 7.4 Tris-HCl, 50 mmol/L pH 8.0 Tris-HCl, 5 × 10^−5 ^mol/L PdCl~2~ and 1.45 × 10^−2 ^mol/L PdCl~2~, 2.9 × 10^−2 ^mol/L HPtCl~6~ and 5.23 × 10^−5^ mol/L RhS solution were prepared. A pH 7.0 Na~2~HPO~4~-citric acid buffer solution was prepared as follows, a 16.5 mL 0.2 mol/L Na~2~HPO~4~ and 3.5 mL 0.1 mol/L citric acid solution were mixed together to obtain a concentrations 0.16 mol/L Na~2~HPO~4~. A 1.0 × 10^−3 ^mol/L VBB solution was prepared as follows, 0.0250 g VBB was dissolved in 5.0 mL ethanol, and diluted to 50 mL with water. The nanosols and ssDNA-AuNP were prepared as in the SI[@b37].
Procedure of HAuCl~4~- nanoparticles -H~2~O~2~ system
-----------------------------------------------------
A 80 μL 0.1% HAuCl~4~ (84 μmol/L), 100 μL 0.01 mol/L HCl, a certain amount of nanoparticles including AuNP~B~, AuNP~c~, AuNR, AgNPs, PdNPs and PtNPs, and 50 μL 0.3% (0.1 mol/L) H~2~O~2~ were added into a 5 mL marked test tube and mixed well, and diluted to 1.5 mL. The mixture was heated at 60 °C for 15 min, cooling with ice water to quench the reaction. A part of the solution was transferred into a 1 cm quartz cell. The RRS spectra were recorded by synchronous scanning excited wavelength *λ*~ex~ and emission wavelength *λ*~em~ (*λ*~ex~−*λ*~em~ = Δ*λ* = 0), a PMT voltage of 400 v, both excited and emission slit width of 5 nm, emission filter of 1%T attenuator on fluorescence spectrophotometer. The RRS intensity at 370 nm (*I*~370 nm~) and the blank value (*I*~370 nm~)~0~ without nanoparticles were recorded. The value of Δ*I*~370 nm~ = *I*~370 nm~−(*I*~370 nm~)~0~ was calculated. 200 μL 1.0 × 10^−5 ^mol/L VBB, 20 μL 5.23 × 10^−5^ mol/L RhS or 100 μL 1 × 10^−4^ mol/L tibetan red T was added in the mixture respectively, The SERS intensity corresponding at 1612 cm^−1^, 1645 cm^−1^, 1370 cm^−1^ and the blank value *I*~*0*~without nanoparticles were recorded. The value of Δ*I* = *I* − *I*~*0*~ was obtained.
Procedure of apt-AuNP~c~-Pb^2+^ system
--------------------------------------
A 500 μL 2 μmol/L Substrate strand, 500 μL 1 μmol/L DNAzyme catalytic strand, 1 mL 50 mM pH 7.4 Tris-Hcl buffer solution and 50 μL 1 mol/L NaCl were mixed well, incubated at 65 °C water bath for 10 min, then gradually cooled to room temperature over 2 h, and hybrid solution was obtained. In a 5 mL marked test tube 120 μL 50 mM pH 8.0 Tris-Hcl buffer solution, 50 μL hybrid solution, a certain amount of Pb^2+^ was added respectively, mixed well and diluted to 1.5 mL. Then the tube was placed at 37 °C water bath for reaction 60 min before cooling with ice water to quench the reaction. After that 250 μL AuNP~c~ and 30 μL 0.5 mol/L NaCl were added in the mixture and mixed well to obtain Pb^2+^ aptamer reaction solution, then a part of the solution was transferred into a 1 cm quartz cell. The RRS spectra were recorded by synchronous scanning excited wavelength *λ*~ex~ and emission wavelength *λ*~em~ (*λ*~ex~−*λ*~em~ = Δ*λ* = 0), a PMT voltage of 450 v, both excited and emission slit width of 5 nm, The RRS intensity at 370 nm (*I*~370 nm~) and the blank value (*I*~370 nm~)~0~ without Pb^2+^ were recorded. The value of Δ*I*~370 nm~ = (*I*~370 nm~)~0~ − *I*~370 nm~ was calculated.
Procedure of apt-nanogold-Pb^2+^ catalytic system
-------------------------------------------------
A 80 μL 0.1% HAuCl~4~ (84 μmol/L),100 μL 0.01 mol/L HCl, 100 μL Pb^2+^ aptamer reaction solution and 50 μL 0.3% (0.1 mol/L) H~2~O~2~ were added into a 5 mL marked test tube and mixed well, and diluted to 1.5 mL. The mixture was heated at 60 °C for 15 min, cooling with ice water to quench the reaction. A part of the solution was transferred into a 1 cm quartz cell. The RRS spectra were recorded by synchronous scanning excited wavelength *λ*~ex~ and emission wavelength *λ*~em~ (*λ*~ex~ − *λ*~em~ = Δ*λ* = 0), a PMT voltage of 400 v, both excited and emission slit width of 5 nm, emission filter of 1%T attenuator on fluorescence spectrophotometer. The RRS intensity at 370 nm (*I*~370 nm~) and the blank value (*I*~370 nm~)~0~ without Pb^2+^ were recorded. The value of Δ*I*~370 nm~ = *I*~370 nm~ − (*I*~370 nm~)~0~ was calculated. 200 μL 1.0 × 10^−5^ mol/L VBB 20 μL or 5.23 × 10^−5 ^mol/L RhS was added in the mixture, The SERS intensity at 1612 cm^−1^ and the blank value *I*~*0*~without Pb^2+^ were recorded. The value of Δ*I* = *I* -- *I*~*0*~was obtained.
Additional Information
======================
**How to cite this article**: Ye, L. *et al*. A novel and highly sensitive nanocatalytic surface plasmon resonance-scattering analytical platform for detection of trace Pb ions. *Sci. Rep*. **6**, 24150; doi: 10.1038/srep24150 (2016).
Supplementary Material {#S1}
======================
###### Supplementary Information
This work supported by the National Natural Science Foundation of China (No. 21267004, 21367005, 21307017, 21465006, 21477025, 21567001, 21567005), the Research Funds of Key Laboratory of Ecology of Rare and Endangered Species and Environmental Conservation of Education Ministry, the Research Funds of Guangxi Key Laboratory of Environmental Pollution Control Theory and Technology, and the Natural Science Foundation of Guangxi (No. 2013GXNSFFA019003, 2014GXNSFAA118059).
**Author Contributions** G.Q., L.L. and A.H. performed the experiment and prepared Figs 1--6 and Fig. 8. L.L., G.Q., H.X., Z.L. and A.H. wrote the main manuscript text and prepared Fig. 7. Z.L., A.H., Q.Y. and G.Q. contributed to the discussion and measurement analysis. All authors contributed to the preparation of the manuscript and reviewed the manuscript.
{#f1}
{#f2}
{#f3}
{#f4}
{#f5}
{#f6}
{#f7}
{#f8}
{#f9}
{#f10}
###### Different nanoparticles catalytic systems analysis feature.
system Detection method The regression equation Linear range (ng/mL NP) The correlation coefficient
------------------------------- ------------------------------ ------------------------- ------------------------- -----------------------------
H~2~O~2~-HAuCl~4~-AuNP~B~ RRS Δ*I* = 131 C + 300 0.038--76 0.9951
Different RhS Δ*I* = 5.9 C + 86 3.8--456 0.9898
probe VBB Δ*I* = 2.9 C−73 19--285 0.9966
SERS Tibetan red T Δ*I* = 1.5 C−9.1 4--190 0.9923
UV Δ*A* = 2.6 × 10^−3^ C+0.0406 9.5--180 0.9825
H~2~O~2~-HAuCl~4~-AuNP~c~ RRS Δ*I* = 51C + 267 0.38--171 0.9941
UV Δ*A* = 2 × 10^−3^ C−0.0407 38--228 0.9808
H~2~O~2~-HAuCl~4~-AuNR~1~ RRS Δ*I* = 0.51 C + 54 32.5--975 0.9840
H~2~O~2~-HAuCl~4~-AuNR~2~ RRS Δ*I* = 0.37 C + 21 32.5--2600 0.9901
H~2~O~2~-HAuCl~4~-AuNR~3~ RRS Δ*I* = 0.24 C + 73 32.5--1950 0.9784
H~2~O~2~-HAuCl~4~-AgNPs RRS Δ*I* = 23 C + 73 3.3--265 0.9971
UV Δ*A* = 0.001 C + 0.0203 13--265 0.9701
H~2~O~2~-AuCl~4~-PdNPs RRS Δ*I* = 0.12 *C* + 2.6 200--9920 0.9939
SERS Δ*I* = 14.71 *C* + 2.06 500--5950 0.9913
UV Δ*A* = 2 × 10^−5^*C* + 0.011 298--1587 0.9939
H~2~O~2~-HAuCl~4~-PtNPs RRS Δ*I* = 0.05 *C* + 21 200--600 0.9729
H~2~O~2~-HAuCl~4~-AuNP~c~-Apt RRS Δ*I* = 70.7 C + 257 0.95--76 0.9936
[^1]: These authors contributed equally to this work.
| {
"pile_set_name": "PubMed Central"
} |
Background
==========
Population health can be viewed as a complex and dynamical system in which the patterns of health and disease exist, persist, and change over geography and time. \[[@B1],[@B2]\] The underlying patterns of exposure that influence health status are the non-random result of interactions between the social, economic, and environmental networks people live within. \[[@B3]\] Therefore, understanding the macro-level effects of environmental determinants of health has become increasingly important. \[[@B4],[@B5]\]
Epidemiologic studies have indicated that people and communities cluster spatially in systematic ways that are highly predictive of disease. \[[@B6]\] Such patterned regularity between groups and communities over time, despite the movement of people in and out of groups, demonstrates a dynamic at the environmental level that accounts for the observed differences in disease rates across spatial and temporal dimensions. \[[@B7],[@B8]\] Epidemiologists have studied this dynamic interaction using complexity theory \[[@B9],[@B2]\], in which populations are considered more than simply a collection of individuals but rather an important context that is fundamental for understanding the causative relationship between determinant and health outcomes. \[[@B3]\] Populations function within a highly composite, complex, adaptive system built up from large numbers of mutually interacting subunits whose repeated interactions result in rich, collective behaviour that feeds back into the behaviour of the individual parts. \[[@B2],[@B10]\] Nonlinearity is the essence of complex systems. \[[@B11]\] Thus, challenges for conducting studies rooted in complexity arise when standard statistical modelling methods are applied to nonlinear and skewed data sets with interactive variables, hierarchical levels of analysis, and feedback mechanisms. The challenge is to understand the environment as it influences health outcomes by using analytical systems that are neither to simplified nor too complex. \[[@B12]\]
Methods for Studying Complex Systems: The Self-Organizing Map
-------------------------------------------------------------
The self-organizing map algorithm (SOM) has been applied in medical research to address the need for non-linear analytical methods to study the multifaceted aetiology of certain diseases. Kohonen developed the algorithm to search for patterns within expansive, multivariate, numerical datasets. \[[@B13]\] SOM fit into the neural network class of methodologies and are tolerant of non-normally distributed data. Multivariate data sets can be developed to represent entities of interest for pattern recognition. Most recently, Oyana et al. applied SOM in a geospatial context to study cases of adult asthma. \[[@B14]\] Valkonen et al. used the SOM to explore the multidimensionality of insulin resistance syndrome. \[[@B15]\] In addition, neural networks have been applied to diagnose myocardial infarction, find patterns in genes, and organize genes according to biological relevance. \[[@B16]-[@B19]\] Beyond clinical applications, Koua and Kraak used the World Bank\'s Living Standards Measurement Survey to analyze factors indicating well-being and estimate health indicators. \[[@B20]\]
The cumulative nature of previous work has demonstrated the SOM as a tool to recognize patterns within data sets measuring clinical health outcomes, social and economic variables, and the physical environment. The algorithm\'s tolerance of nonlinear and nonparametric data presents an opportunity for the SOM methodology to recognize patterns among disease-causing variables within complex, multivariate data sets. Coupling the SOM algorithm\'s pattern recognition capabilities with the spatial analysis capabilities of geographic information systems (GIS) provides a novel approach to study how complex environmental influences affect health outcomes in populations.
The purpose of this study was to explore the potential of a coupled SOM-GIS approach to apply complexity theory to public health research, using community health assessment as an example. Such an approach would enable researchers to overcome challenges of non-linearity and skewed data distributions that have limited research efforts in the past. In this work we classified communities based on social, economic, and physical environmental factors using GIS and SOM methods. We present the results of our work and then discuss the challenges of implementing SOM-GIS for public health research.
Results
=======
Self-Organizing Map Results
---------------------------
The SOM analyzed data describing ninety-two environmental variables for 511 communities representing five counties in New York State. Cluster tuning recognized five significant clusters and communities were categorized according to patterns discovered among variables. Figure [1](#F1){ref-type="fig"} shows the geographic distribution of clusters by county. Cluster 1 included communities characterized by small to mid sized cities distributed throughout Erie, Westchester, and Steuben counties. Cluster 2 contained traditional suburban communities surrounding Buffalo, NY, and suburbs or small cities in Westchester County. Cluster 3 contained rural communities in Erie, Westchester, Steuben, and Hamilton counties. Cluster 4 included the highly urban communities of New York City County; and Cluster 5 represented a few communities in Erie and Westchester counties that were uninhabited or contained extremely few residents.
{#F1}
Test for Spatial Autocorrelation
--------------------------------
Moran\'s I was calculated in ArcGIS to test for spatial autocorrelation. Results provided Moran\'s Index equal to 0.24 and the associated p-value indicated a weakly significant result. The scale is from -1 to 1 where values near 1 are more clustered. Based on this test, the data indicated a low level of positive spatial correlation between the community clusters and, as such, values near one another were similar but not highly dependent on each other for their distribution. Spatial autocorrelation results suggest that major variables were most likely not omitted from the model, as a large Moran\'s Index indicates the potential for an incomplete model.
Analysis of Variance
--------------------
ANOVA evaluated the variance between the weighted observations of disease in communities and the cluster categories assigned to each of the communities (Table [1](#T1){ref-type="table"}). Results indicated that significant differences exist between cluster classes and that there is more variation between clusters than the variation of disease counts within them, demonstrating the value of the grouping variable. For k-1 = 4 and N-k = 506 degrees freedom and Pr\<0.01, the critical F value = 0.07. A significant result with a large ratio of between group variance to within group variance was observed for all health outcomes evaluated.
######
Analysis of variance results according to health outcome.
**DF** **Sum Squares** **Mean Square** **F Value** **Pr\>F** **R**^**2**^
-------------------- ------- -------- ----------------- ----------------- ------------- ----------- --------------
**Outcome 1** Treat 4 0.5953 0.1488 505.36 1 0.7998
Hepatitis A Error 506 0.1490 0.0003
**Outcome 2** Treat 4 0.0138 0.0035 129.11 1 0.505097
TB Error 506 0.0135 0.0000
**Outcome 3** Treat 4 6786.9301 1696.7330 193.14 1 0.604238
Asthma Error 506 4445.2766 8.7851
**Outcome 4** Treat 4 692.6395 173.1599 326.14 1 0.720528
COPD Error 506 268.6550 0.5309
**Outcome 5** Treat 4 0.0826 0.0206 111.02 1 0.4674
Fetal Immaturity Error 506 0.0941 0.0002
**Outcome 6** Treat 4 3519.1946 879.7986 274.99 1 0.684921
Diabetes Error 506 1618.9052 3.1994
**Outcome 7** Treat 4 1.9988 0.4997 171.27 1 0.575182
Influenza Error 506 1.4763 0.0029
**Outcome 8** Treat 4 13301.8047 3325.4512 213.98 1 0.0628464
Atherosclerosis Error 506 7863.7809 15.5411
**Outcome 9** Treat 4 33.4893 8.3723 2.67 0.97 0.020707
Alcohol & Drug Error 506 1583.7708 3.1300
**Outcome 10** Treat 4 188.8172 47.2043 205.34 1 0.61879
Stress/Nonspec Dep Error 506 116.3224 0.2299
Lists the health outcomes studies with ANOVA results for k-1 = 4 and N-k = 506 degrees freedom and Pr\<0.01, the critical F value = 0.07)
Discussion
==========
Principal Findings
------------------
Our study demonstrated the potential of combining SOM and GIS to overcome traditional challenges associated with studying the complexities of the community environment. Kindig and Stoddart state that population health is fundamentally concerned with the interactions between multiple determinants of health outcomes, referring to such interactions as patterns. \[[@B21]\] Results indicated that the methods used were productive for determining the underlying mathematical patterns to group communities according to similar environmental characteristics. The integration of variables from multiple environmental components and the complex relationships considered to link such variables makes it difficult to uncover the significant relationships and sort out similar entities. By searching for patterns to group entities based on observed environmental conditions, it may be possible to discern characteristics of environments that influence community health status in future studies.
Challenges Associated with Data Collection
------------------------------------------
Challenges for testing the hypothesis primarily surrounded obtaining data to represent environmental conditions and health outcomes. To satisfy requirements for the SOM input data needed to be either binary (0,1) or ratio level; additionally, geographic reference was necessary to connect variables with communities. Such conditions presented challenges for developing a diverse inventory of environmental variables since this study used secondary data from multiple sources. \[[@B22]\] The effect of pollutants on health is typically determined by exposure assessment, which is not an uncomplicated process. \[[@B23]\] For purposes of simplification the effects of pollutants were estimated with circular buffers. \[[@B24]\] Integration of exposure modelling within the SOM-GIS method is a natural next step that will improve the quality of input variables. \[[@B25]\] Another simplifying assumption was that Census variables from 2000 represented socioeconomic inputs for the five year period leading up to the Census survey. This assumption did not account for the dynamics associated with demographic variables such as migration or socioeconomic status. The necessity for health data also presented a substantial challenge. Hospital discharge data from the New York State Department of Health (NYSDOH) included conditions serious enough to require inpatient hospitalizations, but did not include data from outpatient services, minor emergency centres, or physician offices and clinics. The study design could not account for the latency between influence of environmental conditions and the onset of symptoms or disease and so the potential for patient migration between communities is of concern when using patient address to assign disease occurrences to communities.
Unanswered Questions and Future Research
----------------------------------------
Our study presented methods that contribute to further research concerning the complexities of environmental systems and their relationship to human health outcome. \[[@B26],[@B27],[@B2]\] Using the SOM-GIS method, patterns relating a large number of variables and their interactions can be analyzed to group communities exhibiting similar data structures. If patterns are observed among the environmental conditions between communities and these patterns correspond significantly to the distribution of various diseases, several questions arise with numerous opportunities for future research. The overarching question is how can the mathematical patterns found among environmental variables be used to understand what is causing differences in observed rates of specific diseases? To determine how environmental conditions influence specific diseases, the variables (single or interactive group variables) that influence pattern structure should be identified. The context of these questions should also be explored to understand how the scale at which systems are studied modifies outcomes and also to determine the influence of nested hierarchical domains on observations at all scales. For example, what is observed at the individual level includes not only individual risk factors, but factors that operate at the population and regional scale, and the way these risk factors change through time. Within the need for contextual studies, research questions should consider the dynamical component of systems and include the temporal dimension to further understand latency between environmental effects and health outcomes.
Conclusion
==========
The significant relationship between SOM classifications and the geographic distribution of population-adjusted rates for selected diseases demonstrated a positive relationship between environmental conditions and health outcomes supporting previous work that described the environment as a determinant of population health. \[[@B28],[@B29]\] This result provides observation-based credibility to conceptual theories suggesting that the environment functions as a complex system; and that environment is correlated with distributions of both chronic and infectious diseases in community level populations. \[[@B3],[@B30],[@B2]\] Given that environmental conditions are related to health outcomes, environmental variables may be useful in estimating population health. Multivariate environmental assessments may be used as proxies for practice-based health assessments in cases where data are limited. Further study is needed to determine the contribution of individual variables (or groups of variables), identify readily available data sets, and to fully investigate the development of a meaningful proxy measure.
Methods
=======
Data Collection and Preparation
-------------------------------
New York State has 62 counties that cover a wide range of landscapes, climate zones, industries, and socioeconomic populations <http://www.labor.state.ny.us/workforceindustrydata/geog.asp>. The counties were categorized based on the level of urbanization such that counties were grouped as highly urban, mixed urban, suburban, rural, and very rural to include a representative of each type of environment. One county was randomly selected from each of these groups; our analysis included five of the 62 counties (8%). The counties selected: New York City (Manhattan), Erie, Westchester, Steuben, and Hamilton.
To identify community boundaries U.S. Census tracts, considered homogeneous groups, were used for the upstate counties. Community boundaries within New York City were adapted from the New York City Department of City Planning. \[[@B31]\] Our intention was to select variables to represent the environmental factors described by previous conceptual models. \[[@B32]\] Environmental variables meeting the following requirements were collected from existing state-wide data sets for the 1995--2000-study period:
1\. Ratio level observations,
2\. Spatially referenced,
3\. Consistent for all counties studied,
4\. Measured at the community level.
Data sets as input variables for the SOM included physical, economic, occupation, housing, education, and demographic information (Table [2](#T2){ref-type="table"}). Figure [2](#F2){ref-type="fig"} shows the geographic distribution of physical variables \[including land use and Toxic Release Inventory (TRI)\]. Data were formatted and pre-processed using SAS, Microsoft Excel, or ArcGIS to achieve spatial and temporal compatibility.
######
Polymorphisms and substitution rates in the L, M and S sequences amplified from field-infected mosquitoes.
**Environment** **Variable** **Description** **Source**
----------------- ------------------ ------------------------------------------------------ -----------------------------------
Physical Air Quality CO, NO, SO2, PM10, VOCU, PMU NYSDEC Air Emission
Physical Toxic Release TRI Waste (Air & Water) US EPA Toxic Release Inventory
Physical Rare Species Presence & Rank of Rare Species NYSDEC Natural Heritage Inventory
Physical Land Use Majority Class (11 21 22 23 41 42 43 81 82 85) USGS NLCD
Economic Household Income Income Distribution US Census ESRI CommunityInfo
Economic Rent Amount Average Contract Rent US Census ESRI CommunityInfo
Economic Home Value Average Value Owner Occupied Housing US Census ESRI CommunityInfo
Occupation Work Location Within County of Residence US Census ESRI CommunityInfo
Occupation Transportation Mode to Work (Auto, Carpool, Public, Bicycle, Walk) US Census ESRI CommunityInfo
Occupation Travel Time Travel Time to Work US Census ESRI CommunityInfo
Occupation Industry Industry of Employment (20 Classifications) US Census ESRI CommunityInfo
Housing Age of Housing Year of Construction (Decadal Increments) US Census ESRI CommunityInfo
Housing Num Households Total Households US Census ESRI CommunityInfo
Housing Household Size Average Household Size US Census ESRI CommunityInfo
Housing Owner Occupancy Owner vs. Renter Occupancy Ratios US Census ESRI CommunityInfo
Housing Vacant Housing Vacant Housing Units US Census ESRI CommunityInfo
Education Education Education Attainment (None thru Doctoral Degree) US Census ESRI CommunityInfo
Demographic Race Racial Group (7 Categories) US Census ESRI CommunityInfo
Demographic Family Family Status (Married, Children in Household), Size US Census ESRI CommunityInfo
Demographic Median Age Median Age US Census ESRI CommunityInfo
Demographic Population Size Total Population US Census ESRI CommunityInfo
{#F2}
Toxic Release Inventory (TRI) maintained by the U.S. E.P.A. included self-reported releases and waste management activity for industrial facilities. Source locations were geocoded using longitude and latitude coordinates provided with the data and cross referenced with facility addresses to ensure positional accuracy. The reported discharge amounts were summed and added as an attribute for each facility in GIS.
Air pollution data was obtained from the New York State Department of Environmental Conservation (NYSDEC) for permitted stationary industrial facilities. Locations were geocoded using address and the accuracy was checked using Google Earth. Facilities ranged from large industrial sources such as refineries and chemical manufacturers to small businesses such as dry cleaning operations and filling stations. Data was originally collected to monitor for permit compliance and contained the amount of each pollutant discharged by location for each year for carbon monoxide, sulphur dioxide, and oxides of nitrogen, volatile organics, total particulate matter, and PM~10~. Annual emission amounts were summed for each of the pollutants by facility and added as an attribute to the facility location in GIS.
The impact area of chemical releases by both TRI and air sources were approximated with a 1-km^2^circular point buffer around the facility. \[[@B33],[@B24]\] The spatial fraction of the buffer contained within the community was multiplied by the discharge quantity from the source. For example, if \"Facility A\" discharged 10,000 pounds of chemical, and .68 of the 1-km^2^area was contained within Community 1, an estimated 6800 pounds of chemical discharge was allocated to Community 1. Other communities containing the remaining fraction of Facility A\'s effect region were assigned the remaining fraction(s) of the emission using the same method. The circular buffers can be seen in Figure [2](#F2){ref-type="fig"}.
The status of rare species and land use represents the ecosystem related data. Land use data were obtained via remote sensing from the United States Geological Survey (USGS). Land use type and percent cover by type were calculated for each community using the zonal statistics function in ArcGIS. The majority land use type is shown in Figure [2](#F2){ref-type="fig"}, represented by shading of communities.
The presence and quality of rare species within a community was selected as a variable to approximate the level of biodiversity, an indicator of ecosystem health. \[[@B27]\] The NYSDEC, Natural Heritage Inventory (NHI) monitors 174 natural community types, 727 rare plant species, and 432 rare animal species across New York, keeping track of more than 11,900 locations where these species and communities are found <http://www.dec.ny.gov/animals/29338.html>. The database includes detailed information on the relative rareness of each species and community, the quality of their occurrences, and descriptions of sites. Data were provided in a de-identified format so that occurrences were listed by location and quality (see website for listing and definition of categories), but the scientific and common names of the organism were omitted for protection. Occurrences were ranked and weighted according to the quality reported by NHI, and the number of ranked occurrences per community was summed in ArcGIS. For example, the presence of a rare species was counted as 1, with added value for the ranked quality of the specimen.
ESRI\'s CommunityInfo product provided the 2000 U.S. Census SF3 survey data to represent the social, cultural, economic, educational, and occupational components of the communities. Each group of variables contained measures of subcategory variables (Table [2](#T2){ref-type="table"}). Given that the study period was 1995--2000, the 2000 Census data were considered representative of the years preceding and leading up to the survey. Every community (such as a Census Tract) was assigned an identification number that was used to join variables using ArcGIS. The resulting table listed each community as a row with corresponding environmental variables occupying the columns.
Self-Organizing Map Analysis
----------------------------
Data were imported from ArcGIS to Viscovery SOMine <http://www.eudaptics.com> for analysis. The number of input variables was multiplied by ten to establish the number of map nodes at 920; map tension influences the neighbourhood radius around nodes and was set at 0.2. \[[@B34]\] Analysis began with map training, or the gradual adaptation of nodes on the grid to resemble the underlying shape of the distribution. Thus, the order of nodes reflected the mathematical neighbourhood inherent in the data. Map training included searching for data clusters, retrieving numerical information, and calculation of cluster statistics. Cluster tuning used the significant breaks between groups of nodes to determine the number of clusters. Results were visualized as a two-dimensional hexagonal grid (or map) that indicated the relationship between nodes and displayed the distribution of data according to clusters. Each community was assigned to a data cluster based on the patterns observed for each corresponding variable. The geographic distribution of community clusters was mapped in ArcGIS using the community ID number to provide spatial reference.
Health Data
-----------
The New York Data Protection Review Board reviewed and approved the use of data from the NYSDOH SPARCS inventory. This project was subject to additional review and approval by the University of Oklahoma IRB for human subjects research prior to the use of health information for this study. All data were used in a manner compliant with agreements between investigators and the NYSDOH and OU IRB.
Ten diseases were selected from the SPARCS database \[[@B35]\] to include infectious and chronic conditions (these are indicated on Table [1](#T1){ref-type="table"} in the results section). Disease occurrences were selected using ICD-9 codes, unique personal identifier codes, county, and year; and data were geocoded using patient address from the medical record. The observed frequency of each disease for every community was scaled according to community population and area density ratios. Analysis of variance (ANOVA) was conducted in SAS to test the relationship between disease frequency and community classification, assuming that all clusters had an equal opportunity for occurrence of disease.
Competing interests
===================
The authors declare that they have no competing interests.
Authors\' contributions
=======================
HB and MY collaborated on the study design, geographical analysis, and statistical analysis. HB obtained and preprocessed data, conducted SOM analysis and drafted the manuscript. MY also helped to draft and edit the manuscript.
Acknowledgements
================
The New York State Department of Health provided health data from the SPARCS program. The New York State Department of Environmental Conservation also provided data for SOM inputs from the Natural Heritage Inventory and Division of Air Resources. The University of Oklahoma Health Sciences Center, College of Public Health provided funds to purchase SPARCS data from the NYSDOH. The authors are grateful for the contributions of these organizations.
| {
"pile_set_name": "PubMed Central"
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Background {#Sec1}
==========
Scientific background {#Sec2}
---------------------
Cognitive health has been consistently quoted as significant for life quality by older people and is regarded as an important contributor to late-life functioning \[[@CR1], [@CR2]\]. Patterns of cognitive change show great variation in healthy aging \[[@CR3]\]. Cognitive training might contribute to enhance or preserve cognitive skills in cognitively healthy older adults (HOA). While improvements in cognition through training have been reported frequently in HOA, the capability to transfer cognitive training gains decreases with age \[[@CR4]--[@CR8]\]. Ideally, cognitive training would not only improve the function of trained cognitive tasks but also of untrained tasks of the same or a different cognitive domain (i.e., transfer of training gains). There is little evidence for successful transfer of cognitive training effects in HOA \[[@CR6], [@CR9]\]. Moreover, although transfer has been the subject of research for many years \[[@CR10]\] the neurobiological mechanisms underlying the complex capability of transfer are still unknown. We recently demonstrated successful transfer of logical reasoning training gains to fluid intelligence immediately after a 4-week training in 71% of the HOA (*N* = 29 out of 41), but this effect persisted only in a subgroup of 22% over a 3-month follow-up period (*N* = 9 out of 41) \[[@CR11]\].
When we assessed structural underpinnings of successful transfer in this previous study \[[@CR11]\], we focused on corpus callosum integrity as a structural prerequisite for successful bihemispheric cooperation, since cross-hemisphere processing results in better performance than within-hemisphere processing in complex cognitive tasks \[[@CR12]\]. Since the callosal structural integrity is related to age (see Fig. [1a](#Fig1){ref-type="fig"}), and age-related disruption of corpus callosum microstructure in HOA impacts the efficiency of bihemispheric processing \[[@CR13]\], we expected that associations between age and transfer might be moderated by the structural integrity of the corpus callosum. While short-term transfer was not related to structural integrity, stable transfer (ST) was predicted by the structural integrity/ connectivity of the corpus callosum \[[@CR11]\]. Based on this preliminary evidence \[[@CR11]\] we hypothesize that bihemispheric processing supported by intact corpus callosum integrity is a precondition of successful transfer in HOA (see Fig. [1a](#Fig1){ref-type="fig"} and [b](#Fig1){ref-type="fig"}). Beside age, several other factors are likely to modulate corpus callosum integrity and other preconditions of successful bihemispheric processing and thereby facilitate or impair the transfer capability in HOA, such as physical activity or physical training, cerebral vascular or cerebral amyloid pathology, genetic factors of risk or resilience, and general intelligence \[[@CR14], [@CR15]\].Fig. 1Age-relation of callosal structural integrity (**a**) and hypothetical association of callosal structural integrity with transfer probability in cognitively healthy elderly (**b**). **a** Fractional anisotropy (FA) of the genu and corpus of the corpus callosum decreases with age in cognitively healthy elderly \[[@CR11]\]; higher FA values indicate better structural integrity. **b** Hypothetical probability of transfer decreases with decreased structural integrity of the corpus callosum
Objectives {#Sec3}
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### Primary objective {#Sec4}
Four types of transfer training interventions have been described: strategy-based (e.g., logical reasoning), multimodal (complex, often social or lifestyle interventions), cardiovascular (i.e., physical training), and specific cognitive process-targeted training interventions (e.g., working memory) \[[@CR16]\]. Transfer effects differ between transfer types but share a common feature: transfer effects decline with age \[[@CR16]\]. To date, little is known about the neural mechanisms of transfer \[[@CR16]\]. Specifically, the mechanisms of either maintenance or age-related decrease of transfer of cognitive training gains in HOA are largely unknown. Increased bihemispheric cooperation can reliably be seen with increasing processing demands in young adults and, as we showed, already on lower levels of cognitive demand in HOA \[[@CR17]\]. This compensatory increase of bihemispheric processing in HOA is known as the concept of Hemispheric Asymmetry Reduction in Older Adults (HAROLD) \[[@CR12]\]. This phenomenon could be the functional link between the observed association of transfer capabilities and structural integrity of the corpus callosum. Beyond pure compensation, HAROLD may serve as a mechanism enabling the structurally and functionally altered aged brain to use alternative neural circuitry and thereby to re-establish more efficient lateralized processing during learning \[[@CR16]\]. Hence, increased bihemispheric cooperation/HAROLD, based on the structural integrity of the corpus callosum, may indeed be a functional mechanism mediating transfer capabilities in HOA.
Main research goal of the AgeGain study is to investigate the neurobiological mechanisms of transfer of cognitive training gains in detail by elaborating brain network capabilities representing transfer using a multimodal-neuroimaging approach in HOA. Specifically, the following primary hypothesis will be tested: Structural integrity of the corpus callosum (high / low) and bihemispheric cooperation (reduced / increased) will predict successful transfer of cognitive training gains in HOA. The association of corpus callosum integrity with transfer capability will be determined by diffusion-tensor imaging (DTI) measures of structural integrity / connectivity, mainly fractional anisotropy (FA) of the corpus callosum, see Fig. [2a](#Fig2){ref-type="fig"}. The association of bihemispheric processing and transfer capability will be determined using task-related functional magnetic resonance imaging (fMRI) (see Fig. [2b](#Fig2){ref-type="fig"}).Fig. 2Joint models of brain structural (**b**) and functional (**a**) mechanisms for the explanation of transfer capability in healthy aging. **a** Fractional anisotropy (FA) of the genu and corpus of the corpus callosum decreases with age in cognitively healthy elderly; higher FA values indicate better structural integrity. Structural integrity of the corpus callosum predicts transfer capability determined by stable success (ST) versus non-transfer (NT) \[[@CR11]\]. Categorial transfer was defined as an increase of fluid intelligence performance (transfer task) beyond the retest effect of untrained healthy elderly after successful training of logical reasoning skills \[[@CR11]\]. Taken the corpus callosum structural integrity (FA) as surrogate of transfer capability, the model delineates a threshold of structural integrity (− − −) dividing ST and NT. Moreover, the model suggests that (e.g., z-standardized) FA values could be taken as dimensional predictors of the transfer amount in single subjects. **b** Increased hemispheric cooperation/HAROLD as measured by BOLD lateralization index \[[@CR70]\] may mediate transfer capabilities in older adults since both are associated with the structural integrity of the corpus callosum. NT subjects may show less hemispheric cooperation compared to ST subjects at baseline thereby predicting less transfer success while both groups show the general pattern of lateralized to bilateral to disengagement of activity with increasing task demand \[[@CR17]\]
### Secondary objectives {#Sec5}
There is evidence that physical activity and cardiorespiratory fitness are associated with reduced brain tissue loss and reduced risk for cognitive impairment in aging humans \[[@CR18], [@CR19]\]. Moreover, aerobic exercise training increased gray and white matter volume in the prefrontal cortex \[[@CR20]\] of older adults and led to significant improvements of executive function \[[@CR21], [@CR22]\]. Further, hippocampal and medial temporal lobe volumes were larger in older adults with greater cardiorespiratory fitness \[[@CR19], [@CR23]\], and larger hippocampal volumes mediated improvements in spatial memory \[[@CR19]\]. Aerobic exercise training increases cerebral blood volume \[[@CR24]\], perfusion \[[@CR25]\] and the adult hippocampal volume. It has been suggested that the aforementioned structural changes are mediated by neurotrophins, in particular the brain derived neurotrophic factor (BDNF) \[[@CR26]\]. We \[[@CR27]--[@CR30]\] and others \[[@CR31], [@CR32]\] demonstrated that exercise induces significant increases in levels of BDNF and other growth factors, and higher serum BDNF after aerobic training was associated with larger anterior hippocampal volume \[[@CR33]\]. Using DTI, more recent studies measured brain structural integrity as a function of cardiorespiratory fitness and aerobic exercise training in HOA. Fractional anisotropy of the corpus callosum was reported to be positively correlated to cardiorespiratory fitness in HOA \[[@CR34]\]. Thus, cardiorespiratory fitness might modulate age-related alterations of corpus callosum integrity. In addition, greater aerobic fitness derived from a walking program was associated with increases in white matter integrity of the frontal and temporal lobes, and improvement of memory performance \[[@CR14]\]. Consistently, using resting state fMRI analyses, it has been shown that cardiorespiratory fitness is positively associated with functional connectivity in the Default Mode Network, and that this in part mediates improved performance on tasks requiring set-shifting, task switching, and spatial working memory \[[@CR35]\]. Furthermore, 1 year of regular walking increased functional connectivity between parts of the frontal, posterior, and temporal cortices within the Default Mode Network and the Frontal Executive Network \[[@CR35]\]. Of note, there is evidence that the beneficial effects of physical activity on cognitive performance is not limited to cardiorespiratory fitness but is also present after resistance \[[@CR36]--[@CR38]\] and coordination exercise training \[[@CR39], [@CR40]\]. Moreover, different types of physical training have been shown to affect different neurocognitive networks \[[@CR40], [@CR41]\]. We hypothesize that coordination exercise training positively modulates cognitive transfer abilities in HOA based on the following rationale: coordination training aims at improving the efficiency of complex body movements including eye-hand coordination, bimanual coordination, leg-arm coordination, and reactions to moving objects \[[@CR40]\]. Such complex movements require bihemispheric interactions via the corpus callosum \[[@CR42]\], and both, size and structural integrity of the corpus callosum typically change after sensorimotor skill acquisition \[[@CR43]\]. These interactions are probably not limited to pure motor processes but rather include selective aspects of cognition necessary for adaptive motor behavior such as attention, inhibitory and facilitatory processes, working memory, and error-related processing. In line with this, it has been demonstrated that rather anterior corpus callosum projections to the prefrontal cortex than middle corpus callosum projections to the primary motor cortex predict bimanual motor learning \[[@CR44]\].
In sum, aerobic exercise -- and coordination exercise training are associated with positive structural and functional brain changes as well as improvements of cognitive performance in HOA. Moreover, cerebral regions targeted by these interventions are primarily involved in age-related cognitive decline including the corpus callosum, the hippocampus, frontal and temporal cortices, and the Default Mode Network. As a secondary outcome, we will determine if physical training, comprising aerobic and coordination components, modulates transfer of cognitive training gains in HOA. Moreover, we will determine the impact of baseline physical activity on transfer in HOA.
Subcortical cerebrovascular lesions reflected in white matter hyperintensities and cortical β-amyloid aggregation are highly prevalent and strongly age associated with central nevous system (CNS) pathologies \[[@CR45]\]. Both findings are suspected to have a deleterious effect on neuronal function. White matter hyperintensities have been found to affect structural connectivity in the brain on the subcortical level \[[@CR46]\]. Amyloid pathology, on the other hand, which is a hallmark of Alzheimer's disease \[[@CR47]\], has been discussed to affect regional synaptic function on the cortical level \[[@CR48]\]. Although both findings have been associated with manifest cognitive decline or dementia, they are known to be present in a significant proportion of the cognitively healthy elderly, potentially leading to subclinical deficits \[[@CR49], [@CR50]\]. White matter hyperintensities frequently observed in the prefrontal cortex in clinically HOA have been linked to subclinical disturbances of executive function, whereas cortical amyloid deposition has been associated with a minor decline in memory function in this population \[[@CR51]\]. Transfer capability represents a multidimensional cognitive function which requires executive and memory skills and presumably depends on highly efficient neuronal pathways including bihemispherical communication. Thus, the two mentioned pathologies are supposed to have a cumulative negative effect on transfer performance, even ahead of other cognitive symptoms. Consistently, as a further secondary outcome, the study aims at investigating white matter hyperintensities and cerebral amyloid burden as potential modulators of transfer of cognitive training gains in HOA.
We and others found that Default Mode Network activity is related to cognitive function and cognitive reserve in HOA \[[@CR52], [@CR53]\]. Default Mode Network activation during cognitive tasks was accompanied by less default suppression with greater task demand and less connectivity among Default Mode Network regions as well as increased frontal activation \[[@CR54]\]. Thus, since cognitive reserve capacity as referring to Default Mode Network might also predict transfer cabilities, resting-state fMRI connectivity measurements will be also conducted \[[@CR55]\]. Moreover, based on the comprehensive data survey the study aims at modeling and analyzing multimodal, high-dimensional datasets with respect to transfer prediction and to build a robust individual index of transfer likelihood.
All secondary outcomes are summarized in Table [1](#Tab1){ref-type="table"}.Table 1Overview of secondary outcomesSecondary objectivesTo evaluate the predictive value of a preceding aerobic and coordination training for transfer of cognitive training gains in healthy older adults (HOA)To evaluate the predictive value of baseline physical activity on transfer in HOA. Baseline physical activity will be measured by a 1-week actigraphy and the Global Physical Activity Questionnaire (see "[Measures](#Sec13){ref-type="sec"}" section below).To evaluate the predictive value of brain vascular lesion (as determined by T2-weighted magnetic resonance imaging (MRI), cortical amyloid burden (as determined by positron-emission tomography), higher default mode network activity (as determined by resting state functional MRI (fMRI) for transfer of cognitive training gains in HOATo model and analyze multimodal, high-dimensional datasets with respect to transfer prediction and to build a robust individual index of transfer likelihood
Methods/design {#Sec6}
==============
Study type {#Sec7}
----------
This is a longitudinal, interventional, parallel-group, multicenter, multimodal-imaging trial.
Study design {#Sec8}
------------
In this 4-year parallel-group, multicenter, multimodal-imaging study, cognitively healthy elderly subjects will be enrolled by three recruiting centers in Germany: Mainz (University Medical Center Mainz - Department of Psychiatry and Psychotherapy), Rostock (University Medical Center Rostock, Clinic of Psychosomatic and Psychotherapeutic Medicine and German Center for Neurodegenerative Diseases (DZNE)), and Cologne (German Sport University Cologne and University Hospital Cologne -- Department of Nuclear Medicine). The experimental protocol can be divided into five phases (see Table [2](#Tab2){ref-type="table"}). In phase 1, inclusion and exclusion criteria will be assessed (screening). In phase 2 (pre-cognitive-training phase), participants will undergo baseline neuropsychological and physical activity assessment, MRI, positron-emission tomography (PET), and genetics. Thereafter, in phase 3 (cognitive-training phase), a cognitive training will be applied over a period of 4 weeks (with three 60-min-long training sessions per week). Directly following, in phase 4 (post-cognitive-training phase), participants will repeat the neuropsychological assessment to determine immediate transfer effects. Finally, in phase 5 (follow-up, after 3 month), a final neuropsychological assessment will be applied to determine ST effects. The study protocol follows the SPIRIT (Standard Protocol Items: Recommendations for Interventional Trials) recommendations (see Additional file [1](#MOESM1){ref-type="media"}). Table 2Trial schedule University Medical Center Mainz and University Rostock^a^Schedule for each subject^b^The Florbetaben-PET can be postponed to the post-cognitive-training phase or the follow-up phaseAbbreviations: *MRI* magnetic resonance imaging, *fMR*I functional magnetic resonance imaging, *PET* positron-emission tomography, *ApoE4* apolipoprotein E4, *CETP* Cholesteryl Ester Transfer Protein, *SNP* single nucleotide polymorphism, BDNF brain-derived neurotrophic factor
To investigate the impact of a physical training (combined aerobic and coordination training, ACT) on transfer of cognitive training gains in HOA (secondary outcome), subjects recruited at the center in Cologne will undergo an extended experimental protocol with two additional phases prior to the pre-cognitive-training phase (pre-ACT-training phase, ACT-training phase). The primary protocol (as shown in Table [3](#Tab3){ref-type="table"}) will be complemented by a 20-week physical training period and an evaluation of the physical fitness, as well as an additional MRI scan evaluation (see Table [3](#Tab3){ref-type="table"}).Table 3Trial schedule German Sport University Cologne/University Hospital Cologne^a^Schedule for each subject^b^The Florbetaben-PET can be postponed to the post-cognitive-training phase or the follow-up phaseAbbreviations: *MRI* magnetic resonance imaging, *PET* positron-emission tomography, *ApoE4* apolipoprotein E4; *CETP* Cholesteryl Ester Transfer Protein, *SNP* single nucleotide polymorphism, *BDNF* brain-derived neurotrophic factor
Population {#Sec9}
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### Sample size {#Sec10}
It is planned to include 237 cognitively healthy elderly subjects (aged 60 years and older) in total distributed over the three trial sites, satisfying the statistical power requirements for different research questions, as described below. Paticipants will be recruited by local newspaper announcements and flyers.
For the investigation of the primary objective, a sample size of 160 subjects has been determined based on a power calculation (see "[Statistics](#Sec23){ref-type="sec"}"). Assuming a dropout rate of about 15%, the recruitment of subjects will be distributed as follows: Mainz: *N* = 60, Rostock: *N* = 60, German Sport University Cologne/University Hospital Cologne: *N* = 72.
For the investigation of the predictive value of a preceding ACT for the transfer of cognitive training gains in HOA (secondary objective), all 72 subjects recruited at the German Sport University Cologne/University Hospital Cologne (who are also part of the investigation of the primary outcome) will undergo an extended experimental protocol, including ACT followed by the cognitive training. The sample size of 72 subjects has been determined based on a power calculation (see "[Statistics](#Sec23){ref-type="sec"}"). Since, so far, no evidence has been published showing that physical activity may have an impact on transfer via mechanisms other than brain structure and function, it is assumed that the preceding physical training in subjects recruited in Cologne will not confound the investigation of the primary objective.
To minimize/avoid bias, the following control subjects will be included: 15 HOA will be enrolled in Cologne undergoing the standard experimental protocol without ACT to assure that the net effect of the cognitive training does not differ from the effects at the other trial sites. To control performance alterations in the neuropsychological examinations for retest effects, an additional sample of 30 HOA, generated in Mainz (*N* = 10), Rostock (*N* = 10), and German Sport University Cologne/University Hospital Cologne (*N* = 10), will undergo the repeated neuropsychological examinations without cognitive training.
Within each trial site, participants will be distributed randomly to experimental or control groups, stratified centrally by the trial coordinator at the trial site University Medical Center Mainz. The study code is centrally managed by this trial site. Participants will be enrolled by their respective trial site.
### Inclusion criteria (for all trial sites) {#Sec11}
The following inclusion criteria will be applied:Age ≥ 60 yearsAbility of subject to understand character and individual consequences of clinical trialSigned and dated informed consent must be available before start of any proceduresSufficient mobility and motivation to be able to participate in the examinations
### Exclusion criteria (for all trial sites) {#Sec12}
Incapability of giving consentCurrent (or history of) psychiatric illnessCurrent (or history of) neurological or cerebrovascular illness, brain lesionsCurrent (or history of) cardiovascular disease (i.e., myocardial infarct, peripheral arterial disease)Secondary disorders restricting individuals' physical capacity (i.e., chronic obstructive pulmonary disease, rheumatism, osteoarthritis, bone fractures)Current (or history of) cognitive illnessDiabetes types 1 and 2Intake of medications that may influence cognitive performanceInsufficient German language skillsParticipation in other clinical trials during the present clinical trial or within the last monthMRI contraindication (pacemaker, metal implants, tattoos, permanent-make-up, chochlear implant, medication pump, acupuncture needles)
Measures {#Sec13}
--------
### Assessment of inclusion/exclusion criteria {#Sec14}
Telephone screeningDemographics, psychiatric screening (diagnostic expert system for psychiatric disorders -- Stamm Screening Interview \[[@CR56]\], International Diagnostic Checklists for ICD − 10 and DSM-IV \[[@CR57]\]Study information and informed consentGeneral study information and informed consent, MRI information and informed consent, PET information and informed consent, genetics information and informed consent
### Neuropsychological examination at baseline (NP I) {#Sec15}
IntelligenceWortschatztest \[[@CR58]\], Hamburg-Wechsler Intelligence Test for adults-revised -- subtests: picture completion, similarities, block design, arithmetic \[[@CR59]\]MemoryVerbaler Lern- und Merkfähigkeitstest \[[@CR60]\], Wechsler-Memory Scale-revised -- subtests: digit span, block span (forward and backward, respectively) \[[@CR61]\], Cogpack -- subtest: remembering \[[@CR62]\]Executive functionLeistungsprüfsystem (comparable to the Raven Matrices): subtest 4 \[[@CR63]\], Tower of London \[[@CR64]\], Trail-Making Test B \[[@CR65]\], Cogpack -- subtest: reasoning \[[@CR62]\].Stimulus interferenceComputerized version of the Stroop TestInformation processing speedTrail-Making Test A \[[@CR65]\], Hamburg-Wechsler Intelligence Test for adults-revised: digit-symbol substitution test \[[@CR59]\]Visual constructionRay-Osterrieth Complex Figure Test -- copy \[[@CR66]\].AttentionTest battery for attention performance -- subtests: alertness, divided attention
### Neuropsychological examination immediately after cognitive training (NP II) {#Sec16}
In the second neuropsychological examination (NP II, post-training phase), the same test battery as in the baseline examination (NPI) will be applied. However, the tests measuring intelligence will only be applied once (at NP I). If available, we will apply parallel test versions to avoid retest effects.
### Neuropsychological examination after 3 months (NP III) {#Sec17}
In the third neuropsychological examination (NP III, follow-up), the same test battery as in NP II will be applied. If available, we will apply parallel test versions to avoid retest effects.
### Neuropsychological training {#Sec18}
Executive functions, memory, information processing speedCogpack: Cogpack is a computerized cognitive training and testing program. The subtests comparisons, searching, logic, anagrams, complete a logical block, and remembering will be appliedAlertnessAttention capacities will be trained using the test battery for attention performance, which permits us to assess/train a variety of attentional aspects. The subtests alertness and divided attention will be trained.Working memoryThe computerized training software TATOOL \[[@CR67]\] will be used to apply a working memory training designed in accordance to Batian et al. \[[@CR68]\]
### Physical activity examination {#Sec19}
ActigraphyObjective physical activity, sleep/wake and energy expenditure measurement solution. The portable wristband (GeneActive, Kimbolton, UK) uses a three-axis accelerometer, a heat flux sensor, a galvanic skin-response sensor, a skin-temperature sensor, and a near-body ambient temperature sensor to capture data for 1 weekGlobal Physical Activity QuestionaireThe Global Physical Activity Questionaire covers several components of physical activity typical of an elderly population. The score takes into account self-reported occupational, household and leisure activities items over a 1-week period (World Health Organization (WHO), <http://www.who.int/chp/steps/GPAQ/en/>)
### Fitness examinations {#Sec20}
Cardiovascular fitnessCardiovascular fitness will be assessed using a modified, graded, exercise-testing protocol in the field. Endurance capacity will be estimated on the basis of walking/running speed, the corresponding concentration of blood lactate and the perceived exertion of each participant.Additionally, the maximum oxygen uptake (VO~2~max) and the peak oxygen uptake (VO~2~peak) will be estimated using the 6-minute Walking Test.Motor fitnessGross motor fitness will be assessed using a test battery comprising the following basic motor skills:Static and dynamic balanceShort Physical Performance BatteryFeet tappingHand tappingKasten-Bumerang TestMovement-Coordination TestAgility TestFine motor fitness will be assessed using the following subtests of the Vienna Test System:Motor performance seriesSensomotor coodinationSpatial orientationReaction timeResponse inhibitionTime/movement anticipation
### Imaging data acquisition {#Sec21}
Structural MRIDTI, T1-weighted structural MRI, FLAIR, high-resolution T2-weighted structural MRIFunctional MRIResting-state MRI, task-related fMRI. For task-related fMRI, participants will perform three runs of the Hybrid Response Inhibition task \[[@CR69]\]. Using identical visual stimulus material the Hybrid Response Inhibition task assesses three subcomponents of response inhibition: response interference, action withholding, and action cancelation (Fig. [3](#Fig3){ref-type="fig"}). Stimuli will be presented in the center of the screen. Participants will be asked to perform a button press according to the pointing direction of an arrow and to refrain from a button press whenever the ellipse surrounding the arrows turns blue (nogo−/stop trials). Event-related fMRI data acquisition will be performed using standard echo planar imaging sequences with whole-brain coverage and isotropic voxels. SPM 12 (<http://www.fil.ion.ucl.ac.uk/spm>) will be used to conduct all image preprocessing and statistical analyses. In addition to standard general linear model analyses, a BOLD lateralization index \[[@CR70]\] will be computed for each subcomponent separately. The lateralization index is hypothesized to mediate transfer capabilities in older adults and to be associated with the structural integrity of the corpus callosum.PETThe amyloid tracer \[18F\]Florbetaben will be applied via a venous cannula. Subjects will be instructed to void their bladder to allow rapid excretion of unbound radioactivity. Subjects will be placed on the scanner approximately 70 min after injection. At 80 min p.i. (post injection) two low-dose computer tomography scans will be acquired for position and attenuation correction. At 90 mins p.i., the PET acquisition will be initiated. PET data will be acquired for 20 min. After the scan the subject will be asked about their well-being and instructed to void their bladder to accelerate excretion of radioactivity. Subjects will be instructed to minimize contact with small children and pregnant women for 12 h after tracer injection. The entire examination, including preparation and scanning procedures, can be finished within 3--3.5 h. Fig. 3The Hybrid Response Inhibition task. Participants are asked to press a button corresponding to the pointing direction of an arrow. Go trials consist of congruent trials; inhibition trials consist of incongruent trials (interference inhibition), occurrence of a no-go stimulus (blue ellipse; action withholding), or of a stop-signal (blue ellipse after a varying stop-signal delay; action cancelation)
### Genetics {#Sec22}
Two samples of 5 ml ethylenediamine tetraacetic acid blood will be collected from participants at the pre-training phase and at the post-training phase in Mainz and Rostock and at the pre-ACT-training phase, post-ACT-training phase, and post-cognitive-training phase in Cologne. Blood samples will be kept on ice for a maximum of 15 min and subsequently stored at − 80 °C in the recruiting centers. Frozen blood samples will be shipped on dry ice to the Laboratory of Molecular Genetics, Institute of Human Genetics, University Medical Center Mainz. There, deoxyribonucleic acid (DNA) extraction from one of the frozen blood samples taken at the pre-training phase will be performed on a chemagic Magnetic Separation Module I using the chemagic DNA Blood Kit special for the automated isolation of DNA from 5 ml whole blood followed by quantification of DNA on a NanoDrop 2000UV-Vis spectrophotometer. Genotyping for the single nucleotide polymorphisms rs7412 and rs429358 in the apolipoprotein E gene, rs5882 and rs1800775 in the *Cholesteryl Ester Transfer Protein* gene, rs6265 in the *BDNF* gene, rs2765 in the *Neurokinin.3 Receptor* gene, rs6439886 in the *Calsyntenin 2* gene, and rs17070145 in the *Kidney and Brain Expressed Protein* gene will be carried out from extracted genomic DNA samples using polymerase chain reaction followed by pyrosequencing on a Pyromark Q96 ID Instrument using PyroMark Gold Q96 SQA Reagents and the Pyromark Q96 ID software. The second blood sample taken at the pre-training phase and both blood samples taken at the post-training phase will be stored at − 80 °C for prospective analyses of training-induced epigenetic and transcriptome changes. Blood samples will be stored in secured rooms at the Institute of Human Genetics of the University Medical Center Mainz for a maximum of 60 years.
Statistics {#Sec23}
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### Sample size {#Sec24}
Primary outcome: the analyses will focus on training gain transfer (defined as a binary criterion for each subject) and simultaneously incorporate measures of corpus callosum integrity and bihemispheric processing. The sample size calculation was performed based on main effects analyses where the effects of both measures will be considered as main effects. Assuming a transfer rate of cognitive training gains of about 15% for participants with low corpus callosum integrity, and a transfer odds ratio of 3 for participants with high corpus callosum integrity, a sample size of 160 provides a power of 0.8 at a significance level of 0.05 for a Wald Test in a logistic regression model. Assuming a dropout rate of about 15%, a total of 192 participants need to be included.
Secondary outcome: for evaluating physical training as a potential modulator of cognitive training gain transfer, ACT-induced fitness changes will be analyzed with respect to changes of corpus callosum integrity, as well as with respect to effects on transfer. The former analysis corresponds to considering the correlation between fitness changes and integrity changes. For the latter, an interaction between corpus callosum integrity and fitness changes with respect to transfer will be considered in a logistic regression model. Such an interaction can be expressed as a correlation between fitness changes and the degree of transfer in a group with low/high corpus callosum integrity. Based on the results by Voss et al. \[[@CR14]\], we assume a correlation of about 0.43 between both measures. At a significance level of 0.05, 30 individuals per (equally sized) integrity group are needed to detect such a correlation with a power of 0.8. As a result, ACT needs to be performed for 60 individuals in total. Assuming a dropout rate of about 15%, 72 subjects at the German Sport University Cologne will undergo the extended experimental protocol.
### Definition and analysis of primary endpoint {#Sec25}
The primary endpoint is defined as the dichotomous outcome training gain transfer (Yes/No). Successful transfer will be defined as: (1) Performance improvement in a logical reasoning training task from the second to the last training session, (2) performance improvement in an untrained fluid intelligence transfer task (Leistungsprüfsystem, subtest 4) from the pre-cognitive-training phase to the post-cognitive-training phase, and (3) maintenance of the performance in the transfer task from post-cognitive-training phase to follow-up. Transfer task performance alterations have to be numerically greater than the mere retest effect of an untrained control group.
The primary endpoint will be analyzed using a logistic regression with measures of corpus callosum integrity and bihemispheric processing as independent variables. As a primary analysis, the effects of both measures will be considered as main effects. In subsequent analyses, we will take into account that bihemispheric processing may not be necessary for all participants, whereby strong training performance might specifically indicate such participants. This will be incorporated into the analyses by interaction terms and by allowing for non-linear effects. The models will be adjusted for age. In the primary analysis subjects with missing values will be excluded. Sensitivity analysis will be performed to evaluate the impact of the missing values.
The primary population is the intention-to-treat population. As a sensitivity analysis, an as-treated analysis will be performed. To investigate the center effects, center number will be additionally incorporated into the models.
### Analysis of secondary endpoints {#Sec26}
For evaluating physical training as potential modulator of cognitive training gain transfer, correlations between fitness changes and integrity changes of the corpus callosum will be applied. Moreover, an interaction between corpus callosum integrity and fitness changes with respect to transfer will be considered in a logistic regression model. Such an interaction can be expressed as a correlation between fitness changes and the degree of transfer in a group with low/high corpus callosum integrity.
To evaluate the predictive value of brain vascular lesion, cortical amyloid load, higher Default Mode Network activity, and genetics for transfer of cognitive training gains in HOA, a logistic regression model with transfer as the dependent variable is used. All effects of the potential predictors are included as main effects. Additionally, to adjust for ACT, ACT and all interactions with ACT are included in the model as independent covariates.
To determine the impact of ACT on the structural integrity of the corpus callosum, Default Mode Network activity, BDNF, motor fitness, and VO~2~peak/VO~2~max, logistic regression models for these variables are fitted. ACT will be included as an independent variable. Additionally, the model will be adjusted for all other variables and the interactions with ACT. For example, the model for structural integrity of the corpus callosum will include ACT, Default Mode Network activity, BDNF, motor fitness, VO~2~peak/VO~2~max and all interactions with ACT as independent covariates.
Similar models will be fitted to examine the association between baseline physical activity and the structural integrity of the corpus callosum, Default Mode Network activity, and BDNF. Here, baseline physical activity and all possible interactions with baseline physical activity will be additionally included as independent covariates.
To build a robust individual index of transfer likelihood based on all potential predictors and interactions, a multimodal, high-dimensional dataset needs to be analyzed. For this purpose a regularized logistic regression for transfer will be performed including all potential predictors and relevant interactions. Specifically, to automatically select relevant covariates, a componentwise likelihood boosting approach will be used. To better detect potentially complex patterns in the multimodal data that are linked to transfer, a deep learning approach will be employed; specifically, deep Boltzmann machines \[[@CR71]\]. This specific approach will potentially allow for the detection of non-linear relations and interactions while still providing type 1 error control, and results in an integrated statistical model for transfer prediction.
Data management {#Sec27}
---------------
### Responsibilities {#Sec28}
The data management in each trial center during the trial will be conducted by one study member of the respective trial site (specified before the start of data collection). The trial coordinator is authorized to contact trial centers to monitor the data management.
### Data collection {#Sec29}
An electronic Case Report Form (eCRF) will be provided for each subject. All trial data will be documented in the subject's source data and in the eCRF. The principal investigators of the trial sites or designated representatives are responsible for ensuring that all sections of the eCRFs of their trial sites are correctly completed and that entries can be verified against source data. All changes in an eCRF entry will be tracked. The investigator, or a designated representative, should complete the eCRF pages as soon as possible after the information is collected. Any outstanding entries must be completed immediately after the final examination. An explanation should be given for any missing data.
### Data handling {#Sec30}
After completion of data entry all data will be collected at the University Medical Center Mainz. The access for data entry will be blocked and checks for plausibility, consistency, and completeness of the data will be performed. Based on these checks, queries will be produced. Any missing data or inconsistencies will be reported back to the respective site and clarified by the responsible investigator. If no further corrections are to be made in the database it will be declared closed and used for statistical analysis. The data checks will be done by the trial coordinator or a designated representative at the University Medical Center Mainz.
Assessment of safety {#Sec31}
--------------------
### Assessment of adverse events (AEs) by investigator {#Sec32}
Subjects must be carefully monitored for AEs by the investigator. The intensity of the AEs and the causal relation to trial medication and/or procedures are to be assessed.
The intensity of an AE will be assessed by the investigator as follows:Mild: temporary event which is tolerated well by the subject and does not interfere with normal daily activitiesModerate: event which results in discomfort for the subject and impairs their normal activitySevere: event which results in substantial impairment of normal activities of the subject
The assessment of the relationship of an AE to trial procedures is a clinical decision based on all available information at the time of the completion of the eCRF. The investigator will evaluate the causal relationship of each adverse event with the trial procedures according to modified criteria of the WHO 1991.
### Documentation of AEs and follow-up {#Sec33}
All AEs (whether serious (SAE) or non-serious) reported by the subject or detected by the investigator will be documented on the "Adverse Event Page" of the eCRF. If an AE is serious, the investigator must complete, in addition to the "Adverse Event Page", a "Serious Adverse Event Form" at the time the SAE is detected. SAEs are required to be reported by the investigator to the sponsor immediately (i.e., no more than 24 h after learning of the event).
All subjects who experience AEs, whether considered associated with the use of the investigational products or not, must be monitored to determine the outcome. The clinical course of the AE will be followed up according to accepted standards of medical practice, even after the end of the period of observation, until a satisfactory explanation is found or the investigator considers it medically justifiable to terminate follow-up, but no longer than 90 days after the end of the trial.
Ethical and legal aspects {#Sec34}
-------------------------
### Good Clinical Practice {#Sec35}
The procedures set out in this trial protocol, pertaining to the conduct, evaluation, and documentation of this trial, are designed to ensure that all persons involved in the trial abide by the quality standards of Good Clinical Practice and the ethical principles described in the Declaration of Helsinki. The trial will be carried out in accordance with all applicable local legal and regulatory requirements.
### Patient information and informed consent {#Sec36}
Before being enrolled into the clinical trial, the subject must consent to participate after being fully informed about the nature, scope, and possible consequences of the clinical trial. The documents must be in a language understandable to the subject and must specify who informed the subject. A copy of the signed informed consent document must be given to the subject. The investigator will retain the original signed consent document. The investigator will not undertake any measures specifically required only for the clinical trial until valid consent has been obtained. After reading the informed consent document, the subject must give consent in writing. The subject's consent must be confirmed by the personally dated signature of the subject and by the personally dated signature of the person conducting the informed consent discussions.
### Confidentiality {#Sec37}
The name of the subject and other confidential information are subject to medical professional secrecy and the regulations of the German Data Protection Act (Bundesdatenschutzgesetz). The name of the subjects and other confidential information will not be supplied to the sponsor. During the clinical trial, subjects will be identified solely by means of an individual identification code (e.g., subject number, randomization number). Trial findings stored on a computer will be stored in accordance with local data protection law and will be handled in strictest confidence. For protection of these data, organizational procedures are implemented to prevent distribution of data to unauthorized persons. The appropriate regulations of data legislation will be fulfilled in their entirety. The investigator will maintain a personal subject identification list (subject numbers with the corresponding subject names) to enable records to be identified.
### Approval of trial protocol {#Sec38}
The study protocol was approved by the local Ethics Committees of all three trial sites (Mainz: Ethics Commission of the Landesärztekammer Rheinland-Pfalz; Cologne: Ethics Commission of Cologne University's Faculty of Medicine; Rostock: Ethics Commission of the Rostock University's Faculty of Medicine). The reference number of the ethics approval of the main trial site Mainz is 837.385.15 (10153). The study was registered at the German Clinical Trials Resgister (ID: DRKS00013077).
Publication policy {#Sec39}
------------------
Any publication of the results, either in part or in total (articles in journals or newspapers, oral presentation, etc.) by the investigators, their representatives, or by the sponsor, will require the approval of all principal investigators of the trial (Prof. Dr. Andreas Fellgiebel, Prof. Dr. Oliver Tüscher, Dr. Andreas Mierau, Prof. Dr. Alexander Drzezga, Prof. Dr. Stefan Teipel, Prof. Dr. Harald Binder). It is planned to publish the results of the trial as original articles in appropriate journals as well as to present the results at congresses.
Discussion {#Sec40}
==========
Knowledge on the mechanisms and possibly modifiable modulators of transfer of cognitive training gains is urgently needed to design future intervention programs for the promotion of cognitive health including lifelong learning in aging. Today, neuroimaging techniques allow a reliable and valid investigation of neuronal mechanisms of transfer based on structural and functional brain network surrogates directly in humans. For this important purpose, the AgeGain Consortium combines all relevant multidisciplinary expertise, including cognitive training and transfer assessments, conduction of multimodal neuroimaging, physical training, and the assessment of subclinical brain pathology that occurs frequently and is known to significantly contribute to incident decline of cognitive function in HOA. As shown by recent publications \[[@CR11], [@CR17], [@CR55], [@CR72], [@CR73]\], the individual research groups assembled in AgeGain are already focusing on these research questions and the first successful European collaborations have been established. The formation of the consortium will enable the development of a strong partnership of basic clinical research in HOA.
The AgeGain study should provide important information for the determination of transfer likelihood in older people, and thus for the identification of HOA, who will most benefit from cognitive training. Findings of this trial should contribute to a better understanding of the neurobiological mechanisms of transfer in aging and will help determining the impact of physical activity and sport as well as of pathological factors (such as cerebrovascular disease and amyloid load) on transfer capability. Specifically, the trial will contribute to an increased understanding of the association of corpus callosum integrity as a structural measure and bihemispheric cooperation as a functional measure (as well as their interaction) with transfer capabilities in older adults. Beyond that, the trial should add important information about the importance of HAROLD for learning in old age. The study results should have a strong impact on future clinical, basic, and healthcare research evaluating lifestyle and training strategies to maintain successful and lifelong learning. A limitation of the study is that it is restricted to the investigation of neurobiological mechanisms of transfer on a macrostructural level. Thus, it will not provide information about microstructural neurobiological mechanisms of transfer.
Trial status {#Sec41}
============
The study is currently in the recruitment phase. Recruitment began in July 2016.
Protocol version: 1.3 (06--21-2016).
Additional file
===============
{#Sec42}
Additional file 1:Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) 2013 Checklist: recommended items to address in a clinical trial protocol and related documents\*. (DOC 121 kb)
ACT
: Combined aerobic and coordination training
AE
: Adverse event
APOE
: Apolipoprotein E
BDNF
: Brain-derived neurotrophic factor
CETP
: Cholesteryl ester transfer protein
DTI
: Diffusion-tensor imaging
eCRF
: Electronic Case Report Form
FA
: Fractional anisotropy
fMRI
: Functional magnetic resonance imaging
GPAQ
: Global Physical Activity Questionnaire
HAROLD
: Hemispheric Asymmetry Reduction in Older Adults
HOA
: Healthy older adult
MRI
: Magnetic resonance imaging
NP
: Neuropsychological examination
NT
: Non-transfer
PET
: Positron-emission tomography
SNP
: Single nucleotide polymorphism
ST
: Stable tansfer
VO~2~max
: Maximum oxygen uptake
VO~2~peak
: Peak oxygen uptake
**Electronic supplementary material**
The online version of this article (10.1186/s13063-018-2688-2) contains supplementary material, which is available to authorized users.
*AgeGain study group*: University Medical Center Mainz: Andreas Fellgiebel, Oliver Tüscher, Bernhard Baier, Dominik Wolf, Bianca Kollmann, Florian Fischer, Alexandra Sebastian; German Sport University Cologne: Heiko Strüder, Andreas Mierau, Kristel Knaepen, David Riedel; University Clinic Cologne: Alexander Drzezga; University Medical Center Rostock: Stefan Teipel, Katharina Brüggen, Judith Henf, Esther Lau; University of Freiburg: Harald Binder.
Ethics approval and consent participate {#FPar1}
=======================================
The study was granted ethical approval by the local Ethics Commitees of all trial sites (Mainz: Ethics Commission of the Landesärztekammer Rheinland-Pfalz; Cologne: Ethics Commission of (1) Cologne University's Faculty of Medicine and (2) German Sport University; Rostock: Ethics Commission of the Rostock University's Faculty of Medicine). Reference number of the ethics approval of the main trial site Mainz is 837.385.15 (10153). Subjects gave written consent to participate in the trial.
Funding {#FPar2}
=======
The AgeGain study is funded by the German Federal Ministry of Education and Research (BMBF) (grant number: 01GQ1425A). The funding body has no influence on the trial whatsoever.
DW and AF contributed to the conception of the assessment and analysis of the structural MRI data. Moreover, DW and AFdrafted the manuscript. OT contributed to the conception of the assessment and analysis of the fMRI data. ST participated in the conception of the assessment and analysis of the DTI and resting-state fMRI data. AM and HS carried out the conception of the physical training. AD carried out the conception and preparation of the assessment and analysis of the PET data. BB contributed to the conception of the assessment and analysis of the white-matter-lesion data. HB participated in the conception of the statistics and carried out power calcuations. All authors contributed significantly to the design of the study. All authors read and approved the final manuscript.
Competing interests {#FPar3}
===================
The authors declare that they have no competing interests.
Publisher's Note {#FPar4}
================
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
| {
"pile_set_name": "PubMed Central"
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Abbreviations {#nomen0010}
=============
1-OHP
: 1-Hydroxypyrene
2-AN
: 2-Aminonaphthalene
3-HPMA
: 3-Hydroxypropylmercapturic acid
4-ABP
: 4-Aminobiphenyl
6MWT
: Six Minute Walk Tests
AE
: Adverse Event
Ae24h
: Amount excreted over 24 Hours
AIx
: Augmentation Index
BoE
: Biomarker of Exposure
CEMA
: 2-Cyanoethylmercapturic acid
CEVal
: N-(2-cyanoethyl)valine haemoglobin adducts
CI
: Confidence interval
CO
: Carbon Monoxide
COPD
: Chronic obstructive pulmonary disease
dTx
: 11-Dehydrothromboxane B2
ET-1
: Endothelin-1
FDA
: Food and Drug Administration
FeNO
: Fractional exhaled nitric oxide
FEV1
: Forced expiratory volume in 1 s
FMD
: Flow-mediated dilation
FVC
: Forced vital capacity
HDL
: High-density lipoprotein cholesterol
HEMA
: 2-Hydroxyethylmercapturic acid
HMPMA
: 3-Hydroxy-1-methylpropylmercapturic acid
HPHC
: Harmful and potentially harmful constituents
LDL
: Low-density lipoprotein cholesterol
LS
: Least squares
MCP-1
: Monocyte chemoattractant protein-1
MHBMA
: Monohydroxybutenyl-mercapturic acid
MRTP
: Modified Risk Tobacco Product
NNAL
: 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol
NNN
: N-nitrosonornicotine
NO
: Nitric oxide
NRT
: Nicotine replacement therapy
o-Tol
: Ortho-toluidine
PRRP
: Potentially Reduced Risk Product
PWA
: Pulse wave analysis
PWV
: Pulse wave velocity
RHI
: Reactive hyperaemia index
sICAM-1
: Soluble intercellular adhesion molecule-1
S-PMA
: S-Phenylmercapturic acid
THP
: Tobacco Heating Product
TNeq
: Total nicotine equivalents
WHO
: World Health Organization
1. Introduction {#sec1}
===============
In the past few decades, smoking prevalence has significantly declined around the world due to regulatory policies and educational campaigns. However, this success has been only partial, and smoking remains one of the most important causes of preventable disease, with the World Health Organization (WHO) estimating that there will be around 1.5 billion smokers worldwide by 2050 \[[@bib1]\].
Pharmaceutical products commercialised as nicotine replacement therapies (NRTs) are designed to assist smokers by relieving cravings from nicotine withdrawal and, hence, to increase the likelihood of smoking cessation. However, the nicotine pharmacokinetic profiles of these products, such as patches, gums, sprays and inhalers, are dissimilar to those of conventional cigarettes, with typically lower C~max~ \[[@bib2],[@bib3]\]. Due to these differences in nicotine pharmacokinetics and rituals associated with smoking, smokers may not find pharmaceutical products as satisfying and have limited effect without behavioural support. Differences in delivery format and pharmacokinetic profiles may be some of the reasons for the limited efficacy of NRT products as aids to smoking cessation \[[@bib4],[@bib5]\].
Policy-makers have used harm reduction approaches to enhance interventions. These approaches can be particularly beneficial when harm cannot be easily eradicated \[[@bib6]\]. It has been suggested that a tobacco harm reduction approach could bring benefits to the overall population by substituting combustible products with products with a lower risk profile \[[@bib7]\]. For inhalable products, these potentially reduced risk products (PRRPs) belong to two main categories: vapour products, which are also known as electronic cigarettes or e-cigarettes and tobacco heating products (THPs).
A number of studies have been carried out on e-cigarettes including analysis of emissions and toxicological analysis in comparison to conventional cigarettes. These studies indicate reductions in chemical toxicants \[[@bib127]\] as well as reduced levels of DNA damage \[[@bib128]\], mutagenicity \[[@bib129],[@bib130]\], cytotoxicity \[[@bib131]\], and carcinogenicity \[[@bib132]\]. In addition, clinical studies have demonstrated that when smokers switch to e-cigarettes there are substantial reductions in exposure to selected cigarette smoke toxicants \[[@bib133],[@bib134]\].
THPs consist of a two-part system comprising a tobacco-containing consumable and an electronic heating device that heats tobacco, typically to temperatures lower than 345 °C, to avoid combustion \[[@bib8]\]. Due to the absence of tobacco combustion, significantly fewer chemical toxicants are formed, however, nicotine is still released with the inhaled aerosol \[[@bib9],[@bib10]\]. Given the presence of a cigarette-like hand-to-mouth action, and the presence of nicotine in the aerosol, THPs are expected to provide a more familiar experience to smokers helping them to transition from combustible cigarettes to a PRRP.
Compared with e-cigarettes, there has been less published research investigating the properties of THPs; however, available data generated from laboratory smoking machines and short-term biomarker of exposure studies have revealed significant reductions in emissions and exposure, respectively, to many chemical toxicants found in cigarette smoke \[[@bib120],[@bib122]\]. Smoking health risks are linked to the amount of cigarettes smoked and the duration that consumers have smoked for \[[@bib11]\], this relationship is not necessarily linear, however, a significant reduction of repeated and sustained exposure to cigarette smoke is expected to have a beneficial effect on health outcomes. If widely adopted and if these products prove to substantially reduce risk, this strategy might offer substantial public health gains by providing smokers, who would otherwise continue to smoke, with alternative sources of nicotine, but with similar rewarding effects, i.e., the substitution of cigarettes by PRRPs.
Measuring biomarkers of exposure (BoEs) associated with cigarette smoke exposure is informative as it could, for example, demonstrate how far using a PRRP instead of smoking reduces a user\'s exposure to certain toxicants found in cigarette smoke. However, how these changes in BoEs translate into changes in risk for smoking related diseases is still unknown. Therefore, additional biomarkers that go a stage further and could potentially indicate changes in disease development and health outcomes are investigated. This report describes the statistical approaches and rationale used to investigate both BoEs and health effect indicators in a 12-month long ambulatory study of smokers who switch from cigarettes to a THP, contextualised against smokers who cease cigarette smoking, and individuals who have never smoked \[[@bib12]\].
2. Methods {#sec2}
==========
2.1. Study design {#sec2.1}
-----------------
The full protocol describing this study has been recently published \[[@bib12]\]. In brief, this is a multi-centre randomised switching study where participants are recruited from three populations:
Subjects in the continue to smoke/THP population will be randomised to continue smoking commercially manufactured filter cigarettes and/or roll your own (n = up to 80, Arm A) or randomised to the THP glo™ coded as THP1.1(RT) (n = 200, Arm B). Subjects in the intend-to-quit population will be allocated to the assisted smoking cessation arm (Arm D). Subjects in the never smoker population will be assigned to the never smoker arm (Arm E). Main endpoint assessments will take place at days 90, 180 and 360 from baseline.
2.2. Sample size determination {#sec2.2}
------------------------------
The target of 50 subjects for Arms A, B and D was based on the primary biomarker, Augmentation Index (AIx), requiring the largest sample size to observe differences between the test and control product. Specifically, the power calculation was based on the number of subjects required to perform a contrast based on the F-statistic with 90% power between the arm using the THP product and the continue to smoke arm at day 360. For this calculation, we assume an expected change in this biomarker of 80% with respect to changes observed in subjects completely quitting smoking \[[@bib13]\]. Hence, the AIx expected means were 25.7% and 17.5% for the smoker and THP arms, respectively, with a common standard deviation of 12.4%. The significance level was adjusted for timepoint multiplicity using the O\'Brien-Fleming sequential approach with α = 0.0471 at the end of the study.
The objective is to complete the study with at least 50 subjects in each arm with the exception of never smokers, for which 30 was considered sufficient to characterise a never-smoker benchmark \[[@bib124]\]. The number of subjects allocated to each group is based on the expected attrition rate in this 12-month ambulatory study. Attrition rates in Arm A (continue to smoke) and Arm E (never smoker) are expected to be low based on our previous experience with ambulatory smoking studies \[[@bib14]\]. However, it has been observed that significant attrition rates occur in smoking cessation studies \[[@bib15]\], and it is expected that switching completely to a new product may also lead to study withdrawal. Therefore, additional subjects were assigned to these study arms to account for the anticipated attrition rates.
The main assessment timepoints are Day 90 (±3 days) for BoE endpoints only and, Day 180 (±2 weeks) and Day 360 (±2 weeks) for all endpoints.
2.3. Randomisation {#sec2.3}
------------------
Some endpoints assessed in this study have been shown to be affected by demographic characteristics such as gender and age. For example, NNAL has been found to be correlated to age and gender \[[@bib126]\]. Aiming to mitigate potential confounding effects due to unbalanced demographics, the study arms are randomized by using four separate randomisation lists for gender combinations and age 40 years as a threshold. This generated four lists of subjects at each centre (males ≤ 40, males \> 40, females ≤ 40 and females \> 40 years). Within each list, blocks of 8 are used to allocate 2 subjects to Arm A (continue to smoke) and 6 to the Arm B (switch from smoking to THP).
2.4. Analysis populations {#sec2.4}
-------------------------
The *randomised population* is defined as all subjects who were assigned to a study arm and had at least one valid assessment of a biomarker variable.
The *per-protocol population* is defined as all subjects who had a valid assessment of a biomarker variable and completed the study without major protocol deviations.
All statistical analyses will be performed on the randomised and per-protocol populations.
2.5. Product compliance {#sec2.5}
-----------------------
Subject compliance is a crucial aspect of every clinical study as it has a large bearing on the outcome. Subject compliance to their assigned arm will be extremely important for the assessment of biomarker changes during this study. Compliance will be particularly important for subjects switching to the THP (Arm B) and ceasing to smoke (Arm D), where a full switch to the THP or complete abstinence from smoking is intended respectively. If subjects fail to comply with this and continue smoking, potentially alongside the investigational product, they are not likely to experience the full change in biomarker levels or may even experience no changes at all.
In such a long ambulatory study, self-reported cigarette consumption is not likely to be a reliable means of determining subjects' cigarette use. Furthermore, the clinical assessments typically used for this purpose have a short half-life and may not be able to detect smoking, even if it has occurred a few days earlier, thus, longer term biomarkers indicative of cigarette consumption are required. To enable identification of potential non-compliance, we will use *N*-(2-cyanoethyl)valine haemoglobin adducts (CEVal), a biomarker of exposure to acrylonitrile. Acrylonitrile is not expected to be present in the THP emissions, or in much lower concentrations than cigarettes \[[@bib9]\], and CEVal has a long half-life, based on the red blood cell life cycle which is between 90 and 120 days in the circulation in healthy individuals. Therefore, it is expected to take several months before concentrations of CEVal fall to levels comparable to those of never smokers. This property could potentially make CEVal a suitable biomarker of compliance for this study.
Based on a previous study where participants were switched to a prototype combustible product \[[@bib14]\], we computed thresholds for CEVal for the main assessment timepoints of the study (data not published). Based on the concentration of acrylonitrile in the emissions of the products, the THP product in the current study is expected to outperform the prototype combustible product used in the previous study, and thus CEVal concentrations are expected to be lower than those observed in the previous study. Thresholds are based on percentiles with the exception of potential THP solus use at Day 360, which has been defined as the maximum concentration among never smokers observed in the previous study \[[@bib14]\] because it was higher than the suggested threshold from switching. Therefore, this is a conservative approach in which some non-compliant subjects may still be classified as potential THP solus users or complete cessation (Arm D). The thresholds proposed to guide assessment of lack of compliance are summarised in [Table 1](#tbl1){ref-type="table"}.Table 1Proposed thresholds to guide assessment of lack of compliance.Table 1CategoryDay 90Day 180Day 360Highly Likely Smoking (H)\>164 pmol/g Hb\>112 pmol/g Hb\>78 pmol/g HbPotential Dual Use (D)\[78, 164 pmol/g Hb\]\[54, 112 pmol/g Hb\]\[35, 78 pmol/g Hb\]Potential Solus Use (S)\<78 pmol/g Hb\<54 pmol/g Hb\<35 pmol/g Hb
These thresholds are speculative and not validated, therefore, it is unknown how many subjects will fall within each category. A minimum of 30 subjects in a category is considered necessary to provide statistical power for most of the BoEs. In order to reach 30 subjects for statistical analysis, a stepwise merging method will be taken, starting in the lower categories (the more likely compliant subjects), and statistical analysis will be performed only if there is a minimum of 30 subjects in a group. Starting from the lowest category, this category will be merged with the next category until the minimum number of 30 subjects is reached. This approach may yield up to two groups (S + D and D + H) if individual categories do not reach n = 30. Note that merging S + D + H would be the same as a per-protocol analysis. If the number of subjects in a group is still below 30 after merging, no further analyses will be performed for that group.
3. Rationale for study outcomes {#sec3}
===============================
Smoking is known to be a risk factor for several chronic diseases \[[@bib111]\]. However, its mechanisms are complex and depend, to a great extent, on exposure and duration of tobacco smoke exposure; nevertheless, other still unknown factors contribute to disease development because not all smokers develop disease \[[@bib11]\]. Within this context, the predictability of disease is limited and currently there is no qualified panel of endpoints that can predict smoking-related disease development.
Epidemiological studies have been able to demonstrate the harm caused by smoking by assessing disease prevalence among people with different smoking habits \[[@bib111]\]. In the longer term, whether PRRPs realise their potential and become reduced risk products is also likely to be revealed through epidemiology. Given that these products have been commercially available only relatively recently, epidemiological data will not be available for many years and it is critical to provide the most complete information to policy-makers and consumers to help them make informed decisions now. Therefore, this study investigates several health effect indicators that are thought to be related to various smoking-related disease pathways.
Due to the long-term complex nature of disease development associated with smoking and the complex nature of tobacco smoke, which contains more than 6500 compounds \[[@bib16]\], a large number of endpoints have been included in this study. The endpoints include BoEs to investigate exposure to cigarette smoke toxicants, whereas the health effect indicators and physiological measures characterise biological functions that are known to be perturbed by tobacco smoke. Where there are no qualified health effect indicators to predict the onset of smoking-related disease, multiple indicators have been included to characterise trends in each area of interest and progressive stages of disease development.
Overall, the study outcomes can be broadly split into BoEs to tobacco smoke constituents ([Table 2](#tbl2){ref-type="table"}) and health effect indicators relating to i) lung cancer risk, ii) cardiovascular risk and iii) obstructive lung disease risk ([Table 3](#tbl3){ref-type="table"}). Comprehensive reviews of many of the biomarkers included in this study and their applicability to the assessment of novel tobacco and nicotine products which serve as good point of reference for a broad overview of the area, have been conducted by Scherer and Peck \[[@bib17],[@bib123]\].Table 2Biomarkers of exposure.Table 2BiomarkerAbbreviationAssociated toxicant/compoundMatrixCarbon monoxide[b](#tbl2fnb){ref-type="table-fn"}COCarbon monoxideExhaled breathTotal nicotine equivalents (nicotine, cotinine, 3-hydroxycotinine and their glucuronide conjugates)[b](#tbl2fnb){ref-type="table-fn"}TN~eq~NicotineUrine (24-h)Total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol[a](#tbl2fna){ref-type="table-fn"}Total NNALMetabolite of the smoke toxicant 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)Urine (24-h)Total *N*-nitrosonornicotine[b](#tbl2fnb){ref-type="table-fn"}Total NNNNNNUrine (24-h)3-Hydroxypropylmercapturic acid[b](#tbl2fnb){ref-type="table-fn"}3-HPMAAcroleinUrine (24-h)3-Hydroxy-1-methylpropylmercapturic acid[b](#tbl2fnb){ref-type="table-fn"}HMPMACrotonaldehydeUrine (24-h)*S*-Phenylmercapturic acid[b](#tbl2fnb){ref-type="table-fn"}S-PMABenzeneUrine (24-h)Monohydroxybutenyl-mercapturic acid[b](#tbl2fnb){ref-type="table-fn"}MHBMA1,3-butadieneUrine (24-h)2-Cyanoethylmercapturic acid[b](#tbl2fnb){ref-type="table-fn"}CEMAAcrylonitrileUrine (24-h)1-Hydroxypyrene[b](#tbl2fnb){ref-type="table-fn"}1-OHPPyreneUrine (24-h)2-Hydroxyethylmercapturic acid[b](#tbl2fnb){ref-type="table-fn"}HEMAEthylene oxideUrine (24-h)4-Aminobiphenyl[b](#tbl2fnb){ref-type="table-fn"}4-ABP4-aminobiphenylUrine (24-h)2-Aminonaphthalene[b](#tbl2fnb){ref-type="table-fn"}2-AN2-aminonaphthaleneUrine (24-h)Ortho-toluidine[b](#tbl2fnb){ref-type="table-fn"}o-TolOrtho-toluidineUrine (24-h)[^1][^2]Table 3Health effect indictors.Table 3Study endpointDisease pathwaySummary descriptionRelated references8-Epi-prostaglandin F2α type III[a](#tbl3fna){ref-type="table-fn"}Oxidative stressSurrogate outcome measure of oxidative burden in the body, which may be indicative of future disease risk. A product of lipid peroxidation \[[@bib25]\] will be measured in urine\[[@bib17],[@bib54], [@bib55], [@bib56], [@bib57], [@bib58]\]4-Hydroxy-2-nonenalOxidative stressA product of lipid peroxidation \[[@bib57]\]\[[@bib17],[@bib60]\]HomocysteineOxidative stressKnown to deplete endothelial antioxidant levels \[[@bib59]\]\[[@bib17],[@bib62]\]3-NitrotyrosineOxidative stressA product of nitrosative stress following the interaction of tyrosine residues with peroxynitrite \[[@bib61]\]\[[@bib17],[@bib64]\]White blood cell countInflammationMarker of general inflammation; elevated blood levels are associated with the development of atherosclerosis\[[@bib17],[@bib54]\]High sensitivity C-reactive proteinInflammationMarker of general inflammation; elevated blood levels are associated with the development of atherosclerosis\[[@bib17]\]Monocyte chemoattractant protein-1 (MCP-1), E-selectin, and soluble intercellular adhesion molecule-1 (sICAM-1)InflammationAssociated with endothelial dysfunction and adhesion of immune cells to the vascular endothelium, a critical step in the formation of atherosclerotic lesions \[[@bib63]\]\[[@bib17],[@bib54]\]Tissue plasminogen activatorCoagulationA serine protease found on endothelial cells that catalyses the conversion of plasminogen to plasmin, a major enzyme responsible for the breakdown of blot clots \[[@bib64]\]\[[@bib65], [@bib66], [@bib67], [@bib68], [@bib69], [@bib70], [@bib71]\]Plasminogen activator inhibitor-1CoagulationA member of the serine protease inhibitor (serpin) family, and a major physiologic inhibitor of serine proteases such as tPA \[[@bib70]\]\[[@bib68],[@bib72], [@bib73], [@bib74]\]FibrinogenCoagulationThe soluble precursor to insoluble fibrin (the major constituent of blood clots); it also supports platelet aggregation \[[@bib73]\]\[[@bib17],[@bib54],[@bib75], [@bib76], [@bib77]\]11-Dehydrothromboxane B2 (dTx)CoagulationA metabolite of TxA2, which is a potent activator of platelets with thrombogenic and vasoconstrictive properties; dTx has been implicated in endothelial dysfunction, atherosclerosis, type II diabetes and hypertension \[[@bib76]\]\[[@bib78], [@bib79], [@bib80], [@bib81]\]; reviewed in Refs. \[[@bib17],[@bib82]\]Augmentation index (AIx) and pulse wave velocity (PWV)[a](#tbl3fna){ref-type="table-fn"}Physiological measures: arterial stiffnessPulse wave analysis (PWA) assesses changes in blood pressure in major arteries during the cardiac cycle. AIx and PWV are two key outputs from PWA with relevance to arterial stiffness\[[@bib17],[@bib83], [@bib84], [@bib85], [@bib86], [@bib87], [@bib88], [@bib89]\]Reactive hyperaemia indexPhysiological measures: endothelial dysfunctionFinger plethysmography will be used to monitor peripheral reactive hyperaemia as a surrogate measure of flow-mediated dilation (a marker of endothelial dysfunction) \[[@bib39]\]\[[@bib90], [@bib91], [@bib92], [@bib93]\]Brachial systolic and diastolic blood pressurePhysiological measures: blood pressureChronically elevated blood pressure defines hypertension, a known risk factor for cardiovascular disease\[[@bib94], [@bib95], [@bib96], [@bib97], [@bib98]\]Endothelin-1 (ET-1)Vascular toneA potent vasoconstrictor released by endothelial cells that acts upon vascular smooth muscle via the Endothelin A receptor to induce prolonged vasoconstriction. It also acts upon endothelial cells via the endothelin B receptor to induce nitric oxide (NO) production (promoting vasodilation), hence acting as a counterbalance to its own primary effects \[[@bib97]\]\[[@bib99], [@bib100], [@bib101], [@bib102], [@bib103], [@bib104], [@bib105]\]6-min walk test (6MWT)Physiological measuresThe 6MWT is a submaximal exercise test used to quantify functional exercise capacity in clinical populations; it is commonly used as an outcome measure for treatment of COPD and cardiovascular disorders\[[@bib106], [@bib107], [@bib108]\]Serum lipids (HDL/LDL/total cholesterol and triglycerides)AtherosclerosisAtherosclerosis is a well-known risk factor for cardiovascular events. Accumulation of LDL cholesterol in blood vessel walls is a hallmark of the condition, while athero-protective HDL cholesterol levels are reduced\[[@bib17],[@bib48], [@bib49], [@bib50], [@bib51],[@bib53],[@bib109],[@bib110]\]Lung spirometry, FEV1, FEV1/FVC ratioLung function: spirometryAirflow limitation is a major characteristic of COPD. Smoking is well-known to accelerate a decline in forced expiratory volume in 1 s (FEV1) over time \[[@bib109]\]; coupled with forced vital capacity FVC (generating the FEV1/FVC ratio), it diagnoses and defines the severity of COPD \[[@bib110]\]\[[@bib17],[@bib113], [@bib114], [@bib115]\]Fractional exhaled nitric oxide (FeNO)Nitric oxide bioavailabilityEndogenous NO plays an important role in the vasculature and the airways, and is generated by NO synthases. Smoking reduces the generation of NO directly by oxidising critical amino acid residues of NO synthases, and indirectly by reducing bioavailability of enzymatic cofactors (e.g. tetrahydrobiopterin), causing uncoupling of the enzymes \[[@bib26]\]\[[@bib17],[@bib116], [@bib117], [@bib118], [@bib119]\][^3]
Three endpoints were chosen as primary endpoints: total NNAL, AIx and 8-Epi-prostaglandin F2α type III (8-Epi-PGF2α type III). Their relevance to tobacco-related disease outcomes are explained in the following sections.
3.1. Biomarkers of exposure {#sec3.1}
---------------------------
To investigate whether reduced exposure is sustained, biomarkers (urinary and exhaled breath) were selected in accordance with the Food and Drug Administration (FDA) list of harmful and potentially harmful constituents (HPHCs) of tobacco and tobacco smoke, with the exception of pyrene, which is used as a surrogate BoE for benzo\[a\]pyrene \[[@bib18],[@bib19]\].
Total NNAL has more commonly been used as a BoE for exposure to 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) a tobacco specific-nitrosamine. However, this was included as a primary end-point in this study because of its potential to cause DNA damage and association with cancer \[[@bib21]\].
3.2. Health effect indicators {#sec3.2}
-----------------------------
Health effect indicators can be broadly split into the areas of i) lung cancer risk, ii) cardiovascular risk, and iii) obstructive lung disease risk. The development of each of these diseases is an on-going process over time and involves many different mechanistic networks; therefore, health effect indicators have been selected that cover various stages of the disease development process. These range from biomarkers of immediate biological response to exposure to chemical toxicants (e.g. oxidative stress) to biomarkers which are related to pathological processes which take longer to manifest (e.g. arterial stiffness). The aim of this approach is to yield data on whether or not shorter-term biological changes are associated with longer term changes of direct relevance for smoking-related disease development. The health effect indicators included in this study are described in further detail below.
### 3.2.1. Biomarkers of oxidative stress {#sec3.2.1}
Oxidative stress has been described as *"an imbalance between oxidants and antioxidants in favour of the oxidants, leading to a disruption of redox signaling and control and/or molecular damage"* \[[@bib22]\], and is reported to be a significant factor behind the development of all three of the above disease risk areas \[[@bib23], [@bib24], [@bib25]\]. Oxidants are known to directly and indirectly damage DNA, which increases the risk of permanent DNA mutations and subsequently neoplasia under suitable local conditions \[[@bib26],[@bib27]\]. Furthermore, oxidative stress is known to contribute to impaired vasodilation of vascular tissue, of relevance to arterial stiffening and hypertension \[[@bib28]\] and chronic inflammatory states in vascular tissue and the lung, of relevance to the development of atherosclerosis and chronic obstructive pulmonary disease (COPD) \[[@bib29],[@bib30]\].
To assess the primary objective of the study, 8-epi-prostaglandin F2α type III (8-epi-PGF2α); an isoprostane and product of lipid peroxidation \[[@bib27]\] will be measured in urine. Given the numerous smoking data available of 8-isoprostanes and smoking, its fairly consistent change upon smoking cessation (and in smaller sample sizes), its decline upon THP use and link to smoking-related diseases including a potential link to hypertension \[[@bib28]\] we included 8-epi-prostaglandin F2α type III as a primary outcome in this study.
### 3.2.2. Biomarkers of inflammation {#sec3.2.2}
Acute and chronic inflammation are hallmarks of tissue damage and the developmental stages of vascular and obstructive lung disease, respectively \[[@bib23],[@bib25]\]. Inflammation also has numerous roles in carcinogenesis and tumour progression \[[@bib31],[@bib32]\]. Persistent exposure to chemical toxicants, radical species, and physical and microbial insults can lead to persistent damage, unresolved inflammation, and subsequently tissue re-modelling over time, as the body adapts to protect itself from chronic noxious stimuli \[[@bib33]\]. Examples of tissue re-modelling are the development of atherosclerotic lesions and arterial stiffening of relevance for cardiovascular disease \[[@bib33]\], metalloproteinase release, fibrosis and emphysema of relevance for obstructive lung disease \[[@bib34]\], squamous cell metaplasia and epithelial to mesenchymal transition of relevance for lung carcinogenesis \[[@bib35]\]. These phenotypes are generally accepted to be pathological in nature and are pre-cursor steps to overt disease.
The inflammatory biomarkers included in this study support the secondary and exploratory objectives of the study.
### 3.2.3. Biomarkers of coagulation {#sec3.2.3}
Coagulation is a critical component of tissue repair in the body, however, in some circumstances such as atherosclerotic plaque rupture, coagulation can be very dangerous. In haemostasis, blood vessel walls are lined with antithrombotic mediators, which inhibit platelet activation and coagulation. However, the subendothelial layer is highly thrombogenic. When damage occurs to the endothelium, these thrombogenic factors can activate platelets and initiate the formation of a thrombus. If thrombi are not broken down, they may become lodged in key blood vessels supplying nutrients to the heart and brain, for example. Such occlusions can often lead to myocardial infarction and stroke \[[@bib36]\]. Hypercoagulability or thrombophilia is the increased tendency of blood to thrombose, placing affected individuals at a greater risk of thrombotic disease \[[@bib37]\]. Tobacco smoking has been reported to induce a hypercoagulation state, where smokers may be more at risk to blood clot formation \[[@bib38]\].
### 3.2.4. Physiological measures {#sec3.2.4}
Pulse wave analysis (PWA) assesses changes in blood pressure in major arteries during the cardiac cycle, and its potential application to clinical research and treatment has been reviewed by Hametner and Wassertheurer \[[@bib39]\]. PWA has been employed mainly in the study of vascular ageing (arterial stiffness) and hypertension \[[@bib40]\]. AIx and pulse wave velocity (PWV) are two key outputs from PWA that have relevance for arterial stiffness and are included as outcomes in this study. Essentially, AIx is a function of the difference between the primary aortic pressure wave just after a heartbeat and the reflected pressure wave from that heartbeat, received back at the central aorta from the distal aortic bifurcation in the pubic area. It is usually normalised to 75 beats per minute to account for variation in heart rate among individuals. Smoking studies involving AIx and PWV are increasing in number of late. Kim et al. \[[@bib87]\] reported that current smokers had significantly higher AIx than never smokers and that ex-smokers had significantly lower AIx compared to current smokers. Xue et al. \[[@bib90]\] reported that both AIx and brachial/ankle PWV decreased in healthy smokers, following a period of 3 months of smoking cessation, and further improved at a 12-month follow-up. Given the links to cardiovascular risk prediction and potential development of hypertension \[[@bib28]\] as well as consistent reversibility upon smoking cessation, AIx was included as a primary outcome in the study. PWV is the speed in m/s at which the pressure waveform traverses major arteries. Taken together, these metrics provide a useful insight into arterial stiffness. PWV was included as a secondary outcome, as although a favourable performance was observed in literature, questions remain over the length of time required to observe meaningful changes in smoking cessation studies.
Finger plethysmography will be used to monitor peripheral reactive hyperaemia in the study groups as a surrogate measure of flow-mediated dilation (FMD; a marker of endothelial dysfunction). Occlusion of the brachial artery for a period of 5 min restricts blood flow to the forearm, resulting in ischaemia. Upon release of the occlusion, the resulting surge in blood flow increases endothelial sheer stress and correspondingly increases production of nitric oxide from endothelial cells, resulting in vasodilation of the distal arteries. This change is captured in the reactive hyperaemia index (RHI), which is calculated on the EndoPAT™ device. In general, RHI values below 2 are categorized as endothelial dysfunction \[[@bib41]\].
The 6-min walk test (6MWT) is a simple and low-cost submaximal exercise test used to quantify the functional exercise capacity in clinical populations and, is commonly used as an outcome measure for treatment of COPD and cardiovascular disorders. Longer distances in the 6MWT are associated with higher exercise capacity \[[@bib42]\]. Both patients with COPD and cardiovascular-related disorders are known to record lower distances in the 6MWT \[[@bib43], [@bib44], [@bib45], [@bib46], [@bib47]\], which is symptomatic to poor oxygen bioavailability in the circulation during exercise.
#### 3.2.4.1. Biomarkers of atherosclerosis {#sec3.2.4.1}
Hypercholesterolaemia is a well-known risk factor for cardiovascular disease, and statin therapy has been shown to normalise low-density lipoprotein cholesterol (LDL) levels and reduce cardiovascular risk. However, many people remain at high risk even when their level of LDL has been reduced by aggressive treatment with statins, and this is thought to be due to low levels of high-density lipoprotein cholesterol (HDL) \[[@bib48]\]. Lipids such as triglycerides and LDL are well known to accumulate in blood vessel walls, forming plaques, during the process of atherosclerosis. This accumulation is associated with macrophage infiltration, smooth muscle cell phenotypic switching (to a macrophage-like phenotype), leading to the formation of foam cells, macrophagic cells packed with lipid deposits, which persist in the plaque \[[@bib49]\]. HDL cholesterol has been reported to be athero-protective in nature. Not only does HDL promote cholesterol efflux from vessel walls \[[@bib50]\], it also has been reported to reduce oxidation and inflammation and improve endothelial function and repair \[[@bib51]\].
### 3.2.5. Biomarkers of lung function {#sec3.2.5}
COPD is a chronic inflammatory condition of the large and small airways \[[@bib112]\], and alveoli that is defined as chronic airflow obstruction that is progressive and only partly reversible \[[@bib52]\]. It is associated with severe airflow limitation, mucous hypersecretion, impaired mucocilliary clearance, coughing, wheezing and poor gaseous exchange. Smoking is a major risk factor for the disease, but other environmental/occupational hazards are also known to contribute to its development \[[@bib53]\].
4. Statistical analysis approaches {#sec4}
==================================
Toxicant emissions of the investigational THP product have been shown to be significantly reduced compared to those from cigarette smoke \[[@bib9]\], therefore, it is expected that the endpoints chosen in this study will change for the participants who switch to the investigational product compared with those who continue to smoke. Additionally, the cessation arm will provide a benchmark to evaluate the direction and magnitude of those changes.
The concentration of biomarkers, especially BoEs, is linked to smoking behaviour and consumption \[[@bib14],[@bib120]\]. To favour comparability between arms, primary and secondary endpoints will be assessed as the absolute change from baseline, where baseline is defined as the last value measured prior to commencement of the subject\'s randomised Arm (Day 0), including unscheduled readings. For each subject, the change from baseline will be calculated by subtracting their individual baseline value from the value at a subsequent timepoint.
Primary and secondary endpoints will be statistically assessed at three time points during the study: Day 90, Day 180 and Day 360. Any primary or secondary endpoints producing statistically significant results at an early timepoint will not be statistically analysed at subsequent timepoints. However, descriptive statistics will be presented for all endpoints at all timepoints.
Urine biomarkers will be expressed as amount excreted over 24h (Ae~24h~) according to the formula:
4.1. Analysis of primary endpoints {#sec4.1}
----------------------------------
Total NNAL is a urinary biomarker and therefore will be reported as Ae~24h~. 8-Epi-PGF2α type III will be analysed in 24h urine collection but it will also be reported as corrected by creatinine, simply by dividing each subject\'s value by their own creatinine concentration measured at the same timepoint. Before statistical analysis, AIx requires normalisation to a heart rate of 75 bpm, which provides AIx75 (%) using the formula:$$AIx75 = AIx - \left( {\left\lbrack \frac{\left( {75\ - HR} \right)}{10} \right\rbrack \ast 4.8} \right).$$
Changes from baseline for all three aforementioned biomarkers will be compared between the THP arm and continue to smoke arm by using specific contrast in a regression model including baseline values and arm as the main effects. Data will be examined and may be transformed to adhere to distributional assumptions associated with statistical tests.
Multiple comparisons adjustments are essential in studies with numerous endpoints to control for Type I error inflation. As explained in section [2.2](#sec2.2){ref-type="sec"}, to retain an overall significance level of 0.05 we considered the "Multiple Endpoints in Clinical Trials" guidance provided by the US Food and Drug Administration (FDA) \[[@bib121]\]. Following this guidance, the overall significance level has been adjusted for each time point using the O\'Brien-Fleming method. This provides significance levels of α = 0.0006, 0.0151 and 0.0471 for Days 90, 180 and 360 respectively. At Day 90, only BoEs are expected to show a significant change; therefore, only total NNAL will be statistically assessed with α = 0.0006. For statistical analyses performed at Day 180 the α level will be equally distributed between primary endpoints. However, if any of the primary endpoints are found to be statistically significant at Day 90 or 180, they will not be assessed in subsequent timepoints, as appropriate, and the assigned α level will be equally distributed between the remaining endpoints. At day 360, α = 0.0001 has been assigned to total NNAL and 8-Epi-PGF~2α~ type III and the remaining alpha level (0.0469) will be used to assess AIx.
Generalisability of outcomes will be explored by performing a sensitivity analysis in which the previous model is adjusted for age and gender. This secondary analysis model includes baseline, age (as continuous), gender, arm, and the interaction of gender and arm as fixed effects. In addition, clinical site will also be included as a random effect. If the interaction effect between gender and study arm is found to be significant, specific differences within arm genders will be assessed by contrasts using available α level for each endpoint and timepoint.
An ancillary analysis will also be performed for categories of product compliance based on CEVal concentrations. Contrasts from a regression model with change from baseline as a dependent variable and independent variables including CEVal categories/groups and the control arm continue to smoke (Arm A) will be used to compare each of the product compliance groups to Arm A.
For all analysis, least squares (LS) means and 95% confidence intervals (CIs) for each group (groups based on CEVal compliance analysis) will be presented, along with differences, CIs and p-value for each comparison. If the data are log-transformed before analysis, the results will be back-transformed to provide geometric LS means and 95% CIs, as well as geometric LS mean ratios and CIs for each comparison.
4.2. Analysis of secondary endpoints {#sec4.2}
------------------------------------
Secondary endpoints can be found in [Table 2](#tbl2){ref-type="table"}, [Table 3](#tbl3){ref-type="table"} marked with \*. Total nicotine equivalents TNeq is a compound endpoint formed as a summation of different nicotine metabolites:$$\begin{array}{l}
{\text{TN}_{\text{eq}}\left\lbrack \text{mg/}24\text{h} \right\rbrack = \left( \text{free}\ \text{nicotine}\ \left\lbrack \mu\text{mol/L} \right\rbrack + \text{nicotine} - \text{glucuronide}\ \left\lbrack \mu\text{mol/L} \right\rbrack \right.} \\
{\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ + \text{free}\ \text{cotinine}\ \left\lbrack \mu\text{mol/L} \right\rbrack + \text{cotinine} - \text{glucuronide}\ \left\lbrack \mu\text{mol/L} \right\rbrack} \\
{\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ + \text{free}\ \text{trans} - \text{3}' - \text{hydroxycotinine}\ \left\lbrack \mu\text{mol/L} \right\rbrack} \\
{\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ + \text{trans} - \text{3}' - \text{hydroxycotinine} - \text{glucuronide}\ \left\lbrack \mu\text{mol/L} \right\rbrack)} \\
{\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \text{*}162.2\ \left\lbrack \mu\text{g/}\mu\text{mol} \right\rbrack \ast \left( \text{urine}\ \text{volume}\ \left( \text{L} \right)/1000 \right)} \\
\\
\end{array}$$
Due to the nature of the assessment and practicalities in clinic, the procedure for exhaled CO, a short-term indicator of cigarette consumption, will be performed at different time points in contrast with the rest of the study endpoints: Days 120, 150, 210, 240, 300 and 330. Means for the paired values 120 + 150, 210 + 240 and 300 + 330 will be calculated and analysed with the other secondary endpoints as the nominal Days 90, 180 and 360, respectively.
Secondary endpoints will be analysed by following the same approaches used for the primary endpoints, however, the significance level used to perform statistical tests will be determined by the α level remaining after statistical analysis of primary endpoints. This approach implies that if none of the primary endpoints yield statistically significant results, then statistical analysis of the secondary endpoints will not be performed. Additionally, further multiple comparisons adjustments will be carried out among secondary endpoints by using Holms' method \[[@bib125]\].
4.3. Handling missing data {#sec4.3}
--------------------------
No data imputation will be performed for missing data. Taking a conservative approach, for biomarker concentrations below the limit of detection or quantification (e.g., \<20 ng/mL), the urine concentration will be replaced by half the limit of detection or quantification respectively (e.g., 10 ng/mL) prior to calculation of the amount excreted. Similarly, if the urine concentration is above the upper limit of quantification (e.g., \>500 ng/mL), then the urine concentration will be replaced by the upper limit of quantification (e.g., 500 ng/mL) prior to calculation of the amount excreted.
4.4. Safety data {#sec4.4}
----------------
Adverse events (AEs) will be classed as occurring in one of two periods:
Pre-randomisation -- any AE that starts after the subject has provided written informed consent and that resolves prior to 06:00 on Day 0, or an AE that starts prior to 06:00 on Day 1 and does not increase in severity after 06:00 on Day 0.
Exposure Period -- any AE that occurs after 06:00 on Day 1 or that is present prior to 06:00 on Day 0 and becomes more severe after 06:00 on Day 0.
All AEs will be listed. Onset times post-product use will be calculated from the last product administered for Arms A to B, and from 06:00 on Day 1 for Arms D and E.
AEs occurring in the exposure period will be summarised by the arm that the subject is randomised to, by severity, and by relationship to the product. The frequency of AEs (i.e., number of AEs, number of subjects experiencing an AE, and percentage of subjects experiencing an AE) will be summarised by the arm the subject is randomised to, and by MedDRA system organ class and preferred term. Summary and frequency AE tables will be presented for all causalities and for those AEs considered to be related to the product (those that have a relationship of possibly related or related). Any severe or serious AEs will be tabulated. If an AE changes severity ratings, it will be included only once under the maximum severity rating in the summaries.
5. Discussion {#sec5}
=============
Most smoking-related diseases are a consequence of continued exposure to tobacco smoke toxicants, but it can take many years for smoking-related diseases to develop in susceptible individuals. Epidemiological substantiation of PRRP efficacy as a less risky alternative to smoking may take decades due to the time lag between exposure and disease outcome. Therefore, studies are required that focus on the potential of PRRPs to deliver, in the shorter term, reduced health effects relative to continued smoking to smokers that ultimately are likely to manifest in a reduction in the incidence of smoking-related disease. The data generated from such studies will provide valuable information to guide public health decision-making in the present day and may help to enable consumers to make a more informed choice on the use of nicotine products available to them.
This report describes the design and statistical analysis approach of a randomised, open-label, parallel group switching study to assess the effects of replacing smoking with a THP for 1 year. The primary objective of the study is to evaluate changes in BoEs and health effect indicators among healthy volunteers using the investigational THP product relative to continuing to smoke over a 1-year period.
The study will investigate three primary endpoints: total NNAL, which has been linked to lung cancer \[[@bib20],[@bib21]\]; 8-Epi-PGF2α type III, which is an indicator of oxidative stress that can lead to various diseases \[[@bib17],[@bib54], [@bib55], [@bib56], [@bib57], [@bib58]\]; and AIx, which has been used as an indicator of arterial stiffness, a risk factor for cardiovascular disease \[[@bib17],[@bib83], [@bib84], [@bib85], [@bib86], [@bib87], [@bib88], [@bib89]\]. Secondary endpoints have been chosen to provide a broad picture of disease mechanisms that are linked to the primary outcomes and have been shown to be negatively affected by smoking (e.g., inflammation, coagulation, blood pressure and lung function). The study also presents a well-established BoE panel to investigate reductions in exposure to known tobacco smoke toxicants. Current literature suggests that reductions in toxicant exposure, combined with favourable changes in health effect indicators, are likely to lead to health benefits for smokers who completely replace smoking with a PRRP \[[@bib7]\]. Ultimately, such changes are likely to indicate a reduction in smoking-related disease risk over time. Given the lack of qualification of these health effect indicators to predict the onset of smoking-related disease, the study will characterise this by measuring them in smokers who quit smoking by conventional recommended methods. Because smoking cessation is a globally recognised approach to reduce smoking-related disease risk, and the epidemiology of the health consequences of quitting are well documented, these data will contextualise the health effect changes in smokers switching to the investigational product.
Great efforts have been made to provide a framework that will facilitate transparency and critical appraisal of the results. For example, the study has been designed to facilitate comparative assessment, including a cessation group as the gold standard. Critically, product compliance is monitored by using both diaries of self-reported product use and biomarkers of compliance. In addition to this statistical analysis plan, the protocol has been published \[[@bib12]\], and we are fully committed to publish the results from the study. Best practices have been followed by providing appropriate sample size calculation to satisfy the main hypothesis and adjusting for multiple timepoint and endpoint assessments.
In conclusion, we present in this manuscript the design and statistical analysis approach of a randomised, open-label, parallel group switching study assessing the effects of replacing smoking with a THP for one year. This study is an essential element to understand the potential health effects as a result of switching from conventional and roll-your-own cigarettes to the investigational THP product glo™ and will be an important addition to the growing evidence evaluating THP products.
Funding {#sec6}
=======
This work has been fully funded by British American Tobacco Investments Ltd.
[^1]: NNAL is included as a primary endpoint due to its association with cancer development \[[@bib20],[@bib21]\].
[^2]: Secondary endpoints.
[^3]: Primary endpoints; All other endpoints are secondary endpoints.
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Background
==========
The development of a fistula between the tracheobronchial tree and the gastric conduit post esophagectomy is a rare and often fatal complication. Most fistulae occur from direct communication between a dehisced anastomosis and adjacent bronchus. Anastomotic leaks are responsible for approximately 40% of post-esophagectomy deaths \[[@B1]\]. Clinically apparent thoracic anastomotic leaks and fistulae are associated with a high rate of mortality despite advances in critical care and endoprostheses \[[@B2]\]. We present herein a particularly rare case, a fistula from the left main bronchus into a cervical esophagogastric anastomosis, and discuss the presentation and the approach to successful conservative management.
Case Presentation
=================
A 68 year old man presented with a five month history of progressive dysphagia and weight loss of 5 kg. An adenocarcinoma arising in Barrett\'s epithelium in the lower third of the esophagus was diagnosed, and staging including CT-PET and endoscopic ultrasound suggested clinical T3N1 M0 staging. He was treated with a standard regimen of neoadjuvant chemoradiotherapy prior to esophagectomy \[[@B3]\]. At surgery, extensive fibrosis was evident, and an en-bloc resection was performed including thoracic duct, part of pericardium, and mediastinal lymph node dissection including complete clearance of the sub-carinal nodes, and a cervical hand-sewn anastomosis was fashioned. Pathology revealed a ypT3N1 tumor, with clear margins, and 7 of 30 glands involved by tumor.
On day four postoperatively he had a neutrophil leucocytosis of 15 × 10^9^/L and evidence of left basal consolidation. This persisted despite antibiotics, and a CT of thorax demonstrated no other abnormalities. He was managed on the ward, and his FiO~2~varied from 0.4 to 0.6. Aspiration pneumonia was considered possible, and his nasogastric tube was left *in situ*. His neck wound was dry with no signs of inflammation or leakage. On day 14 the nasogastric drainage bag dramatically filled with air, and this persisted throughout the day and succeeding days, but without deterioration in respiratory function or evidence of progressive sepsis. A fiberoptic bronchoscopy was performed on day 16 which demonstrated bubbling at a gastro-bronchial fistula in the posterior aspect of the left main bronchus (Fig. [1](#F1){ref-type="fig"}). An endoscopy revealed a healthy gastric tube but a tiny area of granulation tissue in the anterior portion of the anastomosis, the assumed site of fistula communication. A CT scan of the thorax demonstrated air in the mediastinum and the gastric conduit (Fig. [2](#F2){ref-type="fig"})
{#F1}
{#F2}
He was managed conservatively with antibiotics, enteral nutrition via a jejunostomy, and non-invasive respiratory support in the form of humidified oxygen via face mask and chest physiotherapy. The huge amounts of air in the nastogastric bag persisted for a further 9 days. When it subsided, he was introduced to oral diet and progressed well and was discharged. A follow- up bronchoscopy 60 days after the diagnostic bronchoscopy, confirmed spontaneous closure of the fistula (Fig. [3](#F3){ref-type="fig"}).
{#F3}
Conclusions
===========
The development of a fistula between the gastric tube and the tracheobronchial tree represents a very rare but potentially catastrophic complication after esophagogastrostomy for esophageal carcinoma. The commonest cause is a leak from the esophagogastric intrathoracic anastomosis with subsequent mediastinal abscess and rupture into the posterior wall of the tracheobronchial tree. Anastomotic leaks are responsible for approximately 40% of post-esophagectomy deaths \[[@B1]\]. Clinically apparent thoracic anastomotic leaks and fistulae are associated with a high rate of mortality despite advances in critical care and endoprostheses \[[@B2]\].
Most anastomotic leaks result from gastric ischemia, but this does not appear to have been the problem with our patient. Potential mechanisms in this particular case include the rendering vulnerable or ischemic of the tracheobronchial tissue by neoadjuvant chemoradiotherapy, combined with sharp dissection to radically remove all sub-carinal nodal tissue, with consequent injury and delayed rupture. This is the first case to our knowledge to present in this fashion, where the primary source appears to be the bronchus with the secondary consequence at the anastomotic site. Generally, gastro-bronchial fistulae may present in the early postoperative period or relatively late in the follow-up, with management strategy influenced by the site and size of the fistula, the underlying cause, and the clinical presentation \[[@B4],[@B5]\]. It has previously been postulated that untreated gastro-bronchial fistula is usually fatal due to chronic pulmonary sepsis and that conservative treatment is inadvisable \[[@B6]\]. However, surgery is fraught with high morbidity in these patients.
The commonest mode of presentation is cough after swallowing, dyspnea, fever and recurrent pneumonia \[[@B7]\]. These are non-specific symptoms in the post-esophagectomy period. In this case, the dramatic presentation of a naso-gastric bag filling every two hours with air made the diagnosis clinically obvious, and bronchoscopy established the source. Stenting of the bronchus was considered, but the site of fistulation did not lend itself easily to an endo-bronchial prosthesis. Endoscopy of the anastomosis and stomach helped to establish that the stomach was healthy, and predict that this would heal as long as the bronchial leak settled. Self-expanding esophageal endo-prostheses have been used successfully in patients with anastomotic leaks after esophageal surgery \[[@B8]\], usually intra-thoracic leaks. Stent migration has been reported in as many as 37.5% of patients. In this case, the high anastomosis, 3 cm below the cricopharyngeus, and a normal diameter nonstenotic anastomosis were considered to predict a high risk of stent failure so stenting was not seriously entertained.
To our knowledge, this is the first report of successful conservative management of a gastro-bronchial fistula complicating a subtotal esophagectomy. Our experience would suggest that in very carefully selected cases where bronchopulmonary contamination from the fistula is minimal or absent, there is no associated inflammation of the tracheobronchial tree and the patient is stable from a respiratory point of view without evidence of sepsis, there may be a role for a trial of conservative management.
In conclusion, this is the first experience of this high-volume center with such a complication, and to our knowledge the first such case reported. The case highlights how a fistula that is unlikely to have been caused by contamination will resolve itself, and that a conservative approach with no surgery or endoprosthesis resulted in a successful outcome.
Consent
=======
Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.
Competing interests
===================
The authors declare that they have no competing interests.
Authors\' contributions
=======================
**JMS**was involved in the postoperative clinical management and drafted the manuscript; **JL**was involved in the postoperative clinical management and drafted the manuscript; **FOC**provided postoperative respiratory consultation and performed the bronchoscopies; **NR**performed the surgery and oversaw the writing of the manuscript; **JVR**performed the surgery, oversaw patient management and edited the manuscript. All authors read and approved the final manuscript.
Pre-publication history
=======================
The pre-publication history for this paper can be accessed here:
<http://www.biomedcentral.com/1471-2482/9/20/prepub>
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"pile_set_name": "PubMed Central"
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1. Introduction {#sec1-proteomes-06-00051}
===============
Novel methods for proteomic analysis of biological tissues have developed rapidly in the past decade; however, neuroproteomics remains a challenging field of study. The mammalian central nervous system (CNS) is far different from any other organ in the mammalian system, primarily because it is made up of several hundred different cell types \[[@B1-proteomes-06-00051]\]. Each cell type has unique characteristics, and distinct populations of cells are present in different brain regions. For instance, although 40% of all cells in the brain are astrocytes, neurons outnumber astrocytes in the cerebellum, whereas there is an inverse correlation in the cortex \[[@B2-proteomes-06-00051]\]. Furthermore, Herculano-Houzel et al. \[[@B3-proteomes-06-00051]\] determined that almost 70% of the two billion neurons found in the adult rat brain are located in the cerebellum, and five-fold less are present in the cortex. Brain cells also possess region-specific identities and biomarkers that have proven useful in cell-type-specific studies but can also complicate analyses \[[@B4-proteomes-06-00051],[@B5-proteomes-06-00051]\]. In addition, neural cells lack uniformity and make projections to different brain regions, resulting in spatiotemporal regulation of many signaling processes within the brain. Consequently, these factors make separation and isolation of specific cell types from brain challenging.
A second issue is that proteomic analysis of brain cells has lagged behind in comparison to its transcriptomic counterpart, which continues to make rapid advances. The facile method of RNA amplification has enabled over 500 single-cell transcript expression analyses \[[@B6-proteomes-06-00051]\]. In a few years, the field has moved from the use of quantitative reverse transcription-polymerase chain reaction (qRT-PCR) to quantify globin gene expression in human erythroleukemic cells \[[@B7-proteomes-06-00051]\] or measure expression levels of five genes in single cells isolated from mouse pancreatic islets \[[@B8-proteomes-06-00051]\], to methods with greater scope and scale. For example, RNA sequencing (RNA-seq) methods have been used to successfully analyze gene expression in single cells \[[@B9-proteomes-06-00051],[@B10-proteomes-06-00051],[@B11-proteomes-06-00051],[@B12-proteomes-06-00051],[@B13-proteomes-06-00051],[@B14-proteomes-06-00051]\]. One study classified 3005 cells in the mouse cortex and hippocampal CA1 region using single-cell RNA-seq, revealing 47 subclasses from nine known cell types \[[@B13-proteomes-06-00051]\]. A later report used single-nuclei RNA-seq to identify 16 neuronal subtypes from 3227 single-neuron datasets isolated from six different regions of the postmortem human brain \[[@B14-proteomes-06-00051]\]. Recently, a study successfully profiled gene expression in 4347 single cells from mutant human oligodendrogliomas \[[@B10-proteomes-06-00051]\]. Variations of the RNA-seq method have been developed to enable more high-throughput, comprehensive analyses \[[@B15-proteomes-06-00051],[@B16-proteomes-06-00051]\], including a recent study that profiled over 400,000 single-cell transcriptomes from more than 800 mouse cell types using a method termed Microwell-seq \[[@B15-proteomes-06-00051]\]. This rapid, cost-effective method uses an agarose microwell system for single-cell isolation and barcoded magnetic beads for mRNA capture. Drop-seq uses a similar concept but isolates and lyses single cells in nanoliter droplets of liquid prior to barcode labeling \[[@B16-proteomes-06-00051],[@B17-proteomes-06-00051],[@B18-proteomes-06-00051],[@B19-proteomes-06-00051]\]. This method enabled isolation and characterization of over 44,000 transcriptomes from mouse retinal cells, which were ultimately grouped into 39 different cell types \[[@B16-proteomes-06-00051]\]. Drop-seq has also been used to analyze RNA expression levels in 690,000 cells from 9 different adult mouse brain regions \[[@B18-proteomes-06-00051]\]. Though comprehensive transcriptomic analyses have proven useful in the characterization of specific cell types, these methods do not account for differential control of protein synthesis and degradation. Therefore, mRNA expression often does not correlate with protein abundance and may not be reliably used as a predictive tool for proteomics \[[@B20-proteomes-06-00051]\].
Large-scale proteomic studies use mass spectrometry, an approach that continues to improve in terms of accuracy and sensitivity \[[@B21-proteomes-06-00051],[@B22-proteomes-06-00051],[@B23-proteomes-06-00051],[@B24-proteomes-06-00051]\]. However, one major difference between transcriptomic and proteomic profiling is that protein abundance cannot be amplified in the same way that nucleic acids can. Therefore, the protein quantity isolated from a cell population must be above the threshold of detection for mass spectrometry analysis. While highly abundant proteins can be analyzed by mass spectrometry at the single cell level (see below), the protein yields obtained from a single cell are often below the levels necessary for reliable quantitation and therefore do not allow the depth of coverage observed in transcriptomic analyses. Moreover, past and current cell isolation techniques are often inefficient and collect small quantities of cells in a given experiment, which in turn results in low protein yields. Specific to neurons and other CNS cells, due to their non-uniformity of size and subcellular organization, many of the current separation techniques are incapable of retaining cellular structure, often resulting in leakage of cellular contents or loss of cell integrity entirely. Furthermore, protein/peptide loss can occur during sample preparation, either through peptide adsorption to sample tubes and/or during transfer of sample to and from multiple tubes \[[@B25-proteomes-06-00051],[@B26-proteomes-06-00051]\]. Mass spectrometry analysis itself can also influence the number of proteins identified, which can often be attributed to ionization efficiency and instrument sensitivity \[[@B26-proteomes-06-00051]\].
Overcoming the challenges facing cell-type-specific proteomics is of critical importance, as many types of psychiatric, developmental, and neurodegenerative disorders are associated with specific cell types in the brain. Drug addiction is one of these psychiatric disorders in which specific neuronal cell types are implicated. For instance, the psychostimulant, cocaine, regulates the reuptake of the neurotransmitter, dopamine, leading to aberrant signaling in specific sub-types of striatal medium spiny neuron (MSN) in the dorsal and ventral striatum \[[@B27-proteomes-06-00051]\]. While morphologically similar, MSNs can be separated into at least two large subtypes that differentially express D1- or D2-classes of dopamine receptors that are in turn differentially coupled to either increased or decreased cAMP signaling, respectively \[[@B28-proteomes-06-00051],[@B29-proteomes-06-00051]\]. Thus, exposure to cocaine results in opposite patterns of phosphorylation of important intracellular targets such as DARPP-32 in intermixed sub-populations of MSNs \[[@B29-proteomes-06-00051]\]. Biochemical analysis of striatum, in the absence of separation of different MSN cell types, leads to an averaging of the increased or decreased signals, and a loss of important information.
In addition to drug addiction, neurodegenerative disorders like Alzheimer's disease (AD) and Down syndrome (DS) are associated with specific cell types in the brain \[[@B30-proteomes-06-00051],[@B31-proteomes-06-00051]\]. For instance, pathology of both AD and DS patients involves overproduction of amyloid beta peptide, and the development of neurofibrillary tangles and amyloid plaques. Astrocytes, which are a type of glial brain cell, also play active roles in pathogenesis of AD brain tissue \[[@B5-proteomes-06-00051],[@B32-proteomes-06-00051]\]. In mice overexpressing amyloid beta, plaques are surrounded by reactive astrocytes and activated microglia \[[@B33-proteomes-06-00051],[@B34-proteomes-06-00051]\]. Furthermore, brain inflammation caused by glial and microglial activation is observed in brain tissue of AD patients \[[@B33-proteomes-06-00051],[@B35-proteomes-06-00051],[@B36-proteomes-06-00051]\].
Other cell-type-associated disorders include Parkinson's disease (PD), Amyotrophic lateral sclerosis (ALS), and Huntington's disease (HD). In PD subjects, pathology within the *substantia nigra* revealed a loss of a sub-population of dopaminergic neurons, followed by an increase in Lewy body structures within the retained neurons \[[@B5-proteomes-06-00051],[@B37-proteomes-06-00051],[@B38-proteomes-06-00051]\]. The subsequent DA depletion causes cell-specific effects such as hyper- and hypoactivation of D2 and D1 MSNs, respectively \[[@B39-proteomes-06-00051],[@B40-proteomes-06-00051],[@B41-proteomes-06-00051]\]. Astrocytes are also implicated in PD in many animal-based studies \[[@B5-proteomes-06-00051]\]. ALS is a degenerative disease that affects the motor cortex, brain stem, and spinal cord and ultimately results in motor neuron death \[[@B5-proteomes-06-00051],[@B42-proteomes-06-00051],[@B43-proteomes-06-00051]\]. Patients with HD exhibit a preferential loss of D2 MSNs, and an accumulation of the mutant form of Huntingtin (HTT) protein occurs in human neurons and astrocytes \[[@B5-proteomes-06-00051],[@B44-proteomes-06-00051],[@B45-proteomes-06-00051]\].
It is clear from the ongoing list of disorders that a greater focus needs to be placed on biochemical characterization of neural cell types. Though many technologies have advanced in recent years to address the issues of cell separation and isolation as well as increasing the depth of proteomic coverage for cell-type-specific analyses, there are still many aspects that need to be improved. This review will outline the different methods available, while also noting the benefits and limitations of each. Studies which have employed these techniques will also be highlighted, and potential improvements for these methods will be discussed.
2. Cell-Type-Specific Isolation Methods {#sec2-proteomes-06-00051}
=======================================
The nonuniformity and complex networks of different cell populations within the brain often require the use of cell-type-specific markers to improve the accuracy of isolation. This can be accomplished through promoter-directed expression of a reporter protein either through viral transduction (transient) or generation of a transgenic animal (stable). While viral transduction can be useful for some experimental applications (See Proteome labeling methods), expression levels may be variable when compared to transgenic animals, which may ultimately affect proteomic analyses. Though generation of transgenic animals can be time- and resource-intensive, many groups have now successfully developed transgenic tools for characterization of brain cell types \[[@B46-proteomes-06-00051],[@B47-proteomes-06-00051]\]. One of these tools was developed by taking advantage of a bacterial artificial chromosome (BAC) to express a green fluorescent protein (GFP) marker in specific neural cell types \[[@B46-proteomes-06-00051]\]. The same BAC approach was used to generate Ribo-tagged transgenic mice expressing an enhanced green fluorescence protein (EGFP)-L10a ribosomal protein under the control of cell-type-specific promoters \[[@B47-proteomes-06-00051]\]. Along with cell-type-specific visualization, this design has the added advantage of enabling translating ribosome affinity purification (TRAP) to isolate ribosomes from target cell types. Emergence of these tools coupled to cell isolation techniques is useful for proteomic analysis of CNS cell types.
One frequently-used method to isolate specific cell types is fluorescence-activated cell sorting (FACS) ([Figure 1](#proteomes-06-00051-f001){ref-type="fig"}A), which relies on a fluorescent cellular marker that can be endogenously-expressed or immunolabeled for detection. In an early study, 5000--10,000 striatal MSNs were isolated via FACS from fluorescently-labeled neurons expressing EGFP under the *Drd1*, *Drd2*, or *Chrm4* promoter (BAC transgenic mice) \[[@B48-proteomes-06-00051]\]. FACS of tissue from transgenic mice expressing GFP under the control of the parvalbumin-expressing interneuron (*Pvalb*) promoter was later used to isolate approximately 5000 and 10,000 GFP-positive nuclei from striatal and hippocampal tissue, respectively \[[@B49-proteomes-06-00051]\]. Nuclei from different sub-populations of MSNs were also subjected to FACS after acute or chronic cocaine treatment to observe cell-type-specific differential post-translational modification of histones \[[@B50-proteomes-06-00051]\]. FACS has also been used for glutamatergic synaptosomal enrichment by expressing fluorescent VGLUT1 protein in mice, which resulted in identification of 163 enriched proteins after mass spectrometry analysis \[[@B51-proteomes-06-00051]\]. Recently, FACS and subsequent LC-MS/MS was performed on sensory inner ear hair cells, enabling identification of 6333 proteins \[[@B52-proteomes-06-00051]\].
An alternative single-cell isolation method is termed laser capture microdissection (LCM) ([Figure 1](#proteomes-06-00051-f001){ref-type="fig"}A), which uses a microscope equipped with a high-precision laser to dissect small areas within a tissue slice (\>100 µm^2^). Imaging and dissection can be performed in fluorescence or bright-field modes, enabling a variety of experimental applications. For instance, Drummund et al. \[[@B53-proteomes-06-00051]\] performed LCM on neurons isolated from formalin-fixed, paraffin-embedded (FFPE) AD cortical brain tissue, which yielded more than 400 proteins identified by LC-MS/MS analysis. In this study, extensive sample treatment optimization was also performed on tissue isolated via LCM from the temporal cortex. Results from this optimization ranged from 202 to over 1700 proteins identified from approximately 4000--80,000 neurons. Another study identified 1000 proteins from tissue sections of neuromelanin granules isolated from the human *substantia nigra* \[[@B54-proteomes-06-00051]\]. Furthermore, mass spectrometry analysis of four different compartments in FFPE fetal human brain tissue identified a total of 3041 proteins \[[@B55-proteomes-06-00051]\]. Two recent reports isolated cells from human post-mortem tissue using LCM to identify a small number of potential biomarkers from AD \[[@B56-proteomes-06-00051]\] and ischemic stroke \[[@B57-proteomes-06-00051]\] patients via mass spectrometry. LCM was also recently used to quantify approximately 1000 proteins from 10--18 cells (100-µm-diameter) isolated from different rat brain regions \[[@B26-proteomes-06-00051]\]. For these analyses, optimization was first performed with 50 µm (2--6 cells), 100 µm (10--18 cells), and 200 µm (30--50 cells) diameter tissue sections from rat brain cortex, where 180, 695, and 1827 protein groups were identified, respectively.
While LCM clearly offers precision for a variety of experimental workflows, it does have limitations. If an endogenously-expressed fluorescent protein is used as a cell-type-specific marker in the tissue of interest, it must be expressed at an intensity above the threshold of detection for the microscope to accurately dissect. Furthermore, most LCM microscopes are not capable of cooling the tissue specimen during dissection. Therefore, the user must work rapidly to prevent altered protein expression and/or degradation, particularly when using fresh tissue. Moreover, dissection of the tissue can be more tedious and time-consuming than many other isolation methods, which could result in a lower number of cells (and protein) isolated in a given amount of time. Finally, if the tissue must be immunolabeled, the antibody is often processed with the rest of the cellular protein extract. This could ultimately affect proteomic results depending on the amount of antibody used. Despite these potential issues, LCM is clearly a powerful method that can be useful for many types of cell-type-specific applications.
Although animal models are useful for investigative research in neuroscience, results and treatments do not always translate to the human system. It is difficult to obtain brain tissue from human subjects, particularly over a range of development with age-matched controls and within a post-mortem interval short enough to avoid protein degradation and variations in post-translational modifications (PTMs) \[[@B58-proteomes-06-00051],[@B59-proteomes-06-00051],[@B60-proteomes-06-00051],[@B61-proteomes-06-00051]\]. In an effort to address these challenges, researchers have turned to developing specific neuron cell types from induced pluripotent stem cells (iPSCs) ([Figure 1](#proteomes-06-00051-f001){ref-type="fig"}B) \[[@B62-proteomes-06-00051],[@B63-proteomes-06-00051]\]. A major benefit of using iPSCs is that they can be produced from human somatic cells such as dermal fibroblasts (HDF) instead of embryonic stem cells, which have ethical conflicts associated. Furthermore, these iPSCs can be directly reprogrammed to differentiate into virtually any cell type with patient- or disease-specificity \[[@B62-proteomes-06-00051]\]. Many studies have already demonstrated successful production of a variety of region-specific neuronal cell types including ventral forebrain cholinergic, ventral midbrain dopaminergic, cortical glutamatergic, and cholinergic motor neurons \[[@B64-proteomes-06-00051],[@B65-proteomes-06-00051],[@B66-proteomes-06-00051],[@B67-proteomes-06-00051],[@B68-proteomes-06-00051]\]. Recently, iPSCs have undergone proteomic characterization for numerous experimental applications \[[@B69-proteomes-06-00051],[@B70-proteomes-06-00051],[@B71-proteomes-06-00051],[@B72-proteomes-06-00051],[@B73-proteomes-06-00051],[@B74-proteomes-06-00051]\]. For instance, Yamana et al. \[[@B69-proteomes-06-00051]\] compared lysates of iPSCs and fibroblast cells to identify a total of 9510 proteins via mass spectrometry analysis. A later study used quantitative mass spectrometry to identify 2217 total proteins in spinal muscular atrophy (SMA) patient-derived and healthy control motor neurons differentiated from iPSCs \[[@B73-proteomes-06-00051]\]. A comparison of the two groups indicated that 63 and 30 proteins were up-regulated in control and SMA motor neurons, respectively. Recently, three-dimensional neuron-spheroids were derived from AD and control patient iPSCs and subjected to tandem mass tag (TMT) LC-MS/MS analysis \[[@B74-proteomes-06-00051]\], which is a quantitative mass spectrometry approach that uses reporter ions generated during MS/MS fragmentation for quantitation \[[@B75-proteomes-06-00051]\]. Collectively, 1855 proteins were identified in the 3D neuro-spheroid samples that were differentiated from a total of ten iPSC lines between both the AD and control subjects. Furthermore, 8 proteins were found to be up-regulated in AD subjects, while 13 proteins were down-regulated. Another recent study profiled the proteomes of iPSCs, neural progenitor cells (NPCs), and differentiated neurons in cell culture to identify a total of 2875 proteins among all three groups \[[@B55-proteomes-06-00051]\]. Notably, 90, 33, and 126 proteins were unique to iPSCs, NPCs, and neurons, respectively. Although differentiation of iPSCs has demonstrated significant promise for moving closer to a human model system while also improving protein yield, these analyses are still being performed in vitro. It therefore becomes difficult to maintain true neural connectivity, which could ultimately result in altered protein expression compared to what would normally be observed in the human brain. Nevertheless, this approach still has potential for a variety of neurological applications in the future.
3. Proteome Labeling Methods {#sec3-proteomes-06-00051}
============================
Cell-type-specific proteome labeling is a technique that can be used to circumvent the issue of maintaining cellular integrity during isolation. Until recent years, proteome labeling studies were performed primarily using Stable Isotope Labeling with Amino acids in Cell culture (SILAC) \[[@B76-proteomes-06-00051],[@B77-proteomes-06-00051],[@B78-proteomes-06-00051],[@B79-proteomes-06-00051],[@B80-proteomes-06-00051],[@B81-proteomes-06-00051],[@B82-proteomes-06-00051],[@B83-proteomes-06-00051]\]. The obvious caveat to SILAC, however, is that experiments must be performed in cell culture. A variation termed Stable Isotope Labeling with Amino acids in Mammals (SILAM) can be used for quantitation of protein expression in vivo, however, labeling times are long (\~25 d) and it cannot be performed in a cell-type-specific manner. Recent efforts have attempted to make in vivo labeling methods compatible with cell-type-specific applications. One of the first studies to perform in situ proteome labeling over a short, 2 h time course, was termed BioOrthogonal Non-Canonical Amino acid Tagging (BONCAT) \[[@B84-proteomes-06-00051]\]. BONCAT takes advantage of a cell's protein synthesis machinery and enables incorporation of a noncanonical amino acid into the proteome of interest ([Figure 1](#proteomes-06-00051-f001){ref-type="fig"}C). Recently, this method has transitioned to cell-type-specific labeling of proteomes through generation of transgenic mice that express a mutated methionyl-tRNA synthase (MetRS\*) with an expanded amino acid binding site that recognizes the noncanonical amino acid ANL \[[@B85-proteomes-06-00051]\]. Expression of MetRS\* is driven by a cell-specific promoter and enables charging of supplemented ANL onto an endogenous tRNA^Met^, which is then stochastically incorporated into the target cell proteome. After labeling, click-chemistry can be performed to biotinylate ANL residues, followed by enrichment via streptavidin affinity chromatography. Mass spectrometry analysis of ANL-labeled, enriched proteins in hippocampal neurons and Purkinje cells resulted in 2384 and 1687 proteins identified, respectively \[[@B85-proteomes-06-00051]\]. Furthermore, a hippocampal proteome analysis of mice exposed to standard (SC) or enriched (EE) housing environments identified 2384 and 2365 proteins, respectively, of which 225 were significantly regulated after statistical comparison. Not only can click-chemistry be used for biotinylation, but fluorescent probes can be added to the ANL residues, which Dietrich et al. \[[@B86-proteomes-06-00051]\], termed FlUorescent Non-Canonical Amino acid Tagging (FUNCAT) ([Figure 1](#proteomes-06-00051-f001){ref-type="fig"}C). This method can be used for temporal visualization of newly-synthesized proteins, while also enabling post-visualization enrichment by methods such as immunoaffinity chromatography.
A similar technique called Stochastic Orthogonal Recoding of Translation (SORT) has also recently been established to label proteomes in vivo \[[@B87-proteomes-06-00051],[@B88-proteomes-06-00051]\]. Instead of requiring generation of a transgenic animal, SORT uses targeted, viral-mediated expression of an orthogonal pyrrolysyl-tRNA synthetase-tRNA~xxx~ pair that recognizes and incorporates a non-canonical amino acid AlkK into the target proteome of interest ([Figure 1](#proteomes-06-00051-f001){ref-type="fig"}C). Click-chemistry can then be performed in the same way as BONCAT/FUNCAT. Recently, SORT was used to label, biotinylate, and enrich proteins in mouse striatal MSNs prior to mass spectrometry analysis, which resulted in identification of 1780 cell-type specific proteins \[[@B89-proteomes-06-00051]\].
While these methods of cell-type-specific proteome labeling seem advantageous for future studies in neuroproteomics, there are still associated challenges and extensive optimization required for each experiment. For BONCAT/FUNCAT, transgenic animals must be generated and characterized, which is not only time-consuming, but costly. Furthermore, the MetRS\* expression levels may vary depending on the cell-type-specific promoter used, which could result in low labeling efficiency and ultimately low protein yield for mass spectrometry analysis. Similarly, low expression levels of the pyrrolysyl-tRNA synthetase-tRNA~xxx~ pair could also be observed for the SORT method for a variety of reasons including promoter selection, transduction efficiency, and accuracy of injection. Both methods also require supplementation of the non-canonical amino acid, either through drinking water intake or injection. This supplementation also needs to be optimized to ensure equivalent dosages and labeling efficiencies occur between animals. Moreover, the proteomics results from the aforementioned studies \[[@B85-proteomes-06-00051],[@B89-proteomes-06-00051]\] indicate that improvements need to be made to reach a greater depth of proteomic coverage. The observed number of protein identifications is far below the known upper limit of detection (\~12,000 proteins) \[[@B90-proteomes-06-00051],[@B91-proteomes-06-00051]\] and could potentially be improved by a variety of factors such as increasing the number of animals used and/or selecting a promoter that labels at a level above the limit of detection for the assay but does not label proteins at a level that could interfere with cellular processes.
Another labeling approach that takes advantage of the cell's native protein synthesis machinery uses a puromycin analog tag \[[@B92-proteomes-06-00051],[@B93-proteomes-06-00051],[@B94-proteomes-06-00051],[@B95-proteomes-06-00051]\]. The puromycin analog binds the acceptor (A) site of the ribosome and is then incorporated into the nascent polypeptide chain prior to inhibition of protein synthesis. The incorporated puromycin analog can then be chemically modified to enrich for newly synthesized proteins. This method was first demonstrated in cultured cells and mice using *O*-propargyl-puromycin (OP-puro), where newly-synthesized proteins were visualized via fluorescence microscopy after a copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction with a fluorescent azide \[[@B92-proteomes-06-00051]\]. Recently, a similar technique was modified for cell-type-specific labeling of proteomes in vivo \[[@B94-proteomes-06-00051]\]. This modification involves introduction of a cell-type-specific antibody bearing a tetrazing (Tz) tag and a "caged" form of puromycin (TCO-PO), which is unable to be incorporated into the proteome. When the Tz-tagged antibody and a TCO-PO molecule come in contact, a reaction occurs which results in conjugation of TCO to the antibody, rendering the PO molecule "uncaged" and free to incorporate into the proteome of the target cell. From this study, more than 1200 proteins were identified via LC-MS/MS when this method was employed in A431 cells. An earlier study performed a similar type of experiment with cell-type-specific, viral-mediated expression of an enzyme capable of activating a "caged" puromycin analog in mouse pancreatic islets and HEK 293T cells \[[@B95-proteomes-06-00051]\]. Mass spectrometry analysis of the HEK 293T cell proteome resulted in identification of 1165 proteins enriched puromycin-incorporated, enzyme-expressing proteome.
There are several advantages to using a puromycin labeling strategy over the biorthogonal labeling methods. First, the functional concentration of puromycin is much lower than that of noncanonical amino acids, reducing the likelihood of unwanted side-effects \[[@B92-proteomes-06-00051],[@B94-proteomes-06-00051],[@B95-proteomes-06-00051],[@B96-proteomes-06-00051]\]. Furthermore, unlike noncanonical amino acids, methionine does not directly compete with puromycin for incorporation into the proteome. Therefore, animals that undergo puromycin labeling do not require the low-methionine diet which may be necessary for biorthogonal labeling methods and are not subject to potential bias toward proteins with higher methionine content \[[@B92-proteomes-06-00051],[@B93-proteomes-06-00051]\]. Another advantage is that puromycin incorporation may not require use of a genetically modified organism, which does not always represent a true native biological environment \[[@B94-proteomes-06-00051]\]. Moreover, puromycin incorporation displays higher temporal resolution than biorthogonal labeling, which requires charging of the non-canonical amino acid to the tRNA prior to incorporation \[[@B92-proteomes-06-00051],[@B93-proteomes-06-00051],[@B94-proteomes-06-00051]\]. Despite the advantages of in vivo puromycin incorporation, cell-type-specific variations have only been demonstrated in cultured cells to date \[[@B94-proteomes-06-00051],[@B95-proteomes-06-00051]\].
Not only are specific cellular proteomes being labeling for general protein identification, but in situ proximity labeling methods have recently emerged to identify protein-protein interactors within discrete cellular compartments. In general, these methods rely on expression of a promiscuous biotin protein ligase fused to a target protein whose interacting proteins are being investigated. After biotin supplementation, the target interacting proteins are biotinylated by the ligase and can then be enriched and identified using proteomic analysis ([Figure 1](#proteomes-06-00051-f001){ref-type="fig"}D). One of these methods has been termed BioID, which was originally developed by Roux et al. \[[@B97-proteomes-06-00051]\] and used to identify lamin-A (LaA) interacting proteins. In this study, an *E. coli* biotin protein ligase BirA was fused to LaA and expressed in HEK293 cells to identify 122 proteins unique to BioID-LaA via LC-MS/MS. A more recent study used the BioID method to identify interacting proteins of excitatory and inhibitory postsynaptic protein complexes \[[@B98-proteomes-06-00051]\]. Viral-mediated expression of BirA, PSD-95-BirA, or BirA-gephyrin, BirA-collybistin, and BirA-InSyn1 was performed in mouse brain tissue prior to enrichment of biotinylated proteins and subsequent mass spectrometry analysis. For the PSD analysis, PSD-95-BirA interacting proteins were compared to those of the BirA control. In total, 2183 proteins were identified, 121 of which were enriched at least two-fold in PSD-95-BirA samples compared to the BirA control. For the inhibitory protein complexes, gephyrin-, collybistin-, and InSyn1-BirA interacting proteins were compared to those of the BirA control. Mass spectrometry analysis of the samples identified 2533 total proteins with a combined 181 proteins significantly enriched in the three target interactomes compared to the BirA control. More recently, BioID2 was developed, which is a similar method that employs a smaller promiscuous biotin ligase \[[@B99-proteomes-06-00051]\]. This improved method has several advantages to traditional BioID, including increased selectivity of targeting fusion proteins, a reduced amount of biotin required, and enhanced labeling of proximal proteins. TurboID is a similar approach developed recently that takes advantage of a different mutated form of biotin ligase, which is capable of proximity labeling within 10 min \[[@B100-proteomes-06-00051]\]. In this study, TurboID displayed a significantly higher biotin labeling efficiency and a similar proteome coverage of subcellular compartments within HEK293T cells after quantitative LC-MS/MS when compared to BioID.
A second method termed APEX (short for Enhanced APX) uses an engineered ascorbate peroxidase fusion protein for biotin labeling of target interacting proteins. This method was first demonstrated in HEK293 cells, where APEX was targeted to the mitochondrial matrix, and biotinylated interacting proteins were enriched and subjected to LC-MS/MS \[[@B101-proteomes-06-00051]\]. In total, 495 proteins were identified in the mitochondrial matrix proteome. Recently, APEX was used in *C. elegans* to identify tissue-specific and subcellular-localized proteomes \[[@B102-proteomes-06-00051]\]. APEX was targeted to the nucleus or cytoplasm of intestine, epidermis, body wall muscle, or pharyngeal muscle tissues, from which 3180 interacting proteins were collectively identified. A separate study used APEX to identify spatiotemporal interacting proteins of the delta opioid receptor (DOR) in HEK cells \[[@B103-proteomes-06-00051]\]. This study observed changes in DOR interactions over an activation time course of 1--30 min as well as different subcellular compartments, including the plasma membrane (PM) and endosome (Endo). Recently, a modified APEX strategy was used to map proteins at excitatory and inhibitory synaptic clefts of rat cortical neurons, resulting in identification of 199 and 42 proteins, respectively \[[@B104-proteomes-06-00051]\].
Like the other labeling techniques, extensive optimization of these proximity labeling assays is required for optimal performance. Moreover, the amount of starting material needed for adequate protein enrichment for LC-MS/MS analysis is substantial and not feasible for small amounts of tissue or certain cell types. Furthermore, standardization and reproducibility of labeling methods becomes difficult since protein output is often not provided (See [Table A1](#proteomes-06-00051-t0A1){ref-type="table"}) and can vary between organisms. Though these proximity labeling methods are similar in practice, APEX labeling times are much faster (\~1 min) compared to the 24 h labeling time of the BioID method, which could significantly impact proteomics results. Notably, however, APEX has limited stability in heated or reducing environments compared to BioID, and the presence of H~2~O~2~ in the cell can lead to toxicity. Nevertheless, APEX does have great appeal, particularly for those interested in rapid proteomic changes such as altered subcellular localization or metabolic regulation.
4. Mass Spectrometry Methods {#sec4-proteomes-06-00051}
============================
One of the major challenges in workflows related to cell-type-specific proteomics is loss of protein during sample handling, which occurs at various steps between isolation of the single or multiple cell and peptide injection onto the mass spectrometer. Furthermore, enzymatic cleavage is necessary to generate peptides for bottom-up proteomics, but this can result in partial or incomplete digestion depending on the amino acid composition of the protein. Peptides generated from poor cleavage are often too large for ionization and detection via LC-MS/MS, ultimately resulting in loss of information for these specific regions of the protein. Instrument issues also include sensitivity and accuracy as well as chromatographic and spectral reproducibility between sample runs.
Efforts to overcome some of these issues have utilized alternative workflows in an attempt to obtain cell-type level proteome or metabolome analysis ([Figure 2](#proteomes-06-00051-f002){ref-type="fig"}). One such method termed mass spectrometry imaging (MSI) can analyze tissue sections with high spatial resolution to determine relative abundances and distribution of proteins \[[@B105-proteomes-06-00051],[@B106-proteomes-06-00051],[@B107-proteomes-06-00051],[@B108-proteomes-06-00051],[@B109-proteomes-06-00051],[@B110-proteomes-06-00051],[@B111-proteomes-06-00051]\]. Of the MS ionization sources available, matrix-assisted laser desorption/ionization (MALDI) and secondary ion mass spectrometry (SIMS) microprobes are most commonly used for imaging mass spectrometry due to their softer, non-destructive qualities that enable ionization of intact biomolecules at micro- and nanometer resolutions, respectively \[[@B105-proteomes-06-00051],[@B112-proteomes-06-00051],[@B113-proteomes-06-00051]\]. MALDI uses a laser light for desorption and ionization of the sample, and SIMS uses a more focused, accelerated primary ion beam to ionize analytes from the surface of cells. Furthermore, MALDI is particularly useful for detecting higher molecular weight species (2--70 kDa), while SIMS offers detection of molecules below 1 kDa or 2000 *m/z* \[[@B112-proteomes-06-00051],[@B114-proteomes-06-00051],[@B115-proteomes-06-00051],[@B116-proteomes-06-00051]\].
These methods have been used for a range of experimental cell-type-specific applications \[[@B106-proteomes-06-00051],[@B107-proteomes-06-00051],[@B117-proteomes-06-00051],[@B118-proteomes-06-00051],[@B119-proteomes-06-00051],[@B120-proteomes-06-00051],[@B121-proteomes-06-00051]\]. For instance, MALDI-MSI was performed in mouse pituitary gland samples at a spatial resolution of 5 µm to identify ten neuropeptides at up to 2500 *m/z* \[[@B117-proteomes-06-00051]\]. An earlier study identified proteins in over 82 mass ranges in different mouse brain regions as well as 150 proteins in human glioblastoma tissue using MALDI-MSI \[[@B107-proteomes-06-00051]\]. One of the most recent MALDI-MSI applications demonstrated proteomic profiling of over 1000 rat dorsal root ganglia cells, which were classified into three separate groups on a peptide and lipid data basis \[[@B118-proteomes-06-00051]\]. SIMS has also been used for identification of single-cell metabolites, however, the majority of these studies focus on lipidomic analyses \[[@B120-proteomes-06-00051],[@B121-proteomes-06-00051]\]. One study also used both SIMS and MALDI-MSI approaches to investigate the biomolecular and spatial composition of rat spinal cord tissue \[[@B116-proteomes-06-00051]\].
Mass cytometry is another type of MSI method that uses inductively coupled plasma (ICP) as an ionization source. This method is viewed as a targeted approach to MSI and uses metal-conjugated antibodies to enable antigen localization within the tissue or cell of interest, ultimately improving the limits of detection for target proteins. This multiplexing method enables quantitation of 100 target features, simultaneously without spectral overlap \[[@B122-proteomes-06-00051],[@B123-proteomes-06-00051],[@B124-proteomes-06-00051]\]. Bandura et al. \[[@B122-proteomes-06-00051]\] developed a 20-antigen targeted mass cytometry expression assay using lanthanide-tagged antibodies. This assay was then used to label cell lines from human leukemia patients (monoblastic M5 AML and monocytic M5 AML) and model cell lines (KG1a and Ramos) and subsequently map the isotope tag intensity profiles for an average of 15,000--20,000 cells \[[@B122-proteomes-06-00051]\]. A later report used bone marrow aspirates from a total of 46 leukemia and healthy patients to quantify 20 target biomarkers via mass cytometry \[[@B125-proteomes-06-00051]\]. Recently, tissue preparation techniques were compared for mass cytometry analysis of single-cell suspensions of human glioma, melanoma, and tonsil tissues \[[@B124-proteomes-06-00051]\]. A variation on this method was later developed, termed multiplexed ion beam imaging (MIBI), which images metal isotope-labeled antibodies using SIMS \[[@B123-proteomes-06-00051]\]. This method is also capable of imaging up to 100 features simultaneously at a parts-per-billion (ppb) sensitivity and is compatible with fixed tissue. Angelo et al. \[[@B123-proteomes-06-00051]\] used MIBI to quantify 10 biomarker targets in breast cancer biopsy tissue, which performed at the same level or better than other quantitative clinical immunohistochemistry (IHC) methods.
While there are clear advantages associated with MSI methods for single-cell proteomic and metabolic analyses, including sensitivity and multiplexing capabilities, there are still several drawbacks to these methods. As previously mentioned, MALDI-MSI is limited to higher molecular weight species (\>2 kDa), while SIMS is limited to low molecular weight species (\<2 kDa). Furthermore, MALDI is only capable of micrometer resolution and performance is dependent on the assisting matrix \[[@B105-proteomes-06-00051],[@B112-proteomes-06-00051],[@B113-proteomes-06-00051],[@B126-proteomes-06-00051]\]. Mass cytometry is limited by the number of available metal-isotope-labeled antibodies and the specificity of the antibodies to the target antigen(s). Despite the possible disadvantages, advances in these mass spectrometry techniques have enormous potential to significantly improve the quality of data obtained from cell-type-specific proteomic analyses.
5. Future Perspectives {#sec5-proteomes-06-00051}
======================
Cell-type-specific proteomics has undoubtedly made considerable progress in recent years, particularly in the field of neuroscience. Not only have cell isolation methods improved, but the instrumentation used for proteomic analysis has significantly advanced regarding sensitivity and reproducibility. Based on many of the neural cell-type-specific datasets available, however, the average number of proteins identified continues to fall far below the acceptable threshold of previous neural proteomics reports ([Table A1](#proteomes-06-00051-t0A1){ref-type="table"}) \[[@B90-proteomes-06-00051],[@B91-proteomes-06-00051]\]. As discussed, there are several possible reasons for the discrepancy in protein identifications found in brain tissue versus single-cell datasets. One is the lack of organism- and tissue-specific standardization to determine the threshold of cellular material necessary for adequate proteomic analysis. As displayed in [Table A1](#proteomes-06-00051-t0A1){ref-type="table"}, the number of proteins identified in each of the listed techniques varies drastically between studies. Moreover, many of the results listed are lacking experimental information that is necessary for reproduction. For instance, several reports provide the number of cells and/or tissue quantity isolated but do not include the amount of protein extracted from this material or injected onto the mass spectrometer. This calls attention to the benefit of better standardization methods for cell-type-specific proteomics, in order to improve overall reproducibility and quality of datasets. Furthermore, method development for cell-type-specific proteomics in neuroscience needs to continue with increased focus placed on factors such as improving the efficiencies of cell isolation methods and reducing protein loss during sample preparation.
Recent efforts have also been made to improve these issues in the context of FACS for proteomic analysis. For instance, Zhu et al. \[[@B25-proteomes-06-00051]\] identified an average of 670 protein groups from single HeLa cells after integrating FACS and a novel method called nanoPOTS (nano-droplet processing in one-pot for trace samples). After cells are sorted via FACS, the nanoPOTS method relies on robotic liquid handling to perform sample processing in nanoliter volumes to help minimize sample loss. In this study, FACS was noted to have several advantages in a single-cell proteomic workflow such as precise cell counting and enabling removal of unwanted background contamination through cell dilution in PBS \[[@B25-proteomes-06-00051]\].
In addition to FACS-based approaches, development of mass spectrometry-based methods that combine different analytical features have made considerable progress in the advancement of single-cell proteomics. Capillary electrophoresis (CE) is one feature that has been recently coupled to mass spectrometry methods for single-cell analysis \[[@B127-proteomes-06-00051],[@B128-proteomes-06-00051],[@B129-proteomes-06-00051],[@B130-proteomes-06-00051],[@B131-proteomes-06-00051],[@B132-proteomes-06-00051],[@B133-proteomes-06-00051],[@B134-proteomes-06-00051],[@B135-proteomes-06-00051],[@B136-proteomes-06-00051]\]. Benefits of using CE for single-cell analyses include small sample volume accommodation, increased spatial resolution and sensitivity, and reduced matrix effects \[[@B131-proteomes-06-00051],[@B137-proteomes-06-00051],[@B138-proteomes-06-00051],[@B139-proteomes-06-00051]\]. One group recently coupled CE to microflow electrospray ionization mass spectrometry (CE-µESI-MS) to identify metabolites in different cell types of South African clawed frog (*Xenopus laevis*) embryos in three consecutive studies \[[@B129-proteomes-06-00051],[@B130-proteomes-06-00051],[@B131-proteomes-06-00051]\]. In the first of these studies, CE-µESI-MS was used to compare metabolites in three different *Xenopus* blastomere cell types dissected from the dorsal-ventral and animal-vegetal regions of the 16-cell embryo \[[@B130-proteomes-06-00051]\]. In total, 40 metabolites were significantly altered among the three cell types, indicating both specificity and metabolic interconnection. A year later, this group used a similar method to identify 55 unique small molecules in left and right D1 cells isolated from 8-cell *Xenopus* embryos \[[@B131-proteomes-06-00051]\]. After multivariate and statistical analyses, an equal number of five metabolites were found to be significantly enriched in the left and right D1 cells. Recently, this group was able to use CE-µESI-MS for direct analysis of live *Xenopus* embryo cells \[[@B129-proteomes-06-00051]\]. In this study, approximately 230 different molecular features were identified during mass spectrometry analysis of dorsal and ventral 8--32-cell-embryos. Not only has this group identified metabolites using CE-µESI-MS, but they have also performed proteomic analyses. In one report, they identified a total of 438 proteins from 16 ng of protein digest from a single blastomere of a *Xenopus* 16-cell embryo \[[@B132-proteomes-06-00051]\]. In the same year, they also reported identification of a total of 1709 protein groups from 20 ng of *Xenopus* protein digest from three cell types of the 16-cell embryo \[[@B133-proteomes-06-00051]\]. In addition to electrophoresis, capillaries have recently been used for microsampling of biomolecules from single neurons \[[@B140-proteomes-06-00051]\]. This study integrated this technique with downstream ESI-IMS-MS, which had only previously been performed in human carcinoma cells \[[@B141-proteomes-06-00051]\] and *Arabidopsis thaliana* epidermal cells \[[@B142-proteomes-06-00051]\]. Another study developed a neuron-in-capillary method to culture and isolate single *Aplysia californica* bag cell neurons prior to LC-MS/MS analysis \[[@B143-proteomes-06-00051]\].
Recently, a mass spectrometry-based approach called Single Cell ProtEomics by Mass Spectrometry (SCoPE-MS) was developed to address two of the major challenges facing cell-type-specific proteomic analysis: minimizing protein loss that can occur from protein extraction to mass spectrometry analysis and improving quantitation of low-abundant peptides identified from single cells \[[@B144-proteomes-06-00051]\]. To achieve these goals, live single mouse embryonic stem cells were isolated under a microscope prior to mechanical lysis and protein extraction. Next, single-cell protein was added to that of carrier cells to further reduce sample loss and increase the amount of protein injected on the mass spectrometer. To improve quantitation, tryptic peptides were then subjected to TMT labeling prior to LC-MS/MS, which resulted in quantitation of over 1000 proteins.
Despite the many advantages discovery mass spectrometry has to offer, more quantitative MS approaches have become increasingly popular in recent years. Targeted methods such as parallel reaction monitoring (PRM) and data-independent acquisition (DIA) have emerged in recent years in efforts to improve sensitive, accurate, and reproducible peptide quantitation. Though PRM is limited by the number of peptides that can be quantified in a given assay, it enables multiplexing, which can result in quantitation of multiple peptides in a single run for a more high-throughput analysis. Recently, Wan et al. \[[@B145-proteomes-06-00051]\] used PRM to quantify phosphorylation of PINK1 substrates in human and mouse cortical neurons. Data-independent acquisition (DIA) is not as sensitive as PRM, however, it has a much greater assay capacity. For instance, DIA analysis of fractionated mouse hippocampal neurons resulted in identification of 4558 proteins among all fractions \[[@B146-proteomes-06-00051]\]. A similar method to DIA was recently reported termed "BoxCar" which enabled identification of more than 10,000 proteins from mouse brain tissue \[[@B147-proteomes-06-00051]\]. Finally, label-based quantitation is another method that is becoming increasingly popular for neuroproteomic analyses. Recently, 11,840 protein groups were identified across two brain regions of control, AD, PD, and AD/PD human patients using TMT 10-plex labeling \[[@B148-proteomes-06-00051]\]. While these and other results mentioned above using LCM together with fixed tissue or MALDI-MSI are encouraging, there is a need for systematic and comprehensive cell-type-specific LC-MS-MS analyses in human tissue.
Targeted mass spectrometry is also useful for quantitation of protein isoforms, which can have cell-type- and tissue-specific expression profiles. Since the majority of isoform sequences are highly conserved, they can only be distinguished by isoform-specific peptides, which are often lower in abundance than peptides within the conserved regions. If these specific peptides are not detected via discovery LC-MS/MS, the isoforms cannot be distinguished and are consequently grouped by the mass spectrometry search software. This ultimately results in loss of isoform-specific expression profiles. Using a more sensitive targeted approach drastically improves the probability that isoform-specific peptides will be detected and quantifiable. Depending on the protein sequence, however, it may not be possible to identify specific peptides for all isoforms using the targeted mass spectrometry approach. One of the remaining ways to elucidate isoform-specific expression patterns is through mRNA sequencing. mRNA is alternatively spliced prior to protein translation and is therefore a blueprint for the protein sequence. By integrating the mRNA and protein datasets, a more complete picture of the proteome can be generated. Tools to achieve this type of data integration have already been developed, and continue to improve, which could prove useful for future cell-type-specific analyses \[[@B149-proteomes-06-00051],[@B150-proteomes-06-00051]\].
In summary, there is an overwhelming demand for comprehensive and consistent cell-type-specific data in neuroscience, and novel techniques have been evolving rapidly in attempts to fill this gap. This review has outlined methods and technical challenges present in this area of research as well as potential improvements for these analyses. Collectively, these methods are making substantial progress to increase the sensitivity, reproducibility and depth of proteome coverage necessary for future cell-type-specific studies.
Support was also obtained from the State of Connecticut, Department of Mental Health and Addiction Services.
All authors had equal contribution in writing and preparing the manuscript.
We acknowledge support from the NIH (Yale/NIDA Neuroproteomics Center DA018343; DA040454; MH106934; MH16488).
The authors declare no conflict of interest.
proteomes-06-00051-t0A1_Table A1
######
List of cell-type-specific methods for isolation, enrichment, and detection of proteins. The advantages and disadvantages of each technique are listed in Columns 1--3. References (Ref.) which have demonstrated the corresponding technique for a cell-type-specific application are listed in Column 4. Columns 5--8 contain the cell source (5), isolated cell/tissue quantity (6), protein quantity used for MS analysis (7), and number of proteins identified in the MS analysis (8) for each of the listed references. N/A indicates that information was not provided in the reference text.
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Technique Advantages Disadvantages Ref. Cell Source \# Cells/Tissue Quantity Isolated Protein Quantity for MS Analysis \# Proteins Identified from MS Analysis
-------------------------------------------------------------------------------- ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- -------------------------------- ----------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------ ---------------------------------- -----------------------------------------
Fluorescence-activated cell sorting (FACS) Can purify functionally homogenous cell populationsOffers precise cell countingEnables removal of background contamination Cellular integrity can be compromisedLow cell/protein yield if fluorescent expression/signal is low \[[@B151-proteomes-06-00051]\] Human neuronal nuclei 1 g starting tissue, \>5 × 10^6^ nuclei 25 µg 1755
\[[@B52-proteomes-06-00051]\] Mouse inner ear hair cells 199,894 cells 3 µg 6333
\[[@B51-proteomes-06-00051]\] Mouse glutamatergic synapses 485 synapses 8 µg 2044 total, 163 enriched
\[[@B25-proteomes-06-00051]\] HeLa cells 1 cell N/A 670
Laser-capture microdissection (LCM) High-precision laser enables isolation of neurons (\<100 µm^2^)Imaging and dissection can be performed in fluorescence orbright-field modesCompatible with fixed tissue Tissue cannot be kept cold and may endure heat damage from the laser, potentially causing changes in protein expression or post-translational modificationsLimited by the number of cells that can be analyzed per tissue sliceEndogenous expression of fluorescent marker may not be adequate to visualize and dissect \[[@B53-proteomes-06-00051]\] Human cortical neurons from AD patients 4000--80,000 neurons N/A 202 (4k neurons), 1773 (80k neurons)
\[[@B54-proteomes-06-00051]\] Human *substantia nigra* 550,000 µm^2^ neuromelanin (NM) granules 200 ng 1000
\[[@B57-proteomes-06-00051]\] Human neurons and blood brain barrier (BBB) structures 2500 neurons and\ N/A 365 (Neurons), 539 (BBB)
4000 BBB units
\[[@B26-proteomes-06-00051]\] Rat cortical cells 2--6, 10--18, and 30--50 cells N/A 180 (2--6 cells), 695 (10--18 cells), 1827 (30--50 cells)
\[[@B152-proteomes-06-00051]\] Human pancreatic islets 18 islets N/A 3219
\[[@B55-proteomes-06-00051]\] FFPE fetal human brain tissue 36 samples (4 compartments, 8--15 mm^2^/compartment) 10 µg 3041
Induced pluripotent stem cells\ Resembles the human model more than commonly-used rodent modelsCan be differentiated into any cell typeLess ethical challenges than embryonic cells All analyses are in vitroNeural connectivity is lost \[[@B70-proteomes-06-00051]\] iPSCs 10^8^ cells N/A 7952
(iPSCs)
\[[@B69-proteomes-06-00051]\] N/A 4 µg 9510
\[[@B72-proteomes-06-00051]\] N/A 40 µg 673
\[[@B73-proteomes-06-00051]\] 6 × 10^4^ cells 240 µg 2217
\[[@B74-proteomes-06-00051]\] N/A 100 µg 1855
\[[@B55-proteomes-06-00051]\] 2 × 10^7^ cells 10 µg 2875
BioOrthogonal Non-Canonical Amino Acid Tagging\ Enables in situ proteome labelingEnables time-dependent profiling of protein synthesisNon-canonical amino acid administration through drinking wateror via injectionCan be performed in fixed tissue Metabolic incorporation needs to be performed in Met-free media or animals on a low Met dietTemporal resolution is limited by conversion of non-canonical amino acid into aminoacyl-tRNA prior to protein synthesisLabeled peptides are poorly detected with mass spectrometryRequires optimization of labeling efficiency \[[@B84-proteomes-06-00051]\] HEK293T cells N/A 1.95--2.1 mg input 195
(BONCAT)
\[[@B153-proteomes-06-00051]\] HEK293T cells N/A N/A 138
\[[@B85-proteomes-06-00051]\] Excitatory hippocampal neurons, cerebellar Purkinje cells 130--200 k neurons (Purkinje) N/A 2384 (hippocampal), 1687 (Purkinje)
Stochastic Orthogonal Recoding of Translation\ Viral-mediated expression of a modified tRNA(Does not require generation of a transgenic mouse)Can be performed in fixed tissue Viral expression could be variable depending on the promoter usedOptimization is required to determine time-dependent expression levels of tRNA synthase and labeling efficiency \[[@B87-proteomes-06-00051]\] Fly germ cells 500 ovaries 7 mg 299
(SORT)
\[[@B89-proteomes-06-00051]\] Mouse striatal medium spiny neurons (MSNs) N/A N/A 1780
Antibody-assisted cell-type-specific puromycylation Does not require use of transgenic animalDisplays high temporal resolutionFunctions at lower concentrations than noncanonical amino acids Relies on antibody specificity \[[@B94-proteomes-06-00051]\] A431 cells N/A N/A \>1200
\[[@B95-proteomes-06-00051]\] HEK293T cells 2 × 10^7^ cells N/A 1165 enriched
\ \ \ \ \ \
BioID Enables screening of proximal protein interactors in situ Time-consuming (Need to generate and characterize transgenic mice)Extensive assay optimization requiredLabeling times are slow (\~24 h) \[[@B97-proteomes-06-00051]\] HEK293T cells 4 × 10^7^ cells N/A 122
\[[@B154-proteomes-06-00051]\] *Toxoplasma gondii* parasite N/A N/A 19
\[[@B98-proteomes-06-00051]\] Mouse cortical and hippocampal neurons N/A N/A 121 (ePSD), 181 (iPSD)
BioID2 Uses a smaller biotin ligase than BioIDEnables more selective targeting of fusion proteins than BioIDRequires less biotin supplementation than BioIDDisplays enhanced labeling of proximal interacting proteins than BioID Time-consuming (Need to generate and characterize transgenic mice)Extensive assay optimization requiredLabeling times are moderately slow (\~16 h) \[[@B99-proteomes-06-00051]\] HEK293T cells 4 × 10^7^ cells 100 µg 260
TurboID Efficient labeling time (\~10 min)Compatible with TMT labelingEnables labeling of organelle-specific proteomes Can sequester endogenous biotin and cause toxicity \[[@B100-proteomes-06-00051]\] HEK293T cells N/A 3 mg input 314 (mito), 186 (ER), 1455 (nuclear)
Long labeling times can cause toxicity
Engineered ascorbate peroxidase\ Enables screening of proximal protein interactors in situLabeling is very rapid (\~1 min)Applicable for labeling of subcellular compartments Limited stability in heated or reducing environmentsGenerating a transgenic organism is necessaryH2O2 can cause cellular toxicity \[[@B101-proteomes-06-00051]\] HEK293T cells 7--8 million cells 4 mg input 495
(APEX)
\[[@B155-proteomes-06-00051]\] *Drosophila melanogaster* N/A N/A 389
\[[@B102-proteomes-06-00051]\] *C. elegans* L4 larvae 30,000 larval cells 450--500 µg input 3180
Matrix-assisted laser desorption/ionization MS imaging\ Enables spatial quantitation of proteins in tissue sections Low spatial resolution (µm)Broad mass range (\~500--100 kDa) \[[@B107-proteomes-06-00051]\] APP23 transgenic mouse tissue 50 µm resolution N/A 5 Aβ peptides
(MALDI-MSI)
Non-destructive method \[[@B116-proteomes-06-00051]\] Rat spinal cord 20 µm tissue sections N/A 27 peptides
\[[@B117-proteomes-06-00051]\] Mouse pituitary gland 1.5 mm × 2.5 mm tissue sections N/A 10 neuropeptides
\[[@B118-proteomes-06-00051]\] Rat dorsal root ganglia \>1000 cells N/A 26 peptides
Secondary ion mass spectrometry\ Enables spatial quantitation of proteins in tissue sectionsNon-destructive method High spatial resolution (nm)Low mass range (\<1000 Da) \[[@B121-proteomes-06-00051]\] Benign prostatic hyperplasia (BPH), HeLa,\ 25--30 µm diameter tissue (BPH: 180 × 180 µm^2^, HeLa: 88 × 108 µm^2^, cheek cells: 150 × 175 µm^2^) N/A \<10 biomolecule ions
(SIMS) and human cheek cells
\[[@B116-proteomes-06-00051]\] Rat spinal cord 2.3 µm spatial resolution N/A 18 biomolecule ions
\[[@B120-proteomes-06-00051]\] *Aplysia californica* neurons 0.39--2.3 µm resolution N/A 3 biomolecule ions
Mass cytometry Enables multiplexed targeting of 100 target features without spectral overlap Limited by the number and specificity of available metal-isotope-labeled antibodies \[[@B122-proteomes-06-00051]\] Human leukemia cells (monoblastic M5 AML, monocytic M5 AML) and model cell lines\ 15,000--20,000 cells N/A 20 target antigens
(KG1a, Ramos)
\[[@B123-proteomes-06-00051]\] Human breast tumor cells N/A N/A 10 target antigens
\[[@B125-proteomes-06-00051]\] Human bone marrow aspirates 480,000 cells N/A 28 target antigens
\[[@B124-proteomes-06-00051]\] Human glioma, melanoma, and tonsil tissue cells N/A N/A 8 target antigens
Capillary electrophoresis microflow electrospray ionization mass spectrometry\ Accommodates small sample volumesHigh spatial resolution and sensitivityLow matrix effectsCan be temperature controlled to avoid sample heating Extensive optimization required \[[@B143-proteomes-06-00051]\] *Aplysia californica* neurons 1 neuron N/A \>300 metabolites
(CE-µESI-MS)
\[[@B127-proteomes-06-00051]\] *Aplysia californica* neurons 25 B1 and B2 buccal neurons N/A \>300 metabolites
\[[@B130-proteomes-06-00051]\] *Xenopus laevis* 16-cell embryo 15 blastomeres N/A 40 metabolites
\[[@B131-proteomes-06-00051]\] *Xenopus laevis* 8-cell embryo D1 blastomere N/A 55 small molecules
\[[@B132-proteomes-06-00051]\] *Xenopus laevis* 16-cell embryo 1 blastomere 16 ng 438
\[[@B133-proteomes-06-00051]\] *Xenopus laevis* 16-cell embryo 1 blastomere 20 ng 500--800
\[[@B129-proteomes-06-00051]\] *Xenopus laevis* 8--32-cell embryo 1 blastomere N/A 230 molecular features
Single Cell ProtEomics by Mass Spectrometry\ Minimizes protein loss from protein extraction to LC-MS/MSQuantitative MS approach (TMT labeling) Has been demonstrated in few organisms \[[@B144-proteomes-06-00051]\] Mouse embryonic stem cells 1 cell \>1000 proteins
(SCoPE-MS)
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
{#proteomes-06-00051-f001}
{#proteomes-06-00051-f002}
| {
"pile_set_name": "PubMed Central"
} |
###### Strengths and limitations of this study
- The study shows that weather variation could be captured even in a single seasonal transition.
- After factoring in weather variation, the study establishes an association between urban design and the likelihood of children accumulating globally recommended levels of moderate to vigorous physical activity.
- The study highlights that different types of urban design could facilitate active living in children in different weather conditions.
- These findings not only have implications for conducting active living research, but also for policy interventions that focus on creating active urban communities.
- However, the study\'s findings are limited by cross-sectional design.
Introduction {#s1}
============
Physical activity\'s (PA) benefits in preventing non-communicable diseases and promoting mental well-being in children have been well established.[@R1] [@R2] Despite this, physical inactivity has reached pandemic levels, with a majority of children not accumulating recommended levels of moderate to vigorous PA (MVPA).[@R3] Current global PA guidelines recommend that children aged 5--17 years should accumulate at least 60 min of MVPA every day.[@R5] In curbing physical inactivity, as behavioural interventions directed at individuals have not produced a change at the population level, active living interventions that aim to modify urban design and built environment to facilitate PA have gained prominence.
Active living evidence in children thus far indicates some consistent findings, where safety (especially for girls), perception of recreational environment and opportunity for active transportation have been positively associated with PA.[@R7] Recent evidence has revealed a more complex picture, where the roles of multilevel environmental determinants (urban design, neighbourhood built and social environment, school environment, and home environment) on PA have been emphasised.[@R14]
Nevertheless, active living interventions have underexplored a phenomenon that perennially interacts with all other environmental factors---weather variation. The significance of weather variation\'s influence on PA is especially important in temperate and continental climatic zones (Köppen-Geiger climate classification) due to a wide variation in seasonal weather in these regions.[@R17] Studies that have explored seasonal weather variation\'s exclusive influence on PA in populations inhabiting these regions report a fairly stable and expected observation of higher PA in warmer months, among all age groups.[@R18]
In Canada, the majority of the population experiences a wide variation in seasonal temperatures and weather conditions.[@R17] [@R23] [@R24] Within Canada, prairie provinces like Saskatchewan, where this study was conducted, are known for particularly extreme variations in seasonal weather,[@R23] [@R24] and there is evidence to indicate that the relationship between seasonality and PA is stronger in Saskatchewan.[@R22]
However, weather variation can influence PA even within seasons and during seasonal transitions. Research on the influence of weather on PA in children and adolescents provides further insight into influence of weather variation on PA within seasons. Evidence from a Canadian 5-year longitudinal study of adolescent PA showed that higher temperatures increased PA, and higher rainfalls reduced PA.[@R25] Similarly, a study of the impact of rainfall and school break time policies on PA in 9--10-year-old British children revealed that rainfall is negatively associated with PA.[@R26]
Even though evidence on the influence of weather variation on PA points towards warmer weather being positively associated and rainy weather being negatively associated with PA in temperate and continental climatic zones, most studies thus far have exclusively focused on weather, a non-modifiable entity, instead of understanding how diverse environmental exposures influence PA after factoring in weather variation. More importantly, in developing active urban communities, it is imperative that urban planning policy is conceptualised by taking into account how different types of urban design and built environment interact with weather variation to influence PA accumulation in children. Furthermore, in capturing weather variation, there has been an under exploration of the complex and interrelated dynamics of key variables such as temperature, wind and precipitation.[@R18] [@R25]
This study first conceptualises weather as a complex entity consisting of several interrelated elements that can be combined to categorise localised weather patterns. Thereafter, by factoring in localised weather and neighbourhood and household socioeconomic environment, this study hypothesises that urban and built environment significantly influence globally recommended levels of MVPA in children aged 10--14 years.
Methods {#s2}
=======
The study is part of an active living research initiative in Saskatoon, Saskatchewan, Canada (<http://www.smartcitieshealthykids.com>). The study protocol was approved by the University of Saskatchewan\'s Research Ethics Board.
Urban design of Saskatoon {#s2a}
-------------------------
Presently Saskatoon\'s metropolitan area population of 260 600 is spread across 65 well-defined neighbourhoods,[@R28] where the city plays a major role in urban planning including the geographic allocation of commercial, residential and institutional establishments.
The neighbourhoods designed prior to 1930 surround the city centre and follow a traditional grid-patterned street design ([figure 1](#BMJOPEN2015009045F1){ref-type="fig"}-Planning Era 1), typified by higher density, mixed-use neighbourhoods connected by straight, intersecting streets and back alleys. The semisuburban neighbourhoods built between 1931 and 1966 follow a fractured grid-pattern ([figure 1](#BMJOPEN2015009045F1){ref-type="fig"}-Planning Era 2). They are predominantly residential, with lower density and become progressively car-oriented as the distance from the urban centre increases. Finally, the suburban neighbourhoods built after 1967 follow curvilinear street patterns ([figure 1](#BMJOPEN2015009045F1){ref-type="fig"}-Planning Era 3), characterised by low-density, almost exclusively residential and highly car-oriented configurations. Working with the City of Saskatoon\'s Neighbourhood Planning Department, our Smart Cities Healthy Kids research team has validated the three types of neighbourhoods belonging to the three different planning eras.[@R29]
{#BMJOPEN2015009045F1}
Neighbourhood selection and recruitment {#s2b}
---------------------------------------
The neighbourhood selection and recruitment were part of the Smart Cities Healthy Kids initiative. The sampling frame for recruiting children consisted of all 60 residential neighbourhoods in 2010 in Saskatoon categorised into the three types of neighbourhoods ([figure 1](#BMJOPEN2015009045F1){ref-type="fig"}). Working with our public and Catholic school board partners, all schools in Saskatoon situated in all three types of neighbourhoods were invited to participate in the study. The recruitment was conducted through 30 elementary schools that accepted to participate and the total study sample was representative of all 60 neighbourhoods. Working with the schools, we identified four classrooms at each elementary school (grades 5--8) and each school was provided with letters of consent to be given to each potential participant to deliver to their primary caregiver. In order to participate in the study, caregivers returned the signed forms to their child\'s homeroom teacher. It was made explicit in the consent form that caregivers or children would be able to opt out of participating at any time up until the data were pooled. Children were also provided an opportunity to decline participation even after obtaining parental consent. Of the 1610 children aged 10--14 years who agreed to participate in the Smart Cities Healthy Kids initiative, 455 children agreed to participate in accelerometry. This study exclusively focuses on children who participated in accelerometry.
Built environment measures {#s2c}
--------------------------
In 2009, specific built environment characteristics of all residential neighbourhoods in Saskatoon were measured utilising two replicable tools called the Neighbourhood Active Living Potential[@R30] and the Irvine-Minnesota Inventory.[@R31] Together, these two tools were used to measure neighbourhood safety from traffic and crime, density and diversity of destinations, activity friendliness, attractiveness and pedestrian access.
Census-based measures {#s2d}
---------------------
Neighbourhood level socioeconomic variables were derived from 2006 Statistics Canada Census data[@R32] and 2010 G5 Census projections[@R33] to account for neighbourhood social environment. These included variables such as neighbourhood dwelling value, neighbourhood household income and neighbourhood unemployment rate.
Individual and household data {#s2e}
-----------------------------
In 2010, prior to deploying accelerometers, a questionnaire was administered to 455 children to capture their perception of a range of factors (household, parental, peer and neighbourhood) that influence PA. The questionnaire was pilot tested and revised as appropriate prior to field implementation. The questionnaire contained items such as: 'In the last 30 days, how often have your family members provided transportation to a place where you can do PA?' and 'During a typical week, how often did your friends ask you to walk or bike to school or to a friend\'s place?'
Accelerometry {#s2f}
-------------
Actical accelerometers (Mini Mitter Co., Inc., Bend, Oregon, USA) were deployed through schools to capture activity data of 455 children who completed the questionnaire. Children were visited at their respective schools and were asked to wear the accelerometer equipped belt around their waist to maintain proper positioning (ie, posterior to the right iliac crest of the hip) for seven consecutive days. They were advised to remove the accelerometers during night time sleep and during any water-based activities. The devices were operationalised to measure data at 12:00 on the day following device deployment (ie, almost a full day after the device was deployed) to minimise the potential for subject reactivity within the first day of wearing the accelerometer. Accelerometers were preprogrammed to measure movement in 15 s epochs in order to capture the sporadic nature of children\'s activity.
The raw accelerometer data were analysed using KineSoft V.3.3.63 (KineSoft, Loughborough, UK) to derive activity intensities using cut-points specific to the study sample\'s age group---sedentary behaviour (SB): \<100 counts/min; light physical activity (LPA): 100 to \<1500 counts/min; MVPA: ≥1500 counts/min.[@R34] The accelerometers and cut-points used in this study are the same as those used in the 2007--2009 Canadian Health Measures Survey, whose accelerometry results depicted activity patterns in a nationally representative sample of children in Canada.[@R6] Furthermore, using the accelerometer sample of the 2007--2009 Canadian Health Measures Survey, operational definitions and data reduction techniques were developed by Colley *et al*.[@R37] Valid data for our study were derived by utilising these population and device-specific (ie, actical accelerometers) operational definitions and data reduction techniques, and taking into account established evidence in conducting accelerometry on large samples of children.[@R37] [@R38]
Generation of valid data is essential to exclude days of accelerometry from the analysis when the participants do not wear the device for a period of time deemed sufficient to interpret levels of activity.[@R37] A valid day was defined as a day of accelerometry with 10 or more hours of wear-time.[@R38] Daily wear-time was estimated by subtracting non-wear-time of a particular accelerometry day from 24 h. It was determined that non-wear-time would be a period of at least 60 consecutive minutes of zero accelerometer counts, including up to 2 min of counts between 0 and 100.[@R37] The final sample consisted of children with at least four valid days including at least one valid weekend day, that is, the valid sample (N: 331; boys: 166; girls: 165 (Age 10: boys: 42; girls: 28) (Age 11: boys: 41; girls: 50) (Age 12: boys: 40; girls: 45) (Age 13: boys: 29; girls: 35) (Age 14: boys: 13; girls: 8).
However, even within valid data, there is a chance for systematic variation in daily wear-time, within (on different days of accelerometer use) and between participants. The systemic variation occurs because even though participants are asked to wear accelerometers from the time they wake up in the morning until the time they go to bed at night, every participant wears or removes the accelerometer at her/his discretion, thus potentially introducing a random or non-random measurement bias to activity measurement. We have previously developed a methodology to control for wear-time variation and minimise measurement bias by standardisation of valid data.[@R39] [@R40] The same methodology has been replicated in this study to standardise valid data.
Integration of localised weather with cross-sectional accelerometry {#s2g}
-------------------------------------------------------------------
Accelerometer data were obtained in 25 1 week cycles between 28 April and 11 June 2010 (which represented a 45-day transition period from spring to summer). Each 1 week cycle of accelerometry was conducted on a different cohort of children within the total sample, with each cohort consisting of a different set of children. To match the accelerometry period, detailed weather data for the days between 28 April and 11 June 2010, were obtained from Environment Canada.[@R41] [@R42]
Saskatoon experiences four distinct seasons, with average temperatures of 3.4°C in spring, 17.2°C in summer, 3.2°C in autumn and −14°C in winter. The city\'s precipitation levels are relatively low and winds usually blow from the northwest with an average speed of 15 km/h year round.[@R43] [@R44] To capture the transition from Spring to Summer, extensive exploration of the weather data was conducted to identify daily values of key weather variables corresponding to the accelerometry period: maximum temperature (Celsius), precipitation (millimetres), speed of maximum wind gust (km/h) and hours of illumination.[@R27]
Descriptive analyses were conducted to understand the distribution (ie, mean, median, SD of daily values of the selected weather variables during the 45 days of accelerometry. Daily values of each of these weather variables were aggregated to their corresponding 1 week cycle of accelerometry to calculate their mean weekly values. Thereafter, a decision rule was applied where 1SD of the distribution of daily weather values for the 45 days of accelerometry was set as the cut-point. Using this cut-point, mean weekly values for each weather variable were categorised as follows: temperature: ≥1SD=Warm, \<1SD=Cold; precipitation: ≥1SD=Wet, \<1SD=Dry; speed of maximum wind gust: ≥1SD=Windy, \<1SD=Calm.
Finally, based on these six categories, one of the following four localised weather patterns was assigned to each week of accelerometry (weekly weather): Warm-Wet-Calm, Cold-Dry-Calm, Cold-Dry-Windy and Cold-Wet-Calm. Although, mathematically, the possible combination of weather patterns is higher than four, it is important to highlight that the classification of localised weather is based on actual weather recorded during the period of accelerometry. As the range (2.26) and SD (0.69) of hours of illumination during the 45 days of accelerometry was negligible, hours of illumination was excluded from the classification of localised weather patterns and was instead included as an independent variable in multivariable analyses.
Statistical analyses {#s2h}
--------------------
Using data from all the measures mentioned, an extensive set of predictors were derived taking into account the hierarchical nature of data distribution: neighbourhood level variables (level 2) and individual level variables (level 1)---[table 1](#BMJOPEN2015009045TB1){ref-type="table"}. Current PA guidelines recommend that children aged 5--17 years should accumulate at least 60 min of MVPA every day.[@R5] Thus, all analyses were conducted with MVPA as the outcome variable. Analysis of variance was conducted to assess group differences in MVPA between the four types of localised weather patterns (Warm-Wet-Calm, Cold-Dry-Calm, Cold-Dry-Windy and Cold-Wet-Calm), and between children residing in different types neighbourhoods. Thereafter, to determine the odds of children accumulating recommendation levels of PA, daily MVPA was dichotomised at 60 min to build fixed effects multilevel logistic regression models utilising Hierarchical Linear and Nonlinear Modelling software.
######
Hierarchical distribution of predictors
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Hierarchy Type of measures Examples of derived variables Instrument
---------------------------------- ------------------------------------------------------------------------------- ----------------------------------------------------------------------------------------- -----------------------------------------
Neighbourhood level variables Urban design Grid-pattern\ Urban Planning
Fractured grid-pattern\
Curvilinear
Built environment Diversity of destinations\ Observation Tools: Neighbourhood Active Living Potential and Irvine Minnesota Inventory
Density of destinations\
Safety from traffic\
Safety from crime\
Attractiveness\
Pedestrian access\
Universal accessibility\
Activity friendliness
Neighbourhood social environment Dwelling value\ 2006 Statistics Canada Census and G5 2010 Census Projections
Dwellings per acre\
Household income
Individual level variables Children\'s perception of household, neighbourhood, peer and parental factors Transportation support from family\ Smart Cities Healthy Kids Questionnaire
Peer support to walk or bike\
Household socioeconomic status\
Parents' education
Activity measures Moderate to vigorous physical activity\ Accelerometry
Light physical activity\
Sedentary behaviour
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Data obtained from built environment tools, census data and the smart cities healthy kids questionnaire were utilised to derive variables which were distributed on a numerical scale specific to each measure. Thereafter, exploration of each variable\'s distribution was conducted; all variables were converted into categorical variables by uniformly dichotomising each variable\'s scale at the 50th centile.
All the independent variables included in multilevel modelling were significant at the bivariate stage. Model 1 depicts the influence of localised weather patterns (with Warm-Wet-Calm category as the reference) and as well as the influence of other individual level variables. Model 2 is the final model depicting the influence of neighbourhood and individual level variables. Only significant results from the final model are discussed here.
Results {#s3}
=======
A majority of the children did not meet recommended PA guidelines ([figure 2](#BMJOPEN2015009045F2){ref-type="fig"}). The study sample was representative of neighbourhoods with all three types of urban design ([figure 1](#BMJOPEN2015009045F1){ref-type="fig"}-grid, fractured grid and curvilinear), with boys and girls of all age groups (10--14 years) being sampled from each type of neighbourhood. Before factoring in weather variation, descriptive distribution of activity intensities across the three types of neighbourhoods revealed that children in fractured grid-pattern neighbourhoods accumulated lower MVPA and higher sedentary time in comparison with children in the grid-pattern and curvilinear neighbourhoods ([table 2](#BMJOPEN2015009045TB2){ref-type="table"}).
######
Descriptive characteristics of the study sample depicted across urban design
Variables Total Grid Fractured grid Curvilinear
------------------------------------------------ --------------------------- ---------------------------- ------------------------- ---------------------------
Sampled schools 30 6 10 14
Total sample 331 95 100 136
Boys 166 45 53 68
Girls 165 50 47 68
Age 10 70 16 25 29
Age 11 91 32 22 37
Age 12 85 27 26 32
Age 13 64 13 23 28
Age 14 21 7 4 10
Mean age (SD; Min,Max) 11.6 (1.1; 10,14) 11.6 (1.1; 10,14) 11.5 (1.2; 10,14) 11.63 (1.2; 10,14)
Mean body mass index (SD; Min,Max) 19.9 (4; 13.4,35.9) 19.8 (4.2; 14,35.9) 20.3 (4.2; 13.4,34.3) 19.7 (3.7; 14.2,33.8)
Mean accelerometer Wear-time/day (SD; Min,Max) 796.3 (51.1; 653.3,930.2) 794 (53.1; 680.8,930.2) 797 (53.3; 653.3,915) 797.3 (48.1; 684.5,910.6)
Mean MVPA/day (SD; Min,Max) 71.2 (31.8; 8,234.5) 72.8 (33.7; 8,178.1) 67.3 (32.9; 13.3,234.5) 73.1 (29.4; 16.6,182)
Mean SB/day (SD; Min,Max) 540.2 (64.8; 317.4,691.3) 537.8 (68.9; 317.4, 682.6) 546 (70.5; 344, 691.3) 537.3 (57; 379.7,663.4)
Mean LPA/day (SD; Min,Max) 184.7 (38.9; 92.5,311.6) 183.3 (39.1; 104.4,282.5) 183 (40.9; 92.5,311.6) 187 (37.4; 98 294.6)
Accelerometer wear-time, MVPA, SB and LPA values are expressed in minutes.
LPA, light physical activity; Max, maximum; Min, minimum; MVPA, moderate to vigorous physical activity; SB, sedentary behaviour.
{#BMJOPEN2015009045F2}
Between the four types of localised weather patterns, Warm-Wet-Calm weather was associated with significantly higher MVPA, whereas Cold-Dry-Windy weather was associated with significantly lower MVPA (data not shown here). Another significant observation was that the children residing in neighbourhoods with fractured grid-pattern accumulated lower MVPA during all types of localised weather patterns ([table 3](#BMJOPEN2015009045TB3){ref-type="table"}).
######
ANOVA testing group differences in MVPA between different types of neighbourhoods stratified by localised weather patterns
MVPA accumulation---Warm-Wet-Calm MVPA accumulation---Cold-Dry-Windy
--------------------------------------- --------------------------------------- ------------ ------------- ------------- ------------- ------------ -------------
Grid Fractured Curvilinear Grid Fractured Curvilinear
Grid 0.00 3.67 −5.46 Grid 0.00 0.00 NA
Fractured −3.67 0.00 −9.13\*\*\* Fractured 0.00 0.00 NA
Curvilinear 5.46 9.13\*\*\* 0.00 Curvilinear NA NA 0.00
**MVPA accumulation---Cold-Dry-Calm** **MVPA accumulation---Cold-Wet-Calm**
Grid Fractured Curvilinear Grid Fractured Curvilinear
Grid 0.00 3.07 −4.41 Grid 0.00 6.31\*\*\* 3.78
Fractured −3.07 0.00 −7.49\*\*\* Fractured −6.31\*\*\* 0.00 −2.52
Curvilinear 4.41 7.49\*\*\* 0.00 Curvilinear −3.78 2.52 0.00
Each value presented in the tables is a result of subtraction of group MVPA between two types of urban design (values in rows subtracted from values in columns).
\*\*\*p\<0.001; \*\*p\<0.01.
ANOVA, analysis of variance; MVPA, moderate to vigorous physical activity; NA, not applicable.
The multilevel multivariable model depicts the influence urban design and built environment on the likelihood of accumulation of recommended levels of MVPA by factoring in localised weather ([table 4](#BMJOPEN2015009045TB4){ref-type="table"}).
######
Multilevel logistic regression modelling factoring in weather variation to predict the influence of urban design and built environment on MVPA (mean daily MVPA dichotomised at 60 min)
Null model Model 1 Model 2
------------------------------------------ ------------ --------------- -------------- --------------- -------------- ---------------
Intercept 1.72 1.34 to 2.12 1.13 0.61 to 2.87 0.26 0.00 to 16.72
Cold-Dry-Windy vs Warm-Wet-Calm 0.58\* 0.21 to 0.84 0.78\*\* 0.01 to 0.83
Cold-Dry-Calm vs Warm-Wet-Calm 0.67 0.23 to 12.74 0.54 0.24 to 16.35
Cold-Wet-Calm vs Warm-Wet-Calm 0.32\* 0.16 to 0.78 0.44\* 0.01 to 0.92
Boys vs girls 1.42\* 1.27 to 4.28 2.06\*\* 1.27 to 3.33
Age 11 vs age 10 0.63 0.28 to 9.42 0.82 0.41 to 7.63
Age 12 vs age 10 0.81 049 to 9.75 0.86 0.43 to 9.32
Age 13 vs age 10 0.42 0.56 to 19.42 0.67 0.32 to 22.42
Age 14 vs age 10 0.50 0.03 to 44.77 0.53 0.12 to 36.67
Fractured grid vs grid 0.45\*\* 0.22 to 0.93
Curvilinear vs grid 0.59 0.11 to 2.73
Diversity of destinations to high vs low 2.09\*\* 1.14 to 3.83
Model 1 depicts the influence of localised weather patterns (with Warm-Wet-Calm category as the reference) and as well as the influence of other individual level variables. Model 2 is the final model depicting the influence of both neighbourhood and individual level variables. Only significant results from the final model are discussed in the results.
\*p\<0.05; \*\*p\<0.01; \*\*\*p\<0.001.95% CI.
MVPA, moderate o vigorous physical activity.
Boys were more likely to accumulate recommended MPVA than girls and localised weather did play a significant role in influencing MVPA accumulation. In comparison with children who experienced Warm-Wet-Calm weather, children who experienced Cold-Wet-Calm weather were less likely to accumulate recommended MVPA (OR=0.44; 95% CI 0.01 to 0.92). Another factor that reiterated the findings in descriptive analyses was that children who experienced Cold-Dry-Windy weather were less likely to accumulate recommended MVPA (OR=0.78; CI 0.01 to 0.83) in comparison with children who experienced Warm-Wet-Calm weather.
In terms of urban design of Saskatoon, again reiterating descriptive findings, multilevel modelling showed that children residing in fractured grid-pattern neighbourhoods were less likely to accumulate recommended MVPA (OR=0.45; CI 0.22 to 0.93) in comparison with children living in grid-pattern neighbourhoods. Whereas children residing in neighbourhoods with greater diversity of destinations were more likely to accumulate recommended MVPA (OR=2.09; CI 1.14 to 3.83) in comparison with children residing in neighbourhoods with lower diversity of destinations. Overall, after factoring in weather variation, children residing in fractured grid-pattern neighbourhoods had the least likelihood of accumulating recommended MVPA.
Discussion {#s4}
==========
As epidemiological investigations of active living to date have either concentrated exclusively on weather, or built environment, this study\'s objective is the inclusion of weather variation in understanding how urban design and built environment influence the accumulation of globally recommended levels of MVPA in children. In addressing the gap of non-inclusion of weather variation in active living research, the methodological approach in this study focused on conceptualising weather as a complex entity consisting of several interrelated elements that can be combined to categorise localised weather patterns. Localised weather patterns have been effectively utilised in other areas of health research,[@R45] and this study adopted similar approaches by integrating cross-sectional accelerometry with localised weather patterns, a method that has previously not been employed.
Integration of cross-sectional accelerometry with localised weather patterns depicted that weather variation influences children\'s MVPA even in a single seasonal transition (spring to summer). Moreover, after factoring in localised weather patterns, urban design and built environment influenced children\'s MVPA, where MVPA accumulation varied significantly between children residing in different types of neighbourhoods.
Descriptive results depicted a consistent pattern of Warm-Wet-Calm weather being associated with higher MVPA accumulation and Cold-Dry-Windy weather being associated with lower MVPA accumulation. It is apparent that exposure to higher daily temperatures played a role in higher MVPA accumulation. This observation corroborates the established evidence[@R18] [@R25] [@R26] that overall, in temperate climatic zones, a rise in temperature facilitates higher PA. However, the patterns observed by combining individual weather variables to create localised weather portray a more nuanced picture.
For instance, even though studies indicate that PA accumulation decreases with increasing precipitation,[@R25] [@R26] after factoring in precipitation, Warm-Wet-Calm weather was associated with higher MVPA and Cold-Dry-Windy weather was associated with lower MVPA. In Warm-Wet-Calm and Cold-Dry-Windy weather patterns, irrespective of the amount of precipitation (wet or dry), temperature and speed of maximum wind gust influenced MVPA accumulation. These observations not only underline the complexity of weather, but also the need to account for the interrelatedness and dynamics of specific characteristics such as temperature, wind speed and precipitation.[@R27]
When MVPA accumulation was compared between children who experienced the same type of localised weather, a clear pattern emerged. Children residing in neighbourhoods with fractured grid-pattern urban design consistently accumulated less MVPA in comparison with children residing in neighbourhoods with grid-pattern and curvilinear urban design. The grid-pattern neighbourhoods surrounding the city centre, by virtue of their mixed land-use (combination of commercial, residential, institutional establishments), possess greater density and diversity of destinations, are less car-oriented, and more pedestrian friendly.[@R46] The observation that mixed land-use urban design positively influences the accumulation of more MVPA in children aged 10--14 years corroborates findings from a systematic review that showed mixed land-use to be a strong predictor of PA among children and adolescents.[@R14]
However, curvilinear neighbourhoods, which do not have mixed-land use and are distinct from grid-pattern neighbourhoods in being highly car-oriented, were also associated with higher MVPA. Curvilinear neighbourhoods, though highly car-oriented, represent the higher socioeconomic areas of Saskatoon with greater safety from crime and traffic and this could possibly explain the higher accumulation of MVPA in these neighbourhoods. Nevertheless, the findings suggest that two contrasting types of urban design can have a positive influence on MVPA accumulation in children after factoring in localised weather patterns.
This influence of urban design and built environment on MVPA was further explored by factoring in localised weather patterns in multilevel models that depicted the influence of diverse environmental exposures on the accumulation of recommended levels of MVPA in children. Reiterating descriptive results, multilevel modelling showed that children residing in semisuburban fractured grid-pattern neighbourhoods were less likely to accumulate recommended MVPA in comparison with children residing in grid-pattern neighbourhoods located near the city centre.
The usage of the term 'grid-pattern' is due to the structure of road networks in these neighbourhoods that have multiple intersections and interconnected streets, which ultimately provide greater access to the many destinations available due to mixed land-use. Greater density and street connectivity is associated with higher PA in adults,[@R47] however, greater density and street connectivity bring increased volume of traffic, which is associated with lower PA in children.[@R14] In this study, diversity of destinations and perceived safety from traffic were assessed using Irvine Minnesota Inventory.[@R31] Perceived safety was lowest and diversity of destinations was greatest in grid-pattern neighbourhoods, yet the finding that 10--14-year-old children in these neighbourhoods had higher likelihood of accumulating recommended MVPA requires further explanation.
In terms of the influence of weather, multilevel modelling reiterated descriptive results by depicting those children who experienced Cold-Dry-Windy and Cold-Wet-Calm weather were less likely to accumulate recommended MVPA in comparison with children who experienced Warm-Wet-Calm weather. If weather variation could affect PA even during a single seasonal transition, it is apparent that variation of weather throughout the year has a significant influence on active living. Moreover, as depicted in this study, to capture the complexity of weather variation it is imperative to take the interrelated nature of weather into consideration.
After factoring in weather variation and controlling for social environment at the neighbourhood and household level, urban design and built environment emerged as the key factors that influenced PA in children. More importantly, using multilevel logistic regression modelling an association has been established between mixed land-use urban design and the likelihood of accumulating recommended levels of MVPA in children aged 10--14 years. Moreover, after controlling for factors at the individual and neighbourhood level, boys were more likely to accumulate recommended MVPA. This pattern reiterates existing evidence that boys are more active than girls,[@R6] and points towards the development of gender-specific active living programmes and policies that aim to reduce the existing gap in active living between boys and girls.
The study does have limitations, including the cross-sectional design. Even though objective PA data is obtained through accelerometers, the lack of social (ie, with whom the activity is accumulated) and spatial context related to activity accumulation poses difficulty in establishing accurate understanding. Thus, even though associations between PA accumulation and urban design have been established, these findings do not objectively elaborate *how* activity is accumulated within different environmental contexts or *where* (neighbourhood, indoor/outdoor, playground, recreational facility, etc.) activity is accumulated.
For example, obtaining social and spatial context would enable the understanding of independent mobility of children, an active living indicator which is currently poorly understood.[@R3] Studies are now emerging which utilise ecological momentary assessments[@R48] and global positioning systems[@R49] to understand the complex social and spatial associations of activity accumulation. These advances, when combined with accelerometry, would provide the methodological depth to tease out the complex pathways that determine activity accumulation.
In conclusion, weather variation is a critical factor that needs to be accounted for in active living research and this could be achieved by integrating localised weather patterns with existing cross-sectional accelerometry data. Ultimately, as weather is non-modifiable, the focus falls on understanding how diverse environmental exposures, especially urban design and built environment interact with weather variation to influence accumulation of PA throughout the year.
As the health benefits of accumulation of recommended levels of MVPA are well established in all age groups, the evidence generated by this study has been translated to the urban planners in Saskatoon for the development of active urban neighbourhoods in future. Based on our findings, we have found that more than one type of urban design could facilitate active living in different weather conditions, however, the key factors that emerged were mixed land-use and safety. Moreover, urban planners in all jurisdictions should take into account the interaction between their location-specific weather patterns and the different types of urban design in developing neighbourhoods that moderate the influence of adverse weather and facilitate the influence of favourable weather on active living in children.
The authors acknowledge the Smart Cities, Healthy Kids research team and staff. The authors also acknowledge the University of Regina\'s President\'s Publication Fund which enabled the payment of the open access fee.
**Contributors:** TRK conceptualised the study, analysed and interpreted the data, and drafted all versions of the article. TRK is accountable to all aspects of this work and has provided his final approval for the study to be published. NM and DR played a key role in clarifying the conception of the study, interpreting the data and assisting in generating the final draft. NM and DR provide their approval for this work to be published and will be accountable to all aspects of this work.
**Funding:** This work was supported by the Canadian Institutes of Health Research, the Heart and Stroke Foundation of Canada, and Rx&D: Health Research Foundation.
**Competing interests:** None declared.
**Ethics approval:** University of Saskatchewan Research Ethics Board.
**Provenance and peer review:** Not commissioned; externally peer reviewed.
**Data sharing statement:** No additional data are available.
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Introduction {#sec0005}
============
The COVID-19 pandemic exposes the entire medical team and, mainly, anesthesiologists, to a major risk of infection. As we are dealing with a potentially high severity disease, especially to the population at risk, due to the high risk of infection and transmission to others during its asymptomatic period, adoption of preventive measures is required.
Pathophysiological changes and the drugs used for treatment of the disease interact with anesthetics and anesthetic techniques, leading to unfavorable outcomes.
The American Society of Regional Anesthesia -- ASRA,[@bib0005] the European Society of Regional Anesthesia and Pain Medicine -- ESRA[@bib0010] and the European Society of Anesthesiology -- ESA[@bib0015] published guidance on employing regional anesthesia for patients with COVID-19. The Latin American Society of Regional Anesthesia -- LASRA (chapter Brazil) and the Brazilian Society of Anesthesiology -- BSA, carried out a joint review on the guidance to provide practical recommendations to anesthesiologists on safe patient management ([Fig. 1](#fig0005){ref-type="fig"} ). It is important to underscore that, in face of the high incidence of asymptomatic disease carriers, recommendations should also be considered for suspected cases of disease.Figure 1Guidance on employing regional anesthesia for patients with COVID-19.Figure 1
Why regional anesthesia? {#sec0010}
========================
General anesthesia requires approaching airways, a scenario with a major risk of the disease infecting the medical team, mainly anesthesiologists, due to the production of aerosols.[@bib0020], [@bib0025] Aerosol-generation mitigating techniques, such as rapid sequence induction,[@bib0030] entail higher risk of injury, intubation failure and need for desaturation mask-balloon ventilation. The risk of transmission of acute respiratory infection to health professionals during tracheal intubation is 6.6 times higher in the group exposed to the technique.[@bib0010]
General anesthesia does not provide postoperative analgesia, requiring different analgesics to control pain, such as opioids, anti-inflammatory and adjuvant (clonidine, dexmedetomidine, ketamine, magnesium sulphate, lidocaine) drugs. Such medication can interact with the different therapeutic measures currently used for treating COVID-19 and produce side effects that add to pathophysiological changes, with potential adverse effects.
Nausea and vomiting (PONV) are frequent after general anesthesia, enhancing the risk of infection to health professionals and of patient discomfort. Medication for PONV treatment and prophylaxis may present adverse effects on patients with COVID-19. Regional anesthesia attains analgesia over a prolonged period, frequently 24 hours or more, decreasing consumption of analgesics and potentially reducing the incidence of PONV.
To date, there is no evidence in the literature showing that regional anesthesia worsens COVID-19 presentation or that it presents specific adverse events in patients with the disease. Evidence suggests that regional anesthesia, including neuraxial blocks,[@bib0035], [@bib0040] is safe. There is, however, evidence suggesting higher incidence of hypotension after neuraxial blocks,[@bib0045] as we will further discuss in the present article. In this way, regional anesthesia becomes an interesting alternative for patients with COVID-19.[@bib0050], [@bib0055], [@bib0060]
Pre-anesthesia assessment {#sec0015}
-------------------------
Suggestive signs and symptoms of COVID-19, that include dyspnea, fatigue, fever, dry cough and headache, should be recorded, because they allow screening for suspected cases and immediate adoption of protective measures. For confirmed cases, records should be clear and easy to see.
Patients' clinical status in regard to the infection should be recorded on the patient record, as for example, confirmed case, suspected case (including contact with confirmed and suspected cases), and non-suspected COVID-19 case.[@bib0065] Diagnostic test results should be recorded.
Negative tests, mainly in the initial days of disease, do not rule out diagnosis of COVID-19. In case of uncertainty, a patient should be considered as positive until test results that rule out infection are available.
Pre-anesthesia assessment should include all medications that a patient diagnosed with COVID-19 is taking, given they may cause adverse effects. Hydroxychloroquine, for example, can increase the QT interval, having therefore, the potential of causing severe arrythmias and even, cardiorespiratory arrest, mainly in patients that are on other medications with the same adverse effect. Therefore, all medications patients are on should be recorded.
COVID-19 causes acute respiratory failure, with a major change in the ventilation-perfusion ratio[@bib0070] and pulmonary shunt, leading to hemoglobin desaturation and retention of CO~2~. Appropriate assessment of respiratory function should include respiratory rate and hemoglobin saturation count, and signs and symptoms of respiratory discomfort or failure.
Hypotension and hemodynamic instability can occur in patients with COVID-19. Acknowledging medication taken is extremely important, because its addition to some of the current treatments can cause heart abnormalities, such as arrythmias. Cardiocirculatory system assessment should include blood pressure, heart rate, peripheral perfusion, and electrocardiogram. Signs of circulatory failure and shock, such as paleness, change in level of consciousness and in peripheral perfusion, should be recorded. Chen et al reported significant hypotension during epidural anesthesia in pregnant women.[@bib0045] Hypotension episodes did not progress, were of moderate intensity (≤ 30% of reduction in relation to baseline) and were treated effectively with administration of vasopressors (phenylephrine), fluid infusion and uterus displacement to the left. A possible explanation for more frequent episodes of hypotension in patients with COVID-19 is that the SARS-CoV-2 virus binds to the Angiotensin II converting-enzyme receptor, impairing its normal performance. The receptor plays a cardio-cerebral-vascular protective role, regulating blood pressure and presenting anti-atherosclerotic effect.[@bib0075]
COVID-19 can cause thrombocytopenia.[@bib0080] Due to its potential thrombogenic effect, patients diagnosed with COVID-19 are frequently taking anticoagulants. Analysis of blood clotting tests whenever possible is essential, in addition to acknowledging recommendations on use of anticoagulants and regional blocks.[@bib0085]
Liver and kidney failure can occur in more severe cases. Blood tests are useful for diagnosis and follow-up of organ dysfunctions.
Neurological symptoms have been described in patients with COVID-19.[@bib0090] Symptoms can be divided into two categories: 1) Central Nervous System symptoms (CNS), such as headache, dizziness, acute cerebrovascular disease and epilepsy; and 2) Peripheral nervous system symptoms, such as anosmia, hypogeusia, hypopsia and neuralgia. Therefore, when faced with the decision on which anesthetic technique is the most appropriate and safe for confirmed or suspected patients of COVID-19, judicial investigation of neurological symptoms potentially present is mandatory, equally to what is done for the pulmonary and cardiovascular symptoms common in these patients[@bib0095] In this way, we can come across cases in which distinguishing post-dural puncture headache and headache due to SARS-CoV-2 infection is difficult. It can also be difficult to distinguish viral neuralgia from neuralgia caused by mechanical injury after a regional block. Last, during spinal anesthesia, the SARS-CoV-2 virus can potentially be carried by the needle into the CNS. It is important to mention that there is no direct evidence of this means of virus inoculation to present. On the other hand, general anesthesia compromises the blood-brain barrier,[@bib0100] which can facilitate CNS invasion by the virus. Thus, assessment of risk and benefit of regional anesthesia for patients with central or peripheral neurological symptoms should be careful.
Intensive care (ICU) beds may be required for patients with COVID-19. In a retrospective study of 34 patients with confirmed disease, Lei et al found a mortality of 20.5%, and ICU bed required for 44.1% of patients.[@bib0105]
Operation room preparation {#sec0020}
--------------------------
The surgical unit must be prepared to avoid contact and proximity of patients with suspected or confirmed COVID-19 with patients without the disease. The patient should be taken immediately to the OR where assessment, anesthesia, and recovery will take place, avoiding therefore contamination of other rooms and patient remaining in common areas.
Supplies and medication to be used should be packaged individually.
All patients should be transported to the surgical ward wearing a surgical mask. Additional supplies and medication can be kept outside ORs and dispensed by an assistant when required. We recommend restricting the number of individuals in the OR to the minimum possible required.[@bib0055], [@bib0110], [@bib0115]
Routine monitoring should be followed according to Federal Council of Medicine 2017 Resolution 2170.
Procedure for regional anesthesia {#sec0025}
---------------------------------
Neuraxial blocks are contraindicated for patients with clotting disorders. Regional blocks on deep and non-compressible sites are also contraindicated. Regional anesthesia on superficial and compressible sites can be performed, taking into account risk/benefit for patients with mild to moderate clotting disorders.[@bib0120], [@bib0125]
Anesthesia can be performed with routine care for COVID-19 negative patients who are not at risk. The rationale for these patients to wear a surgical mask are false negative tests.
Patients should be kept with a surgical mask whenever possible, and anesthesiologists should wear surgical masks throughout contact with patients, along with cap, goggles and gloves.[@bib0080], [@bib0130] Hand washing for at least 20 seconds is mandatory, but can be replaced by using 70% alcohol.
For patients with confirmed or suspected disease, adoption of personal protection measures is mandatory, that is: impermeable gown (minimum grammage of 30 g.m^-2^), protection gloves, goggles, N95 facial mask or similar, and cap.[@bib0105] Personal Protection Equipment (PPE) should be donned before entering the OR and doffed in the room toward that end, preferably in the presence of an observer attentive to possible contamination.
Despite the recommendation favorable to wearing a surgical mask when in contact with patients with COVID-19 in short and not aerosol-generating procedures,[@bib0135] the possibility of block failures, need for ventilatory care or conversion to general anesthesia should be considered. In these cases, preemptive use of a N95 or similar mask avoids exposure of the team to any possible urgent scenario.[@bib0140] In the event of scarcity of N95 masks, surgical masks are acceptable.
Sedation should be avoided, or when required should be minimal and performed carefully to try to avoid ventilatory depression, hemoglobin desaturation and need for supplementary oxygen. Deep sedation and using a laryngeal mask for oxygen supplementation should be avoided in these cases. The functional pulmonary reserve of patients will be low, increasing the risk of adverse events. We recommend adoption of respiratory function sparing techniques for patients with COVID-19.
Nasal oxygen catheters can be installed under the surgical mask, but high gas flows can increase aerosol dispersion and should be avoided.[@bib0145] Hui et al showed that dispersion distance of exhaled air sideways increases with increase in oxygen flow (20 cm, 22 cm, 30 cm and 40 cm in relation to the sagittal plane, using oxygen flows of 4 L.min^-1^, 6 L.min^-1^, 8 L.min^-1^ and 10 L.min^-1^ respectively).[@bib0150] Cough can also increase dispersion to even longer distances. [@bib0055] Face masks for supplementary oxygen administration replace nasal catheters efficaciously and are preferable. Surgical masks over face masks reduce aerosol dispersion.
Fresh gas flow administered to the patient should be as low as possible to maintain oxygen within normal parameters.
Aseptic techniques should be guaranteed, both for patient and medical team safety.
The SARS-CoV-2 virus has been isolated in the CSF; for this reason, we recommend avoiding dripping during spinal anesthesia.[@bib0155]
COVID-19 carriers, as already mentioned previously, can present hemodynamic instability, mainly after neuraxial blocks, and intense hypotension can occur.[@bib0065], [@bib0160] Vasopressors may be required.
Ultrasound (USG) and neurostimulators during regional anesthesia should be encouraged to improve the quality of blocks, reduce likelihood of failure, and minimize the risks of neurological lesions.[@bib0165]
Assessment of block installation should be performed to guarantee the quality of anesthesia and avoid deep sedation or possible conversion to general anesthesia.
Choosing the appropriate block and performing it in optimal conditions is essential, preferably by the most experienced anesthesiologist in regional anesthesia.
Post-anesthesia recovery should preferably occur in the OR. If not possible, and the patient is sent to the common post-anesthesia recovery unit along with other patients, there should be a minimum distance of 2 meters[@bib0170] among them. We do not, however, recommend the practice.
To date there are no specific recommendations as to management of post-puncture headache in patients with COVID-19. The sphenopalatine lymph node block should not be performed routinely because it is a procedure that possibly produces aerosols, increasing therefore the risk of transmission of SARS-CoV-2 to health professionals. The epidural blood patch should be considered carefully in face of the identification of the virus in the CSF. There is the possibility of a significant introduction of viral load, with possible neurological complications.[@bib0175] If required, it should be postponed to after recovery from infection.
At the end of the procedure, PPEs should be doffed carefully to avoid contamination of the team.[@bib0180] Previous knowledge of the regional anesthesia technique, and of PPE donning and doffing, training team and professionals, and complying with protection measures are important actions in face of COVID-19. A summary of the recommendations previously described are presented in the info graph that follows, adapted from the American and European guidance [@bib0005]
Conclusion {#sec0030}
==========
Regional anesthesia is an interesting alternative to manage patients with COVID-19. Adoption of the appropriate anesthetic technique minimizes adverse effects in the post-operative period and offers safety to patients and to the health team, as long as care described is complied with.
The judicious use of safety techniques and norms is essential. Knowledge of the specificities of the pathophysiology of the disease and its symptoms helps to decide which anesthetic technique is safer and more appropriate for each patient.
Conflicts of interest {#sec0035}
=====================
The authors declare no conflicts of interest.
We would like to thank Dr. Clara Lobo and Dr. Anne Snively for helping us with the approval of the text whithin the ASRA and ESRA Board (European Society of Anesthesiology).We would like to thank Dr. Clara Lobo and Dr. Anne Snively for helping us with the approval of the text.
| {
"pile_set_name": "PubMed Central"
} |
Background
==========
Respiratory allergies, particularly allergic rhinitis, are among the most common allergies within various populations all over the world and the incidence is expected to increase \[[@B1]\]. Prevalence of respiratory allergy has been determined between 10-30% in different studies \[[@B2]-[@B5]\].
Aeroallergens play great role in pathogenesis of respiratory allergic diseases. Pollens, molds and pets are the main sources of allergens \[[@B5],[@B6]\]. The occurrence of allergic rhinitis varies among different countries, as well as different regions within a country, which could be related to the type of allergens existing in those regions \[[@B6]-[@B8]\]. Moreover, for an efficient diagnosis of the specific allergy and an efficient treatment, it is very important to identify common aeroallergens in the area \[[@B3],[@B6],[@B9]\].
Ahvaz, the capital of Khuzestan province, is located in the southwest of Iran with an approximate population of 1.4 millions (census 2006). Ahvaz has a desert climate with long, extremely hot summers and mild, short winters. It is consistently one of the hottest cities in Iran, with summer temperatures regularly at least 45 degrees Celsius, sometimes exceeding 50 degrees Celsius with many sandstorms and dust storms common during the summer period while in winters the minimum temperature could fall around +5 degrees Celsius. Winters in Ahvaz have no snow. The average annual rainfall is around 230 mm. In our region the dust storm was a usual phenomenon in some days in spring and summer but now it may occur almost all of the year \[[@B10]-[@B12]\].
Due to the lack of published studies that have been conducted on sensitization to aeroallergens in the southwest of Iran, the prevalence of common aeroallergens is still not fully understood in the region. The current study was therefore conducted to determine the prevalence of positive skin test for various aeroallergens among allergic patients in the city of Ahvaz. It was hoped that the results would be useful to health system policy-makers in planning respiratory allergic diseases prevention programs in the region.
Methods
=======
Study population
----------------
This study was conducted as the cross-sectional study on candidates participating in Immunology department of Jundishapur University of Medical Sciences and Khuzestan Jahad Daneshgahi Medical Center from July 2010 to September 2011. Two hundred and ninety nine participants with allergic rhinitis (seasonal or perennial) were selected based on ISAAC \[[@B13]\] (The International Study of Asthma and Allergies in Childhood) questioner. The study was conducted according to good clinical practices and the Declaration of Helsinki. The Ethics Committee of Ahvaz Jundishapur University of Medical Sciences was approved the study protocol and an official agreement from all of patients was obtained before the study.
The diagnosis of current allergic rhinitis was established by clinical examination and a combination of having a problem with sneezing or a runny or blocked nose in the absence of a cold or flu and a positive SPT reaction (diameter \>3 mm more than negative control) to at least one of the twenty three aeroallergens. A study questionnaire requesting demographic data, family history of atopy and respiratory symptoms was also administered to each patient.
Skin prick test and total IgE
-----------------------------
Skin prick test using twenty three common allergen extracts (HollisterStier, Spokane, WA, USA) was performed on all patients in accordance with previous study \[[@B6]\] (Table [1](#T1){ref-type="table"}). In this study, allergens were selected based on the plant species existing in our region and other possible allergens were identified from consulting ear, nose and throat specialists \[[@B14]\]. Seven different types of weeds (*Amaranthus palmeri*, *Amaranthus retroflexus*, *Kochia scoparia*, *Chenopodium album*, *Salsola kali*, *Plantago lanceolata*, *Artemisia vulgaris*) and four grasses (*Poa pratensis*, *Cynodon dactylon*, *Lolium perenne*, *Sorghum halepense*) pollen extracts along with four trees (*Acacia longifolia*, *Prosopis juliflora*, *Eucalyptus globulus*, *Fraxinus americana*) extracts; four molds (*Alternaria* Mix, *Aspergillus fumigatus*, *Cephalosporium acremonium*, *Penicillium* Mix), two mites (*Dermatophagoides pteronyssinus*, *Dermatophagoides farinae*), house dust mix, and a mixture of two different cockroach (*Periplaneta americana and Blattela germanica*) extracts were used.
######
Prevalence of positive skin prick test and total IgE among allergic rhinitis patients
**Aeroallergens** **All patients(%)** **Allergic rhinitis** ***p*value**
----------------------------- ------------------------------------- ----------------------- -------------- -------- ---------------
**Weeds**
Russian thistle *Salsola kali* 72.9 79.4 20.6 0.1
Pigweed *Amaranthus retroflexus* 70.9 79.2 20.8 0.2
Carless weed *Amaranthus palmeri* 68.6 79.5 20.5 0.1
Lamb's quarter *Chenopodium album* 67.9 79.3 20.7 0.2
Burning Bush *Kochia scoparia* 66.6 79.9 20.1 0.1
Mugwort *Artemisia douglasiana* 60.9 80.8 19.2 0.07
Plantain *Plantago lanceolata* 55.2 80.0 20.0 0.2
**Any weeds** 89.0 79.4 20.6 0.09
**Grasses**
Kentucky blue grass *Poa pratensis* 54.8 81.1 18.9 0.08
Bermuda grass *Cynodon dactylon* 52.5 80.9 19.1 0.1
Johnson grass *Sorghum halepense* 46.5 80.6 19.4 0.2
Perennial rye grass *Lolium perenne* 36.1 78.7 21.3 0.7
**Any grasses** 75.0 80.6 19.7 0.06
**Trees**
Mesquite *Prosopis juliflora* 65.9 80.7 19.3 **0**.**04**
White Ash *Fraxinus americana* 52.5 80.9 19.1 0.1
Acacia *Acacia longifolia* 48.2 81.9 18.1 0.07
Eucalyptus *Eucalyptus globulus* 21.7 89.2 10.8 **0**.**009**
**Any Trees** 86.0 79.0 21.0 0.2
**Mites**
Mite *Dermatophagoides farinae* 32.1 76.0 24.0 0.6
Mite *Dermatophagoides pteronyssinus* 27.1 69.1 30.9 **0**.**03**
**Any mites** 43.0 75.2 24.8 0.4
**Molds**
Fungus *Cephalosporium acremonium* 11.4 85.3 14.7 0.3
Fungus *Aspergillus fumigatus* 5.0 80.0 20.0 0.5
Penicillium Mix^\*^ Penicillium Mix 9.4 75.0 25.0 0.8
Alternaria Mix^†^ Alternaria Mix 8.0 75.0 25.0 0.7
**Any molds** 24.0 82.0 18.0 0.4
**House dust**
House dust mix^‡^ House dust mix 19.1 82.5 17.5 0.3
**Cockroach**
Cockroach mix *P*. *americana* + *B*. *germanica* 30.8 75.0 25.0 0.4
**Total IgE** (mean, IU/ml) 177.77 172.54 196.39 0.3
^\*^ The extract includes *P*. *digitatum*, *expansum*, *glaucum*, *roseum* and *notatum*. ^†^ The extract includes *A*. *tenuis* and *Hormodendrum cladosporioides*. ^‡^ Pooled collection containing kapok, feather mix (chicken, duck and goose) and mattress dust.
Skin prick tests were performed under physician's supervision. In this test, allergen extracts were put on patients' inner forearms and irritation of the epidermis was caused by prick method using the lancet and the result was observed after 15 minutes. Next, the diameter of patient's skin reaction was measured and compared with negative (glycerin saline) and positive (histamine hydrochloride, 10 mg/ml) controls. Patients with a wheal diameter \>3 mm were considered positive compared with negative and positive controls. Patients using drugs affecting skin test were excluded from the study.
In the end, enzyme immunoassay method (ELISA) was performed to collect the total IgE serum. Serum samples were stored at −20°C until the analysis. ELISA test was performed in duplicate by using the commercial kit (DIPLUS, Canada). Based on manufacturer's instruction, samples with more than 100 IU/ml total IgE was considered elevated.
Statistical analysis
--------------------
In the end, all the data was analyzed by SPSS Version 18.0 software (Version 11.0 of SPSS software) (Chicago, USA). Chi-square test was used to compare the relationship between variables. *p* value less than 0.05 was considered significant.
Results
=======
Demographic characteristics of subjects are presented in Table [2](#T2){ref-type="table"}. The mean duration of allergic rhinitis was 6.78 (±3.89) years. Symptoms of active conjunctivitis and allergic rhinitis were found in 172 (72.8%) of patients (Table [2](#T2){ref-type="table"}). Among patients with positive for SPT, seasonal pattern was seen in 77.7% of the patients, perennial pattern in 22.3%. In addition, 193 patients (64.5%) had positive family history of allergy.
######
Characteristics of patient population
**Characteristics** **No**. **of cases(%)**
------------------------------------------------------------ -------------------------
All patients 299 (100)
The mean duration of allergic rhinitis (±SD): 6.78 (±3.89)
Gender
Male 156 (52.2)
Female 143 (47.8)
Age (mean, range: 32.02, 4--70)
Disease presentation
Sneezing 237 (79.3)
Runny nose 254 (84.9)
Itching and nasal congestion 187 (62.5)
Rhinoconjuctivits 142 (47.5)
Allergic rhinitis (with skin prick test positive)
Seasonal 199 (77.7)
Perennial 57 (22.3)
Family history of allergy 193 (64.5)
The overall frequency of sensitization to any allergen was 85.6% (256/299), whereas 14.4% (43/299) of patients did not react to any of the tested allergens.
In outdoor allergens the most prevalent aeroallergen category was weeds (89 %) followed by tree and grasses, and in indoor allergens, mites (43%) and cockroaches (30.1) were the most and least prevalent aeroallergen types, respectively (Table [1](#T1){ref-type="table"}).
Prevalence of positive skin test to any allergen is shown in Table [1](#T1){ref-type="table"}. Skin reaction to *Salsola kali* was the most common among the allergens (72.9%). Other prevalent weeds were *Amaranthus retroflexus* (70.9%), *Amaranthus palmeri* (68.6%), *Chenopodium album* (67.9%), and *Kochia scoparia* (66.6%). Ninety five percent of patients with positive skin prick test for outdoor allergens were also sensitized to at least to one of these five allergens, which belong to the Amaranthaceae and Chenopodiaceae families.
Among tree pollen, the most prevalent allergen was *Prosopis juliflora* with 65.9% and the least prevalent was *Eucalyptus globulus* with 21.7%.
Among indoor allergens the most prevalent allergen was *Dermatophagoides farinae* (32.1%). Skin reaction to the mixture of cockroach extracts of Americanus and Germanium has been found in 30.8% of patients.
The mean and median numbers of positive test reactions among those with positive test responses were 11.5 and 13.0, respectively. The mean age of men and women with positive SPT were 33.69 ± 14.61 and 31.92 ± 13.05, respectively. However, the differences were not significant (*p*=0.9). Eighty four percent of patients were poly-sensitised (positive skin reaction to more than two allergens) and about 50% of them were sensitised to more than twelve different allergens (Figure [1](#F1){ref-type="fig"}).
{#F1}
As shown in Table [3](#T3){ref-type="table"}, the analysis of the positive skin prick test rate according to age groups revealed, with the exception of the grasses pollen allergens, there is no significant difference between sensitization to selected allergens among 4 age groups and for 4--17 age group compared with 31--45 age groups.
######
Age distribution of patients with positive skin prick test to indoor and outdoor allergens
--------------- --------------------------------------------- ---------------------- --------------------- ---------------------
**Allergens** **% Frequency of positive skin prick test**
**4-17 yr (n= 40 )** **18-30 yr (n= 76)** **31-45 yr (n=96)** **\>45 yr (n= 44)**
**Mites** 30.0 42.12 46.9 45.5
**Molds** 27.5 30.3 16.7 25.0
**Cockroach** 27.5 42.1 32.3 40.9
**Weeds** 82.5 92.1 88.5 90.9
**Grasses** 62.5 76.3 71.9 88.6\*
**Trees** 75.0 86.8 85.4 93.2
--------------- --------------------------------------------- ---------------------- --------------------- ---------------------
\* *p*\<0.05.
Among outdoor allergens sensitization to *P*. *juliflora* and *E*. *globulus* \[(*p*\<0.04) and *p*\<0.009), respectively\], and among indoor allergens skin test reactivity to *D*. *pteronyssinus* (*p*\<0.03) was significantly prevalent in allergic rhinitis patients with seasonal pattern (Table [3](#T3){ref-type="table"}).
The mean total IgE serum was determined as 177.77 IU/ml. The mean total of IgE serum in men (174.31 IU/ml) was significantly greater than women (129.45 IU/ml) (*p*\<0.01). Moreover, the mean total IgE serum among patients with positive SPT was significantly higher the one than in patients without positive SPT (177.77 vs. 4.56 IU/mL, *p* \< 0.001).
Discussion
==========
Based on the results, 85.6% of patients with active rhinitis were responsive to at least one aeroallergen, which is found to be a higher percentage compared to other studies \[[@B1],[@B15],[@B16]\]. In the conducted study, 64.5% of patients with active rhinitis had family history of atopy (allergy), which is in agreement with some studies \[[@B3],[@B15]\]. A family history of atopy is an established risk factor for allergy particularly allergic rhinitis \[[@B3],[@B17]\].
The results of skin prick test in allergic rhinitis patients indicated the high incidence of allergy to plant pollens and the most common was found to be *S*. *kali* (*Russian thistle*) pollen with 72.9%. Allergy to *S*. *kali* pollen was reported as one of the most causes of allergic rhinitis in neighboring regions \[[@B6],[@B18]-[@B21]\]. *Prosopis juliflora* (Mesquite) pollen (65.9%) was the most common sensitizing tree pollen in the present study. This is in accordance with previous studies in countries neighboring to our region \[[@B17],[@B19],[@B21]\]. Mesquite is abundant in Khuzestan and other region with hot and humid climate, where it is planted as shade and ornamental tree or for binding sand. Surprisingly, in spite of very limited presence of *Fraxinus americana* (Ash) tree in Khuzestan province, the Ash pollen was the second most common sensitizing tree pollen (52.5%) in our study. It may be due to confirmed cross-reactivity between pollens of ash and olive \[[@B22]-[@B24]\], a close taxonomical relationship tree, which is cultivated in orchards and sometimes in parks and gardens throughout the area. It is also possible that the high rate of sensitization to ash pollen is due to cross-reactivity among pollens of ash and grasses and weeds, as has been previously reported \[[@B22]-[@B24]\]. It could be supported by our findings that indicated a significant correlation between sensitization to pollens of ash and studied grasses or weeds (*p*\<0.001) (data not shown).
Moreover, among other selected outdoor allergens, allergenic molds were found to be less prevalent, which in accordance to previous studies \[[@B9],[@B17],[@B21],[@B25]\]. However, it seems that the exact prevalence of sensitization to molds is difficult depending on more variability of fungal antigens than other allergens, extracts used, and species tested \[[@B26],[@B27]\].
Around 95% of patients with positive skin prick test were sensitive to at least one pollen type. This finding is also supported by studies conducted in other regions \[[@B3],[@B5],[@B15],[@B25]\]. Recent studies have shown that high incidence of respiratory allergy is related to the global increase of CO2 and other greenhouse gasses, which leads to an increase in the production of pollens by plants, allergenicity of pollens, increase in production period and distribution of pollens, and changes in plant pattern in areas \[[@B28],[@B29]\]. There is no doubt that environmental factors especially air pollution play an important role in the increasing allergies in the worldwide. In recent years, due to severe geographical and climate changing, the dust storm becomes a common phenomenon in our region \[[@B10]-[@B12]\]. To this date, there is no data available to report the effects of Middle East storm on public health particularly allergic diseases in this area. However, recently increasing evidence has been accumulated that exposure to particulate matters which contained various aeroallergens such as pollens and fungal spore, during dust storms could increase respiratory allergic diseases \[[@B11],[@B12],[@B30]-[@B32]\].
In recent years, due to climate change, the dust storm has become a common phenomenon in Khuzestan province \[[@B10]-[@B12]\]. To this date, there is no available data on the probable effects of Middle East storm on public health particularly allergic diseases in this area. Due to the lack of data, further studies are required to investigate the effect of dust storm on hospital visits, admission frequency, respiratory allergic diseases onset and mortality in local resident people.
In our research, prevalence of positive skin prick test to *Dermatophagoides farinae* and *Dermatophagoides pteronyssinus* was found to be 32.1% and 27.1% respectively, which was lower than the results of other studies \[[@B9],[@B15]\]. Regarding to the optimum condition of 60% humidity and 25°C temperature, high incidence of mite allergy is expected in humid regions, such as Singapore \[[@B33]\], Malaysia \[[@B15]\] and Thailand \[[@B34]\]. Unexpectedly, this particular allergy was also found in hot and dry regions, like Sistan and Baluchestan province of Iran \[[@B9]\], Qatar \[[@B35]\] and Kuwait \[[@B17],[@B21]\]. It may be associated with an increase in usage of air conditioners inside the houses which make good environment for mites to grow and increase exposure to indoor allergens \[[@B6],[@B36],[@B37]\].
In addition, prevalence of allergy to cockroach was determined to be 30.8%, which is higher than results of other studies \[[@B6],[@B25],[@B38],[@B39]\], while the sensitization to cockroach allergens is one of the most common indoor allergens in some regions with similar weathers to our region \[[@B17],[@B35],[@B40]\].
The most of SPT positive patients (85%) were reactive to two or more allergens. This is in accordance with earlier reports \[[@B6],[@B17],[@B18],[@B25]\]. It seems multi-allergen sensi-tization may be due to several factors, including cross-reactivity among allergens belonging to close reservoirs, which reflects the presence of common allergenic epitopes in different but botanically close plant species, long-term exposures to close phylogenic source of allergens, and interactions of genetic and environmental factors \[[@B6],[@B17],[@B18],[@B25],[@B37]\]. As the use of a panel of plant allergens belongs to closely related species; it may be an explanation of the high rate of multi-sensitization in our study.
In this study, no significant statistical relationship was found between the positive skin prick test and patient's gender. Our data were similar with some studies \[[@B21],[@B41]\], but in some others higher prevalence of allergy was seen in men compared to women \[[@B3],[@B25],[@B37],[@B42]\]. This phenomenon cannot be explained since there is no evidence showing that men are more exposed to pollens than women. This is considerable since in some countries, such as Kuwait and Iran \[[@B17],[@B43]\], men usually spend more time outdoors than women; therefore, they are more exposed to the outdoor allergens. However, our results show no significant difference between incidence of outdoor allergens and patient's gender.
Traditionally, allergic rhinitis has been subdivided into seasonal and perennial types based on time and duration of symptom occurrence. In general, mites, cockroach and some molds are considered as perennial allergens, whereas weeds, grasses and trees are considered as seasonal allergens. In our studies, there was no significant correlation between selected allergens and pattern of allergenic rhinitis. The same results were obtained in some previous studies \[[@B44],[@B45]\]. This may be due to several possible reasons: (1) it is difficult for patients to differentiate between seasonal or perennial allergies with the common cold and some active infections \[[@B44]\], (2) in tropical area with mild fall and winter, the exposure to some pollens is long standing because of increase in production period and distribution of pollens \[[@B28],[@B29],[@B44]\], (3) nasal inflammation is extended for weeks after pollen contact in patients with seasonal rhinitis \[[@B46]\], (4) the exposure to some perennial allergens is not comparable over the year and symptoms could be of short duration \[[@B44]\], and (5) in our study, the majority of patients (about 85%) are polysensitized to pollen and perennial allergens.
With exception of grasses, the analysis of date showed that there is no difference in the rates of positive skin prick test between younger and older allergic patients. However, some previous studies revealed that sensitization to some indoor allergens such as mites and cockroach is more common in younger patients \[[@B17],[@B36]\]. It should be noted that in hot and dry climate area like Ahvaz city, the people spend most of their time inside buildings which have air conditioner, which may offer an explanation for high prevalence of indoor allergens in adults in present study \[[@B6],[@B37]\].
In this study the mean total IgE serum in men was significantly higher than women (174.31 vs. 129.45) (*p*\<0.01). Moreover, among skin prick test negative population, the average total IgE serum in men was greater than women. Elevated total IgE levels are usually associated to allergy, but it may be depend on various factors; such as parasitic infestations, smoking, pollution, local diet and different genetic background \[[@B25],[@B47]\].
Conclusions
===========
The study revealed that prevalence of the skin prick reactivity to outdoor allergens, particularly weed pollens, is significant in southwest Iran and multiple sensitizations was surprisingly common. Moreover, exposure to components of dust storm such as pollens and fungal spores may affect human health directly through allergic induction of respiratory disorders. However, further studies are needed to identify components of dust particles and define association of this phenomenon with the prevalence or exacerbation of respiratory allergic diseases in local resident population.
Competing interests
===================
The authors declare that they have no competing interests.
Authors' contributions
======================
MA, ASH and AA carried out data collection. MA and AA collated and analyzed statistics. MA drafted the manuscript. ASH finalized the manuscript. All authors read and approved the final manuscript.
Acknowledgment
==============
This study was supported by Ahvaz Jundishapur University of Medical Sciences and Khuzestan Jahad Daneshgahi Medical Center.
| {
"pile_set_name": "PubMed Central"
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1.. INTRODUCTION
================
"CVI is characterized by sudden neurological dysfunction resulting from insufficient blood supply to brain tissue. Stroke can be divided into two groups depending on the etiology: ischemic (83%) and hemorrhagic (17%)" ([@R1]). "Thrombosis is a blood clot that obstructs a blood vessel or develops in the cavity of the heart. Thrombus can originate from anywhere in the cardiovascular system. Often there is damage to the wall of the vessel, with subsequent accumulation of blood platelets" ([@R2]). Transient ischemic attack (TIA) or stroke before the brain. "By definition, a TIA is a short-term disturbance of neurologic function, which is accompanied by disruption of regional cerebral blood flow that may only last a few seconds or that its duration is up to 24 hours. Signs and symptoms of TIA also will depend on which part of the brain is deprived of blood supply" ([@R3]).
Good training of pre hospital services is essential factor in early diagnosis of cerebrovascular stroke, rapid transport to appropriate institutions and thus facilitates early reperfusion brain. Hence there is the need for education of family physicians and the nurses (technicians) as well as emergency medical unit's staff. After receiving a call about the incident, when examining the patient, it is necessary to determine whether this is really the CVI or some other neurological disorder. There are two screening scales for pre hospital stroke identification Cincinnati hospital scale and Los Angeles hospital scale (LAPSS).
In the pre hospital treatment assistance is provided to all those who suffer a stroke and as a first level of urgency. In order to adequately treat the patient with stroke, it is necessary to reach the patient as soon as possible. To such patients then is needed to provide rapid transport to the hospital with a diagnostic CT capabilities and department of neurosurgery (optimum time for the possibility of thrombolytic therapy in indicated patients is up to 3 hours).
1.1.. CLIMATE CONCEPTS
----------------------
"Persons with impaired health, in which some mechanisms do not operate well, have difficulties to adapt to changing weather conditions. In this sense different health problems may arise, sometimes even including myocardial infarction, stroke, perforation of stomach ulcer, etc." ([@R4]). Climate is Sarajevo has mild continental characteristics. "The average altitude of the Sarajevo valley is about 500 meters above sea level. Valley occupies an area of approximately 31.7 km^2^ with a center in the area of Ilidza and Plandiste. It is located in the area of Bosnia river springs in which flows Zujevina and Rakovica, Zeljeznica with Kasindolka, Dobrinja with Tilava and river Miljacka. Average annual relative humidity is 71.6% and varies within the range from 66.3% to 74.8% ([@R5]).
2.. GOALS
=========
The goals of this study are as follows: Identify the importance of the influence of humidity on the stroke incidence;Determine the trend of CVI occurrence in the reporting period and correlate these findings with the trend of change in relative humidity during those days;Correlate the incidence of stroke in relation to gender, age, and selected climatic parameter.
3.. MATERIAL AND METHODS
========================
This paper presents a retrospective study. Subjects were patients of Institute for Emergency Medical Care in Sarajevo, which in the period from 2004 to 2006 have been diagnosed as CVI. Patients with a first episode of CVI or as a repeat stroke, which were included in this study, were analyzed in relation to age, gender, time of diagnosis (day, season and year) and the basic meteorological parameter--relative humidity. Basic meteorological data for days, months and years of the study period was provided by the Federal Meteorological Institute of the Sarajevo Canton (Decision No. 04-33-2-197/06). Relative humidity measurements were performed at 7, 14 and 21 o'clock every day during the investigated period from beginning of 2004 until the end of 2006. Statistical analysis was done in Microsoft Excel and Microsoft Access. Results obtained in the study were statistically analyzed and for all analyzed parameters are made tables and chart with a comment for better visibility of individual factors and their relationships. In the study was used the normal distribution test to test the hypothesis.
4.. RESULTS
===========
During the three-year study (2004, 2005 and 2006) was registered in the working protocols 1930 patients who experienced an acute or recurrent stroke. From these 1930 patients, there were 924 (48.88%) males and 1006 (51.12%) females. Results are grouped according to the analysis of certain parameters that were studied. In a survey for each year are taken two months with the highest stroke incidence and compared the mean relative humidity with the same parameters when there were no strokes. For the sake of precision, were separated two weeks, with the most impact from the given month with the average values of relative humidity and tested the null hypothesis. Also are correlated changes in relative humidity at the day when CVI occurred and the day before. The incidence of patients who experienced an acute stroke by years of research in relation to the total population of the Sarajevo Canton: (2004) 151/100,000, (2005) 147/100,000 (2006) 162/100,000. (Table [1](#T1){ref-type="table"}).
Tabulation for testing of the null hypothesis for selected days from two months with the most strokes in the 2006 and analyzed climate parameters with average values of climatic parameters without CVI in the three-year study. Months with the highest CVI incidence are January and October of the 2006. It was found that at the significance level of 0.05 relative humidity has no influence on the incidence of stroke.
From Table [2](#T2){ref-type="table"} can be seen that the largest number of stroke patients is aged 70-79 years or 801 (41.35%), then in the 60-69 decade 469/24.30%. Separately, men are more represented at the decade 60-69 yrs., and later women. The average age for a three-year period (2004-2006) was 69.31 for men, for women 72.53 and 70.92 for the total sample. Average per day most CVI occurs primarily at very low relative humidity of 43%, then 61%, 75% and a very high of 96%. Average the largest number of stroke per day occurred at a very low relative humidity of 41%, and elevated at 83% in the 2005. The highest average of the CVI per day occurs at extremely lowered average relative humidity of 38% and an extremely increased to 98% and at least with humidity of 50%. The highest number of CVI's has been recorded in winter (26.16%) and the lowest in autumn (22.69%) for the period 2004-2006. By sex, there is a visible deviation by seasons. Number of males with stroke is highest in the spring with 256 cases or 13.26%, and the woman in the winter with 281 cases or 14.55%. Also, women in autumn and spring had a little more CVI's than men. The average number of CVI's in the day increases as the air is extremely dry (38%), and extremely humid (98%). For 60 occurrences of different numbers of strokes classified by grades in the table correlation coefficient is r = 0.29, which makes a slight correlation between CVI and the average humidity during the day. For 21 occurrence of different numbers of strokes classified by grades in the table correlation coefficient is r = -0.07, which makes a slight correlation between CVI and changes in the relative humidity of the day. (Table [3](#T3){ref-type="table"}, Table [4](#T4){ref-type="table"}).
5.. DISCUSSION
==============
During the three-year study carried out in EMS Sarajevo during the 2004, 2005 and 2006th, there was a homogeneous group of patients, and the total number of people who experienced acute stroke (CVI), and who are registered in the operating protocols is 1930. This represents a very large sample survey which was used for test of normal distribution. CVI was diagnosed from adolescents to elderly usually between 70 and 79 years in all three years of the study (2004 -- 40.31%, 2005 -- 41.88%, 2006 -- 42.26%), followed by age group 60-69 years also in all three years of the study: 2004 -- 27.24%, 2005 -- 24.51% and in 2006--21.35%. The youngest patient was 24 years old and the oldest 99 years. The average age of patients in all three years of the study was 70.92 years and separately by gender male--69.31 and female 72.53 years. To the patient's emergency medical assistance was provided by the staff of EMS Sarajevo.
Detailed neurological examinations used by medical doctors in the pre hospital phase are not practical because they delay transport of the patient. Once the acute stroke is suspected time spent outside of the hospital must be minimized. The presence of an acute stroke is an indication for "load and go", because the therapy window is limited. Pre hospital treatment consists of the ABC management and close monitoring of vital signs. Studies worldwide show that more than 20% of the population has meteropathy, or persons who with difficulties tolerate changes in the weather, in which changes provoke the emergence of new or worsening of already present illness. Healthy people have mechanisms that can adapt to the changes in the atmosphere: the autonomic nervous system, skin, endocrine system and balance of body fluids. Disruption of these mechanisms in some chronic disease causing deterioration during weather changes. Positive weather conditions in which a person feels good are mix of multiple climate parameters, and of the investigated ones are the temperature 17-25°C, ideal average temperature of 21°C, barometric pressure in the Sarajevo area of 943 mbar, and at the sea level of 1013 mbar with daily relative humidity below 65% to 50% average relative humidity as it is set also in the air conditioning units. Feigin and colleagues (2001) published the results of a study based on a population that was done in the southern hemisphere (Australia, Oakland) to 783 cases (from 1981 to 1997). This was the first study to show a significant seasonal fluctuations occur in hemorrhagic stroke with the highest number of cases in young people \<65 in the spring and in the elderly ≥ 65 in the winter. During the day the highest incidence is between 8 and 12h and the lowest between midnight and 6am. High blood pressure and low atmospheric temperatures is possible trigger SAH. The results are similar to studies in the Northern Hemisphere ([@R6]). Jakovljevic et al. (1996) published the results of stroke research in Finland based on population n=15449 (period from 1982 to 1992), which was conducted in three areas of Finland: Kuopio, S. Karelia and Turku/Loimaa. The emergence of CVI was usually higher in the winter than during other seasons, but the study could not detect a significant difference between the seasons ([@R7]). Oberg et al. (2000) from University of S. Carolina, USA in research conducted on 72,779 patients over 10 years (1986-1995), found clear evidence of occurrence of ischemic stroke caused by weather. The highest incidence is in mid-May. There is no connection between CVI and the seasons. Area and race did not have any effect ([@R8]). In our study, as in study of Jakovljevic et al. winter was with the most cases of stroke (27%) and there was no significant difference between the seasons in the CVI incidence. As for seasonal fluctuations, as in study by Feign et al., hemorrhagic stroke among young people in spring and elderly people in the winter, when we could not determine because this pre hospital study included all types of stroke, and certain types of stroke may only be determined in hospital studies. In our study was also recorded the highest incidence of stroke in the morning and the lowest after midnight.
In our study, women were also more represented in total (1006) or 52.12% then men 924 or 47.87% as well as in all three years of research individually, but without substantial statistical significance in terms of gender influence on the incidence of CVI. Strokes are more common over 65 years of age and make up 76.17% (1470), while they are the highest in the decade between 70-79 years--801 or 41.50%. Men over 65 are represented with 648 cases or 44.09% and 822 women or 55.91%. In earlier research by Bokonjic R. ([@R9]), most cases were in the 60-69 decade of life, indicating that the extended age by a decade due to the consequences of stroke and its frequency, and that the medicine has advanced in its development, diagnosis and treatment.
Laad et al. (2004) in France analyzed the seasonal variations in the appearance of the stroke and the influence of meteorological factors on their appearance. The study was a population-based Dijon registry of stroke and included 3287 patients from 1985 to 1998. The difference from one season to another was significant only for the total number of strokes, with a minimum from July to September and a maximum in October. Detected are correlations with meteorological data for the total number of strokes. They pointed to the influence of temperature and relative humidity on days with stroke or 1-5 days earlier ([@R10]). In our study, when testing the null hypothesis about the effects of relative air humidity on days with and without CVI's, (where the null hypothesis denies the influence of the facto and working confirms the influence of humidity) compared to the average values of relative humidity is monitored in two months with the highest stroke incidence and four weeks with most strokes within these months, there was no effect of the average relative humidity on the incidence of stroke. It was found only by this test in 2005 the average relative humidity had influence on the incidence of CVI's. The influence of relative humidity showed the increased incidence of stroke and at extreme values: first at a very low average humidity (38%), followed by a very high (98%). The influence of fluctuations in relative humidity is negligible during the day r = 0.07 (7 am to 14 pm), and there is a mild correlation compared to the average humidity of the day r = 0.29 to the emergence of CVI when we include all the cases. In extreme situations, with an increased number of stroke and no significant correlation between relative humidity, but only a mild correlation with changes in the average humidity of the day in relation to the previous day. Fluctuations for all analyzed parameters during the day was analyzed by comparing measurements in the period from 7 am to 14 pm, when it was most strokes during a three-year research and where it is found that there is no significant correlation between relative humidity and CVI's. Given that the measurements made in terms of 7h, 14h and 21h, analyzes were performed in these terms, but without significant results.
6.. CONCLUSIONS
===============
Patients gender in all three years of the study did not significantly influence the incidence of CVI's, women were represented slightly more (1006) or 52.12%, and men 924 or 47.87% of the cases. When testing the existence of statistical differences in CVI incidence by seasons it showed no statistically significant differences between seasons on the incidence of stroke and individually by years of research and in all three years of research together. During the study from the 2004 to 2006, when testing the null hypothesis for specific days with stroke compared to days without CVI and relative humidity this showed no statistically significant effect on the incidence of stroke, except in the year 2005 in two months with the highest CVI in years of research in relation to values of humidity in the days without CVI's. We found that the incidence of stroke in three years combined 2004-2006 and individually for each year of research increases at extremely low or high values of relative humidity. Age of the patient affected the incidence of stroke and stroke occurred the most, as far as age concerned, in the older age group in the three-year of study at age from 70-79 years (41.31%) in 2004, (41.88%) in 2005, (42.26%) in 2006 and for all three years combined at average of 41.35%. The correlation between CVI and the average relative humidity of the day in all cases was determined a slight correlation with r = 0.29 for the average humidity of the day. In examining the correlation of CVI and changes in the relative humidity of the day (7 am to 14 pm) when most strokes occurred were found insignificant correlation r = 0.07.
Conflict of interest
====================
None declared.
######
Selected days in the typical months
--
--
######
Number of strokes by gender and age in 2004-2006
--
--
######
Correlation of the number of strokes and the average humidity during certain day
--
--
######
Correlation of the number of strokes and humidity changes in a day
--
--
| {
"pile_set_name": "PubMed Central"
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Introduction {#sec1-1}
============
Brachial plexus tumor is recognized as a very rare clinical problem. Benign tumors include schwannoma and neurofibroma, whereas malignant tumors include malignant schwannoma, fibrosarcoma, neuroblastoma, as well as metastatic tumors of plexus, such as lymphoma, leukemia, lung cancer, and breast cancer. There is no sex predilection for this tumor,\[[@ref1]\] and the age of onset is young adulthood. Further, this type of tumor is more common in patients with von Recklinghausen\'s disease.
In patients with brachial plexus tumors, pain tends to be constant and burning, and paresthesia is a common occurrence. Muscle wasting depends on the involved muscle groups in this condition.\[[@ref1][@ref2]\] Pain is usually not affected by activity and cannot be relieved even with significant narcotics. Patients with brachial plexus tumors often present with a mass and local or radicular pain.\[[@ref1][@ref3]\] Such patients do not generally have neurologic deficits, but in case of neurologic deficits, surgery or biopsy is contraindicated.\[[@ref4][@ref5][@ref6][@ref7]\]
According to another classification, brachial plexus tumors are divided into benign peripheral nerve sheath tumors (e.g., schwannoma and neurofibroma), benign nonneural tumors (e.g., lipoma and desmoid tumor), and malignant nonneural sheath nerve tumors (e.g., sarcoma). Overall, in soft tissue masses, tumor size, location, mobility, and tenderness to percussion, in addition to the presence or absence of Tinel\'s sign, are evaluated.\[[@ref1][@ref8]\]
In paraclinical evaluation of brachial plexus tumors, chest X-ray, neck X-ray for cervical spine involvement, computed tomography (CT) scan, or magnetic resonance imaging (MRI) for delineation of tumor location and anatomy are used. On the other hand, in malignant tumors, metastasis workups, such as abdominal and pelvic CT scans and myelography, are applied. In addition, electrodiagnostic studies and tissue detection are important in this area.\[[@ref9][@ref10]\]
Case Report {#sec1-2}
===========
The patient was a 29-year-old woman, who had presented with a 6 × 8 cm soft tissue mass in the left deltopectoral groove eight years ago. The mass was compatible with a brachial plexus tumor located in the infraclavicular region. It was firm, consistent, and immobile, although it was not painful. There was no tenderness or Tinel\'s sign on percussion test. In addition, there was no thrill, bruit, or history of trauma. The only chief complaint of the patient was enlargement of the soft tissue mass. She underwent MRI, ultrasound, and CT angiography, which confirmed juxtaposition with the subclavian artery and brachial plexus.
Finally, the patient underwent surgery through classic incision of the infraclavicular brachial plexus. However, the findings during surgery indicated hypervascularity and bleeding, along with adhesion to the brachial plexus, which had invaded the infraclavicular region. The mass was resected completely. There were no neurologic or vascular deficits during the postoperative period. Permanent pathology showed a large venous hemangioma \[Figures [1](#F1){ref-type="fig"}-[3](#F3){ref-type="fig"}\].
{#F1}
{#F2}
{#F3}
Discussion {#sec1-3}
==========
Brachial plexus hemangioma is recognized as a very rare clinical problem. Extraneural brachial plexus hemangioma usually presents with the signs and symptoms of a peripheral nerve sheath tumor. The signs and symptoms include pain, paresthesia, and weakness. Meanwhile, use of nonspecific imaging findings for preoperative diagnosis is difficult.
Brachial plexus hemangioma is usually diagnosed as a sarcoma or peripheral nerve sheath tumor. The final step in paraclinical evaluation is needle biopsy or open biopsy, which can cause some complications; therefore, biopsy results may be confounding. In our patient, fine needle aspiration was performed, which involved fibro-fatty tissues and collagen fibers \[[Figure 4](#F4){ref-type="fig"}\].\[[@ref11][@ref12][@ref13]\]
{#F4}
Conclusion {#sec1-4}
==========
Extraneural brachial plexus hemangioma is a very rare condition. If suspected on MRI findings, CT angiography or angiography can be helpful. For reducing the risk of bleeding, especially in patients with a tumor larger than 3 cm on angiography, embolization is very useful \[[Figure 2](#F2){ref-type="fig"}\].
Declaration of patient consent {#sec2-1}
------------------------------
The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Financial support and sponsorship {#sec2-2}
---------------------------------
Nil.
Conflicts of interest {#sec2-3}
---------------------
There are no conflicts of interest.
| {
"pile_set_name": "PubMed Central"
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1. Introduction {#s1}
===============
The growth of older population around the world in the last few decades has caused an increase in problems which can be associated to deteriorated mechanical properties of bone; osteoporosis is the clearest example of one such condition.
Computer models have been extensively employed to evaluate the mechanical response of bone and bone-implant systems under a range of loading scenarios (Pankaj, [@B34]). Previous studies have assumed bone to be homogeneous (Completo et al., [@B8]; Conlisk et al., [@B9]), i.e., its properties do not vary from point to point in space or heterogeneous (Helgason et al., [@B17]; Schileo et al., [@B39]; Tassani et al., [@B47]), i.e., its properties vary with location (these are typically assigned on the basis of grey-scale values observed in micro-computed tomography scans). However, in the large majority of studies, bone is assumed to be linear elastic and isotropic, i.e. its properties at a certain point in space are the same in all directions. It is well- recognized that the cellular microstructure of trabecular bone renders it anisotropic (Turner et al., [@B48]; Odgaard et al., [@B31]), i.e., properties at a point in space vary in different directions. Finite element (FE) analysis of the bone microstructure, in which the solid and pore phases are explicitly modeled, has been used to evaluate the homogenized anisotropic linear elastic properties of bone in the past two decades. Morphology-elasticity relationships that use bone density and fabric have also been established, with fabric typically measured through the mean intercept length (MIL) fabric tensor (Harrigan and Mann, [@B16]). These relationships establish links between density, fabric, and the components of the stiffness tensor (Zysset, [@B55]). More recently, some studies have attempted the evaluation of homogenized yield behaviour (Cowin, [@B10]; Wolfram et al., [@B52]; Levrero-Florencio et al., [@B26]).
Homogenized FE models of the whole bone can include microstructural information at the continuum (macroscopic) level and can thus improve the assessment of the behavior of bone and bone-implant systems in clinical scenarios. Homogenization relies on averaging the strains and stresses over a representative volume element (RVE) of the considered material; it is the most widely used multiscale approach to study the macroscopic behavior of trabecular bone. Homogenization of an RVE in the post-elastic regime requires examining its response to a wide range of loading scenarios (Bayraktar et al., [@B2]; Levrero-Florencio et al., [@B26], [@B24]). It is important to note that, in experiments, it is not possible to test multiple load cases after a certain load threshold has been surpassed because permanent deformations and/or damage caused during the first loading case will affect the behavior in subsequent loading cases. Therefore, computational means provide an attractive alternative. Nonetheless, the need for fine resolution to recreate a biofidelic geometry of the bone microstructure leads to micro-FE (μFE) systems of several tens of millions of degrees of freedom. The need to undertake multiple load cases each in non-linear regime requires the usage of high performance computing (HPC) platforms and software which can take advantage of them.
Although the damage behavior of bone has been considered in a few studies (Keaveny et al., [@B21]; Garcia et al., [@B14]; Shi et al., [@B43]; Schwiedrzik and Zysset, [@B41]; Lambers et al., [@B23]), there are apparent limitations to most of the employed mathematical formulations. For example, most macroscopic damage models of trabecular bone employ an isotropic damage evolution, i.e., a "basic," or single scalar isotropic formulation, as mentioned in Carol et al. ([@B7]), and do not take into account that the development of damage may be related to the load case being considered (Levrero-Florencio et al., [@B24]). The authors have previously conducted a series of uniaxial simulations which show that damage develops differently in tension−compression, and in normal−shear (Levrero-Florencio et al., [@B24]).
This study has a number of aims. Firstly, it extends the study performed in Levrero-Florencio et al. ([@B24]) by adding 12 biaxial macroscopic cases in the normal strain space. The second aim is to examine the suitability of certain damage mechanisms by fitting different damage laws to the damaged macroscopic stiffness tensors. The study then investigates the possible relationships between the macroscopic damage behavior of trabecular bone and its density and fabric description, by including these micro-architectural indices as additional data in the fitting procedure. The data for these formulations is obtained computationally through homogenization-based multiscale simulations run on a HPC platform with an *in-house* developed parallel implicit FE code.
2. Notation {#s2}
===========
The mathematical operators defined in this section largely follow the notation used in Wu and Li ([@B53]), Schwiedrzik et al. ([@B40]), and Levrero-Florencio et al. ([@B26]). Compact tensor notation is used throughout this study, with indicial notation within brackets being used in this section to clarify certain tensorial operations, or in specific sections where further clarification might be required.
As a general rule, scalars are denoted with Greek or Latin italic characters (e.g., λ or *a*, respectively); vectors, or first-order tensors, are denoted by Latin bold lower-case characters (e.g., **a**); second-order tensors are denoted with Greek or Latin bold upper-case characters (e.g., σ or **A**, respectively); and fourth-order tensors are denoted by Latin double-barred upper-case characters (e.g., 𝔸).
Tensorial operations are denoted as follows. Single contraction of tensorial entities may appear as **a**·**b** (*a*~*i*~*b*~*i*~), **a**·**B** (*a*~*i*~*B*~*ij*~), **Ab** (*A*~*ij*~*b*~*j*~), or **AB** (*A*~*ik*~*B*~*kj*~), note that the scalar product symbol (·) only appears when the first entity to be contracted is a first-order tensor; double contraction of tensorial entities may appear as **A**:**B** (*A*~*ij*~*B*~*ij*~), 𝔸:**B** (*A*~*ijkl*~*B*~*kl*~), **A**:𝔹 (*A*~*ij*~*B*~*ijkl*~), or 𝔸 : 𝔹 (*A*~*ijmn*~*B*~*mnkl*~). Different tensor products have been defined, which include **a** ⊗ **b** (*a*~*i*~*b*~*j*~), **A** ⊗ **B** (*A*~*ij*~*B*~*kl*~), **A**[⊗]{.ul}**B** (*A*~*ik*~*B*~*jl*~), **A**$\overline{\underline{\otimes}}$**B** (*A*~*il*~*B*~*jk*~), or $\mathbf{A}\underline{\overline{\otimes}}\mathbf{B} = \frac{1}{2}(\mathbf{A}\underline{\otimes}\mathbf{B} + \mathbf{A}\overline{\otimes}\mathbf{B})$ ($\frac{1}{2}\left\lbrack {A_{ik}B_{jl} + A_{il}B_{jk}} \right\rbrack$).
Curly brackets {·} are used to represent vector projections of second-order tensors, such as
{
A
}
=
{
A
11
A
22
A
33
A
12
A
13
A
23
}
T
.
Square brackets \[·\] are used, in conjunction with parentheses (·), to indicate priority in the order of mathematical operations; an important exception occurs when square brackets are used to represent the matrix projection of a fourth-order tensor, such as
\[
𝔸
\]
=
\[
A
1111
A
1122
A
1133
A
1112
A
1113
A
1123
A
2211
A
2222
A
2233
A
2212
A
2213
A
2223
A
3311
A
3322
A
3333
A
3312
A
3313
A
3323
A
1211
A
1222
A
1233
A
1212
A
1213
A
1223
A
1311
A
1322
A
1333
A
1312
A
1313
A
1323
A
2311
A
2322
A
2333
A
2312
A
2313
A
2323
\]
.
Double vertical bars \|\|(·)\|\| are used to represent the Frobenius norm of the matrix (·), such as the Frobenius norm of the following 3 × 3 symmetric matrix,
‖
\[
A
\]
‖
=
A
11
2
\+
A
22
2
\+
A
33
2
\+
2
A
11
2
\+
2
A
13
2
\+
2
A
23
2
.
3. Materials and methods {#s3}
========================
3.1. Computational methods
--------------------------
This section follows the "Materials and Methods" section in Levrero-Florencio et al. ([@B24]). The authors used μCT images of trabecular bone samples to create detailed FE models, which ranged from 10 to 30 million elements, representing the solid phase of bone for a cubic trabecular bone samples (which includes both solid phase and pores) of size 5 mm. In the study conducted by Levrero-Florencio et al. ([@B24]), plasticity and damage were considered for the solid phase post-elastic properties and nine uniaxial strain cases were investigated (load cases 1 to 9 of Table [1](#T1){ref-type="table"}) representing: three tensile cases (+ε~11~, +ε~22~, and +ε~33~), three compressive cases (−ε~11~, −ε~22~, and −ε~33~), and three shear cases (ε~12~, ε~13~, and ε~23~). The macroscopic damage behavior was studied by using an appropriate homogenization-based multiscale technique, which is explained later.
######
Description of the performed strain-controlled load cases.
**Load case** **Description**
------------------------------- -----------------------------------
1 ε~11~ \> 0; ε~22~ = ε~33~ = 0
ε~12~ = ε~13~ = ε~23~ = 0
2 ε~22~ \> 0; ε~11~ = ε~33~ = 0
ε~12~ = ε~13~ = ε~23~ = 0
3 ε~33~ \> 0; ε~11~ = ε~22~ = 0
ε~12~ = ε~13~ = ε~23~ = 0
4 ε~11~ \< 0; ε~22~ = ε~33~ = 0
ε~12~ = ε~13~ = ε~23~ = 0
5 ε~22~ \< 0; ε~11~ = ε~33~ = 0
ε~12~ = ε~13~ = ε~23~ = 0
6 ε~33~ \< 0; ε~11~ = ε~22~ = 0
ε~12~ = ε~13~ = ε~23~ = 0
7 ε~11~ = ε~22~ = ε~33~ = 0
ε~12~ \> 0; ε~13~ = ε~23~ = 0
8 ε~11~ = ε~22~ = ε~33~ = 0
ε~13~ \> 0; ε~12~ = ε~23~ = 0
9 ε~11~ = ε~22~ = ε~33~ = 0
ε~23~ \> 0; ε~12~ = ε~13~ = 0
10 ε~11~ = ε~22~ \> 0; ε~33~ = 0
ε~12~ = ε~13~ = ε~23~ = 0
11 ε~11~ \> 0; ε~22~ \< 0; ε~33~ = 0
ε~12~ = ε~13~ = ε~23~ = 0
12 ε~11~ \< 0; ε~22~ \> 0; ε~33~ = 0
ε~12~ = ε~13~ = ε~23~ = 0
13 ε~11~ = ε~22~ \< 0; ε~33~ = 0
ε~12~ = ε~13~ = ε~23~ = 0
14 ε~11~ = ε~33~ \> 0; ε~22~ = 0
ε~12~ = ε~13~ = ε~23~ = 0
15 ε~11~ \> 0; ε~33~ \< 0; ε~22~ = 0
ε~12~ = ε~13~ = ε~23~ = 0
16 ε~11~ \< 0; ε~33~ \> 0; ε~22~ = 0
ε~12~ = ε~13~ = ε~23~ = 0
17 ε~11~ = ε~33~ \< 0; ε~22~ = 0
ε~12~ = ε~13~ = ε~23~ = 0
18 ε~22~ = ε~33~ \> 0; ε~11~ = 0
ε~12~ = ε~13~ = ε~23~ = 0
19 ε~22~ \> 0; ε~33~ \< 0; ε~11~ = 0
ε~12~ = ε~13~ = ε~23~ = 0
20 ε~22~ \< 0; ε~33~ \> 0; ε~11~ = 0
ε~12~ = ε~13~ = ε~23~ = 0
21 ε~22~ = ε~33~ \< 0; ε~11~ = 0
ε~12~ = ε~13~ = ε~23~ = 0
Trabecular bone is an anisotropic material; its anisotropy may be quantified with a fabric tensor, which indicates how directionally distributed a material is. The Mean Intercept Length (MIL) fabric tensor is used in this study because it is widely used in trabecular bone studies, and it performs slightly better than other fabric measures (Kabel et al., [@B19]; Zysset, [@B55]). The magnitude of an eigenvalue of the MIL fabric tensor denotes the proportion of material which is aligned in the direction expressed in the correspondent eigenvector. The fabric tensors are normalized by a trace equal to 3 (Zysset, [@B55]).
In this study, 10 out of the 12 samples employed in Levrero-Florencio et al. ([@B24]) were subjected to 12 additional biaxial strain cases in the normal strain space (Table [1](#T1){ref-type="table"}, cases 10--21). Kinematic uniform boundary conditions (i.e., conditions in which displacements, or macroscopic strains, are controlled) were used for all analyses; these are known for providing an upper bound for the macroscopic stiffness tensor and macroscopic yield surface of trabecular bone (Wang et al., [@B51]; Panyasantisuk et al., [@B35]). An example of how boundary conditions are implemented can be seen in Figure [1](#F1){ref-type="fig"}, which corresponds to load case 4 in Table [1](#T1){ref-type="table"}. The morphological indices of these samples are shown in Table [2](#T2){ref-type="table"}. BV/TV stands for bone volume over total volume and it is a surrogate for density, DOA stands for degree of anisotropy and it is the ratio of the highest to the lowest eigenvalues of the MIL fabric tensor, and SMI stands for structure model index and it ranges from rod- (SMI = 3) to plate-shaped (SMI = 0) microstructure.
{ref-type="table"}). **(Right)** Rendered image of one of the used trabecular bone specimens; this particular sample led to a FE mesh of \~21M degrees of freedom.](fphys-09-00545-g0001){#F1}
######
Morphological indices of the 10 used specimens.
**Specimen** **BV/TV (%)** **DOA** **SMI**
-------------- --------------- --------- ---------
1 30.3 2.67 0.52
2 18.1 3.47 1.33
3 14.8 2.65 1.59
4 16.5 2.13 1.37
5 17.7 2.59 1.40
6 22.2 3.47 0.84
7 24.6 2.85 0.88
8 20.3 1.61 1.16
9 23.1 2.10 0.98
10 26.9 2.55 0.79
The 10 samples were aligned with the MIL fabric tensor eigenvectors, with the eigenvalues sorted in descendent order (*m*~1~ \> *m*~2~ \> *m*~3~). The samples were then meshed with trilinear hexahedra and subjected to the aforementioned strain-controlled load cases; the largest mesh consisted of \~27M degrees of freedom, leading to square sparse stiffness matrices of up to 27M×27M elements. The considered constitutive law at the tissue level was isotropic with coupled plasticity and damage (the former captures irrecoverable deformations while the latter takes accounts for stiffness reduction), meaning that damage and plasticity interact with each other and evolve at the same time; the considered yield surface was Drucker-Prager (Tai et al., [@B46]; Carnelli et al., [@B5]; Panyasantisuk et al., [@B35]) with yield values corresponding to 0.41% strain in tension and 0.83% strain in compression (Bayraktar and Keaveny, [@B3]). Linear isotropic hardening corresponding to 5% of the undamaged elastic slope (Wolfram et al., [@B52]; Sanyal et al., [@B38]) was used. At the tissue level, damage evolution was assumed to be isotropic and it was obtained from Schwiedrzik and Zysset ([@B41], [@B42]). The maximum damage was capped at 0.9 (90% isotropic stiffness reduction) to avoid numerical difficulties related to the loss of positive-definiteness of the stiffness matrix; this was performed by using
D
(
ε
p
)
=
D
max
(
1
−
e
−
k
p
ε
p
)
where ε^*p*^ = \|\|**ε**^*p*^\|\| is the accumulated plastic strain, *D*~max~ is the maximum damage, and *k*~*p*~ is a parameter obtained from Schwiedrzik and Zysset ([@B42]).
The μFE simulations were run on a Cray XC30 supercomputer hosted by ARCHER (UK National Supercomputing Service), with an *in-house* version of ParaFEM (Smith et al., [@B44]; Levrero-Florencio et al., [@B25]) which solves implicit quasi-static finite strain elastoplasticity problems in a highly scalable message passing interface-based (MPI) parallel fashion. Each simulation took from 40 to 120 min when using 1,920 cores, depending on the considered load case, with biaxial compression-compression load cases taking the longest. In order to improve the convergence aspect of the local (constitutive level, i.e., at each integration point) Newton--closest-point projection method (Newton-CPPM), two additional schemes were implemented: (a) a line search as in the primal-CPPM scheme described in Pérez-Foguet and Armero ([@B36]) and (b) an improved trial predictor (Bićanić and Pearce, [@B4]; de Souza Neto et al., [@B13]). In the latter scheme, if the first Newton-CPPM fails to converge, it is restarted but this time with the initial guess for stress as **σ**^proj^, which is the stress returned to the frozen yield surface, i.e., no hardening or damage evolution. If these two mechanisms do not work, to ensure that a possible local lack of convergence does not influence the results of the μFE simulations, lack of convergence of the CPPM scheme is broadcasted to all MPI processes in order to cut down the time increment to half of its value. The initial, and maximum, step size corresponded to 0.1% macroscopic strain Frobenius norm and was allowed to decrease to a minimum of 0.001%, if global (structural level, i.e., the global stiffness matrix) or local convergence was not achieved. The global solution scheme employed was Newton-Raphson, and a Jacobi, or diagonally, preconditioned conjugate gradients method was used as the linear algebraic solver.
The macroscopic elastic stiffness tensor was calculated at each time increment by using the homogenization procedure described by van Rietbergen et al. ([@B50], [@B49]), in which the macroscopic elastic stiffness tensor 𝔼 is
𝔼
=
1
V
∫
Ω
(
1
−
D
μ
)
𝔼
μ
:
𝕄
d
V
,
which, in a FE setting, is equivalent to $$\mathbb{E} = \frac{1}{V}{\sum\limits_{i = 1}^{\text{n}_{\text{els}}}{\sum\limits_{j = 1}^{\text{n}_{\text{ips}}}{(1 - D_{\mu ij})}}}\mathbb{E}_{\mu ij}:\mathbb{M}_{ij}\text{det}(\mathbf{J}_{ij})w_{j},$$
and where *V* is the volume of the cubic region (5 × 5 × 5 = 125 mm^3^), *D*~μ~ is the damage at the solid phase, 𝔼~μ~ is the solid phase undamaged stiffness tensor, n~els~ is the total number of elements in the considered mesh, n~ips~ is the number of integration points in a trilinear hexahedron, det(**J**~*ij*~) is the determinant of the Jacobian of the transformation from normal to natural coordinates, *w*~*j*~ is the weight of the corresponding integration point, and 𝕄 is the local structure tensor, which relates the solid phase strain **ε**~μ~ to the average strain tensor **ε**, such that $$\mathbf{\varepsilon}_{\mu} = \mathbb{M}:\mathbf{\varepsilon}.$$
This tensor 𝕄 was determined by solving six completely linear FE systems for six macroscopic uniaxial strain cases (three tensile or compressive and three shear). For each of these cases, the tissue strains calculated represent one of the six columns of the matrix projection of 𝕄 (Hollister and Kikuchi, [@B18]). The assumption made was that the samples are aligned in their orthotropic axes as they were aligned with the MIL fabric tensor eigenvectors (Odgaard et al., [@B31]). Macroscopic strain points were defined by using the 0.2% strain criterion (Wolfram et al., [@B52]; Levrero-Florencio et al., [@B24]), and it was extended to define further 0.3, 0.4, and 0.5% strain levels. The corresponding damaged slope to calculate these strain points is determined at each time step, depending on the load case. The following is an example for the biaxial tensile case ε~11~ = ε~22~ \> 0 (load case 10 in Table [1](#T1){ref-type="table"}). Since the macroscopic strains are small, the assumption of linear kinematics can be considered at the macroscale; thus, the homogenized infinitesimal stress can be obtained through the macroscopic infinitesimal strain and the macroscopic stiffness tensor, such as $$\begin{array}{l}
{\left\{ \begin{array}{l}
\sigma_{\text{hom},11} \\
\sigma_{\text{hom},22} \\
\sigma_{\text{hom},33} \\
\sigma_{\text{hom},12} \\
\sigma_{\text{hom},13} \\
\sigma_{\text{hom},23} \\
\end{array} \right\} = \left\lbrack \begin{array}{llllll}
E_{1111} & E_{1122} & E_{1133} & E_{1112} & E_{1113} & E_{1123} \\
E_{2211} & E_{2222} & E_{2233} & E_{2212} & E_{2213} & E_{2223} \\
E_{3311} & E_{3322} & E_{3333} & E_{3312} & E_{3313} & E_{3323} \\
E_{1211} & E_{1222} & E_{1233} & E_{1212} & E_{1213} & E_{1223} \\
E_{1311} & E_{1322} & E_{1333} & E_{1312} & E_{1313} & E_{1323} \\
E_{2311} & E_{2322} & E_{2333} & E_{2312} & E_{2313} & E_{2323} \\
\end{array} \right\rbrack\left\{ \begin{array}{l}
\varepsilon_{11} \\
\varepsilon_{22} \\
0 \\
0 \\
0 \\
0 \\
\end{array} \right\}} \\
{\text{~~~~~~~~~~~~~} = \left\{ \begin{array}{l}
{E_{1111}\varepsilon_{11} + E_{1122}\varepsilon_{22}} \\
{E_{2211}\varepsilon_{11} + E_{2222}\varepsilon_{22}} \\
0 \\
0 \\
0 \\
0 \\
\end{array} \right\},} \\
\end{array}$$
where **σ**~hom~ is the homogenized stress tensor, leading to $$\left\| \mathbf{\sigma}_{\text{hom}} \right\| = \sqrt{E_{1111}^{2}\varepsilon_{11}^{2} + E_{1122}^{2}\varepsilon_{22}^{2} + E_{2211}^{2}\varepsilon_{11}^{2} + E_{2222}^{2}\varepsilon_{22}^{2}},$$
with the damaged slope being (note that in the considered biaxial cases \|ε~*ii*~\| = \|ε~*jj*~\|) $$K_{\text{dam}} = \sqrt{E_{1111}^{2} + E_{1122}^{2} + E_{2211}^{2} + E_{2222}^{2}}.$$
3.2. Theoretical framework of damage
------------------------------------
The previously described μFE simulations, together with the homogenization-based multiscale procedure, were used to derive the damaged macroscopic stiffness tensors of the considered samples, for different load scenarios (Table [1](#T1){ref-type="table"}) and load levels (0.2, 0.3, 0.4, and 0.5% strain norm). These stiffness tensors were used as data points for a minimization procedure (described in the following subsections), which was used to fit the macroscopic damage behavior to several theoretical damage models: single scalar isotropic formulation, three scalars anisotropic formulation, and isotropic/anisotropic combined formulation with tension/compression asymmetry.
Coupled damage and plasticity were considered for the μFE simulations. However, the focus of this study is on the macroscopic damage behavior of trabecular bone and therefore no plasticity is assumed at the macroscale. This is why, in the following, the total strain **ε** is used instead of the elastic strain **ε**^*e*^.
### 3.2.1. Basic concepts and description of the baseline model
Let us consider the theoretical framework of elastic degradation by using state variables, from which the different damage constitutive models are derived (Carol et al., [@B6], [@B7]; Murakami, [@B29]). The starting point of the theoretical framework is the assumption of a Helmholtz free energy potential per unit reference volume ψ of the considered material, from which the state equations are derived. The free energy potential may be expressed as
ψ
(
ε
,
D
k
,
R
k
)
=
ψ
e
(
ε
,
D
k
)
\+
ψ
D
(
R
k
)
=
1
2
ε
:
𝔼
(
𝔼
0
,
D
k
)
:
ε
\+
1
2
∑
k
=
1
l
K
k
R
k
2
,
where **ε** is the infinitesimal strain tensor, 𝔼 and 𝔼~0~ are, respectively, the damaged and undamaged stiffness tensors, *D*~*k*~ are a set of *l* scalar damage variables; *R*~*k*~ and *K*~*k*~ are, respectively, a set of *l* variables and *l* parameters controlling the size and hardening of the (damage) dissipation potential functions *F*~*k*~ (Equation 16).
Time derivative of Equation (11) yields
ψ
˙
=
∂
ψ
∂
ε
:
ε
.
\+
∑
k
=
1
l
∂
ψ
∂
D
k
D
˙
k
\+
∑
k
=
1
l
∂
ψ
∂
R
k
R
˙
k
,
which, when used in the Clausius-Duhem inequality for isothermal processes
σ
:
ε
.
−
ρ
ψ
˙
≥
0
,
gives rise to the dissipation inequality
ϕ
=
(
σ
−
ρ
∂
ψ
∂
ε
)
:
ε
.
−
∑
k
=
1
l
ρ
∂
ψ
∂
D
k
D
˙
k
−
∑
k
=
1
l
ρ
∂
ψ
∂
R
k
R
˙
k
=
∑
k
=
1
l
Y
k
D
˙
k
\+
∑
k
=
1
l
B
k
R
˙
k
≥
0
,
where ρ is the density of the considered material, $\mathbf{\sigma} = \rho\frac{\partial\psi}{\partial\varepsilon}$, $Y_{k} = - \rho\frac{\partial\psi}{\partial D_{k}}$, and $B_{k} = - \rho\frac{\partial\psi}{\partial R_{k}} = K_{k}R_{k}$.
The evolution equations of *D*~*k*~ and *R*~*k*~ are derived from the corresponding dissipation potential functions *F*~*k*~, leading to
D
k
.
=
γ
˙
k
∂
F
k
∂
Y
k
;
R
˙
k
=
γ
˙
k
∂
F
k
∂
B
k
,
where ${\overset{\cdot}{\gamma}}_{k}$ are indeterminate multipliers. Since *F*~*k*~ also delimit the undamaged region of the considered material, the non-negativeness of Equation (14) is assured (Murakami, [@B29]). Linear, a priori uncoupled, criteria for *F*~*k*~ are considered in this study (each *D*~*k*~ is related to a single *F*~*k*~), such that
F
k
(
Y
k
,
B
k
)
=
Y
k
−
(
B
k
\+
B
k
,
0
)
=
Y
k
−
(
K
k
R
k
\+
B
k
,
0
)
≤
0
,
where *B*~*k*,\ 0~ are the initial sizes of *F*~*k*~, i.e., when *R*~*k*~ = 0. These linear functions are considered for the sake of simplicity and also because data on additional strain points is needed so that more complex, non-linear, evolution expressions of the dissipation potentials may be taken into account.
Energy equivalence is adopted here since it automatically induces major symmetry in the stiffness and compliance tensors. This leads to
ψ
e
(
ε
,
D
k
)
=
1
2
ε
:
𝔼
(
𝔼
0
,
D
k
)
:
ε
=
1
2
ε
:
𝕄
T
(
D
k
)
:
𝔼
0
:
𝕄
(
D
k
)
:
ε
=
1
2
ε
eff
(
ε
,
D
k
)
:
𝔼
0
:
ε
eff
(
ε
,
D
k
)
,
where 𝕄 is the fourth-order damage effect tensor which depends on the considered damage formulation, and 𝔸^T^ is defined so that $\mathbb{A}^{\text{T}} \equiv A_{ijkl}^{\text{T}} = A_{klij}$.
### 3.2.2. Numerical solution of the damage models
Equations (15, 16) are integrated with Backward Euler. Residual equations for each of the variables to be sought can be formulated, with a format similar to that of CPPM equations of computational plasticity (Armero and Pérez-Foguet, [@B1]; Pérez-Foguet and Armero, [@B36]), so that
{
R
D
,
k
R
R
,
k
F
k
}
=
{
D
k
,
n
\+
1
−
D
k
,
n
−
Δ
γ
k
,
n
\+
1
∂
F
k
∂
Y
k
\|
n
\+
1
R
k
,
n
\+
1
−
R
k
,
n
−
Δ
γ
k
,
n
\+
1
∂
F
k
∂
B
k
\|
n
\+
1
Y
k
,
n
\+
1
−
(
K
k
R
k
,
n
\+
1
\+
B
k
,
0
)
}
where *n* stands for the *n*th time increment, and the vertical bar means "evaluated at".
The resulting set of non-linear equations (Equation 18) can be solved with a numerical scheme, for instance a Newton-Raphson approach. The first step is to calculate the Jacobian of the system, and therefore the residuals (Equation 18) are linearized, leading to (time subscripts are dropped for convenience from now onwards)
{
0
0
0
}
=
{
d
D
j
(
δ
j
k
−
Δ
γ
k
∂
∂
D
j
∂
F
k
∂
Y
k
)
−
d
Δ
γ
k
∂
F
k
∂
Y
k
d
R
k
−
d
Δ
γ
k
∂
F
k
∂
B
k
d
D
j
∂
F
k
∂
D
j
\+
d
R
k
∂
F
k
∂
R
k
}
.
where $\delta_{ij} = \left\{ \begin{array}{l}
{0\text{~if~}i \neq j} \\
{1\text{~if~}i = j} \\
\end{array} \right.$ is the Kronecker delta. The specific expressions for the derivatives of the Jacobian are presented for each of the considered damage models in the following sections.
The resulting Newton-Raphson scheme to solve for *D*~*k*~, *R*~*k*~, and Δγ~*k*~ is
{
D
k
R
k
Δ
γ
k
}
m
\+
1
=
{
D
k
R
k
Δ
γ
k
}
m
−
\[
δ
j
k
−
Δ
γ
k
∂
∂
D
j
∂
F
k
∂
Y
k
0
−
∂
F
k
∂
Y
k
0
1
−
∂
F
k
∂
B
k
∂
F
k
∂
D
j
∂
F
k
∂
R
k
0
\]
m
−
1
{
R
D
,
k
R
R
,
k
F
k
}
m
where *m* stands for the *m*th iteration of the Newton-Raphson scheme.
### 3.2.3. Damage models
This section describes the three main models, and their variants, used in this study. The first two models, single scalar isotropic model (section 3.2.3.1) and three scalars anisotropic model (section 3.2.3.2) are mainly used to assess the BV/TV and fabric eigenvalue dependencies of macroscopic damage models of trabecular bone. The proposed model (section 3.2.3.3), we believe, is a considerable improvement upon the usually employed single scalar isotropic formulation.
#### 3.2.3.1. Single scalar isotropic formulation
In this simple damage formulation a single scalar damage variable *D* equally affects all the components of the stiffness tensor, i.e., all directions are equally affected by damage. The damage effect tensor is
𝕄
=
(
1
−
D
)
𝕀
sym
,
where $\mathbb{I}_{\text{sym}} = \mathbf{I}\underline{\overline{\otimes}}\mathbf{I}$.
The Helmholtz free energy potential for this model is
ψ
(
ε
,
D
,
R
)
=
1
2
ε
:
(
1
−
D
)
2
𝔼
0
:
ε
\+
1
2
K
R
2
,
which leads to the following expressions for the conjugate thermodynamic associated variables
σ
=
(
1
−
D
)
2
𝔼
0
:
ε
Y
=
−
1
2
ε
:
∂
𝔼
∂
D
:
ε
=
ε
:
(
1
−
D
)
𝔼
0
:
ε
B
=
K
R
and to the following expressions for the derivatives in Equation (20)
∂
∂
D
∂
F
∂
Y
=
0
∂
F
∂
Y
=
1
∂
F
∂
D
=
∂
Y
∂
D
=
−
1
2
ε
:
∂
2
𝔼
∂
D
2
:
ε
=
−
ε
:
𝔼
0
:
ε
∂
F
∂
R
=
−
K
.
BV/TV dependence is included in this model by defining $K = K_{0,\text{iso}}\rho^{o}$ and $B = B_{0,\text{iso}}\rho^{p}$, where ρ is the BV/TV of the considered sample, and *o* and *p* are the exponents expressing the BV/TV dependency.
#### 3.2.3.2. Three scalars anisotropic formulation
In the anisotropic damage formulation a damage scalar for each principal direction of the sample is considered (*D*~1~, *D*~2~, and *D*~3~), meaning that each of these three orthogonal directions has a different damage behavior (as previously stated, these orthogonal directions are parallel to the axes of the cubic sample). Since the range of post-elastic strains applied to the sample is relatively small, it is assumed that no rotation of the orthotropic axes occurs. The damage effect tensor is
𝕄
=
(
𝕀
sym
−
𝔻
)
,
where
∂
𝔻
∂
D
1
=
\[
1
α
α
0
0
0
α
0
0
0
0
0
α
0
0
0
0
0
0
0
0
β
0
0
0
0
0
0
β
0
0
0
0
0
0
0
\]
;
∂
𝔻
∂
D
2
=
\[
0
α
0
0
0
0
α
1
α
0
0
0
0
α
0
0
0
0
0
0
0
β
0
0
0
0
0
0
0
0
0
0
0
0
0
β
\]
;
∂
𝔻
∂
D
3
=
\[
0
0
α
0
0
0
0
0
α
0
0
0
α
α
1
0
0
0
0
0
0
0
0
0
0
0
0
0
β
0
0
0
0
0
0
β
\]
.
in which α and β are parameters which determine how the components of the stiffness tensor are affected by the different damage scalars.
The Helmholtz free energy potential is
ψ
(
ε
,
D
k
,
R
k
)
=
1
2
ε
:
𝔼
(
𝔼
0
,
D
k
)
:
ε
\+
1
2
∑
k
=
1
3
K
k
R
k
2
,
which leads to the following expressions for the conjugate thermodynamic associated variables
σ
=
𝔼
:
ε
=
\[
(
𝕀
sym
−
𝔻
)
:
𝔼
0
:
(
𝕀
sym
−
𝔻
)
\]
:
ε
Y
k
=
−
1
2
ε
:
∂
𝔼
∂
D
k
:
ε
B
k
=
K
k
R
k
and to the following expressions for the derivatives in Equation (20)
∂
∂
D
j
∂
F
k
∂
Y
k
=
0
∂
F
k
∂
Y
k
=
1
∂
F
k
∂
D
j
=
∂
Y
k
∂
D
j
=
−
1
2
ε
:
∂
∂
D
j
∂
𝔼
∂
D
k
:
ε
∂
F
k
∂
R
k
=
−
K
k
∂
𝔼
∂
D
k
=
−
\[
∂
𝔻
∂
D
k
:
𝔼
0
:
𝕄
\+
𝕄
:
𝔼
0
:
∂
𝔻
∂
D
k
\]
∂
∂
D
j
∂
𝔼
∂
D
k
=
∂
𝔻
∂
D
k
:
𝔼
0
:
∂
𝔻
∂
D
j
\+
∂
𝔻
∂
D
j
:
𝔼
0
:
∂
𝔻
∂
D
k
Fabric eigenvalue dependencies are included in this model by defining $K_{k} = K_{0,\text{aniso}}m_{k}^{q}$ and $B_{k} = B_{0,\text{aniso}}m_{k}^{r}$, where *m*~*k*~ is the MIL fabric eigenvalue corresponding to the *k*th orthotropic direction of the sample; and *q* and *r* are the exponents expressing the fabric eigenvalue dependency.
#### 3.2.3.3. Combined formulation with tension/compression asymmetry
We propose a combined isotropic/anisotropic damage formulation, which consists of four damage scalars: a single scalar defines the isotropic part of the model (*D*~iso~); and three scalars define the anisotropic part of the model, one for each of the three orthotropic directions (*D*~1~, *D*~2~, and *D*~3~). As in the previous cases, the isotropic damage scalar equally affects all directions, while each of the three orthotropic damage scalars only affect their corresponding orthogonal direction. It is assumed that there is no rotation of the orthotropic axes. The tension/compression asymmetry is included in the damage effect tensor, such that
𝕄
=
𝕀
sym
−
𝔻
iso
−
∑
i
=
1
3
\[
1
\+
η
H
(
−
m
i
·
ε
m
i
)
𝔻
aniso
,
i
\]
,
where
𝔻
iso
=
(
1
−
D
)
𝕀
sym
,
𝔻
aniso
,
i
=
∂
𝔻
∂
D
i
D
i
with $\frac{\partial\mathbb{D}}{\partial D_{i}}$ being defined in Equation (26), η is the parameter governing the tension/compression asymmetry, **m**~*i*~ is the *i*^th^ fabric tensor eigenvector, and H(·) is the Heaviside function defined as
H
(
·
)
=
{
1
if
(
·
)
\>
0
0
if
(
·
)
≤
0
.
The Helmholtz free energy potential for this model is
ψ
(
ε
,
D
k
,
R
k
)
=
1
2
ε
:
𝔼
(
𝔼
0
,
D
k
)
:
ε
\+
1
2
∑
k
=
1
4
K
k
R
k
2
,
BV/TV and fabric eigenvalue dependencies are included in this model by defining $K_{\text{iso}} = K_{0,\text{iso}}\rho^{o}$; $K_{k,\text{aniso}} = K_{0,\text{aniso}}\rho^{t}m_{k}^{q},k \in \left\{ {1,2,3} \right\}$; $B_{\text{iso}} = B_{0,\text{iso}}\rho^{p}$; and $B_{k,\text{aniso}} = B_{0,\text{aniso}}\rho^{u}m_{k}^{r},k \in \left\{ {1,2,3} \right\}$, where *o* and *p* are the exponents expressing BV/TV dependency of the isotropic part of the model; and *t*, *u*, *q*, and *r* are, respectively the exponents expressing BV/TV and fabric eigenvalue dependencies of the anisotropic part of the model. The rest of expressions in the model are the same to those in section 3.2.3.2.
3.3. Fitting of the different damage laws
-----------------------------------------
The different damage constitutive models described in the previous section are fitted to the macroscopic damage response obtained from the homogenization-based multiscale μFE simulations. The constitutive laws were fitted by using a particle swarm optimization scheme (`particleswarm`, MATLAB R2017b, MathWorks Inc.), followed by a gradient-based scheme (`fmincon`, MATLAB R2017b, MathWorks Inc.) to enhance the final tuning of the parameters, as it is assumed that when `particleswarm` finishes, the solution is already within the proximity of a minimum. The minimization problem is thus defined as
min
∑
i
=
1
n
(
‖
\[
𝔼
pred
(
θ
s
)
−
𝔼
μ
FE
\]
‖
i
)
2
,
where *n* is the number of samples×load cases×strain levels, which means that the damage results for each sample, each considered load case, and each considered strain level (i.e., 0.2, 0.3, 0.4, and 0.5%) are used in the parameter fitting procedure; \|\|\[𝔼~pred~\]\|\| is the Frobenius norm of the matrix projection of the damaged stiffness tensor predicted by the considered theoretical damage model, \|\|\[𝔼~μFE~\]\|\| is the Frobenius norm of the matrix projection of the damaged stiffness tensor calculated through homogenization, and θ are the *s* different parameters of the considered damage model.
This minimization problem (Equation 35) involves the fitting of parameters which govern the size of the damage dissipation potentials (i.e., the surface containing the elastic regime, in which damage does not develop; it is the damage analog to the yield surface in plasticity), and therefore the solution of the CPPM scheme may involve negative Δγ~*k*~, which are not physical solutions. The CPPM scheme is used in computational plasticity and/or damage contexts to solve the corresponding non-linear equations (Equation 20). If the loading state of a sample is found within the elastic regime (i.e., inside of the yield surface in a plasticity context, or inside the damage dissipation potential in a damage context), no equations need to be solved as plasticity and/or damage related quantities would not further develop. Thus, these undesired values of Δγ~*k*~ will arise only if the loading state of the considered sample is not outside of the damage dissipation potential. In order to avoid these, the minimization problem is modified with a penalty term to avoid such unwanted situations, such that
min
∑
i
=
1
n
\[
(
‖
\[
𝔼
pred
(
θ
s
)
−
𝔼
μ
FE
\]
‖
i
)
2
\+
∑
k
=
1
l
H
(
−
Δ
γ
i
,
k
)
K
pen
(
e
\|
Δ
γ
i
,
k
\|
−
1
)
\]
,
where *K*~pen~ is a large (penalty) constant.
The initial choice of a solver not based on gradients is because the addition of this penalty term breaks the *C*^1^ continuity of the functional to be minimized, and its global non-convexity is assumed *a priori*. The specific choice of particle swarm optimization over other methods not based on gradients, such as genetic algorithm, is established on the superior computational efficiency of particle swarm optimization over the genetic algorithm (Panda and Padhy, [@B33]).
The goodness of the fitting procedure was analyzed with the standard error of the estimate (SEE). This is calculated as
SEE
(
\%
)
=
100
∑
i
=
1
n
(
‖
\[
𝔼
pred
−
𝔼
μ
FE
\]
‖
i
)
2
∑
i
=
1
n
(
‖
\[
𝔼
pred
−
𝔼
0
\]
‖
i
)
2
.
4. Results {#s4}
==========
4.1. Evaluation of the μFE results
----------------------------------
For all load cases in Table [1](#T1){ref-type="table"}, the considered samples were subjected to several strain levels, leading to different damage levels. The resulting macroscopic damaged stiffness tensors and the macroscopic strain Frobenius norms were measured at 0.2, 0.3, 0.4, and 0.5% strain levels by using the 0.2% strain criterion (Wolfram et al., [@B52]). This theoretically leads to damage and macroscopic strain Frobenius norms being evaluated, respectively, at 0--0.3% (with 0% being considered as macroscopic yield) macroscopic plastic strain Frobenius norms. The macroscopic strain Frobenius norms at 0.5% strain level for each load case are shown in Figure [2](#F2){ref-type="fig"} in the form of boxplots. It can be seen from this figure that within each group (T, C, S, or MA), higher macroscopic strain Frobenius norms correspond to compression-dominated load cases (load cases 4--6, 13, 17, and 21 in Figure [2](#F2){ref-type="fig"}).
{#F2}
Damage is evaluated by subtracting the damaged stiffness tensor from the undamaged stiffness tensor and calculating the Frobenius norm of its matrix projection (\|\|\[𝔼~0~ − 𝔼~dam~\]\|\|). The values of these norms for each of the considered load cases are shown in Figure [3](#F3){ref-type="fig"}; the damage shown corresponds to the 0.5% strain level. Due to the alignment of the samples and ordering of their fabric eigenvalues (*m*~1~ \> *m*~2~ \> *m*~3~), it can be seen from this figure that within each group (T, C, S, or MA), higher damage values are seen where the fabric tensor eigenvalues are the largest (i.e., load cases 1, 4, 7, and 10--13 in Figure [3](#F3){ref-type="fig"}). Moreover, higher damage values are also seen in load cases that are compression-dominated (load cases 4--6, 13, 17 and 21). These higher damage values in uniaxial compression, or in compressive-dominated multi-axial load cases, compared to tension load cases indicate a possible tension/compression asymmetry in the damage behavior at the macroscopic level. It is important to mention that, although damage values were measured at the same strain levels according to the 0.2% strain criterion, the macroscopic strain Frobenius norms (Figure [2](#F2){ref-type="fig"}) were considerably larger in compression than in tension.
![Boxplots showing \|\|\[𝔼~0~ − 𝔼~dam~\]\|\| at 0.5% strain level, for each load case: uniaxial tension (T), uniaxial compression (C), shear (S), and multi-axial in the normal strain *XY* plane (MAXY).](fphys-09-00545-g0003){#F3}
Multi-linear regressions in log-log space were performed to establish possible relationships between damage and the micro-architectural indices of the considered samples. These regressions were between \|\|\[𝔼~0~ − 𝔼~dam~\]\|\| at 0.5% strain level, BV/TV, fabric eigenvalues and macroscopic strain Frobenius norms, such as
log
(
‖
\[
𝔼
0
−
𝔼
dam
\]
‖
)
=
A
\+
B
log
(
BV/TV
)
\+
C
log
(
m
1
)
\+
D
log
(
m
2
)
\+
E
log
(
‖
ε
0
‖
)
where *m*~1~ and *m*~2~ are the fabric eigenvalues corresponding to directions 1 and 2 (only shear and multi-axial load cases have two directions); *A*, *B*, *C*, *D*, and *E* are the constants in the regression. These regressions were performed separately for the following sets of load cases: uniaxial tension, uniaxial compression, combined uniaxial tension and uniaxial compression, shear, and multi-axial load cases in normal strain space. The results from these regressions can be seen in Table [3](#T3){ref-type="table"}. Table [3](#T3){ref-type="table"} shows that both BV/TV and fabric eigenvalues have a significant effect (*p* ≤ 0.05), and that damage expressed as per Equation (38) is directly proportional to the micro-architectural indices, with the slopes for BV/TV being substantially larger than those for the fabric eigenvalues. The coefficients of determination (*R*^2^) show that only the multi-linear model of the multi-axial load cases in normal strain space behaves poorly in comparison to the rest.
######
Results from the multi-linear regressions between \|\|\[𝔼~0~ − 𝔼~dam~\]\|\| at 0.5% strain level, BV/TV, fabric eigenvalues, and macroscopic strain Frobenius norms, in log-log space.
**Load case** ***B* (MPa)** ***C* (MPa)** ***D* (MPa)** ***p*-value (BV/TV)** ***p*-value (*m*~*k*~)** ***R*^2^**
--------------- --------------- --------------- --------------- ----------------------- -------------------------- ------------
T 1.50 0.31 → 0 0.004 0.91
C 2.03 0.36 → 0 0.002 0.90
T∪C 1.76 0.66 → 0 → 0 0.90
S 1.99 0.53 0.45 → 0 0.001 0.79
MA 1.71 0.62 0.15 → 0 0.001 0.63
*Regressions were performed for uniaxial tension (T), uniaxial compression (C), combined uniaxial tension and uniaxial compression (T∪C), shear (S), and multi-axial (MA) in normal strain space*.
The component-wise fraction between the matrix projection of 𝔼~0~ − 𝔼~dam~ at 0.5% strain level and the matrix projection of *E*~0~ (i.e., the *i*-th and *j*-th component of 𝔼~0~ − 𝔼~dam~ is divided by the *i*-th and *j*-th component of 𝔼~0~) leads to the 6 × 6 matrix with components
\[
D
\]
i
j
=
\[
𝔼
0
−
𝔼
dam
\]
i
j
\[
𝔼
0
\]
i
j
.
This matrix depicts the component-wise ratio of the damaged and undamaged coefficients for each sample and load case. The component-wise mean of \[**D**\]~*ij*~ over all the considered samples was calculated and then normalized from 0 to 1 for each of the considered load cases, forming another 6 × 6 matrix (e.g., the new matrix *i*-th and *j*-th component is the mean of the **D**~*ij*~ components of all the samples); the components in 𝔼~0~ which are zero are ignored and not considered in the normalization, i.e., the non-orthotropic coefficients. The resulting 21 normalized matrices are shown in Figure [4](#F4){ref-type="fig"}. These plots suggest that macroscopic damage in trabecular bone is actually anisotropic and dependent on the considered load case. In uniaxial tensile and compressive load cases, it can be observed that the normal components of the stiffness tensor which are related to the considered load case are the most affected ones (e.g., in the load case ε~11~ \> 0, components *E*~1111~, *E*~1122~, *E*~1133~, and the corresponding symmetric counterparts are more affected than the rest). In shear load cases, the corresponding shear component is the most affected one. Considering multi-axial load cases in normal strain space we find that in tension-tension and compression-compression load cases, the most affected components are in the off-diagonals of the matrix---the components that are related to the plane which is being loaded (e.g., in the load case ε~11~ = ε~22~ \> 0, components *E*~1122~ and *E*~2211~ are more affected than the rest); in tension-compression/compression-tension load cases, the most affected components are in the matrix diagonal - the components that are related to the plane which is being loaded (e.g., in the load case ε~11~ = −ε~22~ \> 0, components *E*~1111~ and *E*~2222~ are more affected than the rest).
![Graphical representation of the normalized component-wise means of \[**D**\]~*ij*~. The represented load cases are shown in Table [1](#T1){ref-type="table"}: **(A--C)** correspond to uniaxial tension, **(D--F)** correspond to uniaxial compression, **(G--I)** correspond to shear, and **(J--U)** correspond to multi-axial in normal strain space.](fphys-09-00545-g0004){#F4}
4.2. Effect of BV/TV and MIL fabric tensor on the damage behavior
-----------------------------------------------------------------
The effect of BV/TV and fabric on the macroscopic damage behavior of trabecular bone was assessed by (1) considering the single scalar isotropic damage model in section 3.2.3.1 with and without considering the effect of BV/TV and then comparing the respective values of SEE; and (2) considering the anisotropic damage model in section 3.2.3.2 with and without considering the effects of BV/TV and fabric eigenvalues and then comparing the respective values of SEE. In the anisotropic scenario, in the case in which fabric eigenvalues were not included, the order of fabric eigenvalues was randomized to maximise the effect of including fabric in the comparison (the ordering no longer corresponds to *m*~1~ \> *m*~2~ \> *m*~3~; the corresponding stiffness and strain tensors were reordered accordingly). The minimization scheme was run for five times to ensure that a suboptimal solution was not chosen. This comparison is shown in Table [4](#T4){ref-type="table"}.
######
SEEs, BV/TV, and fabric eigenvalue exponents for the isotropic and anisotropic models.
**Model** **SEE (%)** **Exp $K_{0} \cdot \text{BV}/\text{T}\text{V}^{k}$** **Exp $B_{0} \cdot \text{BV}/\text{T}\text{V}^{k}$** **Exp $K_{0} \cdot m_{2}^{k}$** **Exp $B_{0} \cdot m_{1}^{k}$**
----------- ------------- ------------------------------------------------------ ------------------------------------------------------ --------------------------------- ---------------------------------
1 37.03
2 33.03 1.71 1.35
3 35.21
4 32.05 1.71 2.35
5 34.06 0.51 0.37
*Models 1 and 2 are isotropic with and without BV/TV dependency, respectively; models 1, 2, and 3 are anisotropic and: (1) without BV/TV and fabric eigenvalue dependencies, (2) with BV/TV dependency only, and (3) with fabric eigenvalue dependency only*.
Note that the values of SEE of the anisotropic cases are not considerable lower than those of the isotropic cases. This is because even if the damage is higher in the components related to the considered load case, all the components of the stiffness tensor are damaged, and \|\|\[𝔼~0~ − 𝔼~dam~\]\|\| takes into account the reduction of all the components of the stiffness tensor. The exponents that express BV/TV dependency are considerably larger than those expressing fabric eigenvalue dependency.
4.3. Macroscopic damage model for trabecular bone
-------------------------------------------------
A damage model which incorporates both isotropic/anisotropic damage progression and tension/compression asymmetry was implemented and its efficacy in evaluating the macroscopic damage behavior of trabecular bone was assessed. BV/TV and fabric eigenvalue dependencies were considered; BV/TV dependency was included in the isotropic part of the model while both BV/TV and fabric eigenvalue dependencies were included in the anisotropic part. Tension/compression asymmetry was included as shown in section 3.2.3.3. The SEE and the value of the parameters of the model are shown in Table [5](#T5){ref-type="table"}.
######
Value of the parameters and SEE of the combined isotropic/anisotropic model with tension/compression asymmetry.
**Parameter** **Value**
--------------- -----------
*K*~0,iso~ 211.59
*B*~0,iso~ −1.77
*p* 1.98
*l* 1.31
α 0.12
β 0.29
η −0.25
*B*~0,aniso~ 0.00
*K*~0,aniso~ 160.62
*u* 4.01
*r* 3.70
*t* 1.75
*q* 0.94
SEE (%) 21.68
This considered model reduces the SEE in more than 15% with respect to the single scalar isotropic model (SEE = 37.03%). Despite the 13 parameters, a considerably larger number in comparison with the two parameters of the isotropic model, the values of some of these parameters suggest that not all of them need to be considered. For instance, the value of *B*~0,aniso~ is very small, which means that these parameters, together with the corresponding exponents expressing BV/TV and fabric eigenvalue dependencies (*u* and *r*) could be ignored, reducing the number of parameters to 10. It is important to point out the negative values of η and *B*~0,iso~.
5. Discussion {#s5}
=============
The macroscopic damage behavior of trabecular bone has been researched in a few studies, but these are usually restricted to uniaxial load scenarios which only permit the assessment of stiffness reduction in the direction of loading (Keaveny et al., [@B22]; Zioupos et al., [@B54]; Garcia et al., [@B14]). Consequently these studies are unable to provide a comprehensive constitutive model that can be included in whole-bone simulations. This study investigated the possible relationship between damage at the tissue level and the macroscopic multi-axial damage behavior, by employing a homogenization-based multiscale approach to samples with a relatively wide range of BV/TV and fabric tensor eigenvalues, subjected to multiple loading scenarios. The macroscopic damage behavior of trabecular bone was approximated via different continuum damage models: isotropic and anisotropic; with and without BV/TV and fabric eigenvalue dependencies; and with and without tension-compression asymmetry. From the results, it can be concluded that the macroscopic damage behavior of trabecular bone has the following features: BV/TV and fabric eigenvalue dependencies; tension/compression asymmetry; a combined isotropic/anisotropic behaviour. The first two of these features are not unexpected as they play a key role in the evaluation of elastic stiffness (Odgaard et al., [@B31]; Zysset, [@B55]), however, the previously unexplored, last feature indicates that damage in trabecular bone is best represented by using both isotropic and anisotropic damage variables. This is likely to be true for most cellular materials.
This study assumed an isotropic model with coupled damage and plastic behavior at the tissue level, which was deemed appropriate as the isotropy assumption at this level is known to result in little to no error in macroscopic results (Cowin, [@B11]). Isotropic damage at the solid phase level leads to an anisotropic macroscopic damage response with a dependency on the considered load case (Levrero-Florencio et al., [@B24]). The variation in the components of the stiffness tensor shows anisotropic damage which depends on the considered load case (Figure [4](#F4){ref-type="fig"}). Shi et al. ([@B43]) suggested that there is a larger proportion of damaged tissue in the longitudinal trabeculae (direction of loading) for uniaxial load cases, which is in agreement with the results presented here, as the most damaged components of the macroscopic stiffness tensor are always the on-axis components. An issue which may make validation of these results very challenging is the use of kinematic uniform boundary conditions; these boundary conditions are extremely difficult, not to say impossible, to reproduce experimentally, especially for the more complex load cases. Most previous studies involving damage in trabecular bone have used isotropic models (Garcia et al., [@B14]; Schwiedrzik and Zysset, [@B41]), which may be acceptable for proportional loading scenarios, but not for changing loads or cyclic loading scenarios, such as those arising during physiological activities.
The results show that the macroscopic strain Frobenius norms were considerably larger in macroscopic compression than in macroscopic tension. This is important in the considered context of damage modeling as the thermodynamic stress-like variables governing damage evolution (*Y*~*k*~) directly depend on the macroscopic strain values, which could explain the higher damage values in compression without the explicit need of modeling tension/compression asymmetry. However, this asymmetry is taken into account because it still leads to a better fit of the damage model and it only consists of one additional parameter. The fact that damage values are higher in compression-dominated load cases compared to tension load cases could be related to the more heterogeneous stress distributions at the solid phase level occurring during macroscopic compression, which includes tensile stresses at the tissue level due to bending and buckling of trabeculae (Stölken and Kinney, [@B45]). Another important factor to take into account is that the considered model at the tissue level is ductile (i.e., fracture is not incorporated). If fracture was considered at a critical damage threshold, the tension/compression asymmetry would probably be different as tissue damage is more diffused in compression than in tension (Lambers et al., [@B23]), and therefore a significant decrease of load carrying capacity would occur in tension.
The variation in the components of the stiffness tensor shows anisotropic damage which depends on the considered load case (Figure [4](#F4){ref-type="fig"}). Shi et al. ([@B43]) suggested that there is a larger proportion of damaged tissue in the longitudinal trabeculae (direction of loading) for uniaxial load cases, which is in agreement with the results presented here, as the most damaged components of the macroscopic stiffness tensor are always the on-axis components. An issue which may make validation of these results very challenging is the use of kinematic uniform boundary conditions; these boundary conditions are extremely difficult, not to say impossible, to reproduce experimentally, especially for the more complex load cases. Most previous studies involving damage in trabecular bone have used isotropic models (Garcia et al., [@B14]; Schwiedrzik and Zysset, [@B41]), which may be acceptable for proportional loading scenarios, but not for changing loads or cyclic loading scenarios, such as those arising during physiological activities.
Multi-linear regressions between \|\|\[𝔼~0~ − 𝔼~dam~\]\|\|, BV/TV, fabric eigenvalues and macroscopic strain Frobenius norms (from Table [3](#T3){ref-type="table"}). It shows that both BV/TV and fabric eigenvalues are statistically significant. The coefficients of determination suggest that only the regression of \|\|\[𝔼~0~ − 𝔼~dam~\]\|\| of the multi-axial load cases in normal strain space behaved poorly in comparison to the others. The slopes of BV/TV are significantly higher than those of fabric eigenvalues, suggesting that BV/TV plays a more important role in these regressions; they also suggest that the higher the BV/TV and fabric eigenvalues, the higher the damage is. Results in Levrero-Florencio et al. ([@B24]) showed that the damage in the orthotropic coefficients of the macroscopic stiffness tensors do not have significant dependencies on BV/TV or fabric, for each of the considered load cases. In this study the Frobenius norm \|\|\[𝔼~0~ − 𝔼~dam~\]\|\| is used instead, which takes into account the damage of all the components of the macroscopic stiffness tensor. Therefore, the slopes and *p*-values in Table [3](#T3){ref-type="table"} suggest that lower BV/TV samples have a more anisotropic damage behaviour in the sense that the longitudinal trabeculae are more damaged than the oblique, and that higher BV/TV samples have a more isotropic behavior, or are more damaged in general. Even if fabric eigenvalues have a significant effect on \|\|\[𝔼~0~ − 𝔼~dam~\]\|\|, the considerably lower slopes suggest that their relevance is significantly lower than that of BV/TV.
The standard errors of the estimate (SEE) and the exponents with respect to BV/TV and fabric eigenvalues of five different damage models indicate that the SEEs are not substantially different in all these considered models, this is because, despite the anisotropic damage behavior, all the components of the stiffness tensor are damaged (Levrero-Florencio et al., [@B24]), suggesting that while a combined isotropic and anisotropic model is most suitable for simulating the macroscopic damage behavior of trabecular bone, an isotropic model is not necessarily poor. The SEEs of the models with dependencies are not substantially lower to those without the dependencies, suggesting that the considered BV/TV and fabric eigenvalue dependencies may not be needed. Nonetheless, the results of the multi-linear regressions (Table [3](#T3){ref-type="table"}) show significance of BV/TV and fabric eigenvalues when modeling damage. Furthermore, since these five assessed damage formulations only partially model some of the features of the macroscopic damage behavior of trabecular bone mentioned earlier, the dependencies are maintained in the combined isotropic/anisotropic model with tension/compression asymmetry.
It is apparent that the model with a combined isotropic/anisotropic behavior and tension/compression asymmetry is a substantial improvement over the single scalar damage formulation since the SEE is reduced by more than 15% (Table [5](#T5){ref-type="table"}). Nonetheless, it is important to mention that this model has 13 parameters instead of 2, though the value of the parameter *B*~0,aniso~ indicates that this parameter and the associated exponents expressing BV/TV and fabric eigenvalue dependencies can be ignored. The negative value of η suggests that if tension-dominated cases had similar strains to those in compression-dominated cases, the damage values would be higher in tension, as a negative value of η implies crack-closure, which is expected as bone could be considered a quasi-brittle material (Hambli, [@B15]; Mayya et al., [@B28]). The negative value of *B*~0,iso~ suggest that, when modeling the damage progression with a linear model, there is an initial presence of damage, which has been previously observed in Levrero-Florencio et al. ([@B24]) (the intercepts of the *y*-axis of the damage-accumulated plastic strain plots are not zero).
This study has a number of limitations. As previously mentioned, bone at the solid phase level is assumed to be ductile, i.e., while reduction in stiffness due to damage is included, fracture is not. This is perhaps appropriate for the considered level of loading, but it is indeed not applicable if large strains are applied, as complete fracture of trabeculae can occur. Nawathe et al. ([@B30]) shows that ductile tissue behavior overestimates the experimental yield properties. Another limitation, previously stated in Levrero-Florencio et al. ([@B24]), is that although there is plenty of experimental data on uniaxial load cases (Keaveny et al., [@B20]; Bayraktar and Keaveny, [@B3]; Sanyal et al., [@B37]; Manda et al., [@B27]), these physical experiments do not allow evaluation of stiffness for samples subjected to different load cases and the effect of loading in one direction on the behavior in the others. Therefore, a study completely based on numerical simulations is the only alternative even though the results cannot be currently validated experimentally. The use of kinematic uniform boundary conditions in the μFE analyses could also be considered a limitation, as they are known for providing an upper bound of the stiffness tensor (Pahr and Zysset, [@B32]; Wang et al., [@B51]) or macroscopic yield (Panyasantisuk et al., [@B35]), and may also affect the damage morphology when compared to the *in situ* case (Daszkiewicz et al., [@B12]). We also assume that the orthotropic directions do not rotate during loading, which may be a valid assumption for the considered range of strains.
Use of a large number of load cases (21) and samples (10) shows that the evolution of the damaged macroscopic stiffness tensor is based on the loading history. By examining relationships between bone microstructural indices (such as BV/TV and fabric) with macroscopic damage constitutive laws, we show that the proposed combined isotropic/anisotropic damage law with tension/compression asymmetry is a viable superior alternative to the widely used single scalar isotropic damage formulation as it reduces the fitting error from 37 to 22%; it does, however, require specification of a larger number of material parameters. The relationships of damage progression with bone\'s micro-architectural indices (density and fabric) developed in this study provide an approach for the creation of macroscale continuum models that incorporate damage and will, therefore, improve clinical predictions of the behavior of bone and bone-implant systems.
Author contributions {#s6}
====================
FL-F designed the study, performed the FE simulations and parameter fittings, and analyzed the data; PP contributed to the design of the study. Both authors contributed to the critical writing and revision of the manuscript.
Conflict of interest statement
------------------------------
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We gratefully acknowledge Dr. Lee Margetts, from The University of Manchester, for his assistance with the implementation and development of the used version of ParaFEM.
**Funding.** This work was supported by funding from the Engineering and Physical Sciences Research Council grant EP/K036939/1. The authors gratefully acknowledge ARCHER, UK National Supercomputing Service, for access to their Cray XC30 supercomputer under the project "Modelling the nonlinear micromechanical behaviour of bone".
[^1]: Edited by: Alfons Hoekstra, University of Amsterdam, Netherlands
[^2]: Reviewed by: Bradley John Roth, Oakland University, United States; Kumari Sonal Choudhary, University of California, San Diego, United States
[^3]: This article was submitted to Computational Physiology and Medicine, a section of the journal Frontiers in Physiology
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Clinical Case Reports 2017; 5(11): 1785--1788
Introduction {#ccr31184-sec-0001}
============
The *GJB2* gene encodes the connexin 26 (CX26) protein, which is a major structural component of gap junction channels between cells, and plays a vital role in hearing physiology [1](#ccr31184-bib-0001){ref-type="ref"}. To date, more than 150 different variants have been reported in the *GJB2* gene [2](#ccr31184-bib-0002){ref-type="ref"}, of which many have been identified as pathogenic (e.g., c.35delG, c.235delC) or benign mutations in nonsyndromic hearing loss based on clinic and genetic evidence. However, little is known about those rare alleles of *GJB2* pertaining to their clinic implications due to lack of documentation. Here, we report two heterozygous carriers of c.464A\>G variation (dbSNP ID: rs776335087) in the *GJB2* gene in a Han Chinese family. Our case report may provide information to clinicians and genetic counselors.
Case Report {#ccr31184-sec-0002}
===========
A three‐generation Han Chinese family was referred to our institute for genetic counseling (Fig. [1](#ccr31184-fig-0001){ref-type="fig"}). The young couple, patients II‐3 and II‐4 presented with severe hearing loss and were unable to communicate orally. According to their medical records, the possibility of drug‐induced deafness cannot be excluded. Prior to their visit to our institute, patients II‐3 and II‐4 received targeted gene sequencing in a local genetics hospital. In terms of the report, the diagnostic panel was designed to target exons of 143 genetic deafness‐associated genes, six deafness‐associated mitochondrial DNA regions, and three microRNAs (Tables [S1--S3](#ccr31184-sup-0001){ref-type="supplementary-material"}). Targeted sequencing reveals that patient II‐3 is heterozygous for c. 464A\>G variation in *GJB2*, and patient II‐4 is heterozygous for both c.235delC and c.176‐191del16 mutations in *GJB2*. No other mutations were detected by the diagnostic panel.
{#ccr31184-fig-0001}
To find out whether c.464A\>G variation in patient II‐3 is a *do novo* mutation or parentally inherited, we recommended Sanger sequencing of exons of the *GJB2* gene for the family. Written informed consent was received from the family and five members (I‐1, I‐2, II‐2, II‐3, and III‐1) provided periphery venous blood samples for analysis. Based on sequencing results (Fig. [2](#ccr31184-fig-0002){ref-type="fig"}) and the pedigree, the genotypes of all participants are deduced (Fig. [1](#ccr31184-fig-0001){ref-type="fig"}), and we consider that patient II‐3 most likely inherited the c.464A\>G allele from I‐2, his mother.
{#ccr31184-fig-0002}
Discussion {#ccr31184-sec-0003}
==========
Mutations in the *GJB2* gene are the most common causes for autosomal recessive nonsyndromic hearing loss (NSHL) [3](#ccr31184-bib-0003){ref-type="ref"}, [4](#ccr31184-bib-0004){ref-type="ref"}. In the study by Zheng et al. which involved 1067 Han Chinese subjects, mutations in the *GJB2* gene are responsible for approximately 34.96% of NSHL, and c.235delC is the most frequently observed pathogenic mutation [5](#ccr31184-bib-0005){ref-type="ref"}. In this report, multiple variations in the *GJB2* gene have been detected in a Chinese family. ClinVar records (<https://www.ncbi.nlm.nih.gov/clinvar>) show that c. 79G\>A variation is generally benign, whereas c.109G\>A variation is pathogenic or likely pathogenic. In a recent study, homozygosity of c.109G\>A variation is reported to associate with a broad spectrum of hearing phenotypes in Chinese, which can be mild to profound hearing loss or totally normal hearing [6](#ccr31184-bib-0006){ref-type="ref"}. In this study, both individuals I‐1 and III‐1 are heterozygous carrier of c.109G\>A variation and have normal hearing based on self‐report.
Exome Aggregation Consortium has deposited c.464A\>G variation of the *GJB2* gene in its online database (ExAC, <http://exac.broadinstitute.org/>), with one c.464A\>G allele being detected among a total of 121,052 alleles from subjects representing diverse ethnicities, equaling to an allele frequency of 8.26 × 10^‐6^. Here, we observed two heterozygous carriers of c.464A\>G allele in a Chinese family namely the proband and his mother. The proband\'s mother also carries c.79G\>A variation and exhibits normal hearing. To the best of our knowledge, this is the first report of c.464A\>G variant of *GJB2* in Han Chinese. However, our findings are far insufficient to link c.464A\>G allele with hearing phenotype and more pedigrees carrying this allele are needed to address this issue.
The c.464A\>G variation in the *GJB2* gene leads to substitution of tyrosine 155 by cysteine in its encoding protein. With the well‐annotated 3D structure of GJB2 protein (<http://www.ebi.ac.uk/pdbe/entry/pdb/5ER7>), it is easy to know that tyrosine 155 localizes in the C‐terminal within a helix structure containing amino acid residues 136--156, where tyrosine 155 is engaged with multiple neighbor residues via immediate atomic contacts (Fig. [3](#ccr31184-fig-0003){ref-type="fig"}). We speculate that cysteine substitution may interrupt the residue interactions that tyrosine 155 holds. However, it is currently unknown whether the substitution results in a loss‐of‐function or even pathogenic protein. It will be an interesting study to test this mutant in cell line models in the future.
{#ccr31184-fig-0003}
Collectively, our case report shows two heterozygous carriers of c.464A\>G variation in the *GJB2* gene, and the pathological significance of c.464A\>G allele remains to be ascertained.
Conflict of Interest {#ccr31184-sec-0005}
====================
The authors have no conflict of interest to declare.
Authorship {#ccr31184-sec-0006}
==========
GHZ: designed the study and analyzed data. HYS: performed experimental studies. HYZ: performed experimental studies. YC: designed the study and interpreted data. FZ: performed physical examination. BN: acquired samples. WQX: analyzed data, made figures, and wrote the manuscript.
Supporting information
======================
######
**Table S1**. List of genetic deafness‐associated genes in targeted gene sequencing.
**Table S2**. List of deafness‐associated mitochondrial DNA regions.
**Table S3**. List of deafness‐associated microRNAs.
######
Click here for additional data file.
This work was supported by Projects (81570928, 81400457, 31470948, 81602389, and 81100716) from the National Nature Science Foundation of China and the Development and Reform Commission of Hunan Province.
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Dejun Yu (Te-tsun Yu, Feb. 1, 1908--July 14, 1986) was a famous plant taxonomist and horticulturist in China as well as an expert on botanical gardens (Fig. [1](#Fig1){ref-type="fig"}). As a patriotic scientist, Dejun Yu refused a position in Britain and returned to China without hesitation in 1950. He devoted his whole life to the botanical sciences. His contributions went far beyond advancing botany, however, extending to construction of botanical gardens and the exploration of plant resources, especially fruit trees in China. His research was closely connected to the scientific and economic development of his country.Figure 1Dejun Yu (Te-tsun Yu, Feb. 1, 1908--July 14, 1986)
Dejun Yu began his career in plant taxonomy at the Fan Memorial Institute of Biology upon his graduation from the Department of Biology at National Beiping Normal University in 1931. His first important collecting task was three-year plant hunting in western Sichuan province from 1932 to 1934. He started his second hunting journey with his team to northwest Yunnan province, which went from 1937 to 1939, three years later. The regions he went to in the 1930s suffered from warlord conflicts, serious poverty, isolation, poor transportation and complicated natural environment, but also had high biodiversity. Such explorations, therefore, meant a lot of dangers and challenges on one hand, and great contributions to the progress of plant taxonomy and investigation in China on the other hand. Dejun Yu often hired local people as carriers, guides and translators, and got along well with them. A villager from the Dulong River valley, Zhiqing Kong, once shared their stories. Dejun Yu was the first botanist collecting plants around Dulong River region. Sometimes he even risked his life to collect species. He also always worked hard on recording and organizing specimens after long trudges in the jungle. He established deep friendship with local people including Zhiqing Kong, teaching him mandarin and helping him enter school, which made Kong become the first educated Dulong person (Kong & Zhou, [@CR2]).
Dejun Yu was both a field botanist and an armchair botanist, and often shifted between the two when he focused on his taxonomic research, including families of Rosaceae, Leguminosae, Begoniaceae and Theaceae. He and his students spent over 20 years on the taxonomy of Rosaceae plants, organizing and checking nearly 300,000 specimens collected all over the country. However, this did not mean they completely relied on specimens. They often travelled to investigate plants *in situ*, especially to identify new species. In 1980, Dejun Yu and his student Chaoluan Li found a new genus named *Taihangbia* by identifying a specimen. They affirmed it by travelling to Taihang Mountain and finding its type species *Taihangbia rupestris*, and then published this genus. His team finally compiled three volumes of *Flora Republicae Popularis Sinicae* (FRPS) on this family, Vol. 36--38 (Anonymous, [@CR1]). Supported by the Kew in London, UK, Dejun Yu did research and received advanced training in taxonomy of higher plants, horticulture, establishment and management of botanical gardens at the Royal Botanic Garden Edinburgh and the Kew from 1947 to 1950. In Britain, he was inducted into International Camellia Society because of his highly praised paper *Camellia plants and their cultivars in Yunnan Province*, *China*. From 1978 to 1986, he served as the acting editor-in-chief and then editor-in-chief of FRPS, as many as 35 volumes of which were published during that period. His another contribution to taxonomy was his research on fruit trees. In 1979 he compiled *Taxonomy of Chinese Fruit Trees*, collecting 59 families and 670 species, including detailed information on morphology, chromosome number, phenology, distribution, and cultivars. This monograph also won him the first prize of "National Excellent Monographies of Science & Technology" in 1982.
During his visit in Britain, Dejun Yu realized how important role botanical gardens played in research, economic development and science popularization. He devoted himself to the foundation of the Beijing Botanical Garden (BG) as soon as he returned to China. In order to find a proper place for the new garden, Dejun Yu led a team of experts to explore a dozen places all over Beijing city and compare them, and finally built it at the foot of Xiangshan Mountain. He was then appointed as the director of Beijing BG, engaged in the design, planting, building and daily management of it. Besides founding Beijing BG, he also greatly helped with the establishment of quite a few botanical gardens in China including: Lushan BG, Wuhan BG, Xishuangbanna Tropical BG, Hangzhou BG, etc. (Fig. [2](#Fig2){ref-type="fig"}). Along with the development of botanical gardens in China, he compiled *Handbook of Botanical Gardens*, an important guide to the building of modern botanical gardens in China, started the journal *Collections of Plant Introduction and Domestication*, to introduce research and progress achieved by these newly built gardens, and published the album of painting *Chinese Botanical Gardens*. As far as the introduction of plants is concerned, Dejun Yu proposed six principles of recording for each plant: 1. Introducing year, number and origin; 2. Accurate Chinese and Latin names; 3. Picture of planting and ID card; 4. Complete phenological records; 5. Detailed biological description; 6. Photo, seed samples and exsiccate (Anonymous, [@CR1]). These requirements were identified with his ideas on the aims of botanical gardens, especially those under the administration of Chinese Academy of Sciences, namely research first, followed by protection of plants species and especially cultivation of economic plants for the need of agriculture.Figure 2Bronze Statue of Prof. Dejun Yu located in South China Botanical Garden, Chinese Academy of Sciences (SCBG, CAS)
Besides the taxonomy and botanical gardens, Dejun Yu's interests in botany also extended to plant resources, especially research and cultivation of fruit trees and ornamental plants, both closely related to his taxonomic research. During Dejun Yu's life, China experienced wars, deep poverty and the "Great Cultural Revolution". He was very sensitive to species that may be cultivated as economic or ornamental plants. Many species of Rosaceae and Theaceae, the two families he specialized in, have been cultivated in agriculture and horticulture. According to his research, Chinese plants had been greatly introduced around the world. There were over two hundred fruit species around the world native to China. Various Chinese flower plants were blooming in European gardens, which won China a name of "Mother of Gardens" (Yu, [@CR4]). Specifically, he contributed to the exploration of plant resource by means of collecting wild species with potential for cultivation during his plant hunting, exploring fruit species all over the country and trying to improve them, cultivating new species in the botanical garden, inviting experts from the Soviet Union to China to perform research and give advice on cultivating fruit trees, carrying research on fruit trees and publishing monography on the taxonomy, etc.
Dejun Yu also stood firm to protect the science and resources of his own country. He told the history of plant exploration and plunder by western imperialists in China, and expressed his anger in an article. He mentioned that, plant hunters often collected seeds, specimens and even living economic and ornamental plants for their own countries. They even stole processing technology of plant products (such as tea making) and information of natural resources and environment, and sold plants or their seeds directly. Beautiful Chinese plants still living in European gardens, type specimens kept in their herbariums and Latin names of Chinese plants named after plant hunters are outstanding examples of their imperialism. Because of the imperial botany, China was greatly influenced on the development of plant resources and the progress of botanical sciences (Yu, [@CR3]). Given this situation, Yu and other Chinese botanists tried their best to study specimens of Chinese plants during their visits to Europe, especially type ones.
Dejun Yu passed away on the afternoon of July 14, 1986. His last wish was that there would be no farewell ceremony for him, and his body would be donated to a hospital for pathological anatomy. His name was carved on a marble tombstone as the founder of Beijing BG. He will also be remembered in the history of botany and botanical gardens.
Figure [1](#Fig1){ref-type="fig"}. is reproduced by permission of Institute of Botany, the Chinese Academy of Sciences. Figure [2](#Fig2){ref-type="fig"}. is kindly provided by Miss. Ruilan Huang, SCBG, CAS.
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Introduction {#s1}
============
Acute kidney injury (AKI) occurring in the setting of intensive care medicine (ICU) complicates the clinical course of approximately 40% of ICU patients \[[@SFS070C1], [@SFS070C2]\]. Its incidence is rising due to more aggressive diagnostic and therapeutic interventions in an ageing population with multiple comorbid diseases \[[@SFS070C3]\]. On average, 5% of ICU patients with severe AKI require renal replacement therapy (RRT) \[[@SFS070C4], [@SFS070C5]\].
AKI rarely develops as isolated organ dysfunction, but presents with a broad spectrum of severity, and has heterogeneous unpredictable clinical outcomes \[[@SFS070C6]\]. Undoubtedly, this patient population has a poor short-term outcome with excessive in-hospital mortality rates (40--60%), prolonged ICU and hospital stay as well as reduced health-related quality of life \[[@SFS070C7]\].
Prior to the era of chronic kidney disease (CKD) staging, it was generally accepted that patients who survive an episode of AKI had a 'good' renal outcome as assessed by a rapid return of renal function towards baseline values in most patients and by a low incidence of end-stage renal disease (ESRD) \[[@SFS070C8]\]. Traditionally it was thought that AKI was reversible and, as a consequence, survivors of AKI were not followed up. As recently as 2005, an epidemiologic surveillance study reported that 'although the majority of patients with severe AKI will die, most survivors will become independent from RRT within one year', but it did not mention the level of kidney function regained \[[@SFS070C9]\].
The systematic review and meta-analysis reported by Schmitt *et al.* \[[@SFS070C10]\] found that there is impaired recovery of kidney function after AKI in the aged (65 years and older). The recognition of age-related functional and structural alterations of human kidneys is of clinical importance, as the prevalence of elderly individuals admitted to hospital continues to increase over time, as does the incidence of AKI in these patients.
More recently, however, a number of studies assessing large-scale database cohorts of patients demonstrated that patients who survive AKI have a significant risk for the development of advanced stages (4/5) of CKD. In one of the first analyses linking AKI to progressive CKD, Ishani *et al*. \[[@SFS070C11]\] assessed a random sample of Medicare beneficiaries. They found that the adjusted hazard ratio for developing ESRD was 41 for patients with AKI on CKD relative to those patients without either acute or CKD. It was 13 for patients with AKI and without previous CKD, and 8.4 for CKD without AKI. Because this study utilized diagnostic codes, it is difficult to determine the proportions of patients who suffered AKI and then progressed directly to ESRD when compared with patients who suffered AKI, recovered renal function and then progressed to ESRD.
Lo *et al.* \[[@SFS070C12]\], utilizing a database from the health insurer Kaiser Permanente, evaluated retrospectively the risk of progressive CKD in a cohort of patients with baseline estimated glomerular filtration rate (eGFR) of at least 45 mL/min/1.73 m^2^. In this study, the investigators found that an episode of dialysis-requiring AKI was associated with a 28-fold increase of developing advanced CKD and a 2-fold increase in mortality compared with patients who did not suffer dialysis-requiring AKI.
Extending these lines of investigations, Bucaloiu *et al*. \[[@SFS070C13]\] retrospectively analysed the impact of AKI on long-term mortality and progressive kidney disease using a hospital database. The authors limited their analyses to patients with no evidence of pre-existing kidney disease who manifested complete recovery of kidney function after AKI. A total of 1610 discharged patients with reversible AKI (mostly AKIN stage I) were successfully matched with 3652 control patients who had not experienced AKI. Reversible AKI was associated with significant risk of *de novo* CKD and death. The authors identified numerous predictors of *de novo* CKD, including increasing age, lower baseline eGFR, burden of comorbidities and severity of AKI. However, it is important to recognize that retrospective database studies use codes for AKI and do not give the causes of AKI. They often lack actual baseline eGFR (within two months prior to renal injury), do not differentiate between CKD stages and underestimate the burden of comorbid disease and its effects on renal function. The recent meta-analysis published by Coca *et al.* \[[@SFS070C14]\] identified AKI as an independent risk factor for CKD, ESRD, long-term non-renal morbidity and death.
Palevsky \[[@SFS070C15]\] suggested two alternative models for the relationship among patient risk factors, development of AKI, and *de novo* CKD. According to the first model, risk factors predispose to the development of AKI. The development of AKI mediates the development of subsequent CKD, which increases mortality risk. The alternative model suggests that risk factors for the development of AKI are also risk factors for the subsequent development of CKD and mortality. In the second model, AKI may be either a direct mediator for the development of CKD and mortality or only a marker of risk. Whether or not AKI is a marker or mediator, there is a need for prospective follow-up for patients who sustain an episode of AKI.
The aims of our investigations were to quantify long-term mortality and incidence of *de novo* CKD in critically ill patients with AKI necessitating RRT. The analyses were based on the 10-year follow-up summary data from our prospective cohort study, the interim analyses at 1 and 5 years have been published \[[@SFS070C16], [@SFS070C17]\].
Materials and methods {#s2}
=====================
Study design {#s2a}
------------
This prospective single-centre 10-year follow-up observational study was carried out according to the principles of the Declaration of Helsinki. The nature of the study was explained in detail to the patients or their next of kin; they all consented to participate in the investigations. Great attention was paid to the use of non-identifiable data exclusively. The internal ethical board approved the study protocol \[[@SFS070C16]\].
### Study cohort {#s2a1}
Inclusion criteria, renal characteristics of patients, indication for initiation of RRT as well as the performance of RRT have been previously described in detail \[[@SFS070C16]\]. Briefly, 425 critically ill patients with severe AKI necessitating RRT at the dialysis unit of the University Hospital Munich-Innenstadt (1990--2001) formed the cohort. None of the patients had radiological signs of CKD or persisting functional abnormalities (urine microscopy, urine dipstick proteinuria, microalbuminuria defined as more than 30 mg/L at two evaluations within 3 months prior) or decreased glomerular filtration rate (GFR \<90 mL/min/1.73 m^2^). The GFR values were measured by creatinine clearance from 24-h urine or calculated by the Cockroft-Gault formula. The values were obtained from measurements performed within 3 months prior to initiation of RRT.
Only patients with a clinical diagnosis of acute tubular necrosis (ATN) participated in the cohort study. Presumed ATN was differentiated from other causes of AKI by detailed and accurate medical history, thorough physical examination, laboratory tests and imaging studies. A fractional excretion of sodium of more than 2% and the presence of granular casts on microscopic examinations of the urine were considered typical for ATN. None of the study patients underwent a kidney biopsy. A total of 143 patients with pre-existing CKD, or with AKI due to other causes than ATN, were excluded from the investigations. The severity of AKI at initiation of RRT was defined by the RIFLE classification scheme. All patients had either an acute increase in serum creatinine three times above baseline or anuria for 12 h (RIFLE Class F).
The indications for the initiation of RRT were volume overload with pulmonary oedema inadequately controlled with diuretics; hyperkalaemia refractory to conservative measures; the need for hyperalimentation in the presence of insufficient urinary output; uraemic signs or symptoms for which uraemia could not be ruled out as a precipitating cause; and/or a blood urea nitrogen concentration \>100 mg/dL \[[@SFS070C15]\].
### Outcome parameters {#s2a2}
The primary outcome variables of the study were specified in advance as (i) in-hospital all-cause mortality, (ii) long-term survival, (iii) recovery of renal function at hospital discharge and (iv) development of *de novo* CKD.
Clinical data during hospital stay were recorded at the day of initiation of RRT. In-hospital outcome (death, survival) and recovery of renal function at discharge were taken from the patient\'s medical record. Information related to long-term outcome (survival, eGFR) was obtained from the family doctor annually for a follow-up period of 10 years.
The follow-up of patients was performed by (a) annual calls to the patients\' family and (b) by an annual questionnaire sent to their treating physicians.
All patients were informed at initiation of the study period about the purpose and the necessary data collection for follow-up. The large majority of the survivors (226 patients) lived in the area in and around Munich. The death of the patient was either confirmed by the family or doctor or taken from the death register. Around 50% took up the offer to have their initial follow-ups performed in the out-patient department of our university. Almost all patients felt that the frequent follow-up contacts functioned for them as continued specialized care. Very few patients moved from Munich to another area in Germany. Their addresses were given to the follow-up centre. The follow-up at 10 years was mostly performed by the treating physicians (general practice, internal medicine practice, nephrologists in those with kidney dysfunction) of the patients.
Definition of recovery of renal function from AKI {#s2b}
-------------------------------------------------
Recovery of renal function was defined as complete if the decreased glomerular filtration rate returned to the pre-AKI baseline level of eGFR. Partial recovery of renal function was defined as persistent change of serum creatinine concentrations to more than 0.3 mg/dL above baseline, but without continued requirement for RRT. CKD was defined and staged according to the National Kidney Foundation (NKF) Clinical Practice Guidelines for CKD \[[@SFS070C18]\]. ESRD was defined as stage 5 of CKD maintained on regular dialysis.
Statistics {#s2c}
----------
Statistical analyses were performed with SPSS for Windows (Version 15, SPSS, Chicago, IL, USA). Continuous variables were reported as mean ± SD. Categorical data were expressed as proportions (percentage). Fisher\'s exact test was used to analyse the categorical data. Multivariate logistic regression analysis was performed to identify independent determinants of long-term mortality of hospital survivors. A two-sided *P*-value of \<0.05 was considered statistically significant.
Results {#s3}
=======
Characteristics of the cohort at initiation of RRT {#s3a}
--------------------------------------------------
The cohort consisted of 425 medical and surgical ICU patients with AKI secondary to clinically diagnosed ATN and a need for RRT (RIFLE Class F). The study population was characterized by a high mean age, heavy burden of comorbid diseases (cardiovascular, metabolic, pulmonary) and severe underlying illness (Table [1](#SFS070TB1){ref-type="table"}). None of the patients had pre-existing kidney disease. They all had normal GFR. Clinically diagnosed ATN arose in the presence of ischaemia (cardiovascular surgery, septic or cardiogenic shock), sepsis or nephrotoxins or often combined insults. However, no patient had more than one episode of AKI during the hospital stay. AKI presented in all patients as part of a multiple organ failure syndrome. In total, 75% of the patients required mechanical ventilation, 58% needed vasopressor support and 48% of the patients had oliguria at initiation of RRT. The main mode of RRT was intermittent haemodialysis (Table [1](#SFS070TB1){ref-type="table"}). RRT was initiated when one of the classical indications was present. Its frequency was prescribed according to the need of the patient (either alternate days or daily). The prescribed doses were a single-pool *K*~t~/*V* urea of 1.3 ± 0.3 (delivered dose sp *K*~t~/*V* 1.1 ± 0.3) for intermittent haemodialysis (IHD) and an effluent volume rate of 24.0 ± 2.4 mL/kg/h (delivered 20.5 ± 3.5 mL/kg/h) for continuous veno-venous haemodialysis (CRRT). Table 1.Patient characteristics at initiation of RRT^a^Number of patients425Age (years)65 ± 12Gender (M)66%Comorbid conditionsHypertension48%Diabetes mellitus22%COPD14%Chronic heart failure18%Setting of AKISurgery41%Medicine59%Severity of illnessAPACHE III88 ± 29Number of failed organs2.4 ± 0.2Renal dataGFR (mL/min/1.73 m^2^)^b^113 ± 12Presumed cause of ATNIschaemia60%Sepsis33%Nephrotoxins7%Markers of AKISerum creatinine (mg/dL)4.3 ± 1.1Oliguria48%RIFLE Class F100%Mode of RRTIHD68%CRRT13%IHD/CRRT19%[^1][^2]
Completeness of follow-up {#s3b}
-------------------------
None of the study patients was lost for follow-up.
Mortality of critically ill patients during in-hospital stay and long-term follow-up (Figure [1](#SFS070F1){ref-type="fig"}). {#s3c}
-----------------------------------------------------------------------------------------------------------------------------
The overall in-hospital mortality was 47%; the 1-, 5- and 10-year mortality rates were 65, 75 and 80%, respectively (Table [2](#SFS070TB2){ref-type="table"} and Figure [2](#SFS070F2){ref-type="fig"}). The corresponding mortality rates of survivors were 34, 53 and 62% (Table [2](#SFS070TB2){ref-type="table"}). Major clinical causes of deaths in survivors were congestive heart failure, acute myocardial infarction and sudden death (65%). Table 2.Long-term outcomes of critically ill patients surviving AKI requiring RRT1 year (%)5 years (%)10 years (%)Mortality rateCohort of AKI patients657580Survivors of AKI345362Renal recoveryNormal eGFR748786CKD 21130CKD 31133CKD 4343CKD 5138[^3] Fig. 1.Outcome of the study cohort. ICU, intensive care unit; CRR, complete renal recovery; PRR, partial renal recovery; NRF, normal renal recovery; CKD, chronic kidney disease. Fig. 2.Survival curve of the cohort of critically ill patients with AKI requiring RRT.
Long-term renal function in survivors of an AKI episode requiring RRT {#s3d}
---------------------------------------------------------------------
At hospital discharge, recovery of renal function was complete in 56%, i.e. 126 of the 226 survivors (assessed by eGFR). None of these 126 patients developed CKD during the 10 year follow-up period. Partial recovery of renal function was documented in 100 of the patients discharged from hospital (44%). During the first year of follow-up of these patients, low eGFR improved in 26 patients, normalized in 10 patients, showed no change in 56 patients and progressed in 8 patients. Thereafter, impaired eGFR remained stable or showed progression. In the subgroup of 90 patients with initial partial recovery of renal function from AKI, in 12 patients ESRD occurred within the follow-up period (Figure [3](#SFS070F3){ref-type="fig"}). None of these patients had suffered a second episode of AKI. Fig. 3.Number of survivors from AKI (RIFLE class F) developing end-stage renal disease during a 10-year follow-up period. ESRD, end-stage renal disease.
There were significant differences in the prevalence of chronic arterial hypertension among patients with normal renal function and those with CKD (45 vs. 76%, *P* \< 0.001). At the end of the 10-year follow-up, 85 patients of the 226 survivors were still alive. The majority of long-term survivors had normal renal function, five patients had CKD stages 2--4 and eight patients underwent maintenance dialysis.
The mortality rate of survivors regaining normal eGFR after AKI was significantly better than that of survivors with CKD (46 vs. 83%, *P* \< 0.001). Multivariate logistic regression analysis incorporating the variables age, gender, APACHE II score, comorbid disease index, mode of RRT, setting of AKI showed that independent predictors of long-term mortality were *de novo* CKD, extra-renal comorbid disease and surgical setting of AKI (Table [3](#SFS070TB3){ref-type="table"}). It should be noted that the subgroup of patients with sepsis was relatively small (35%) and the majority of these patients died during the hospital stay. Thus these survivors were too small a cohort to reach significant conclusions. Table 3.Independent predictors of long-term mortality of survivors of ICU AKIVariableOdds ratio (95 % confidence Interval)*P*-value*De novo* CKD4.3 (2.9--6.2)0.001Comorbidity2.9 (1.9--4.5)0.001Surgery1.5 (1.1--2.2)0.01[^4]
Discussion {#s4}
==========
Our prospective single-centre cohort study of 425 consecutive critically ill patients without evidence for pre-existing CKD demonstrates that AKI requiring RRT has significant long-term negative implications. AKI *per se* drives the excess in-hospital mortality of these patients and also increases the risk for long-term death. There is no doubt that AKI is causally associated with long-term outcomes and not simply a marker for unmeasured risk factors in a sicker patient population. The analysis of renal recovery from AKI in ICU patients without prior kidney disease clearly showed that partial recovery of renal function at hospital discharge was followed by progression to CKD.
AKI requiring RRT is associated with poor survival in ICU patients. The present report is the first dealing prospectively with the association between long-term mortality after critical illness and AKI requiring RRT. We found fatality rates in our ICU patients of 75% at 5 years and 80% at 10 years after initiation of RRT. These results are in line with published survival rates of 16--47% at 5 years \[[@SFS070C19]--[@SFS070C22]\] or of 17% at 10 years \[[@SFS070C23]\]. Variations in the long-term survival among studies utilizing different research methods are likely caused by the combined effects of differences in case mixture and demographic variables (severity of illness and burden of comorbidity). Furthermore, published survival analyses varied in the extent of loss to follow-up, restriction to homologous groups (cardiac surgery, \>65 years), and the use of administrative codes for AKI, acute illness or pre-existing comorbidity \[[@SFS070C24]\]. Our cohort consisted only of critically ill patients with defined extra-renal pre-existing diseases, a clear classification of AKI (RIFLE Class F) necessitating RRT, and a complete follow-up.
The survival analysis of our ICU cohort suggested a three-phase pattern of death after AKI requiring RRT. Phase 1, during the index ICU and hospital stay, was associated with a dismally high case-fatality rate. In phase 2, from hospital discharge and lasting several years, there is an excess of deaths in survivors compared with comparable ICU patients without AKI \[[@SFS070C22]\]. The duration of this phase differs among studies depending on the cause of critical illness. In phase 3, up to 10 years observation, the survival curves of our ICU patients flattened. Major determinants of long-term mortality of survivors of an AKI episode were *de novo* CKD, comorbidities and setting of AKI in our study population and in retrospective analyses \[[@SFS070C8], [@SFS070C19]\]. Although in our patients who died, no autopsy was performed, a significant proportion of the deaths are believed to have been caused by cardiac diseases. This observation is in accordance with findings reported by other authors that myocardial infarction and heart failure are more frequent in this population \[[@SFS070C22], [@SFS070C25]\].
Traditionally, AKI was considered a reversible condition, as GFR recovered in most of the survivors discharged from hospital. However, recovery of renal function is directly related to the cause of AKI \[[@SFS070C26], [@SFS070C27]\] and to a large extent to pre-existing CKD \[[@SFS070C28]\]. The frequency of ESRD in survivors of AKI due to ATN has been shown to be low, if pre-insult glomerular filtration had been low in the normal range \[[@SFS070C27], [@SFS070C29]\]. None of our patients had pre-existing CKD; they all had clinically diagnosed ATN as a cause of AKI. At hospital discharge, recovery of renal function was complete in 56% of surviving patients as assessed by serial determinations of eGFR. None of the patients discharged with normal eGFR showed any deterioration in renal function during their follow-up. ESRD secondary to progressive CKD occurred in 12 patients (3%) of the total cohort or 5% of all survivors. Serial measurements up to 10 years documented that in progressive CKD, eGFR declined continuously rather than abruptly.
There is broad experimental support for a causal link between AKI and post-AKI--CKD. Numerous studies demonstrate that AKI can induce permanent damage to the microvasculature and subsequent abnormalities in kidney structure and function. Through inflammation and fibrosis, the residual kidney damage can lead to progressive structural kidney damage, which may then predispose to faster decline of GFR by worsening hypertension \[[@SFS070C30], [@SFS070C31]\] which are well-known risk factors for cardiovascular disease.
The prospective design, long-term follow-up and completeness of data are the strength of our analysis. The data set may be of value for treatment decisions in the ICU or the discussions with patients or their relatives about the prognosis of AKI, at least in patients with normal GFR prior to the renal insult(s).
However, our investigations have some limitations. By nature, observational studies cannot prove that AKI plays a causal role in long-term outcomes of critically ill patients. However, the data obtained by our cohort study provide several lines of evidence, that AKI is not a short-term illness, but a renal syndrome with truly linked long-lasting effects on patient outcomes. Secondly, as this was a long-standing single-centre study, results may not be replicable in other settings. However, our cohort of aged surgical and medically critically ill patients with a burden of comorbid diseases reflects well the characteristics of current patient populations treated in mixed ICUs showing similar short-term outcomes \[[@SFS070C26], [@SFS070C32]\]. Randomized studies are needed to provide definitive proof for the AKI--CKD nexus.
There is an unmet need for strategies to prevent the development of AKI, to hasten the recovery of renal function and to minimize the adverse outcomes following AKI. Survivors of an episode of AKI should be closely followed-up after discharge to detect early development of CKD and its detrimental cardiovascular effects on long-term survival \[[@SFS070C33]\].
*Conflict of interest statement*. None declared.
[^1]: ^a^Results are given as mean ± SD or percentage. AKI, acute kidney injury; ATN, acute tubular necrosis; CKD, chronic kidney disease; GFR, glomerular filtration rate; RRT, renal replacement therapy; IHD, intermittent haemodialysis; CRRT, continuous veno-venous haemodialysis.
[^2]: ^b^Measured by creatinine clearance from 24-h urine or calculated by the Crockroft--Gault formula.
[^3]: AKI, acute kidney injury; CKD, chronic kidney disease; eGFR, estimated glomerular filtration rate; RRT, renal replacement therapy.
[^4]: CKD, chronic kidney disease.
| {
"pile_set_name": "PubMed Central"
} |
FDA Status: Not Cleared
**Summary:** Cartilage defects are frequently present in patients with FAI. Several arthroscopic treatment options are available including MFx and AMIC. In the present study tha AMIC technique using a collagen type I/III bilayer matrix (Chondro-Gide®) is having advantages in maintaining clinical improvements over time with less complications when compared to MFx in FAI.
**Purpose:** The aim of this study is a single-centre retrospective analysis of the efficacy of arthroscopic autologous matrix induced chondrogenesis (AMIC) compared to microfracture (MFx) for the treatment of acetabular cartilage defects in femoro acetabular impingement (FAI) at 7 years follow-up.
**Materials and Methods:** 50 patients (35 male, 15 female) were treated with arthroscopic MFx and 59 patients (27 male, 32 female) were treated with arthroscopic AMIC with the application of a collagen type I/III bilayer matrix (Chondro-Gide®). Patients were evaluated pre- and post-operatively by the modified Harris Hip Score (mHHS) al 6, 12 months and then yearly until final follow-up. Data are reported as mean±standard deviation. Mean patient age at surgery was 38±10 (range 19-54 years) for MFx and 39±9 (range 18-50 years) for AMIC. All patients presented grade III to IV chondral defects (ICRS classification) with a mean lesion size of 3.6±1.4 cm^2^ (range2-8 cm^2^) for MFx and 3.5±1.5 cm^2^ (range 2-8 cm^2^) for AMIC. 20 patients in the MFx an 24 patients in the AMIC group had a concomitant chondropaty of the femoral head treated with microfracture only.
**Results:** Baseline mHHS was 48±5 for MFx and 44±6 for AMIC. Major improvement was observed and comparable in the first 6 and up to 12 months after surgery, MFx 86±9 and AMIC 83±8. In the AMIC group this result was maintained over time with a mHHS of 81±7 at 7 years, while outcome in the MFx group deteriorated to 75±10. This deterioration was even more prominent in patients with large (=4 cm^2^) lesions. The mean mHHS improvement at 7 years follow-up with respect to pre-operative level was 26±10 for MFx ad significantly higher with 37±7 for AMIC. 9 patients in the MFx group subsequently required total hip arthroplasty (THA) compared to none in the AMIC group.
**Conclusions:** MFx and AMIC both led to clinical short-term improvement in patients with acetabular cartilage defects in FAI. AMIC, however, gave significantly better results which were maintained at a mean of 7 years follow-up, particularly in patients with large (=4 cm^2^) lesions. The conversion rate to THA was higher in the MFx group, with 9 patients (18%) requiring THA subsequently, compared to none in the AMIC group.
| {
"pile_set_name": "PubMed Central"
} |
Background
==========
The Effort-Reward Imbalance (ERI) model, a recent model of occupational stress, focuses on a negative trade-off between experienced \'costs\' and \'gains\' at work. In this model, high ratio of occupational effort spent relative to rewards received in turn in terms of money, esteem, job security, and career opportunities, elicits sustained stress responses and ill health \[[@B1]-[@B4]\]. This contractual reciprocity is frequent in cases where people have no alternative choice in the labor market or where they are exposed to heavy competition \[[@B5]\]. In addition to effort and rewards, the ERI model includes a third component, overcommitment, which refers to a set of attitudes, behaviours, and emotions reflecting excessive striving in combination with a strong desire to be approved of and esteemed. However, the evidence of adverse health effects is stronger for high efforts and low rewards (i.e., high ERI) than for overcommitment \[[@B6],[@B7]\].
In addition to work-related morbidity, the model assumes that high ERI promotes lifestyle risk factors, such as smoking, high alcohol consumption, unhealthy dietary habits, and sedentary behavior. However, empirical research to support this hypothesis is scarce. There is some evidence suggesting a relation of high ERI or some of its components with smoking \[[@B8]-[@B10]\], alcohol consumption or dependence \[[@B11],[@B12]\], and higher body mass index (BMI) \[[@B13]\].
Lifestyle risk factors tend to aggregate \[[@B14]\], and they may reinforce each other in their effects. The combined effects of adverse lifestyle factors have been demonstrated to be synergistic rather than additive \[[@B15]\]. No reports, to our knowledge, are available on the association between ERI and the co-occurrence of lifestyle risk factors.
We examined the relationship of ERI and its components to the co-occurrence of lifestyle risk factors in a sample of Finnish public sector employees. Our study aimed at adding to prior research in four ways. First, this is the first study to examine the ERI model in relation to the co-occurrence of lifestyle risk factors. Second, most previous studies have assessed ERI with individual-level self-reports. To control for individual differences in response styles, such as a personal disposition to answer negatively to questionnaires, we used occupational and organizational -level aggregated scores (in addition to individual-level scores) to model the effect of ERI. Aggregated scores were based on the responses of all the workers in the same occupation and organizational unit. Third, several third factors can confound associations in observational studies. We controlled for several confounders or possible predictors of risk factor co-occurrence, such as socioeconomic status (SES), age, marital status, and type of job contract (permanent vs. temporary). Fourth, very large data sets are required to examine co-occurring risk factors as co-occurrence of multiple risk factors is rare. To ensure statistical power, we focused on a large population covering over 36,000 employees in more than 1000 combinations of organizations and occupations. Indeed, this is one of the largest studies of the ERI model.
Methods
=======
Study design and study sample
-----------------------------
We used cross-sectional data from a large employee sample participating in an ongoing prospective Finnish Public Sector Study conducted in ten towns and 21 hospitals \[[@B16],[@B17]\]. All workers employed in these organizations were invited to participate. Participation was voluntary. 39,255 women and 9337 men aged 17 to 65 were examined through self-administered questionnaires in 2000--2002. Response rate was 68% and the sample did not substantially differ from the eligible population. In the ten town sub sample, figures for participants vs. eligible population (N = 47,351) were as follows: mean age 44.9 vs. 44.5 years, proportion of women 77% vs. 72%, proportions of upper non-manual, lower non-manual and manual employees 34%, 46%, 20% vs. 35%, 42% and 22%, respectively. The corresponding figures for the hospital sub sample (N = 23,610) were: mean age 43.1 vs. 43.1 years, proportion of women 87% vs. 84%, proportions of upper non-manual, lower non-manual and manual employees 16%, 77%, 8% vs. 13%, 81% and 7%, respectively.
Respondents who did not provide information about all four lifestyle risk factors were excluded (N = 3636). We also excluded those with missing data on age (N = 27). Moreover, the survey instrument for the personnel of seven hospitals did not contain the ERI measure and there were also some other missing cases for ecological level ERI (N = 9911) and for the covariates (N = 1032). In consequence, the data set of the present study comprised 28,844 women and 7233 men who provided complete data with respect to age, ecological ERI, all four examined risk factors, and all covariates (74% of the respondents to the baseline survey).
In the Finnish Public Sector Study, written consent was obtained from the participants for linking register-based information on sickness absences with survey responses. Regarding the questionnaire survey (the present data), written consent was not obtained as the study was approved by the Ethics Committee of the Finnish Institute of Occupational Health.
Measurements
------------
The standard measure of ERI in Finnish was not available in this study. The questionnaire used included one question about effort in work and three questions about rewards. These measures were used to construct the proxy measure of ERI.
Effort in work was measured with the following question: \"How much do you feel you invest in your job in terms of skill and energy?\" Rewards were assessed with a scale containing three questions about feelings of getting in return from work in terms of (1) income and job benefits, (2) recognition and prestige, and (3) personal satisfaction (Cronbach\'s Alpha = 0.64) \[[@B10]\]. Response format for all the questions was a five-point Likert scale ranging from 1 = \"very little\" to 5 = \"very much\". Rewards were assessed as a mean score of the three rewards questions. If half or more of the component items were missing, a value of missing was recorded in the total reward score. To measure ERI, a ratio of effort (numerator) and the mean of rewards (denominator) was computed in accordance with the procedure used in recent publications \[[@B3],[@B10],[@B18]\].
The mean scores for effort, rewards and ERI were additionally calculated for each occupational group (Statistics Finland codes) within each workplace (town or hospital), a total of 1049 groups each including 10 or more employees. The mean scores (ecological scores) were applied to all members of the group. For both the individual-level measures and ecological measures, we divided the participants into tertiles according to the distribution of effort and rewards scores in the total study population. Similarly, we constructed tertiles of the effort-reward ratio to identify a high-risk group in terms of the upper tertile, while the lowest tertile indicated the most advantageous position of low effort relative to rewards.
Risk factors assessed in this study included current smoking, heavy drinking, being overweight as defined by body mass index (BMI) ≥25 kg/m^2^\[[@B19]\], and physical inactivity. The four risk factors were dichotomized as to be adherent to the public health recommendation (no risk referred to non-smoking, non- or moderate drinking, BMI \<25 kg/m^2^, and physical activity ≥2 metabolic equivalent task hours per day).
Smoking was assessed by a question on whether the respondent was a current smoker or not. The respondents reported their habitual frequency and amount of beer, wine, and spirits intake. One unit of pure alcohol (12 g) is equal to a 12-cl glass of wine, a single 4-cl measure of spirits, or a 33-cl bottle of beer. A dichotomous variable was created to represent heavy drinking, with a cut-off point corresponding to an average weekly consumption ≥190 g \[[@B20]\] of absolute alcohol for women and \>275 g for men \[[@B21]\].
Participants reported the average amount of time spent per week on leisure and on the journey to and from work in physical activity corresponding to the activity intensity of walking, vigorous walking, jogging, and running. The time spent at each activity in hours per week was multiplied by its typical energy expenditure, expressed in metabolic equivalent tasks (METs). Activity MET index was expressed as the summary score of MET-hours/week. Participants whose volume of activity was \< 2 MET-hours/day were classified as being physically inactive \[[@B22]\].
Co-occurrence of lifestyle risk factors was defined as the number of risk factors for which an individual participant in question had high-risk values. If a participant belonged to the high risk group for all four risk factors, the corresponding co-occurrence score was 4; if the participant belonged to three \"high risk\" groups, the co-occurrence score was 3, etc. Since the number of employees having all four risk factors was very low \[0.3% (N = 98) of the women and 1% (N = 96) of the men\], we collapsed the co-occurrence score into four categories, ranging from 0 (having none of the four risk factors) to 3 (having three or four risk factors).
Potential confounders measured were: sex, age group (17--34, 35--50, and 51--63 years), marital status (married or cohabiting vs. single, divorced, or widowed), socioeconomic position, and type of job contract (permanent vs. fixed term). Information on sex, age, occupational title, and type of job contract was obtained from the employers\' records. The occupational titles, expressed as five-digit codes of Statistics Finland, were categorized into the socioeconomic positions of manual, and lower and upper non-manual work following the Statistics Finland classification.
Statistical analysis
--------------------
Multinomial logistic regression models were calculated to investigate the relationship of ecological and individual level effort, rewards, and ERI with the co-occurrence of lifestyle risk factors. Participants with one, two, and ≥3 risk factors were compared with those with no risk factors. We used the employees in the most favorable tertiles for each of the ERI dimensions as reference groups.
All models were fit separately for women and men. Due to different cut-off points of heavy drinking, exact numerical comparison of the results between women and men is not possible. The analyses were made in two phases. Firstly, the associations were examined adjusting only for age. Then, marital status, socioeconomic position, and type of job contract were added to the model as covariates to see how this affected the associations. To further test the association and to take into account the fact that individual employees were nested within work units, we performed the logistic regression analysis with generalized estimating equations (GEE) method \[[@B23]\] comparing those with ≥3 risk factors with those with 0 to 2 risk factors.
Binary logistic regression analyses were performed to examine the associations between ecological ERI and the likelihood of single risk factors.
The results are presented as odds ratios (ORs) and their 95% confidence intervals (CIs). Data were examined with SPSS for Windows 12.0.1 (SPSS, Inc., Chicago, Illinois) and SAS V8 (SAS Institute, Cary, NC) program packages.
Results
=======
Participant characteristics
---------------------------
The mean number of lifestyle risk factors by socio-demographic variables, and the prevalence of single risk factors are presented in Table [1](#T1){ref-type="table"}. The mean number of risk factors was 0.9 (SD = 0.9) for the women and 1.2 (SD = 0.9) for the men. Four percent of the women and 9% of the men had ≥3 risk factors. The proportion of women with none of the four risk factors was 38%, and the corresponding figure for the men was 24%. In both women and men, the mean number of risk factors was significantly higher among older people, manual workers, permanent employees, and among participants living without a partner. (p \< 0.001 in all cases.)
Association between ERI and the co-occurrence of risk factors
-------------------------------------------------------------
Adjusted ORs (95% CIs) for co-occurring risk factors among the women are presented in Table [2](#T2){ref-type="table"}. Exposure to high ecological ERI was associated with a higher likelihood of risk factor co-occurrence, and the excess risk persisted although lowered after adjustment for marital status, socioeconomic position, and type of job contract. In the fully adjusted model, the women with high ecological ERI (OR = 1.44, 95% CI 1.23--1.69) were more likely than their counterparts with low ERI to have ≥3 risk factors (vs. 0 risk factors). When examined separately, both components of ecological ERI, low effort and low rewards, were also associated with a higher likelihood of risk factor co-occurrence. The analyses using individual-level measures of ERI largely replicated the results.
Table [3](#T3){ref-type="table"} shows that the results for the men were mostly in the same direction as those for the women. In the fully adjusted model men with the greatest disparity between ecological effort and rewards had a 1.4-fold odds for ≥3 risk factors (vs. 0 risk factors) compared with the men with low ecological ERI. Low effort was statistically significantly associated with the co-occurrence of ≥3 risk factors before but not after adjustment for all of the covariates. However, low ecological effort was associated with the co-occurrence of 2 risk factors also in the fully adjusted model. Low ecological rewards were associated with the co-occurrence of risk factors irrespective of adjustments. The analyses using individual measures of ERI largely replicated these results. However, the association between low effort and a higher likelihood of the co-occurrence of ≥3 risk factors (vs. 0 risk factors) remained significant in the fully adjusted model.
With the exception of ecological effort and individual rewards in the men, among both the women and the men the associations for the comparisons of 1 vs. 0 risk factors and 2 vs. 0 risk factors were weaker than the associations for the comparisons of ≥3 vs. 0 risk factors.
The results of logistic regression analyses with GEE method were in the same direction as the results presented here suggesting that the hierarchical structure of the data was an unlikely source of major bias in this study.
Association between ERI and single risk factors
-----------------------------------------------
Table [4](#T4){ref-type="table"} presents the associations between ecological ERI and single dichotomous risk factors. After adjustment for age, marital status, socioeconomic position, and type of job contract, high ERI was associated with a higher likelihood of smoking (OR = 1.45, 95% CI: 1.33--1.58), physical inactivity (OR = 1.07, 95% CI: 1.00--1.15), and BMI ≥25 kg/m^2^(OR = 1.08, 95% CI: 1.02--1.15) among the women. Although high ERI was also related to these risk factors among the men in the age adjusted models, a significant association was detected only for physical inactivity (OR = 1.23, 95% CI: 1.07--1.41) in the fully adjusted models. Of the ERI components, lower effort and lower reward were associated with a higher likelihood of smoking among the women. Moreover, among both sexes, lower effort was associated with a higher likelihood of physical inactivity and being overweighted and lower rewards were associated with a higher likelihood of physical inactivity.
Discussion
==========
According to the effort-reward imbalance model, high effort-reward imbalance would be expected to be associated with a higher likelihood of the lifestyle risk factor co-occurrence. The present results from a sample of 36,127 Finnish employees gave moderate support for this hypothesis. High ERI at the ecological occupational and organizational level was associated with 40% higher odds of having ≥3 lifestyle risk factors (vs. 0 risk factors) as compared with jobs with low ERI. The results with the individual level scores were in the same direction than those obtained with the ecological scores.
When the ecological ERI components were examined separately, low rewards and low effort were associated with an elevated prevalence of risk factor co-occurrence. The first finding was in line with the hypothesis, whereas the latter result was unexpected as according to the theory of ERI, low effort should be associated with a lower rather than a higher likelihood of risk factor co-occurrence. As a result, the ERI model was only partially supported by our findings. Furthermore, given the unexpected findings for high efforts, the adverse effects of the total ERI measure may solely be due to the adverse effects of low rewards.
The measurement of effort with a single item not included in the standard assessment instrument may have contributed to an unexpected finding. Further research should preferable use the original ERI measures for efforts. Moreover, in our data, effort and rewards were significantly positively correlated (*r*= .24, p \< 0.001). Low effort might represent passive life orientation or lifestyle, which was manifested also with single risk factors: in our data, low effort was associated with a higher likelihood of sedentary lifestyle and being overweight. If our measurement of high effort tapped active lifestyle rather than work overload, then these results should not be interpreted as counterevidence for the ERI model.
The associations between ERI, its components and co-occurring risk factors were independent of individual-level confounders such as age, marital status, socioeconomic position, and type of job contract. Therefore, although the possibility of confounding by an unknown factor can never be totally excluded, a major bias in our study is unlikely. However, compared with the age adjusted model, the association weakened after adjustments and further analysis showed that the attenuation was mostly due to socioeconomic position. The effects of socio-economic and psychosocial factors are often difficult to separate. Rewards gained from work highly differ between socioeconomic groups. Employees with lower socioeconomic position typically report higher ERI at work and they suffer more from the adverse consequences of ERI with respect to cardiovascular risk \[[@B4]\]. Including socioeconomic position as a covariate in the model when studying the health effects of ERI may thus lead to an over-adjustment.
This is apparently the first study to show that high ERI at work is associated with the co-occurrence of multiple lifestyle risk factors. Our findings rely on a large survey with a reasonable response rate. The respondents represented the target population satisfactorily in terms of mean age and the distribution by socioeconomic position. Demographic information obtained from the employers\' registers was available to practically all participants. Findings were replicated using both individual and ecological measures of ERI. In cross-sectional data common method variance may artificially inflate relationships between variables and may bias the results concerning bivariate associations \[[@B24]\]. By using ecological approach, which is only possible in such large data sets as ours, problems related to common-method variance were largely avoided.
High cost -- low gain conditions at work, such as having a demanding but an unstable job, or achieving at a high level without being offered any promotion prospects \[[@B2]\] could generate feelings of frustration, negative attitudes toward work, low job satisfaction, as well as general passivity and apathy. The alienation from work could be associated with adoption of unhealthy behaviors \[[@B25]\]. It is possible that some workers with jobs and workplaces characterized by high ERI use unhealthy behaviors as a response for their unsatisfactory job conditions. For example, they may eat for comfort \[[@B26]\] and use smoking and excess drinking as a means of coping \[[@B27]\]. In addition, emotional stress may be an obstacle to initiate or maintain exercise behavior \[[@B28]\], or it may postpone decisions to quit smoking. Earlier research has indicated an association of mental distress \[[@B29]\] and depression \[[@B14]\] with the number of lifestyle risk factors. In a similar way, ERI has been associated with depression \[[@B30]\]. These and similar factors can potentially be among the mechanisms linking ERI with co-occurrence of lifestyle risk factors.
Earlier evidence, which is scarce and which relates to single risk factors, is characterized by relatively weak associations. In the present study, the associations of ecological ERI with single risk factors were less marked than those with the co-occurrence of risk factors with the exception that in women high ERI and low rewards were associated with current smoking as strongly as with the co-occurrence of ≥3 risk factors and among the men low rewards were more strongly associated with physical inactivity than with the co-occurrence of ≥3 risk factors.
There may be individual differences in behavioral reactions to stress, some people eating more and gaining weight during stress, some smoking more intensively when suffering from stress and some people becoming physically inactive during stressful periods of life. For this reason, the associations of ERI with specific risk behaviors are expected to be relatively weak, whereas the associations of ERI with having one or some of the risk factors (irrespective of their combination) are expected to be stronger. Risk factors tend to cluster in same individuals and the novel finding of this study is that high ERI may increase the likelihood of such a clustering. This finding emphasizes the importance of focusing on the co-existence of risk factors. Moreover, it has been shown that the co-occurrence of multiple lifestyle risk factors greatly influences morbidity and mortality and that the effects of negative health practices are cumulative. For example, Meng et al. \[[@B15]\] detected a positive synergistic effect between smoking and BMI in regard to mortality: whereas the sum of the excess risk ratios of current smoking and BMI for male total mortality was only 1.3, but the observed combined risk ratio was 2.4.
Limitations of the study
------------------------
We used a large-scale sample, but the study was based on a cross-sectional design. Therefore we do not claim that the observed associations are evidence of a causal relationship. Although high ERI may lead to an increased likelihood of the co-occurrence of lifestyle risk factors, such unsatisfactory job conditions may sometimes also reflect these adverse behaviors, either as the effects of behaviors itself or as the effects of being a smoking, heavily drinking, overweight, and physically inactive employee. Workers with multiple unhealthy behaviors may not invest as much on their work and career than their healthy-living counterparts. Unhealthy lifestyle and poor health may lead to downward drift to low status jobs with high contractual non-reciprocity and job insecurity. However, that is a less likely explanation for the results based on ERI indicator derived from ecological scores.
The original ERI measure was not available in this study. In particular, our measure did not include overcommitment, which refers to a personal pattern of coping with work demands -- excessive striving in combination with a strong desire to be approved of and esteemed. Overcommitment is hypothesized to amplify the adverse health effects produced by ERI, because overcommitted workers exaggerate their efforts beyond the normally considered appropriate \[[@B1],[@B3]\].
However, the study by Fahlen et al. \[[@B31]\] showed that the approximate and the original ERI instrument pointed to the same direction and in general both studies using original and proxy measures have found support for the ERI model, indicating an effect of ERI regardless of the measure being used \[[@B6]\]. Furthermore, previous reports of this study cohort have shown an association between high ERI and increased body mass index \[[@B13]\] and smoking intensity \[[@B10]\], an indication of the predictive validity of our ERI measure. In spite of this, there is a possibility that our measure did not fully capture the ERI model and the Cronbach\'s Alpha for the rewards scale was moderate. These issues may have underestimated the associations observed. Further research with original ERI measures is therefore needed to confirm the present findings.
The magnitude of the associations was rather small. However, this study was not based on the assumption that ERI is the major determinant of lifestyle-related risk factors and their co-occurrence, but we rather hypothesized that ERI might be one of the factors influencing these behaviors \[[@B32]\]. Besides, cumulative or chronic ERI over a long period of time is associated with higher risk of disease compared to single assessment \[[@B33]\].
Although the use of aggregated data reduced problems related to common method variance and response bias, this ecological approach has a potential weakness. In aggregated scores the subjective component is largely excluded, and it is possible that the essence of the ERI model is just this subjective component, i.e., only perception of ERI is likely to elicit an adverse effect on health and health behaviors. The individual-level analysis might better capture that perceptual component and thus ecological analysis is likely to provide a conservative estimate of the true association between ERI and co-occurring risk factors.
The dichotomization of risk factors enabled assessment of co-occurring risk factors but may have reduced statistical power thereby underestimating the strength of the associations. If we had chosen lower cut-off points, the proportion of high-risk individuals would have increased notably. Moreover, the self-report nature of the data makes them subject to recall and response bias. Nonresponse and misclassification are likely to influence different behaviors to differing degrees. For example, self-reported current smoking is probably more precise than self-reported alcohol use \[[@B34]\].
Although the sample size was large, the present data were female-dominated and from the public sector. The respondents were representative of Finnish public sector employees in terms of sex and age, but the female predominance did not correspond to the sex distribution of the Finnish general working population. Therefore, the findings should be interpreted with caution until they are validated in studies using other samples.
Finally, we did not have sufficient information to include all important lifestyle risk factors. In particular, information on dietary habits was lacking. However, since BMI is influenced by energy intake (as well as physical activity), inclusion of it has probably partially accounted for dietary habits.
Conclusion
==========
To summarize, findings from a large public sector employee population indicate that failed reciprocity in work may be associated with the co-occurrence of the most important potentially preventable lifestyle-related risk factors that contribute to cardiovascular disease and other chronic diseases \[[@B35]\]. However, an unexpected association between low effort and a higher likelihood of risk factor co-occurrence as well as the absence of data on overcommitment (and thereby a lack of full test of the ERI model) warrant caution in regard to the extent to which the entire ERI model is supported by our evidence.
If confirmed by prospective studies with other study populations, this moderately supportive evidence implies that the reduction of effort-reward imbalance at work could help efforts to improve health behaviors among working population.
List of abbreviations
=====================
BMI = body mass index; ERI = effort-reward imbalance MET = metabolic equivalent task; OR = odds ratio.
Competing interests
===================
The author(s) declare that they have no competing interests.
Authors\' contributions
=======================
AK designed the study, carried out the data analyses and was the principal author of the paper. MK and JV are the directors of the Finnish Public Sector Study and the second principal investigators of the study; they helped in designing the study, the data analysis, interpreting the results, and writing the paper. MV and AL collected the data, contributed to interpretation of the results, and edited the manuscript. JP constructed the ecological measures and supervised the data analyses. TH and ME contributed to interpretation of the results and manuscript writing.
All authors read and approved the final manuscript.
Pre-publication history
=======================
The pre-publication history for this paper can be accessed here:
<http://www.biomedcentral.com/1471-2458/6/24/prepub>
Acknowledgements
================
This study was supported by grants from the Academy of Finland (projects 104891 and 105195), the Finnish Work Environment Fund (project 103432), the Finnish Association for Promotion of Occupational Health (AK), and the participating towns and hospitals.
Figures and Tables
==================
######
Descriptive statistics and the mean number of risk factors by sociodemographic variables and Effort-Reward Imbalance (ERI)
**Women (N = 28,894)** **Men (N = 7233)**
------------------------------------------------ ------------------------ -------------------- ----------- ------------
**Age group (yr)**
17 to 34 4913 (17) 0.7 (0.8) 1181 (16) 1.0 (0.9)
35 to 50 14,878 (51) 0.9 (0.9) 3543 (49) 1.2 (0.9)
51 to 64 9103 (32) 1.0 (0.9) 2509 (35) 1.3 (0.9)
p \< 0.001 p \< 0.001
**Marital status**
Married or cohabiting 21,506 (75) 0.9 (0.8) 5834 (81) 1.2 (0.9)
Single, divorced or widowed 7311 (25) 0.9 (0.9) 1399 (19) 1.3 (1.0)
p \< 0.001 p \< 0.001
**Socioeconomic position**
Manual 3579 (12) 1.1 (0.9) 2404 (33) 1.4 (0.9)
Lower non-manual 17,119 (59) 0.9 (0.8) 2057 (28) 1.2 (0.9)
Upper non-manual 8196 (28) 0.8 (0.8) 2767 (38) 1.0 (0.9)
p \< 0.001 p \< 0.001
**Type of job contract**
Permanent 23,833 (83) 0.9 (0.9) 6282 (87) 1.2 (0.9)
Fixed term 5061 (18) 0.8 (0.8) 951 (13) 1.0 (0.9)
p \< 0.001 p \< 0.001
**Effort-Reward Imbalance (ecological score)**
Low 9291 (32) 0.8 (0.8) 2826 (39) 1.1 (0.9)
Intermediate 10,202 (35) 0.8 (0.8) 2059 (28) 1.2 (0.9)
High 9710 (33) 1.0 (0.9) 2417 (33) 1.4 (0.9)
p \< 0.001 p \< 0.001
**Effort-Reward Imbalance (individual score)**
Low 9001 (31) 0.9 (0.8) 2657 (37) 1.2 (0.9)
Intermediate 11,085 (38) 0.9 (0.8) 2429 (33) 1.2 (0.9)
High 8758 (30) 0.9 (0.9) 2151 (30) 1.3 (1.0)
p \< 0.001 p \< 0.001
**Lifestyle risk factors**
Current smoker 4978 (17) 1715 (24)
Heavy drinker\* 2210 (8) 890 (12)
Physically inactive^†^ 7065 (25) 1950 (27)
Body mass index ≥25 kg/m^2^ 11,454 (40) 4252 (59)
**No. of risk factors**
Zero 10,958 (38) 1731 (24)
One 11,437 (40) 2918 (40)
Two 5323 (19) 19537(27)
Three or four 1176 (4) 647 (9)
Only participants with no missing data in any of the variables were included. (In analyses for the individual level ERI score, N = 28,544 for women and N = 7170 for men.)
SD, standard deviation
p values from Chi Square test.
\*Average weekly consumption ≥190 g of absolute alcohol for women and \>275 g for men.
^†^\< 2 MET hours.
######
Associations between Effort-Reward Imbalance (ERI) dimensions and co-occurrence of risk factors in women (N = 28,894)
**Age adjusted model** **Fully adjusted model\***
----------------------------- ------------------------ ---------------------------- ---------------------- ---------------------- ---------------------- ----------------------
**[Ecological score]{.ul}**
Effort (component of ERI)
Low 1.00 1.00 1.00 1.00 1.00 1.00
Intermediate **0.88**(0.82--0.94) **0.82**(0.75--0.89) **0.78**(0.67--0.90) 0.96 (0.90--1.03) 0.93 (0.86--1.01) 0.93 (0.80--1.09)
High **0.79**(0.74--0.84) **0.65**(0.60--0.70) **0.59**(0.51--0.69) 0.94 (0.87--1.01) **0.81**(0.74--0.89) **0.80**(0.67--0.95)
Rewards (component of ERI)
High 1.00 1.00 1.00 1.00 1.00 1.00
Intermediate 1.05 (0.98--1.11) **1.12**(1.03--1.21) 1.08 (0.92--1.26) 0.97 (0.95--1.11) 1.03 (0.94--1.12) 0.98 (0.84--1.16)
Low **1.26**(1.18--1.35) **1.61**(1.48--1.75) **1.90**(1.63--2.20) 1.03 (0.95--1.11) **1.22**(1.11--1.35) **1.35**(1.13--1.60)
Effort-Reward Imbalance
Low ERI 1.00 1.00 1.00 1.00 1.00 1.00
Intermediate ERI 1.00 (0.93--1.06) **1.10**(1.01--1.19) 1.05 (0.89--1.23) 0.98 (0.92--1.05) 1.07 (0.99--1.16) 1.02 (0.87--1.19)
High ERI **1.21**(1.13--1.29) **1.51**(1.39--1.64) **1.83**(1.57--2.12) **1.07**(1.00--1.15) **1.25**(1.15--1.37) **1.44**(1.23--1.69)
**[Individual score]{.ul}**
Effort (component of ERI)
Low 1.00 1.00 1.00 1.00 1.00 1.00
Intermediate **0.87**(0.80--0.95) **0.78**(0.69--0.86) **0.72**(0.60--0.87) **0.91**(0.84--1.00) **0.84**(0.77--0.96) **0.80**(0.67--0.97)
High **0.86**(0.78--0.94) **0.77**(0.70--0.87) **0.70**(0.58--0.86) 0.93 (0.85--1.02) **0.86**(0.77--0.96) **0.82**(0.67--1.00)
Rewards (component of ERI)
High 1.00 1.00 1.00 1.00 1.00 1.00
Intermediate **1.09**(1.03--1.16) **1.11**(1.03--1.20) 1.13 (0.97--1.31) **1.07**(1.00--1.14) 1.08 (1.00--1.16) **1.09**(0.93--1.27)
Low **1.17**(1.10--1.26) **1.33**(1.23--1.45) **1.69**(1.45--1.96) **1.11**(1.04--1.19) **1.22**(1.12--1.33) **1.51**(1.30--1.76)
Effort-Reward Imbalance
Low ERI 1.00 1.00 1.00 1.00 1.00 1.00
Intermediate ERI 1.01 (0.95--1.07) 0.99 (0.91--1.07) 0.98 (0.84--1.14) 1.02 (0.96--1.09) 1.01 (0.93--1.09) 1.01 (0.87--1.17)
High ERI **1.10**(1.02--1.17) **1.19**(1.10--1.30) **1.36**(1.17--1.59) **1.07**(1.00--1.15) **1.15**(1.06--1.26) **1.31**(1.12--1.52)
Only participants with no missing data in any of the covariates or ecological ERI were included in these models. (In the analyses for the individual ERI score, N = 28,544.)
ERI, Effort-Reward Imbalance; OR, odds ratio; CI, confidence interval
\*Adjusted for age, socioeconomic position, job contract, and marital status.
^†^Statistically significant at 95% confidence level or better bolded.
Risk factors are current smoker, BMI ≥25 kg/m^2^, physically inactive, and heavy drinker, where inactive individuals have \<2 MET-hours/day, and heavy drinkers are women who consume on average \>190 g of absolute alcohol per week.
######
Associations between Effort-Reward Imbalance (ERI) dimensions and co-occurrence of risk factors in men (N = 7233)
**Age adjusted model** **Fully adjusted model**\*
----------------------------- ------------------------ ---------------------------- ---------------------- ------------------- ---------------------- ----------------------
**[Ecological score]{.ul}**
Effort (component of ERI)
Low 1.00 1.00 1.00 1.00 1.00 1.00
Intermediate **0.77**(0.65--0.92) **0.57**(0.47--0.68) **0.47**(0.36--0.62) 1.00 (0.82--1.23) 0.86 (0.69--1.08) 0.80 (0.58--1.11)
High **0.60**(0.52--0.68) **0.43**(0.37--0.49) **0.36**(0.29--0.45) 0.88 (0.72--1.08) **0.75**(0.60--0.95) 0.78 (0.56--1.08)
Rewards (component of ERI)
High 1.00 1.00 1.00 1.00 1.00 1.00
Intermediate **1.50**(1.27--1.78) **1.65**(1.37--1.99) **1.65**(1.24--2.18) 1.15 (0.95--1.39) 1.24 (1.00--1.53) 1.15 (0.84--1.58)
Low **1.55**(1.36--1.77) **2.35**(2.03--2.72) **2.95**(2.39--3.64) 0.96 (0.78--1.18) **1.30**(1.04--1.63) **1.46**(1.06--2.01)
Effort-Reward Imbalance
Low ERI 1.00 1.00 1.00 1.00 1.00 1.00
Intermediate ERI 1.05 (0.91--1.21) 1.06 (0.91--1.25) 1.11 (0.88--1.40) 0.99 (0.86--1.15) 0.99 (0.84--1.16) 1.00 (0.79--1.26)
High ERI **1.41**(1.22--1.64) **1.93**(1.64--2.25) **2.32**(1.87--2.88) 1.06 (0.89--1.25) **1.22**(1.01--1.46) **1.36**(1.06--1.74)
**[Individual score]{.ul}**
Effort (component of ERI):
Low 1.00 1.00 1.00 1.00 1.00 1.00
Intermediate 0.85 (0.72--1.01) **0.72**(0.60--0.85) **0.60**(0.48--0.76) 0.95 (0.80--1.12) 0.85 (0.71--1.02) **0.75**(0.60--0.95)
High **0.80**(0.67--0.96) **0.60**(0.50--0.74) **0.44**(0.34--0.58) 0.96 (0.80--1.16) **0.81**(0.66--1.00) **0.64**(0.48--0.85)
Rewards (component of ERI)
High 1.00 1.00 1.00 1.00 1.00 1.00
Intermediate 1.13 (0.98--1.31) **1.35**(1.15--1.58) **1.30**(1.02--1.64) 1.03 (0.89--1.19) 1.16 (0.98--1.37) 1.06 (0.84--1.35)
Low **1.36**(1.17--1.57) **1.94**(1.65--2.28) **2.31**(1.85--2.89) 1.13 (0.97--1.32) **1.46**(1.23--1.73) **1.61**(1.27--2.04)
Effort-Reward Imbalance
Low ERI 1.00 1.00 1.00 1.00 1.00 1.00
Intermediate ERI 1.07 (0.93--1.23) 1.02 (0.87--1.19) 0.84 (0.67--1.06) 1.05 (0.91--1.21) 1.00 (0.85--1.17) 0.82 (0.66--1.03)
High ERI **1.23**(1.06--1.43) **1.45**(1.23--1.71) **1.53**(1.23--1.91) 1.12 (0.96--1.31) **1.26**(1.07--1.49) **1.29**(1.03--1.61)
Only participants with no missing data in any of the covariates or ecological ERI were included in these models. (In the analyses for the individual ERI score, N = 7170.)
OR, odds ratio; CI, confidence interval
\* Adjusted for age, socioeconomic position, job contract, and marital status.
^†^Statistically significant at 95% confidence level or better bolded.
Risk factors are current smoker, BMI ≥25 kg/m^2^, physically inactive, and heavy drinker, where inactive individuals have \<2 MET-hours/day, and heavy drinkers are men who consume on average ≥275 g of absolute alcohol per week.
######
Associations between ecological Effort-Reward Imbalance (ERI) dimensions and single risk factors (adjusted\* odds ratios, 95% confidence intervals)
---------------------------- ---------------------- ----------------------- --------------------------- ----------------------
**Smoking** **Heavy drinking**^†^ **Physical inactivity ‡** **BMI ≥25 kg/m**^2^
**Women (N = 28,894):**
Effort (component of ERI)
Low 1.00 1.00 1.00 1.00
Intermediate 1.01 (0.93--1.09) 1.00 (0.88--1.13) **0.91**(0.85--0.98) 0.99 (0.93--1.05)
High **0.89**(0.81--0.97) 1.11 (0.98--1.27) **0.82**(0.76--0.88) **0.93**(0.87--0.99)
Rewards (component of ERI)
High 1.00 1.00 1.00 1.00
Intermediate **1.17**(1.08--1.28) **0.88**(0.79--0.98) 1.01 (0.94--1.08) 0.96 (0.90--1.02)
Low **1.42**(1.30--1.56) 0.92 (0.82--1.05) **1.12**(1.03--1.21) 1.03 (0.96--1.11)
Effort-Reward Imbalance
Low ERI 1.00 1.00 1.00 1.00
Intermediate ERI **1.21**(1.11--1.31) 1.05 (0.94--1.16) 0.96 (0.89--1.02) 0.98 (0.92--1.04)
High ERI **1.45**(1.33--1.58) 0.99 (0.88--1.11) **1.07**(1.00--1.15) **1.08**(1.02--1.15)
**Men (N = 7233):**
Effort (component of ERI)
Low 1.00 1.00 1.00 1.00
Intermediate 0.97 (0.81--1.17) 0.96 (0.75--1.23) **0.80**(0.67--0.96) **0.84**(0.71--0.99)
High 0.92 (0.75--1.12) 1.05 (0.82--1.34) **0.81**(0.67--0.97) 0.96 (0.82--1.13)
Rewards (component of ERI)
High 1.00 1.00 1.00 1.00
Intermediate 0.97 (0.80--1.17) 1.04 (0.82--1.31) 1.16 (0.97--1.38) 1.09 (0.93--1.29)
Low 1.13 (0.93--1.37) 1.12 (0.88--1.43) **1.43**(1.19--1.72) 1.12 (0.96--1.31)
Effort-Reward Imbalance
Low ERI 1.00 1.00 1.00 1.00
Intermediate ERI 0.93 (0.80--1.07) 1.10 (0.93--1.32) 1.01 (0.88--1.15) 1.00 (0.89--1.13)
High ERI 1.13 (0.98--1.30) 1.08 (0.89--1.31) **1.23**(1.07--1.41) 1.08 (0.95--1.23)
---------------------------- ---------------------- ----------------------- --------------------------- ----------------------
Only participants with no missing data in any of the covariates, risk factors or ecological ERI were included in these models.
\* Adjusted for age, socioeconomic position, job contract, and marital status. Statistically significant at 95% confidence level or better bolded.
^†^Average weekly consumption ≥190 g of absolute alcohol for women and \>275 g for men.
‡ \<2 MET-hours/day
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#sec1-1}
============
Loss of permanent teeth among humans is always implicated in progression of dental caries and periodontal diseases in the surrounding teeth. Furthermore, tooth loss can effect individual\'s psychological, social, and physical impairment thereby declining the quality of life.\[[@ref1]\]
The World Health Organization (WHO) Global Oral Health Programme has identified dental caries, periodontal diseases, and dental trauma as the main causes of tooth loss.\[[@ref2]\] Previous studies have highlighted early tooth loss in primary and permanent dentitions.\[[@ref1][@ref3][@ref4]\] A recent study found tooth loss of 47.4% among adolescents in Eastern province of Saudi Arabia.\[[@ref5]\] Contextual variables such as socioeconomic conditions, access to dental care, unhealthy diet, tobacco use, clinical oral health status, oral health knowledge, and behavioral factors have been implicated in prevalence of tooth loss in Saudi Arabia.\[[@ref1][@ref5][@ref6][@ref7]\]
Oral-health-related quality of life (OHRQoL) is a multidimensional concept that incorporates physical, psychological, and social well-being components.\[[@ref8]\] Patient-based outcome measures are being used widely to get insight into people\'s perceptions and feelings about their health status to make provision of treatment of oral conditions and rehabilitation of tooth loss.\[[@ref8][@ref9][@ref10][@ref11]\] Of all the instruments developed to measure the OHRQoL, the 14-item Oral Health Impact Profile (OHIP-14)\[[@ref12]\] is the most commonly used to evaluate the impact of oral health on quality life in adults and the elderly.\[[@ref11]\] Recent systematic reviews have pointed out that the tooth loss has an impact on quality of life, irrespective of the type of instrument being used to measure the quality of life.\[[@ref13][@ref14]\]
Several studies have examined the impact of tooth loss on OHRQoL among adults and elderly population.\[[@ref15][@ref16][@ref17][@ref18]\] But none of the studies has reported the impact of tooth loss on OHRQoL of adults from Saudi Arabia. Hence, the main purpose of this study was to assess the impact of tooth loss on OHRQoL in adult patients seeking dental care in private university dental hospital in Saudi Arabia.
Materials and Methods {#sec1-2}
=====================
A cross-sectional study was conducted among the dental patients attending dental clinics of College of Dentistry, Riyadh Elm University (REU), Riyadh, Saudi Arabia, from September to December 2018. The study was registered with the research Centre of the Riyadh Elm University (FUGRP/2018/156) and ethical approval (RC/IRB/2018/1180) was obtained from the Institutional Review Board of REU (IRB approval received on 07-10-2018). Patient participation in the research was voluntary and an informed consent was obtained before start of the examination.
Sample selection {#sec2-1}
----------------
Only adult male and female patients attending Namuthajiya, Munasiya, and Olaya clinics were selected using convenient sampling methodology. Overall, 201 dental patients were screened, and of these 152 volunteers were invited to participate in the survey after meeting the inclusion criteria of having at least 18 years of age and at least one missing permanent tooth.
Sample size calculation {#sec2-2}
-----------------------
Considering effect size of *F*-test = 0.25, α error probability = 0.05, and power of the study 0.79 resulted in a sample size of 152 subjects. The sample size calculation was performed using G \* 3.1.9.4 power sample size calculator.
Oral examination {#sec2-3}
----------------
All the oral health examination was carried out by two trained examiners. Training and calibration sessions were held on 10 patients to unify the examination method and to understand the criteria for recording various dental indices.
Plaque index (PI) (Silness and Loe), gingival index (GI) (Loe and Silness), and complete periodontal examination were performed. Numbers of teeth present and missing were noted.
Assessment of OHRQoL {#sec2-4}
--------------------
The impact of tooth loss on health-related quality of life was assessed using Arabic version of OHIP-14,\[[@ref19]\] which consisted of 14 items with responses rated using a Likert-type scale (0 = never, 1--4 = very often). In addition, socioeconomic, sociodemographic, oral health data, and self-rated oral health were recorded.
Total OHIP-14 score was calculated by addition of all responses of 14 items with scores ranging between 0 and 56. OHIP-14 subscale scores for seven dimensions were obtained by summing the scores for the two items in each subscale. The questionnaire was self-administered.
Statistical analysis {#sec2-5}
--------------------
All the data analysis was performed using SPSS version 25.0 (SPSS® Inc., IBM Corp., Armonk, NY, USA) for Windows. Descriptive statistics of frequency distribution, percentages, and mean ± standard deviation (SD) values were calculated for the sample characteristics and OHIP-14 scores. Inferential statistics was done using Mann--Whitney U-test, Kruskal--Wallis *H*-test, and Spearman\'s correlation test. Level of statistical significance was set at probability values of less than 0.05.
Results {#sec1-3}
=======
Most of the study participants were females \[83 (54.6%)\], age 40--49 years \[46 (30.3%)\], working in government sector \[88 (57.9%)\], having college level of education \[85 (55.9%)\], with income of 5000--10000 SAR \[64 (42.1%)\]. The study participants brushed their teeth twice daily \[65 (42.8%)\] using toothbrush and paste (69.7%), 65.1% visited the dentist within the past 6 months, and 76.3% visited for treatment reasons. Self-rated oral health varied among the study subjects, with majority mentioning fair oral health \[69 (45.4%)\] with more than half \[78 (51.3%)\] lost 6--10 teeth \[[Table 1](#T1){ref-type="table"}\].
######
Characteristics of the study participants (*n*=152)
Variables *n* Percentage
----------------------------------------------- ---------------------------- ------- ------------
Age (years) 18-29 40 26.3
30-39 44 28.9
40-49 46 30.3
≥50 22 14.5
Gender Male 69 45.4
Female 83 54.6
Occupation sector Government 88 57.9
Private 64 42.1
Education ≤High school 67 44.1
College 85 55.9
Income (SAR) Less than 5000 53 34.9
5000-10,000 64 42.1
Above 10,000 35 23.0
Oral hygiene material Toothbrush with paste only 106 69.7
Miswak only 23 15.1
Tooth brush and floss 23 15.1
Frequency of tooth brushing Once/day 59 38.8
Twice/day 65 42.8
Thrice/day 28 18.4
Duration since last visit to dentist (months) 1-6 99 65.1
7-12 30 19.7
\>12 23 15.1
Reason for last visit Pain 29 19.1
Checkup 7 4.6
Treatment 116 76.3
Self-rated oral health Good 51 33.6
Fair 69 45.4
Poor 32 21.1
Severity of tooth loss 1-5 teeth loss 61 40.10
6-10 teeth loss 78 51.30
More than 10 teeth loss 13 8.60
The GI score (1.31 ± 0.73), PI score (1.16 ± 0.60), number of teeth present (25.07 ± 3.64), mean number of teeth lost (6.89 ± 3.45), clinical attachment loss (2.45 ± 0.77), and overall OHIP-14 score (12.96 ± 10.93) were observed in the study sample \[[Table 2](#T2){ref-type="table"}\].
######
Descriptive statistics of clinical dental variables and overall OHIP-14 scores
Clinical variables Mean SD Minimum Maximum
-------------------------- ------- ------- --------- ---------
GI score 1.31 0.73 0.00 3.00
PI score 1.16 0.60 0.00 2.30
Number of teeth 25.07 3.64 7.00 31.00
Tooth loss 6.89 3.45 2.00 19.00
Clinical attachment loss 2.45 0.77 1.19 6.09
Overall OHIP-14 score 12.96 10.93 0.00 50.00
OHIP-14: 14-item Oral Health Impact Profile; SD: standard deviation; GI: gingival index; PI: plaque index
The mean and SD of OHIP-14 scores were compared across different age groups (*P* = 0.209), gender (*P* = 0.99), workplace (*P* = 0.797), education (*P* = 0.52), and income (*P* = 0.522) and they did not show any significant differences \[[Table 3](#T3){ref-type="table"}\].
######
Comparison of overall mean OHIP-14 score among different socioeconomic variables
Variables *n* Mean SD SE 95% CI for mean Min Max *P*
-------------- -------------- ----- ------- ------- ------ ----------------- ------- ------ ------- -------
Age (years) 18-29 40 11.48 11.24 1.78 7.88 15.07 0.00 50.00 0.209
30-39 44 12.95 12.63 1.90 9.12 16.79 0.00 43.00
40-49 46 13.33 10.39 1.53 10.24 16.41 0.00 42.00
50 above 22 14.91 7.65 1.63 11.52 18.30 0.00 29.00
Total 152 12.96 10.93 0.89 11.21 14.71 0.00 50.00
Gender Male 69 12.74 10.50 1.26 10.22 15.26 0.00 43.00 0.99
Female 83 13.14 11.34 1.24 10.67 15.62 0.00 50.00
Total 152 12.96 10.93 0.89 11.21 14.71 0.00 50.00
Workplace Government 88 12.65 10.76 1.15 10.37 14.93 0.00 50.00 0.797
Private 64 13.39 11.23 1.40 10.58 16.20 0.00 43.00
Total 152 12.96 10.93 0.89 11.21 14.71 0.00 50.00
Education ≤High school 67 13.67 11.17 1.36 10.95 16.40 0.00 43.00 0.52
College 85 12.40 10.78 1.17 10.08 14.72 0.00 50.00
Total 152 12.96 10.93 0.89 11.21 14.71 0.00 50.00
Income (SAR) ≤5000 53 11.72 10.18 1.40 8.91 14.52 0.00 38.00 0.522
5000-10,000 64 14.41 11.99 1.50 11.41 17.40 0.00 50.00
\>10,000 35 12.20 9.97 1.69 8.78 15.62 0.00 35.00
Total 152 12.96 10.93 0.89 11.21 14.71 0.00 50.00
OHIP-14: 14-item Oral Health Impact Profile; SD: standard deviation; SE: standard error; CI: confidence interval
Physical pain (38.20%) was the most common response observed among the study participants followed by psychological disability (29.60%), with the least reported being functional limitation (5.90%).
The mean ± SD of OHIP-14 functional limitation subscale scores for 1--5, 6--10, and \>10 teeth loss were found to be 0.03 ± 0.18, 0.04 ± 0.19, and 0.31 ± 0.48, respectively. When the severity of teeth loss is compared with the mean subscale OHIP-14 score, functional limitations showed statistically significant differences (*P* = 0.000). Functional limitation was significantly higher among participants with \>10 teeth loss compared with the study subjects with 1--5 and 6--10 teeth loss. The severity of teeth loss in different categories compared with the mean social disability subscale OHIP-14 showed statistically significant differences (*P* = 0.044) \[[Table 4](#T4){ref-type="table"}\].
######
Mean subscale OHIP-14 scores and frequencies of "fairly often" or "very often" responses in relation to the number of missing teeth
OHIP-14 items Distribution of "often" or "very often" responses (%) Mean subscale OHIP score (±SD) 1-5 Severity of teeth loss
-------------------------- ---------------------------------------------------- ------------------------------------------------------- ------------------------------------ ------------------------ ----------------- ----------------- -----------
Functional limitation 1\. Trouble pronouncing any words 5.90% 0.06 (±0.24) 0.03^a^ (±0.18) 0.04^a^ (±0.19 0.31^b^ (±0.48) 0.000
2\. Sense of taste has worsened
Physical pain 3\. Had painful aching in your mouth 38.20% 0.47 (±0.65) 0.36 (±0.61) 0.50 (±0.64) 0.77 (±0.83) 0.116
4\. Uncomfortable to eat any foods
Psychological discomfort 5\. Been self-conscious 21.00% 0.22 (±0.45) 0.18 (±0.43) 0.24 (±0.46) 0.31 (±0.48) 0.449
6\. Felt tense
Physical disability 7\. Diet has been unsatisfactory 16.40% 0.20 (±0.49) 0.13 (±0.34) 0.24 (±0.56) 0.31 (±0.63) 0.523
8\. Had to interrupt meals
Psychological disability 9\. Difficult to relax 29.60% 0.36 (±0.59) 0.28 (±0.52) 0.36 (±0.58) 0.69 (±0.85) 0.176
10\. Been a bit embarrassed
Social disability 11\. Been a bit irritable with other people 22.40% 0.30 (±0.61) 0.21^a^ (±0.49) 0.29^a^ (±0.61) 0.77^b^ (±0.93) **0.044**
12\. Had difficulty doing your usual jobs
Handicap 13\. Felt that life in general was less satisfying 13.80% 0.16 (±0.44) 0.13 (±0.43) 0.14 (±0.35) 0.46 (±0.78) 0.114
14\. Been totally unable to function
Significant for bold values *P*\<0.05. OHIP-14: 14-item Oral Health Impact Profile; SD: standard deviation. Note: Different letters (a, b) in the same row indicate significant differences between groups (*P*\<0.05), and same letter in the single row indicates no significant differences (*P*\>0.05). ^¶^Kruskal--Wallis test
Comparison of the overall OHIP-14 score among different categories of tooth loss showed statistically significant differences (*P* = 0.005). Study participants with more than 10 teeth loss showed significantly higher overall OHIP-14 scores compared with the 6--10 and 1--5 teeth loss. While study participants with 6--10 teeth loss showed significantly higher overall mean OHIP-14 score compared with the 1--5 teeth loss \[[Figure 1](#F1){ref-type="fig"}\].
{#F1}
The overall OHIP-14 score showed a significant positive correlation (*r* = 0.325, *P* = 0.001) with tooth loss and clinical attachment loss (*r* = 0.346, *P* = 0.001) \[[Table 5](#T5){ref-type="table"}\].
######
Correlation between overall OHIP-14 score and clinical variables
Variables Correlation coefficient Sig. (two-tailed)
------------ ------------------------- -------------------
Tooth loss 0.325\*\* 0.001
GI score 0.027 0.745
PI score 0.125 0.125
CAL 0.346\*\* 0.001
\*\**P*\<0.01. OHIP-14: 14-item Oral Health Impact Profile; GI: gingival index; PI: plaque index; CAL: Clinical attachment loss
Discussion {#sec1-4}
==========
Studies conducted elsewhere in the past have shown an impact of tooth loss on OHRQoL.\[[@ref20]\] However, this concept is new with few studies being published from Saudi Arabia, especially on tooth loss and OHRQoL.
The findings of this study revealed that tooth loss has a definite impact on OHRQoL of the patients. The severity of impact on OHRQoL increased with higher number of teeth loss leading to greater oral impairment. Study participants with more than 10 teeth lost showed highest OHIP-14 score indicating higher oral impairment. Tooth loss was related to the gradient of OHIP severity based on the number of teeth lost as shown in [Figure 1](#F1){ref-type="fig"}. This result is similar to the study reported by Batista *et al*., in which the impact on OHRQoL was higher with loss of more than 13 teeth. Furthermore, the same study reported that tooth loss of up to 12 teeth including anterior teeth also had higher impact on OHRQoL compared with fully dentulous adults.\[[@ref15]\] Similar findings of impaired subjective oral health were more frequently reported among individuals with fewer natural teeth.\[[@ref21]\]
In this study, physical pain, psychological disability, psychological discomfort, social disability, and physical disability are the most common oral impacts affecting 38.2%--16.40% of the participants. Functional limitations and handicaps were the least severe impacts. This finding is in line with other reported study.\[[@ref9]\]
While other studies have reported substantial impact of socioeconomic factors on self-perceived OHRQoL\[[@ref15][@ref22]\] that was not seen in this study. In this study, females perceived higher effects on OHRQoL to a greater extent compared with males.
In this study, we observed that the total OHIP-14 score was significantly higher in subjects with more than 10 teeth loss compared with 6--10 and 1--5 teeth loss. This implies that as the number of teeth loss increased, the OHIP-14 score also increased. Presence of adequate number of functional teeth has positive relationship with chewing ability of an individual. Hence any conciliation in chewing ability might have negative affect on nutritional intake, OHRQoL, and improper food habits leading to poor general health outcomes.\[[@ref23]\]
We consider convenient sampling methodology and relatively small number of patients selected from single-university dental clinics and self-reported responses to the questionnaire are the limitations of our study.
Tooth loss significantly impacts the OHRQoL. Certain oral health awareness-related policies and camps should be organized so that people can retain their natural dentition for longer periods. This study highlights the need for more stringent primary preventive measures such oral health education and oral health promotion by the dentists to reach wider population base.
Conclusion {#sec1-5}
==========
Within the limitations of the study, it can concluded that tooth loss has a definite negative impact on OHRQoL of dental patients. As the severity of teeth loss increased, the OHIP-14 score also amplified indicating higher oral health impairments. Functional limitations and social disability were the most affected domains of OHRQoL among the dental patients with teeth loss. Hence, dentist should be well-aware of the consequences of teeth loss while treating the patients.
Financial support and sponsorship {#sec2-6}
---------------------------------
Nil.
Conflict of interest {#sec2-7}
--------------------
There is no conflict of interest.
| {
"pile_set_name": "PubMed Central"
} |
Background {#Sec1}
==========
The adverse cardiovascular and vasculotoxic effects of long-term exposure to high levels of inorganic arsenic in drinking water have been well characterized \[[@CR1]\]. Recent studies have demonstrated an increased risk of cardiovascular disease (CVD), ischemic heart disease (IHD) and mortality from low-moderate drinking water inorganic arsenic (iAs) exposure (10--20 µg/L) common in the United States (U.S.), particularly for patients with diabetes mellitus (DM) \[[@CR2]\]. Recent prospective cohort study data indicates the vasculotoxicity and cardiovascular disease risk of environmental pollutants, including inorganic arsenic, may be greater for individuals with diabetes \[[@CR2], [@CR3]\]. However, the mechanism of this increased risk of environmental exposures for diabetic vasculopathy has not been studied. Pathological and clinical studies consistently demonstrate that platelets play a key role in atherothrombosis \[[@CR4]\], and have shown the importance of the platelet-megakaryocyte hemostatic axis for vascular disease and CVD events \[[@CR5]--[@CR7]\]. Patients with DM exhibit increased platelet activity both in vitro and in vivo, and heightened platelet function may contribute to excess macrovascular risk in patients with DM \[[@CR8]\]. A previous in vitro study of iAs and atherothrombosis used very high concentrations of sodium arsenite and did not examine the effects of hyperglycemia on thrombotic risk \[[@CR9]\]. We examined whether glucose concentrations common in DM potentiate the effects of iAs on in vitro measures of platelet and megakaryocyte adhesion and activity.
Methods {#Sec2}
=======
Subjects {#Sec3}
--------
Whole blood was collected from healthy donors in the fasting state. Subjects were not on any antiplatelet therapy nor did they have any history of cardiovascular disease, metabolic syndrome or DM. All human experiments were performed in accordance with institutional and state guidelines. Phlebotomy was performed after 10 min of quiet rest. Blood was collected following a clean, problem-free venipuncture, using a 21-gauge needle after a 5 cc discard (a tourniquet was used to obtain access and was removed before blood collection). Blood was collected into vacutainer tubes containing 3.2% (0.105 mol/l) sodium citrate for platelet activity measurements. After collection, each tube was gently inverted 3 times and immediately transferred to the laboratory for processing.
Reagents {#Sec4}
--------
Sodium arsenite was dissolved in dH~2~0 for a stock concentration of 1000 µM then added to whole blood at a concentration of up to 10 µM for a total of 30 min, similar to prior studies \[[@CR10], [@CR11]\]. Similar procedures were performed to achieve concentrations of 0.1, 1, 5 µM sodium arsenite. [d]{.smallcaps}-glucose was dissolved in dH~2~O for a stock concentration of 500 mM then added to whole blood and megakaryocytes at concentrations of 5, 15 or 25 mM to approximate euglycemia (5 mM [d]{.smallcaps}-glucose ≈90 mg/dl blood glucose) to a range of hyperglycemia common in DM (15 mM ≈ 270 mg/dl, 25 mM ≈ 450 mg/dl).
Flow cytometry {#Sec5}
--------------
To examine the effect of iAs on platelet activity, we first measured platelet activation by assessing platelet P-selectin exposure and the presence of monocyte and lymphocyte platelet aggregates (MPA and LPA, respectively) in whole blood samples. We began with a 10 µM concentration of sodium arsenite used in prior in vitro models with aortic endothelial \[[@CR10], [@CR11]\] and vascular smooth muscle cell cultures \[[@CR12], [@CR13]\], a concentration 50--75% less than that used in prior studies of arsenic and thrombosis \[[@CR9]\]. P-selectin expression (CD62P) is a cell surface marker primarily expressed by activated platelets and involved in platelet adhesion. To identify platelet specific P-selectin, we performed flow cytometry on whole blood with CD42b and CD61 to constitutively expressed platelet glycoproteins 1b (GP1b) and IIIa (GPIIIa), respectively. Flow cytometric analysis was performed using the BD Accuri flow cytometer (C6 Flow Cytometer). Whole blood was incubated in the dark for 30 min at room temperature with APC-conjugated mouse antibody specific for CD42b (glycoprotein Ib) and FITC---conjugated mouse antibody specific for CD62P (P-selectin) (BD Biosciences) before the mean fluorescence intensity of P-selectin--bound antibody per 10,000 events was measured. P-selectin is a component of the alpha granule membrane of resting platelets that is only expressed on the platelet surface membrane after alpha granule secretion. In-vivo circulating degranulated platelets rapidly lose their surface P-selectin, but continue to circulate and function \[[@CR14]\]. Monocyte and leukocyte platelet aggregates provide complementary information on in vivo platelet activation; are independently associated with cardiovascular disease events; \[[@CR15], [@CR16]\] and were assessed as events positive to markers CD14-APC and CD45-APC, respectively, in addition to platelet marker CD-61. MPAs were defined as events positive to both monocyte markers (CD14-APC \[BD Biosciences\]) and the platelet marker CD61-FITC (Dako). Monocytes were identified by their staining with CD14-APC and by their characteristic orthogonal light scatter. Monocytes with adherent platelets were identified by CD14-APC positivity. LPAs were defined as events positive to both leukocyte markers (CD45-APC \[BD Biosciences\]) and the same platelet marker CD61-FITC (Dako). The leukocytes with adherent platelets were identified by CD45-APC positivity. Appropriate color compensation was determined in singly labeled samples and matched nonspecific antibody controls (Mouse IgG1 FITC \[BD Biosciences\]). For the co-incubation experiments, whole blood was first incubated with 5 and 15 mM [d]{.smallcaps}-glucose for 30 min. We then used lower concentrations of sodium arsenite at 0, 0.1, 1 and 5 µM which were added to solution and incubated for an additional 30 min. P-selectin expression with unstimulated and stimulated with thrombin 0.025 IU/ml (Sigma) was then assessed.
Cell culture and megakaryocyte gene expression {#Sec6}
----------------------------------------------
Meg-01 cells were purchased from American Type Culture Collection (VA) and cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen, CA, USA) at 37 °C in a 5% CO~2~ humidified atmosphere, consistent with prior studies \[[@CR17], [@CR18]\]. For adhesion assays, 18 mm glass coverslips (Fisher Scientific) coated with collagen (Helena Laboratories, Beaumont, TX, USA) were blocked with 1% BSA in 12-well plates \[[@CR17], [@CR18]\]. Meg-01 cells were stained for 10 min with 1 µM DiOC6 (Fisher Scientific), washed and incubated at 2.5 10^5^ cells/ml for 3 h with and without addition of iAs (0, 1, 5 and 10 µM) in presence of 5 or 25 mM [d]{.smallcaps}-glucose. After the supernatant was aspirated, adherent cells were gently washed with FBS. For each well, five random fields were captured and area of coverage was quantified using Image J (National Institutes of Health, Bethesda, MD).
Nuclear transcription factor kappa B (NFκB) gene expression was measured because of its roles in inflammation, platelet activation, and arsenic vasculopathy \[[@CR18]--[@CR20]\]. Other genes measured include monocyte chemoattractant protein-1 (CCL2) and CD36 that have also been associated with platelet degranulation, diabetes and inflammation. To measure these genes, total RNA was isolated from Meg-01 cells using the Direct-zol RNA Miniprep kit (ZymoResearch, Irvine, CA, USA) and quantified using a Nanodrop ND-2000 spectrophotometer (Wilmington, DE, USA). RNA was converted to cDNA using the iScript cDNA synthesis kit (BioRad). Gene expression of GAPDH and NFκB1 using the Sso fast Evagreen Supermix (BioRad) was assessed with real-time PCR (iCycler Real-Time Detection System, Eppendorf). The sequences of the NFκB1, CCL2 and CD36 primers used for qRT-PCR were CAGATGGCCCATACCTTCAAA and TTGCAGATTTTGACCTGAGGG, CCCAAAGAAGCTGTGATCTTCA and GCAGATTCTTGGGTTGTGGA, and CTATTGGGAAGGTCACTGCGA and CAGGTCTCCCTTCTTTGCATT, respectively.
Statistical analysis {#Sec7}
--------------------
All experimental values are represented as mean ± standard error of the mean (SEM). Differences in selected categorical variables between the respective comparison groups were analyzed with the χ^2^ test of statistical significance. Unpaired two-tailed *t* tests and ANOVA were used to examine differences in continuous variables overall and at each time point under study in the different comparison groups. A value of *P* \< 0.05 was considered statistically significant.
Results {#Sec8}
=======
We first examined the effect of a 10 µM iAs concentration used previously in endothelial and smooth muscle cell culture to assess the effects of inorganic arsenic exposure \[[@CR10]--[@CR13]\]. There was a clear increase in platelets expressing P-selectin by flow cytometry following incubation with 10 µM iAs (Fig. [1](#Fig1){ref-type="fig"}a, b). Compared to 0 µM iAs, the mean fluorescence intensity of P-selectin expression increased significantly after incubation with 10 µM iAs for both unstimulated and thrombin-stimulated platelets (Fig. [1](#Fig1){ref-type="fig"}c, d). We subsequently examined the effect of iAs on monocyte and leukocyte platelet aggregation (MPA and LPA, respectively) a different measure of platelet activity predictive of CVD events \[[@CR15]\]. Compared to 0 µM, incubation with 10 µM iAs significantly increased both MPA and LPA (Fig. [1](#Fig1){ref-type="fig"}e, f). These experiments demonstrate that sodium arsenite concentrations below those used in prior studies of platelet activation have significant effects on multiple measures of platelet activity \[[@CR9], [@CR21]\].Fig. 1Unstimulated (**a**, **c**) and thrombin-stimulated (**b**, **d**) platelet activitation by flow cytometry (**a**, **b**), mean fluorescence intensity (**c**, **d**) and platelet aggregation (**e**, **f**) to 0 and 10 μM sodium arsenite
Consistent with prior data \[[@CR8]\], platelet activity increased with increasing [d]{.smallcaps}-glucose concentrations (Fig. [2](#Fig2){ref-type="fig"}a, b). To investigate whether glucose and arsenic had a synergistic effect on platelet activation, we coincubated euglycemic (5 mM ≈ 90 mg/dl) and hyperglycemic (15 mM ≈ 270 mg/dl) concentrations of [d]{.smallcaps}-glucose with lower concentrations of sodium arsenite than used to demonstrate platelet activation without glucose coincubation. After incubation at hyperglycemic conditions, exposure to 0.1, 1 and 5 µM sodium arsenite led to marked increases in platelet activation. In contrast, these sodium arsenite concentrations did not potentiate platelet activation at euglycemic concentrations of [d]{.smallcaps}-glucose (Fig. [2](#Fig2){ref-type="fig"}a, b).Fig. 2Unstimulated (**a**) and thrombin-stimulated (**b**) percent p-selectin expression to 5 and 15 mM glucose with increasing concentrations of sodium arsenite
Hyperglycemia may induce prothrombotic changes in megakaryocyte function and platelet thrombogenesis \[[@CR6]\]. To test whether glucose and iAs also had a synergistic effect on megakaryocyte adhesion, we coincubated megakaryocytes at euglycemic (5 mM) and hyperglycemic (25 mM) concentrations of [d]{.smallcaps}-glucose with 0, 1, 5 and 10 mM concentrations of sodium arsenite. Similar to the results observed for platelet activation, exposure to subthreshold sodium arsenite concentrations below 10 µM induced significantly greater megakaryocyte adhesion after incubation with a hyperglycemic compared to a euglycemic concentration of [d]{.smallcaps}-glucose (Fig. [3](#Fig3){ref-type="fig"}a, b). Prior studies have demonstrated megakaryocyte nuclear transcription factor kappa B (NFκB) gene expression is an important regulator of inflammation and platelet activation \[[@CR18], [@CR19]\], and may also be an important transcriptional factor for the vascular effects of inorganic arsenic exposure \[[@CR20]\]. To verify the prothrombotic effect of iAs, we measured the gene expression of NFKB1 in Meg-01 cells. monocyte chemoattractant protein-1 (CCL2) and CD36, genes involved in platelet activation and degranulation \[[@CR22], [@CR23]\], were also measured \[[@CR22], [@CR23]\]. Following coincubation of hyperglycemic [d]{.smallcaps}-glucose with 5 and 10 µM sodium arsenite, Meg-01 cells NFκB1 expression increased significantly compared to coincubation with euglycemic [d]{.smallcaps}-glucose (Fig. [4](#Fig4){ref-type="fig"}). There were additional non-significant increases in MCP-1 (CCL2) and CD36 (data not shown). No deleterious effects on Meg-01 cell toxicity were observed within the range of concentrations of sodium arsenite used in this study (0--10 µM) up to concentrations 100-fold greater (Appendix, Fig. [5](#Fig5){ref-type="fig"}).Fig. 3Megakaryocyte adhesion (% area, **a**) and photomicrograph (**b**) to 5 and 25 mM [d]{.smallcaps}-glucose with increasing concentrations of sodium arsenite Fig. 4GAPDH Normalized NFκB Gene Expression to 5 and 25 mM [d]{.smallcaps}-glucose with increasing concentrations of sodium arsenite
Discussion {#Sec9}
==========
There are four primary findings of this report. First, we show for the first time that a concentration of [d]{.smallcaps}-glucose common in DM potentiates sodium arsenite-induced platelet activation. Second, we demonstrate that hyperglycemia also potentiates the effects of sodium arsenite on megakaryocyte adhesion, a marker of atherothrombotic risk \[[@CR18]\]. Third, we demonstrate that lower concentrations of sodium arsenite than previously studied are associated with increased platelet activation and aggregation. Finally, we show that Meg01 NFκB transcription as a marker of megakaryocyte activation increases following exposure to hyperglycemia and sodium arsenite. These findings suggest that alterations in the platelet-megakaryocyte axis may be a pathway through which exposure to environmental toxicants such as iAs increase CVD risk, particularly for patients with DM.
Despite advances in effective medical therapy to reduce CVD events, nearly 70% of patients with DM will die of CVD \[[@CR24]\]. The etiology of this excess CVD risk for DM patients remains unclear. The vasculotoxicity and cardiovascular disease risk of environmental pollutants, including iAs, may be greater for individuals with diabetes \[[@CR2], [@CR3]\], and suggests that low-level environmental exposures may be a novel risk factor for CVD risk in DM. Environmental pollutants enhance inflammation and the generation of reactive oxygen species, steps also important in the pathogenesis of diabetic vasculopathy \[[@CR25]\]. While prior studies have indicated that environmental exposures increase oxidative stress and platelet activation \[[@CR26], [@CR27]\], to our knowledge this is the first report to describe a potential link between diabetic hyperglycemia and enhanced atherothrombotic risk to iAs exposure.
There are a number of pathways of platelet activation shared between hyperglycemia and iAs exposure. Hyperglycemia and diabetes is associated with platelet hyperreactivity, and coupled with enhanced levels of thromboxane, may partially explain increases in cardiovascular disease morbidity and mortality seen among patients with DM \[[@CR8]\]. High levels of drinking water inorganic arsenic (500 ppb) increase platelet thromboxane formation and adhesion protein expression \[[@CR28]\]. Other synergistic pathways between hyperglycemia and iAs exposure include increases in aldose reductase activity and oxidative stress signaling. During hyperglycemia aldose reductase activity increases significantly, leading to abnormal activation of the polyol pathway and enhanced oxidative and osmotic stress \[[@CR8]\]. In turn aldose reductase increases thromboxane formation and platelet activation \[[@CR8]\]. Inorganic arsenic has also been shown to increase aldose reductase activity \[[@CR29]\]. Taken together enhanced aldose reductase activity and thromboxane generation may represent a synergistic pathway of thrombotic risk for both hyperglycemia and inorganic arsenic exposure. Platelet and endothelial mitochondrial function may be another synergistic pathway of risk for iAs exposure in diabetes. Recent studies have indicated the importance of platelet mitochondrial function in cardiovascular disease \[[@CR30]\], and have suggested that alterations in platelet mitochondrial function may increase the risk of diabetic atherothrombosis \[[@CR31]\]. Inorganic arsenic has also been shown to alter endothelial cell mitochondrial function \[[@CR13]\]. Future studies might consider the synergy of inorganic arsenic exposure and diabetes on mitochondrial function in platelets and vascular endothelium as novel pathways of cardiovascular disease risk.
Strengths of the current study include the use of multiple validated measures of the platelet-megakaryocyte axis associated with incident CVD; use of sodium arsenite concentrations below those used in previous models of iAs-induced atherothrombosis; and an investigation of the synergy between hyperglycemia and iAs exposure on atherothrombotic risk. Although we used a lower sodium arsenite concentration than previous atherothrombosis studies \[[@CR9], [@CR21]\], we recognize the concentrations of sodium arsenite used may not correspond to current levels of iAs exposure in the U.S. Future studies should further investigate effects at very low concentrations corresponding to levels more prevalent in human populations. The discrepancy between exposure levels relevant to naturally contaminated drinking water and in vitro concentrations of sodium arsenite may reflect the lack of an accepted biomarker of internal iAs dose. Other limitations include the use of in vitro models and the inability to model in vivo differences in hyperglycemia and insulin resistance seen in type 1 and 2 diabetes. Further study is also needed to better estimate internal inorganic arsenic dose relevant for in vitro modeling; to examine the effect of environmental exposures on the platelet-megakaryocyte axis across the spectrum of diabetes control; and to study the effects of iAs and hyperglycemia on mitochondrial function in platelets and other relevant systems. Treatment studies could consider the use of aldose-reductase inhibitors to attenuate platelet activation and megakaryocyte adhesion \[[@CR8]\].
Conclusion {#Sec10}
==========
Our findings suggest that increased platelet activation and megakaryocyte adhesion may be pathways through which hyperglycemia in DM can enhance the vasculotoxicity of inorganic arsenic exposure. While intensive glycemic control has failed to significantly reduce macrovascular risk in DM, exposure to environmental toxicants such as inorganic arsenic may represent a novel class of modifiable CVD risk factors, particularly for patients with diabetes. Future studies should investigate platelet activation in patients with and without diabetes, at varying levels of glycemic control, following exposure to environmentally relevant concentrations of inorganic arsenic and other environmental exposures.
Appendix {#Sec20}
========
See Fig. [5](#Fig5){ref-type="fig"}.Fig. 5Meg-01 cell toxicity index with 5 and 25 mM [d]{.smallcaps}-glucose with increase concentrations of sodium arsenite
iAs
: inorganic arsenic
CVD
: cardiovascular disease
DM
: diabetes mellitus
MPA
: monocyte-platelet aggregation
LPA
: leukocyte-platelet aggregation
Meg-01
: megakaryocyte
NFκB
: nuclear transcription factor kappa B
FBS
: fetal bovine serum
RNA
: ribonucleic acid
GAPDH
: glyceraldehyde 3-phosphate dehydrogenase
PCR
: polymerase chain reaction
SEM
: standard error of the mean
ANOVA
: analysis of variance
U.S.
: United States
JDN lead and corresponding author, had full access to the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. He wrote the majority of the manuscript. CTE performed many of the experiments, made substantial critical revisions and aided with interpretation. YMO aided substantially with experimental conditions and data acquisition. EM aided substantially with experimental conditions and data acquisition. YC provided critical revisions to the manuscript and aided substantially in the preparation of the revised submission. EAF provided input into study design and made substantial critical revisions to the manuscript. JSB provided crucial laboratory support and input into experimental design and analysis, and made substantial critical revisions to the manuscript. All authors read and approved the final manuscript.
Acknowledgements {#FPar1}
================
Not applicable.
Competing interests {#FPar2}
===================
The authors declare that they have no competing interests.
Availability of data and materials {#FPar3}
==================================
Data and materials will be held by the corresponding author, Dr. Jonathan Newman, and are available on request.
Funding {#FPar4}
=======
This study was funded by the National Institute of Diabetes and Digestive and Kidney Disease (NIDDK) of the National Institute of Health (NIH, U24DK076169-09, subcontract 25732-60). Dr. Newman was partially funded by the National Heart, Lung, and Blood Institute (NHLBI) of the NIH (K23HL125991) and the American Heart Association Mentored Clinical and Population Research Award (15MCPRP24480132). Dr. Berger was partially funded by the NHLBI of the NIH (HL114978). Funders had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; and preparation, review, or approval of the article.
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#sec1}
============
Spontaneous subcapsular or perinephric haemorrhage is a relatively uncommon but diagnostically challenging condition. In the evaluation of this condition, the differential diagnosis includes renal tumours, vascular disorders and pyelonephritis. While computed tomography would readily identify tumours, the vascular disorders may pose greater diagnostic challenges. The major vascular causes of perinephric haematoma include polyarteritis nodosa, renal aneurysm, arteriovenous malformation, Wegener\'s granulomatosis, renal infarction and pre-eclampsia.
In this report, we present a case of spontaneous perinephric haemorrhage in pregnancy associated with cocaine intoxication. The patient was managed conservatively, with clinical improvement until she left the hospital against medical advice. She returned 10 weeks later with acute renal failure, pre-eclampsia, fetal demise and cocaine intoxication. We discuss the presentation, the diagnosis and the conservative management of this syndrome and its relationship to cocaine intoxication in pregnancy.
Case Report {#sec2}
===========
A 36-year-old African American woman at 19 weeks of gestation was admitted through the emergency room due to a severe right lower quadrant pain radiating to her back. She denied fever, vaginal bleeding, or bowel or urinary symptoms. Her medical history was significant for hypertension. Her obstetric and gynaecologic history included four normal-term vaginal deliveries and a caesarean section delivery at 24 weeks due to placental abruption with perinatal death. She denied any trauma but had a 10-pack-year smoking history as well as marijuana and cocaine use. She conceded to have used crack cocaine several times on the day of presentation.
On physical exam, she was acutely ill and pale. Her blood pressure was 182/107 mmHg with a pulse of 83/min which increased to 102/min on standing. She had tenderness to palpation of her right flank and periumbilical region, with no rebound. Her abdomen was distended with the uterine fundus felt just below the umbilicus, compatible with 19-week gestation. No uterine contractions were noted, and the fetal heart rate was 144 beats per minute. The cervix was closed, and there was no evidence of vaginal bleeding or rupture of membranes.
Laboratory studies showed a haemoglobin level of 6.7 g/dL (67 g/L), a white blood cell count of 10.1 × 10^3^/μL (10^9^/L) and a platelet count of 184 × 10^3^/μL (10^9^/L). The prothrombin time (PT) was 10.8 s \[international normalized ratio (INR) 0.97\], with partial thromboplastin time (PTT) of 27 s. The serum creatinine level was 1.0 mg/dL \[88 μmol/L; estimated glomerular filtration rate (GFR) 91.7 mL/min/1.73 m^2^\]. The blood urea nitrogen was 6 mg/dL (2.14 mmol/L), and the urine sediment showed 3+ blood, 2+ protein and 30--50 red blood cells per high-power field. Serum uric acid was 6 mg/dL. The lactate dehydrogenase level (LDH) was 161 IU/mL. The urine drug screen was positive for cocaine metabolite. Urine culture was negative. A renal ultrasound showed normal-sized kidneys with a prominent echogenic material of 3--4-cm thickness surrounding the right kidney without hydronephrosis, tumour or calculi ([Figure 1](#fig1){ref-type="fig"}). A pelvic ultrasound confirmed the presence of a single viable intrauterine gestation. A computed tomography (CT) scan done to better delineate the lesion showed a large complex retroperitoneal fluid collection surrounding the right kidney extending into the right paracolic gutter and the surrounding portions of the inferior vena cava, duodenum and pancreas ([Figure 2](#fig2){ref-type="fig"}).
{#fig1}
{#fig2}
She was emergently resuscitated with intravenous fluids and transfused two units of packed red cells with improvement on her haemoglobin level to 9.1 g/dL (91 g/L). Her hypertension was controlled with intravenous hydralazine and oral amlodipine. Because of improvement in her pain control, stable haemodynamics and good fetal heart tones, a conservative approach was recommended. The patient made a clinical improvement indicated by an adequate pain control and an improved blood pressure control as well as a stable haemoglobin level. However, she signed out of hospital against medical advice on the fourth day of admission.
She again presented at 29-week gestation through the emergency department complaining of abdominal pain, decreased fetal movement and blurred vision. Her blood pressure was markedly elevated at 223/133 mmHg. Her urinalysis was again significant for microscopic haematuria (10--15 red blood cells per high-power field), 2+ proteinuria and a positive urine drug screen for cocaine. Her serum creatinine level was increased to 1.8 mg/dL (158.4 μmol/L; estimated GFR 37.6 mL/min/1.73 m^2^). Her uric acid level had risen to 9.4 mg/dL. A repeat renal ultrasound ([Figure 3](#fig3){ref-type="fig"}) showed a smaller right perinephric fluid collection consistent with a resolving haematoma. Because of her presentation with severe pre-eclampsia, intravenous magnesium sulphate and hydralazine were given, and she had urgent delivery of a non-viable fetus by caesarean section. A bilateral tubal ligation was simultaneously performed. At the time of her discharge home after 5 days of admission, she remained clinically stable, her blood pressure was 135/85 mmHg and her serum creatinine level decreased to 1.0 mg/dL (88 μmol/L) ([Table l](#tab1){ref-type="table"}).
{#fig3}
######
Laboratory findings at 18- and 29-week gestation
Test 19th week 29th week
----------------------------------- ---------------------- ----------------------
Haemoglobin (g/dL) 6.7 11
Platelets 241 52
Aspartate aminotransferase (IU/L) 22 26
Alanine aminotransferase (IU/L) 21 23
LDH (IU/L) 161 730
Serum creatinine (mg/dL) 1.0 1.7
Estimated GFR
Blood urea nitrogen (mg/dL) 6 14
Albumin (g/dL) 2.9 2.3
24-h urine protein (mg/dL) 665 1231
Urine red blood cells
Urine culture Negative Not done
PT (s) 10.8 9.2
INR 0.97 0.84
PTT (s) 27 24
Uric acid (mg/dL) 6.0 9.4
Urine drug screen Positive for cocaine Positive for cocaine
Discussion {#sec3}
==========
The syndrome of spontaneous subcapsular renal bleeding with dissection of blood into the subcapsular and/or perinephric space is a rare, life-threatening condition that is usually caused by renal tumours, vascular disorder, pyelonephritis or pre-eclampsia \[[@ref1]\]. It was first described by Wunderlich in 1856 \[[@ref3]\].
Our patient presented initially with spontaneous perinephric haemorrhage and hypertensive crisis in pregnancy in association with cocaine intoxication. Because of the high morbidity and potential mortality associated with this condition, nephrectomy may sometimes be recommended for uncontrollable haemorrhage or if a tumour is identified. However, a conservative strategy, with follow-up radiologic imaging, has been tried by some with good outcome \[[@ref4]\]. This patient had been stabilized on a conservative approach before she left the hospital against medical advice. On her readmission at the 29th week of gestation, she had a picture of severe pre-eclampsia with hypertensive crisis, blurry vision, epigastric pain and acute renal failure again with cocaine intoxication. This mandated the urgent caesarean section delivery of the dead fetus. The repeat renal ultrasound confirmed the interval reduction in size of haematoma. She showed the common features of this syndrome including acute flank pain, worsening hypertension and proteinuria \[[@ref1]\].
The cause of acute renal failure (ARF) on her second presentation is unclear. It may be due to severe pre-eclampsia as two other cases of this syndrome and ARF in patients with severe pre-eclampsia have been reported. Interestingly, cocaine use has also been associated with pre-eclampsia \[[@ref9]\]. The rapid improvement of renal function after the caesarean delivery and the achievement of good blood pressure control support that pre-eclampsia was more likely the cause of ARF. In three other reports of this syndrome, ARF was also observed, and renal function improved with a conservative management \[[@ref4]\]. Though the precise cause of the spontaneous perinephric haemorrhage in our patient is unclear, the temporal relationship between heavy cocaine use and her presentation supports a causal rather than a casual relationship. There was no evidence of a renal tumour, vasculitis or coagulopathy. We and others reviewed the numerous nephrotoxic effects of cocaine abuse about a decade ago \[[@ref11]\]. How cocaine might induce perinephric haemorrhage/haematoma is unclear, but several mechanisms may be involved including malignant hypertension, inflammation and deranged platelet function. In other cases of this syndrome associated with pre-eclampsia, severe hypertension was proposed to play a role in pathogenesis of spontaneous perinephric haemorrhage \[[@ref5]\]. Furthermore, severe hypertension has been implicated in haemorrhage in other vascular beds like the lungs in both cocaine-abusing patients \[[@ref13]\]. Cocaine use may also cause perinephric haemorrhage if there is a rupture of the renal capsule. In a case reported recently, spontaneous subcapsular kidney rupture and haemorrhage occurred in a patient after rupture of swallowed intestinal packets of cocaine, with massive absorption in a drug dealer \[[@ref15]\]. The patient, in that report, suffered renal infarction. Our patient had no biochemical or radiologic evidence of renal infarction. Spontaneous perinephric haemorrhage may itself cause hypertension. In one case, a hyperreninemic hyperaldosteronism state was shown \[[@ref16]\].
In conclusion, the present case shows that cocaine abuse in pregnancy may lead to severe spontaneous perinephric haemorrhage and haematoma, probably due to severe hypertension. If conservative measures achieve haemodynamic stability with cessation of haemorrhage, nephrectomy may be avoided. This syndrome must be entertained in the differential diagnosis of acute abdomen and haematuria in a pregnant patient with severe hypertension. This report also expands the spectrum of renal complications of cocaine abuse to include spontaneous perinephric haemorrhage.
We thank Dr. Glenfield Knight and Dr. Richard Desruisseau of the radiology unit of Nashville General Hospital for their assistance with radiologic images.
*Conflict of interest statement.* M.F. receives research support as a medical director of Dialysis Clinics Incorporated (DCI) Meharry Clinic in Nashville, TN, USA. All the other authors have no conflict of interest to declare.
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Correction to: *Scientific Reports* 10.1038/s41598-017-18236-7, published online 20 December 2017
The original version of this Article contained an error in the Abstract.
"Over 200 mutations in 60 different genes have been shown to cause RP."
now reads:
"Many mutations in 60 different genes have been shown to cause RP."
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1. Introduction {#sec1}
===============
Hypertension is highly prevalent globally. In 2005, the global burden of hypertension was estimated to rise from nearly 1 billion in the year 2000 to 1.6 billion in 2025 \[[@B1]\]. By 2010, the global burden of hypertension was estimated at about 1.4 billion, and this will potentially exceed 1.6 billion sooner than 2025 \[[@B2]\]. In Thailand, the Thai Burden of Disease study in 2009 reported hypertension as in the top 3 risk factors for disability-adjusted life-years (DALYs) in both males and females. According to the latest National Health Examination Survey conducted in 2015, one out of four Thais of 15 years of age and older had hypertension \[[@B3]\].
Hypertension is a complex disease caused by both genetic and environmental factors. The renin-angiotensin-aldosterone system (RAAS) is a hormone system that regulates blood pressure (BP) and fluid and electrolyte balance. It is commonly targeted for the treatment of hypertension \[[@B4], [@B5]\]. Therefore, on the basis of prior knowledge on biological functions, polymorphisms in candidate genes of the RAAS have been extensively studied, aiming to investigate the influence of RAAS genetic variability on hypertension \[[@B6]--[@B8]\]. While the roles of genes in the RAAS, including angiotensin-converting enzyme (ACE), angiotensinogen (AGT), angiotensin II receptor type 1 (AGTR1), and aldosterone synthase (CYP11B2) genes in hypertension, have been widely studied across different ethnicities \[[@B9]--[@B11]\], there have been limited investigations in Thai population.
In this study, we examined 4 polymorphisms in the RAAS selected because of their roles in the pathogenesis of hypertension. The reference SNP identification (rs) of ACE, rs1799752, an insertion/deletion (I/D) polymorphism, has been associated with many diseases. The D allele, which has increased activity, not related to increased generation of angiotensin II, is associated with increased risk of hypertension and preeclampsia, among others \[[@B11]\]. AGT is converted by ACE to angiotensin II, a potent vasoconstrictor; M235T (rs699) is a nonfunctional polymorphism, but 235 is in linkage equilibrium with −6A \[[@B12]\]. AGT haplotype 1, which contains the variants −217A, −6A, +507G, and +1164A, is associated with increased BP in humans and transgenic mice \[[@B12]\]. The prohypertensive effect of angiotensin II occurs by occupation of AGTR1, resulting in vasoconstriction and sodium retention \[[@B13]\]. Polymorphisms of AGTR1 such as rs5186 are associated with hypertension \[[@B14]--[@B17]\]. The ability of aldosterone to increase BP is caused not only by increasing renal sodium transport but also by increasing vascular smooth muscle contractility, among others, via mineralocorticoid and nonmineralocorticoid receptors \[[@B18]\]. Aldosterone synthase, which is needed to synthesize aldosterone, has a genetic polymorphism, CYP11B2 rs1799998 \[[@B18]\], that is associated with hypertension \[[@B19]\]. However, a recent meta-analysis was not able to show the association of the SNPs of ACE, AGT, and CYP11B2 genes and hypertension \[[@B20]\]. Because the associations between these SNPs and hypertension could be ethnic-dependent and the associations of these SNPs and hypertension have not been studied in the Thai population, we investigated the associations between these SNPs and hypertension in 6463 Thais.
2. Methods {#sec2}
==========
2.1. Data and Study Design {#sec2.1}
--------------------------
The subjects were employees of EGAT (the Electricity Generating Authority of Thailand) who volunteered to participate in a health survey. In 1985, 3499 workers of EGAT (half of the total employees) were randomly enrolled as EGAT 1 cohort. In 1998, 2999 employees were randomly enrolled as EGAT 2 cohort. The age range of 35--54 years was selected in both EGAT 1 and EGAT 2. Both EGAT cohorts were surveyed in 1997-1998 with every 5-year follow-up. During the follow-up in 2002-2003, blood for genotyping was also drawn after a 12-hour fast. DNA was extracted from whole blood, and one SNP per gene was previously selected and genotyped using fluorescent probe melting analysis for rs1799752 (ACE), rs699 (AGT), rs5186 (AGTR1), and rs1799998 (CYP11B2). BP was measured twice after 10-minute rest in a seated position, using a validated automatic device. Individuals were classified as hypertensive when systolic BP (SBP) \> 140 mmHg and diastolic BP (DBP) \> 90 mmHg. More details of the EGAT study cohorts and the study protocols can be found in the study by Vathesatogkit et al. \[[@B21]\].
2.2. Inclusion and Exclusion Criteria {#sec2.2}
-------------------------------------
All samples with genotyping data were included in our analyses. All individuals were included in analyses of hypertension. Individuals taking antihypertensive medication were excluded from analyses of BP. This was done to avoid BP levels that are artificially lowered regardless of the genetic background.
2.3. Statistical Analyses {#sec2.3}
-------------------------
GAS (Genetic Association Study Power Calculator) was used to perform power calculations \[[@B22]\]. For inputs, the GAS requires the number of cases and controls, disease model, disease prevalence, allele frequency, estimated genotype relative risk, and target significance level after adjusting for the number of markers tested for association.
We first tested associations between rs1799752, rs699, rs5186, rs1799998, and hypertension using multivariate logistic regression. This was done using glm() function in R. Sex, age, and BMI were adjusted as potential confounders. An additive genetic model was used to assume an additive risk of disease for an additional effect allele. For example, the genotype for rs1799752 is coded as II = 0, ID = 1, and DD = 2, where D is an effect allele. A significant threshold of 0.0125 was used after accounting for Bonferroni correction with 4 independent candidate SNPs investigated in our study.
Associations between rs1799752, rs699, rs5186, rs1799998, and BP were further explored to investigate these genetic effects in more detail. Quantile regression (QR) was applied to examine these genetic effects on the entire distribution of both SBP and DBP. The quantile regression allows the change across the *i* ^th^ quantile of BP to be tested. This allows the specific threshold currently used to classify individuals under hypertension to be relaxed under this investigation. The rq() function from the quantreg package in R was used to perform quantile regression.
2.4. Systematic Search for Previous Evidence of Associations {#sec2.4}
------------------------------------------------------------
Systematic search for previous evidence of association between the 4 SNPs and hypertension-related traits across populations was performed using the GRASP search engine \[[@B23]\] and the UK Biobank recently made publicly available \[[@B24]\].
The GRASP search software (v2.0.0.0) searches GWAS catalog data housed at the National Center for Biotechnology Information (NCBI). With periodic updates, the current version of GRASP includes available genetic association results from 2,082 GWAS papers, their supplements, and web-based contents. All associations with *P* \< 0.05 from GWAS defined as ≥25,000 markers tested for 1 or more traits are included in the database.
UK Biobank is the largest prospective study in the UK following about 500,000 participants aged from 40 to 69 years. Recently, summary statistics of association across a wide range of phenotypes were publicly made available, including hypertension, SBP, and DBP. Logistic and linear regressions were applied in hypertension and BP, respectively, with an adjustment for sex and 10 principal components.
3. Results {#sec3}
==========
3.1. Characteristics of Study Population {#sec3.1}
----------------------------------------
In this study, we focused on hypertension from the combined EGAT 1 and EGAT 2 data collected in 1997-1998. This first wave of data collection allows the largest sample size possible. Out of the total of 6498 individuals collected in 1997-1998, up to 6463 individuals were genotyped and used in our analyses. The summary of the characteristics of EGAT data is shown in [Table 1](#tab1){ref-type="table"}.
3.2. Genetic Analyses {#sec3.2}
---------------------
The summary of the genotype data in the EGAT study is shown in [Table 2](#tab2){ref-type="table"}. Using a genome browser Ensembl, we compared frequencies of the SNPs of interest between Thai and other populations ([Table 3](#tab3){ref-type="table"}). Frequencies of rs699 and rs1799998 reported in East Asian population are shown to be the same as in EGAT data, while these are different from those in South Asian, European, American, and African populations. For rs1799752 and rs5186, information from East Asian population was not reported. The data from the aggregated populations from the Exome Aggregation Consortium show that rs1799752 is rare, while it is a common SNP in Thai population. rs5186 is reported to have the same allele frequency in Thais and African Americans but much less than that in European Americans.
In the EGAT control group, all 4 polymorphisms are in the Hardy--Weinberg equilibrium which indicates no evidence of genotyping errors, i.e., P_hwe = 0.38 for rs1799752, P_hwe = 0.20 for rs699, P_hwe = 0.18 for rs5186, and P_hwe = 0.32 for rs1799998.
3.3. Power Calculation {#sec3.3}
----------------------
GAS was used to perform power calculations. Hypertension prevalence was 23% on average in the Thai population \[[@B3]\]. Allele frequencies and a number of cases and controls are shown in Tables [2](#tab2){ref-type="table"} and [3](#tab3){ref-type="table"}. Assuming that hypertension is a complex disease with many variants of small effect, the maximum genotype relative risk of 1.1 was used in this calculation \[[@B25]\]. Under an additive model, the power is 0.23 for rs699, 0.11 for rs5186, 0.48 for rs1799998, and 0.44 for rs1799752.
3.4. Association with Hypertension {#sec3.4}
----------------------------------
We investigated the association between rs699, rs5186, rs1799998, rs1799752, and hypertension in up to 4150 individuals with 1331 cases and 2819 controls. Using multivariate logistic regression, no evidence of association was observed under an additive genetic model ([Table 4](#tab4){ref-type="table"}).
3.5. Association with SBP and DBP {#sec3.5}
---------------------------------
We further examined the role of these 4 polymorphisms in more detail in the entire distribution of BP. The summary of DBP and SBP across quantiles is shown in [Table 5](#tab5){ref-type="table"}.
ACE is a target of drugs for hypertension. However, no association was found between ACE rs1799752 and BP across an entire range using multivariate quantile regression ([Figure 1](#fig1){ref-type="fig"}). Similarly, variants of the other drug target genes, AGT (rs699), AGTR1 (rs5186), and CYP11B2 (rs1799998), were not associated with BP (Supplementary Materials ([available here](#supplementary-material-1){ref-type="supplementary-material"})).
We also studied the influence of risk factors for hypertension included in the model as potential confounders, i.e., age, BMI, and sex, on an entire distribution of BP when rs1799752 was included in the model. All risk factors were significantly associated with SBP and DBP. SBP and DBP were higher in males than those in females. We found that the influence of age on BP increased at the higher level of BP ([Figure 2](#fig2){ref-type="fig"}). This indicates possible nonlinear relations between age and each BP measurement, and a quadratic effect of age should be examined in the analyses by fitting the age-squared term. We further included the age-squared term in our model. The age-squared term was significantly associated with SBP and DBP in all models; however, this did not significantly change the results previously observed.
3.6. Evidence of Association in Publicly Available GWAS Studies {#sec3.6}
---------------------------------------------------------------
We systematically searched for evidence of association between our 4 SNPs of interest and hypertension-related traits across populations using the GRASP search engine \[[@B23]\] and a large UK Biobank recently made publicly available \[[@B24]\]. Unfortunately, we did not observe GWAS studies of hypertension and BP in Asian population with publicly available results. For this systematic search, we reported summary statistics from association analyses obtained mainly from Caucasian ancestry although we are aware of the difference in allele frequencies across populations, as previously shown in [Table 3](#tab3){ref-type="table"}.
Using the GRASP search (v2.0.0.0) on GWAS catalog data, we found that only rs699 was associated with hypertension at the GWAS significant threshold (*P* \< 5 × 10^−8^), with the sample size of 84,467 individuals of European ancestry \[[@B26]\].
We further used recently made available UK Biobank results which followed about 500,000 participants from 40 to 69 years of age. Self-reported hypertension was recorded in 27% of the whole UK Biobank population. Three out of our 4 SNPs of interest are available in UK Biobank. rs799752, previously shown as a rare SNP, was neither available in UK Biobank nor in an rAggr web-based application (<http://raggr.usc.edu/>) to search for its proxy SNPs. rs699 and rs1799998 were associated with SBP and DBP but not with hypertension ([Table 6](#tab6){ref-type="table"}). By removing age and BMI from our previous models, we further reported effect sizes and effect directions from the EGAT data under the same models as in UK Biobank without an adjustment for principal components, which cannot be calculated under the limited SNPs. However, because of nonsignificant results, a comparison cannot be made.
4. Discussion {#sec4}
=============
This study aimed to investigate the association between 4 polymorphisms in RAAS genes (i.e., rs1799752 (*ACE*), rs699 (*AGT*), rs5186 (*AGTR1*), and rs1799998 (*CYP11B2*)) and hypertension in a Thai population. In addition, their roles in SBP and DBP levels were investigated. However, we did not observe any evidence of associations between these polymorphisms and hypertension, SBP, or DBP.
The same allele frequencies of rs699 and rs1799998 were observed between Thai and East Asian populations ([Table 3](#tab3){ref-type="table"}). rs1799752 is not in the 1000 Genomes and HapMap projects, that may be related to its low allele frequency in Caucasians; that is, a frequency less than 0.01 was reported only in the Exome Aggregation Consortium \[[@B27]\]. While rs1799752 in the ACE gene is rare in Caucasians, we observed a high frequency of deletion (MAF = 0.32) at rs1799752 in our Thai population.
ACE has a wide range of insertion/deletion regions \[[@B28]\]. The polymorphism of the ACE gene is known for the presence or absence of a 287 bp element on intron 16 on chromosome 17. Hypertension-related traits and genetic mechanisms may vary across races and ethnicities \[[@B29]\]. In contrast to Caucasian populations, there are a limited number of studies in Asian populations on the relationship of the ACE polymorphisms and hypertension. While there are associations between ACE I/D and hypertension in some Chinese \[[@B30], [@B31]\] and Indian \[[@B32], [@B33]\] populations, lack of such associations has also been reported \[[@B34], [@B35]\]. Other genes in the RAAS including AGT, AGTR1, and CYP11B2 have also been widely studied \[[@B36]\]. However, there has been no investigation on the association of polymorphisms in the RAAS and hypertension in a Thai population. In our present study on a Thai population, the association of polymorphisms in the RAAS (rs1799752 (ACE), rs699 (AGT), rs5186 (AGTR1), and rs1799998 (CYP11B2)) and hypertension was not observed.
The motivation for using QR is that it assesses how conditional quantiles of BP vary with respect to measured covariates. There is no theoretical reason to assume that the effect of the covariates is the same at different quantiles of the distribution. In our case, we observed a quadratic effect of age on BP. Because QR considers the entire conditional distribution of the dependent variable and not only its mean as in linear regression, it could provide a more complete picture of the conditional distribution than a single estimate of the center. QR also avoids the need to decide an arbitrary threshold to define the "extremes" \[[@B37]\]; that is, the cutoff of 140 mmHg SBP and 90 mmHg DBP of hypertensive cases can be relaxed. However, in our study, QR did not reveal an evidence of association at any particular quantile, and large confidence intervals across the range were observed.
In 2017, Ji et al. also reported a systematic search on the GWAS catalog for association between a number of polymorphisms in the RAAS and hypertension at the significant threshold level of 5 × 10^−8^. Many polymorphisms did not show an evidence of association across studies, while other polymorphisms associated with traits that have no direct connection with hypertension \[[@B38]\]. Nevertheless, analysis of a very large UK Biobank that was recently made available revealed an association between polymorphisms in RAAS genes and hypertension-related traits at the significant threshold level of 5 × 10^−8^, e.g., rs699 in AGT with both SBP and DBP and rs4308 in ACE with DBP \[[@B39]\]. This could imply that a very large sample size might be required to have an adequate statistical power to detect association in hypertension-related traits, such as shown in AGT and ACE genes.
In conclusion, we did not observe any association between 4 polymorphisms in the RAAS and hypertension in a Thai population. An effect on SBP and DBP in the entire distribution was also not found. While our study is the first and largest study to investigate the role of different polymorphisms in RAAS-related genes in hypertension in a Thai population, the sample size still restricted the statistical power. Our study suggests that either there is no association between these 4 polymorphisms in RAAS-related genes and hypertension or a much larger sample size is required to detect if there is a true association. In our study design, at least 3500 cases and 3500 controls are required to obtain an 80% power to detect if there is a true association between rs1799752 in ACE and hypertension. In addition, denser polymorphisms across these 4 genes are needed to provide a better coverage in the regions of interest.
This study was supported by the Thailand Research Fund (grant no. MRG6280088) and Mahidol University.
Data Availability
=================
The data used to support the findings of this study are available from the corresponding author upon request.
Ethical Approval
================
All procedures performed in this study involving human participants were in accordance with the ethical standards of the Institutional Research Committee, Faculty of Medicine, Ramathibodi Hospital, Mahidol University (ID-05-51-19V), and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.
Conflicts of Interest
=====================
All authors declare no conflicts of interest.
Supplementary Materials {#supplementary-material-1}
=======================
######
Supplementary Figure 1: point estimates and 95% confidence bounds (grey areas) for the increase in SBP (left) and DBP (right) per rs699 risk allele. The dots represent specific BP percentiles in the quantile regression model with adjustment for sex, age, and BMI. The nonzero horizontal lines represent the linear regression coefficients and their 95% confidence intervals. Supplementary Figure 2: point estimates and 95% confidence bounds (grey areas) for the increase in SBP (left) and DBP (right) per rs5186 risk allele. The dots represent specific BP percentiles in the quantile regression model with adjustment for sex, age, and BMI. The nonzero horizontal lines represent the linear regression coefficients and their 95% confidence intervals. Supplementary Figure 3: point estimates and 95% confidence bounds (grey areas) for the increase in SBP (left) and DBP (right) per rs1799998 risk allele. The dots represent specific BP percentiles in the quantile regression model with adjustment for sex, age, and BMI. The nonzero horizontal lines represent the linear regression coefficients and their 95% confidence intervals.
######
Click here for additional data file.
{#fig1}
{#fig2}
######
Characteristics of EGAT data.
Male Female
--------------- -------- --------
*N* 4891 1572
Age 48.34 47.10
BMI 24.33 24.01
Hypertension 39.13% 21.18%
BP medication 10.39% 7.63%
######
Selected SNPs in the RAAS-related genes in EGAT data.
Chr Location Gene Effect allele Noneffect allele *N* of genotyped samples *N* of genotyped samples with hypertension
----------- ----- ----------- --------- --------------- ------------------ -------------------------- --------------------------------------------
rs699 1 230710048 AGT A G 3572 1112
rs5186 3 148742201 AGTR1 C A 4108 1305
rs1799998 8 142918184 CYP11B2 G A 4150 1331
rs1799752 17 63488529 ACE D (deletion) I (insertion) 3674 1171
######
Effect allele frequencies of selected SNPs across different populations.
Effect allele frequency
------------------- ------------------------- ------ ------ ------ ------ ------ ------ ------ --------
rs699^*∗*^ 0.15 0.15 0.36 0.59 0.37 0.10 --- --- ---
rs5186^*∗∗*^ 0.06 --- --- --- --- --- 0.06 0.29 ---
rs1799998^*∗∗∗*^ 0.3 0.29 0.45 0.49 --- 0.18 --- --- ---
rs1799752^*∗∗∗∗*^ 0.32 --- --- --- --- --- --- --- \<0.01
^*∗*^1000 Genomes Project Phase 3: East Asian (EAS: CDX, CHB, CHS, JPT, KHV), South Asian (SAS: BEB, GIH, ITU, PJL, STU), European (EUR: CEU, FIN, GBR, IBS, TSI), American (AMR: CLM, MXL, PEL, PUR), and African (AFR: ACB, ASW, ESN, GWD, LWK, MSL, YRI). ^*∗∗*^NHLBI Exome Sequencing Project allele frequencies. ^*∗∗∗*^HapMap Project: East Asian (EAS: CHB, JPT), South Asian (SAS: GIH), European (EUR: TSI, CEU), and African (AFR: ASW, LWK, YRI). ^*∗∗∗∗*^Aggregated populations from the Exome Aggregation Consortium (ExAC).
######
Odds ratios of developing hypertension among samples with an effect allele, as compared to samples with a noneffect allele, including their *P* values.
Additive model
----------- ------------------- ------
rs1799752 1.03 (0.92, 1.17) 0.60
rs699 0.91 (0.78, 1.06) 0.24
rs5186 1.07 (0.87, 1.32) 0.51
rs1799998 0.98 (0.87, 1.09) 0.67
######
SBP and DBP across quantiles.
The *i* ^th^ quantile
------------ ----------------------- -------- -------- -------- --------
SBP (mmHg) 76.33 113.50 124.50 137.54 236.50
DBP (mmHg) 44.00 69.00 76.50 84.50 149.00
######
Summary statistics from UK Biobank and EGAT data.
Hypertension SBP DBP
----------- -------------- ------ ---------------- ------ ------- ------ ---------------- ---------------- ------- ------ --------------- ----------------
rs699 0.11 0.16 −5.72 × 10^−5^ 0.55 0.76 0.23 −0.02 7.28 × 10^−11^ 0.04 0.93 −0.02 4.15 × 10^−10^
rs5186 −0.23 0.05 5.31 × 10^−5^ 0.60 −0.28 0.75 2.89 × 10^−3^ 0.28 −0.71 0.20 1.25 × 10^−3^ 0.64
rs1799998 −0.08 0.18 3.79 × 10^−5^ 0.69 −0.27 0.57 −9.61 × 10^−3^ 1.03 × 10^−4^ −0.25 0.39 −0.01 1.30 × 10^−6^
[^1]: Guest Editor: Maha Abdalla
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1. Introduction {#sec1-sensors-19-00293}
===============
The security and safety aspects of global navigation satellite systems (GNSSs) have been receiving significant attention from researchers and the general public, because the use of GNSSs has been increasing in modern society \[[@B1-sensors-19-00293]\]. Because the power of a GNSS signal coming from the ground is very low, the signal is exposed to different types of radio interferences \[[@B2-sensors-19-00293]\]. Moreover, in contrast to military signals, safety and security issues are not considered for civilian GNSS signals. A civilian signal is not encrypted, and the details of such a signal are open \[[@B3-sensors-19-00293]\]. In other words, anyone can intentionally transmit a fake signal to deceive the user.
Some of the types of intentional interferences include jamming, meaconing, and spoofing \[[@B4-sensors-19-00293],[@B5-sensors-19-00293],[@B6-sensors-19-00293]\]. The aim of jamming is to prevent a user from receiving the authentic signal by transmitting another signal with a significantly greater power than that of the authentic signal. A meaconing attack involves transmitting another signal collected at a different location or time. If a meaconing attack is successful, the receiver would end up providing navigation information, such as the location and time, at which the meaconing signal was collected. The most dangerous type of interference is a spoofing attack. If the receiver captures a spoofing signal, the navigation solution can be controlled by the spoofer \[[@B7-sensors-19-00293]\].
There are two main technological approaches in spoofing studies: spoofing attacks and anti-spoofing techniques. Many spoofing attack tests have been conducted over the past few years. A portable GPS spoofer was developed, and a spoofing attack test was demonstrated for a target receiver \[[@B8-sensors-19-00293]\]. Although this experiment was conducted with a very short distance between the spoofer and the target receiver, it was possible to develop a practical spoofer with low cost. Moreover, successful spoofing tests were carried out against an unmanned aerial vehicle \[[@B9-sensors-19-00293]\], a ship \[[@B10-sensors-19-00293]\], and a mobile device \[[@B11-sensors-19-00293]\]. These studies have shown that spoofing attacks could be executed in real situations. Moreover, many anti-spoofing techniques have been studied for receiver security and safety. A maximum likelihood estimation-based positioning technique was applied to the detection of spoofing signals and correction of navigation solution \[[@B12-sensors-19-00293]\]. In another study, a cross-correlation approach between two GNSS receivers was used to detect the spoofing signal \[[@B13-sensors-19-00293]\]. In Ref \[[@B14-sensors-19-00293]\], an extended coupled amplitude delay lock loop (DLL) architecture was applied to spoofing detection. A pseudorange difference-based anti-spoofing algorithm was introduced \[[@B15-sensors-19-00293]\]. In Ref \[[@B16-sensors-19-00293]\], spoofing detection was performed using a machine learning algorithm such as a neural network. In other studies, antenna-aided techniques \[[@B17-sensors-19-00293]\] and inertial measurements unit-aided techniques \[[@B18-sensors-19-00293]\] have been developed.
Although the aforementioned studies report on spoofing attacks and anti-spoofing techniques, few have analyzed the conditions and circumstances required for a successful spoofing attack. In \[[@B19-sensors-19-00293]\], the spoofing attack results were presented considering the time, position, and power offset. However, only the effects of the spoofing parameters on the spoofing attack results were studied. In \[[@B9-sensors-19-00293]\], a spoofing signal with a 10 dB greater power than that of the authentic signal was transmitted to successfully deceive a drone. However, to avoid as much as possible the detection of a spoofing signal at the victim receiver, it is better to transmit the signal with the minimum power possible for a successful spoofing attack.
In this paper, we analyzed the conditions for a successful spoofing attack in the code domain. The spoofing parameters considered in this study are the spoofing signal strength, spoofing sweep velocity (Doppler offset), DLL order, and bandwidth. With the increase in the spoofing signal strength or DLL bandwidth, the probability of a successful spoofing attack increases. If the sweep velocity increases, the probability of a successful spoofing attack is reduced because of the increase in the Doppler offset between the authentic signal and the spoofing signal. However, for a specific spoofing signal, it is difficult to determine whether a spoofing attack would be successful when the bandwidth is more than a certain level. It is also difficult to determine the correlation between each parameter for a successful spoofing attack. In this research, we develop a spoofing process equation (SPE) for the entire spoofing process. Generally, to determine whether a specific spoofing signal would be successful, it is necessary to perform an iterative DLL calculation during the entire spoofing process. The concept of the SPE is to reduce the number of iterative DLL tracking calculations during the spoofing process by increasing the integration time. Moreover, we express the entire DLL calculation process in the form of an nth order polynomial. The spoofing attack results could be obtained in one single calculation through the SPE. The following are the contributions of this study:We develop an SPE that can be used to express the entire spoofing process in the form of an nth order polynomial.We obtain the spoofing results in one single calculation using the SPE and determine the correlation between each parameter based on the boundary line which distinguishes between successful and unsuccessful spoofing attacks.For a particular receiver, the minimum power of a spoofing signal for a successful spoofing attack could be estimated via the SPE.
The remainder of this paper is organized as follows: [Section 2](#sec2-sensors-19-00293){ref-type="sec"} introduces the auto correlation function (ACF) models of the authentic and spoofing signals. [Section 3](#sec3-sensors-19-00293){ref-type="sec"} presents simulations of a spoofing scenario using the ACF models. [Section 4](#sec4-sensors-19-00293){ref-type="sec"} presents the derivation process of the SPE. [Section 5](#sec5-sensors-19-00293){ref-type="sec"} shows the simulation results obtained using the SPE. Finally, the discussions and conclusions are provided in [Section 6](#sec6-sensors-19-00293){ref-type="sec"} and [Section 7](#sec7-sensors-19-00293){ref-type="sec"}, respectively.
2. Authentic and Spoofing Signal ACF Model {#sec2-sensors-19-00293}
==========================================
In this section, the authentic and spoofing signal models are presented. To generate a spoofing signal aligned with the authentic signal, the spoofer should estimate the position and velocity of the target receiver. [Figure 1](#sensors-19-00293-f001){ref-type="fig"} shows a brief illustration of a spoofing scenario. First, the spoofer estimates the position and velocity of the victim receiver using radar \[[@B6-sensors-19-00293]\]. The spoofer can then calculate the aligned spoofing signal by compensating for the spoofer processing delay and transmission delay. Moreover, the power of the spoofing signal should be greater than that of the authentic signal. Therefore, it is necessary to compensate for the propagation loss depending on the distance between the spoofer and the victim receiver. The signal received at the victim receiver antenna can be represented using a complex baseband model as follows:$$s(t) = C\lbrack t - \tau_{a}(t)\rbrack\exp(j\phi_{a}(t)) + \sqrt{P_{s}}C\lbrack t - \tau_{s}(t)\rbrack\exp(j\phi_{s}(t)) + n(t).$$ where, $s(t)$ denotes the total received signal;$C$ denotes the pseudorandom code;$\tau_{a}(t)$ is the code phase of the authentic signal;$\tau_{s}(t)$ is the code phase of the spoofing signal;$P_{s}$ is the spoofing power advantage;$\phi_{a}(t)$ is the carrier phase of the authentic signal;$\phi_{s}(t)$ is the carrier phase of the spoofing signal;$n(t)$ is the complex zero-mean white Gaussian noise (AWGN).
In the receiver, a correlation process is implemented to track the input signal $s(t)$. [Figure 2](#sensors-19-00293-f002){ref-type="fig"} shows the ACF model of $s(t)$. The blue triangle indicates the ACF of the authentic signal, whereas the red triangle indicates the ACF of the spoofing signal. The horizontal axis represents the chip offset, and the vertical axis represents the normalized correlator output (the amplitude of the authentic signal is 1). The parameters, shown in [Figure 2](#sensors-19-00293-f002){ref-type="fig"}, can be expressed as follows:$$\begin{array}{l}
{y_{1} = \left\{ \begin{array}{l}
{x + 1,\text{ } - 1 < \tau \leq 0} \\
{0,\text{ }{else}} \\
\end{array} \right.} \\
{y_{2} = \left\{ \begin{array}{l}
{- x + 1,\text{ }0 < \tau < 1} \\
{0,\text{ }{else}} \\
\end{array} \right.} \\
{R_{a}(\tau) = y_{1} + y_{2}} \\
{D = \tau_{s} - \tau_{a}} \\
{y_{s1} = \left\{ \begin{array}{l}
{a_{s}(x + 1 - D),\text{ } - 1 + D < \tau \leq D} \\
{\text{ }0,\text{ }{else}} \\
\end{array} \right.} \\
{y_{s2} = \left\{ \begin{array}{l}
{a_{s}( - x + 1 + D),\text{ }D < \tau < 1 + D} \\
{\text{ }0,\text{ }{else}} \\
\end{array} \right.} \\
{R_{s}(\tau) = y_{s1} + y_{s2}} \\
{R(\tau) = R_{a}(\tau) + R_{s}(\tau)} \\
\end{array}$$ where:$y_{1}$ indicates the left line of the ACF of the authentic signal;$y_{2}$ indicates the right line of the ACF of the authentic signal;$R_{a}(\tau)$ is the ACF of the authentic signal;$D$ is the difference in the code phases between spoofing and authentic signals;$y_{s1}$ indicates the left line of the ACF of the spoofing signal;$y_{s2}$ indicates the right line of the ACF of the spoofing signal;$a_{s}$ is the slope of the ACF of the spoofing signal;$R_{s}(\tau)$ is the ACF of the authentic signal;$R(\tau)$ is the ACF of the total signal;*XE* is the accumulation result with the replica code separated 0.5 chip early;*XP* is the accumulation result with the replica code;*XL* is the accumulation result with the replica code separated 0.5 chip late.
The *XP* could be written as \[[@B20-sensors-19-00293]\]:$$\begin{array}{l}
{XP\lbrack n\rbrack = R(\Delta\tau_{a}\lbrack n\rbrack) \cdot \frac{\sin(\pi\Delta f_{a}\lbrack n\rbrack \cdot T)}{\pi\Delta f_{a}\lbrack n\rbrack \cdot T}\exp(j\Delta\phi_{a}\lbrack n\rbrack)} \\
{\text{ } + R\lbrack\Delta\tau_{s}\lbrack n\rbrack\rbrack \cdot \frac{\sin(\pi\Delta f_{s}\lbrack n\rbrack \cdot T)}{\pi\Delta f_{s}\lbrack n\rbrack \cdot T}\exp(j\Delta\phi_{s}\lbrack n\rbrack)} \\
\end{array}$$ where:$\Delta\tau_{a}\lbrack n\rbrack$ is the code phase difference between the local replica and the authentic signal;$\Delta\tau_{s}\lbrack n\rbrack$ is the code phase difference between the local replica and the spoofing signal;$\Delta f_{a}\lbrack n\rbrack$ is the Doppler frequency difference between the local replica and the authentic signal;$\Delta f_{s}\lbrack n\rbrack$ is the Doppler frequency difference between the local replica and the spoofing signal;$\Delta\phi_{a}\lbrack n\rbrack$ is the carrier phase difference between the local replica and the authentic signal;$\Delta\phi_{s}\lbrack n\rbrack$ is the carrier phase difference between the local replica and the spoofing signal.
In the next section, we explain the change in the replica code phase, $\tau$, depending on the success or failure of a spoofing attack. In our simulation, we assume that the code phase and Doppler frequency of the replica are perfectly aligned with the authentic signal before the spoofing signal approaches. This implies that $\Delta\tau_{a}\lbrack 1\rbrack$ and $\Delta f_{a}\lbrack 1\rbrack$ are zero.
3. Spoofing Scenario Simulation Using ACF Model {#sec3-sensors-19-00293}
===============================================
Using the ACF models explained in [Section 2](#sec2-sensors-19-00293){ref-type="sec"}, we conduct the spoofing simulation. We assume that the authentic signal is stationary and that the spoofing signal is moving from right to left with a static velocity. This simulation is done without noise. In general, the DLL discriminator is used to calculate the feedback output using *XE* and *XL* and thereby track the incoming signal. The replica code phase gradually aligns with the code start point of the incoming signal during DLL code tracking. In our spoofing simulation, the DLL initially tracks the authentic signal. When the spoofing signal approaches and overlays with the authentic signal, the ACF changes. [Figure 3](#sensors-19-00293-f003){ref-type="fig"} shows the sequential ACF variation during the spoofing simulation. The green circle indicates the *XP*, which is the prompt of the DLL. The position of *XP* in each figure is different. The position of *XP* is determined by the shape of the ACF and the positions of previous *XP* and DLL settings.
[Figure 4](#sensors-19-00293-f004){ref-type="fig"}a,b show the $\tau$ histories of the two spoofing simulations. The only difference between the two simulations is the DLL bandwidth. In [Figure 4](#sensors-19-00293-f004){ref-type="fig"}, the black lines indicate the code phase distance between the authentic and spoofing signals. Because the authentic signal is fixed at zero, this can be considered the position of the spoofing signal relative to the authentic signal in the code domain. We now focus on [Figure 4](#sensors-19-00293-f004){ref-type="fig"}a. The green arrow indicates the start point of $\tau$. At the start of the simulation, $\tau$ is zero. The spoofing signal approaches the authentic signal from a distance of two chips. When the spoofing signal reaches a distance of 1.5 chips, $\tau$ starts to gradually increase, as the spoofing signal starts to affect *XL*. A blue arrow indicates the point where $\tau$ is increasing. The peak point of the total ACF $R(\tau)$ is always the same as that of the spoofing ACF $R_{a}(\tau)$. Therefore, $\tau$ moves to the peak point of $R(\tau)$ until the discriminator output becomes zero. After the spoofing signal passes the authentic signal, the peak point of $R(\tau)$ is located on the negative side, as shown in [Figure 3](#sensors-19-00293-f003){ref-type="fig"}c. Finally, $\tau$ follows the spoofing signal. The orange arrow represents the final value of the $\tau$. In the case of [Figure 4](#sensors-19-00293-f004){ref-type="fig"}a, $\tau$ follows the spoofing signal, and therefore, the spoofing attack is successful. [Figure 4](#sensors-19-00293-f004){ref-type="fig"}b shows the other spoofing simulation case. In [Figure 4](#sensors-19-00293-f004){ref-type="fig"}b, the final value of $\tau$ returns to zero. $\tau$ seems to chase the spoofing signal, as indicated using the dotted black line, but eventually returns to its location. This implies that the spoofing attack is a failure. The difference between the two simulations is that the DLL bandwidth. The DLL bandwidth is 5 Hz in the first simulation, whereas it is 3 Hz in the second simulation. As shown in the simulation results, the greater the bandwidth of the receiver, the more vulnerable it is to a spoofing attack. Moreover, the higher the strength of the spoofing signal, the higher is the probability of a successful spoofing attack. The faster the spoofing signal sweeps, the more likely it is that the spoofing attack will fail. [Table 1](#sensors-19-00293-t001){ref-type="table"} lists the changes in the spoofing attack results with respect to increases in the bandwidth, signal strength and sweep velocity. However, it is difficult to determine how strong a signal should be for a successful spoofing attack. It is also difficult to obtain a correlation between the different parameters for a successful spoofing attack.
4. Development of Spoofing Process Equation {#sec4-sensors-19-00293}
===========================================
4.1. Conventional Approach for τ Calculation {#sec4dot1-sensors-19-00293}
--------------------------------------------
*XP* is calculated through DLL using the ACF and previous *XP*. The first-order DLL can be expressed as follows:$$\begin{array}{l}
{\Delta\tau\lbrack n\rbrack = \frac{XE\lbrack n\rbrack - XL\lbrack n\rbrack}{2}} \\
{\tau\lbrack n + 1\rbrack = \tau\lbrack n\rbrack - \omega_{0} \cdot T \cdot \Delta\tau\lbrack n\rbrack} \\
{\omega_{0} = \frac{B}{4}} \\
\end{array}$$ where $\Delta\tau$, $T$, and $B$ indicate the discriminator output, integration time, and DLL bandwidth, respectively. In general, the spoofing attack results can be obtained by determining which signal the DLL is tracking after the spoofing signal completely sweeps the authentic signal. In other words, if the integration time of the receiver is 1 ms, it is necessary to repeatedly calculate the equation thousands of times to obtain the spoofing attack results. This calculation can be expressed as follows:$$\begin{array}{l}
{k = \frac{\omega_{0} \cdot T}{2}} \\
{\tau\lbrack 2\rbrack = \tau\lbrack 1\rbrack - \omega_{0} \cdot T \cdot \Delta\tau\lbrack 1\rbrack = \tau\lbrack 1\rbrack - k \cdot \{ R_{1}(\tau\lbrack 1\rbrack - \frac{1}{2}) - R_{1}(\tau\lbrack 1\rbrack + \frac{1}{2})\}} \\
{\tau\lbrack 3\rbrack\, = \tau\lbrack 2\rbrack - \omega_{0} \cdot T \cdot \Delta\tau\lbrack 2\rbrack = \,\tau\lbrack 2\rbrack - k \cdot \{ R_{2}(\tau\lbrack 2\rbrack - \frac{1}{2}) - R_{2}(\tau\lbrack 2\rbrack + \frac{1}{2})\}} \\
{\text{ }.} \\
{\text{ }.} \\
{\text{ }.} \\
{\tau\lbrack n\rbrack = \tau\lbrack n - 1\rbrack + \omega_{0} \cdot T \cdot \Delta\tau\lbrack n - 1\rbrack = \,\tau\lbrack n - 1\rbrack - k \cdot \{ R_{n - 1}(\tau\lbrack n - 1\rbrack - \frac{1}{2}) - R_{n - 1}(\tau\lbrack n - 1\rbrack + \frac{1}{2})\}} \\
{\tau\lbrack n\rbrack = \tau\lbrack 1\rbrack - (n - 1) \cdot k \cdot {\sum\limits_{m = 1}^{n - 1}{\{ R_{m}(\tau\lbrack m\rbrack - \frac{1}{2}) - R_{m}(\tau\lbrack m\rbrack + \frac{1}{2})}}\}} \\
\end{array}$$
For a specific spoofing attack scenario, a lot of computations are required to calculate the final replica code phase $\tau\lbrack n\rbrack$. Moreover, it is necessary to know $\tau$ and ACF at all previous epochs. Thus, the final $\tau$ value can be written as follows:$$\tau\lbrack n\rbrack = f_{\tau}(\tau\lbrack 1\rbrack,\text{ }\tau\lbrack 2\rbrack,\text{ }\tau\lbrack 3\rbrack\text{ },\,,,\text{ }\tau{\lbrack n - 1\rbrack}_{,}R_{1},R_{2},R_{3},,,R_{n - 1}).$$
4.2. Proposed Approach for τ Calculation {#sec4dot2-sensors-19-00293}
----------------------------------------
In this subsection, we propose a method to compute the spoofing attack results by calculating each epoch at a certain chip interval (CI). The entire spoofing process is summarized in a mathematical equation, i.e., the SPE, and the spoofing results are obtained by one calculation using the SPE. [Figure 5](#sensors-19-00293-f005){ref-type="fig"} shows the results of the $\tau$ estimation with respect to the CI. The blue lines indicate the calculation results of $\tau$ per *1* ms. Red circles indicate the calculation results of $\tau$ per specific CI. The equation for calculating the time interval (TI) in terms of the chip interval can be expressed as follows:$$TI = \frac{293}{V_{s}} \cdot CI,$$ where $V_{s}$ denotes the spoofing sweep velocity (m/s), and the number 293 indicates the wavelength of the C/A code in meter-scale. [Table 2](#sensors-19-00293-t002){ref-type="table"} lists the calculated TI with respect to each CI in case of the spoofing sweep velocity is 80 m/s. $\tau$ error decreases with the decrease in the CI. However, additional calculations are required to estimate the final $\tau$ when CI is low. The concept of CI is similar with sampling interval. CI determines how often the SPE calculates the replica code phase. In our research, we set the CI to 0.125 considering the complexity of the equation and $\tau$ error.
4.3. Spoofing Attack Success or Failure Criteria {#sec4dot3-sensors-19-00293}
------------------------------------------------
[Figure 6](#sensors-19-00293-f006){ref-type="fig"}a shows the $\tau$ results of DLL when the spoofing signal sweeps the authentic signal with constant velocity. The blue line indicates the case of a successful spoofing attack, whereas the red line indicates the case of a failed spoofing attack. Generally, the success or failure of a spoofing attack can be determined from the type of signal the DLL tracks when the spoofing signal completely sweeps the authentic signal. In both the simulations, the only difference is the bandwidth of the DLL.
The success or failure of a spoofing attack can be determined by looking at the absolute value of $\tau$ at the point where *D* is −1. [Figure 6](#sensors-19-00293-f006){ref-type="fig"}b shows the region enclosed in the black box shown in [Figure 6](#sensors-19-00293-f006){ref-type="fig"}a. If the spoofing attack is successful, the absolute value of $\tau$ exceeds 0.5 at the point where *D* is −1, and if it fails, the absolute value of the prompt is lower than 0.5.
[Figure 7](#sensors-19-00293-f007){ref-type="fig"}a shows the $\tau$ estimates for various spoofing parameters listed in [Table 3](#sensors-19-00293-t003){ref-type="table"}. It is noteworthy that the absolute value of $\tau$ at *D* is −1. As shown in [Figure 7](#sensors-19-00293-f007){ref-type="fig"}b, if the absolute value of $\tau$ exceeds 0.5 chip when *D* is −1, the DLL tracks the spoofing signal.
The following analysis shows that the criterion used for determining the spoofing result is reasonable. If the spoofing sweep velocity is very low or if the bandwidth is very high in the spoofing simulation, there will be sufficient time or control input for the DLL to track the peak point of the ACF. In this case, the discriminator output would become zero and *XP* would be located at the point where *XE* equals *XL*. [Figure 8](#sensors-19-00293-f008){ref-type="fig"} shows a series of snapshots where the discriminator output is zero with respect to the ACF. The $\tau$ value for the case, shown in [Figure 8](#sensors-19-00293-f008){ref-type="fig"}a, can be derived as follows:$$\begin{array}{l}
{XE = y_{1} + y_{s1}} \\
{= x + 1 + a_{s}(x + 1 - D)} \\
{= (a_{s} + 1)x + a_{s} - a_{s}D + 1} \\
{= (a_{s} + 1)(\tau - \frac{1}{2}) + a_{s} - a_{s}D + 1} \\
{XL = y_{2} + y_{s2}} \\
{= - x + 1 + a_{s}( - x + 1 + D)} \\
{= - (a_{s} + 1)x + a_{s} + a_{s}D + 1} \\
{= - (a_{s} + 1)(\tau + \frac{1}{2}) + a_{s} + a_{s}D + 1} \\
{XE = XL} \\
{(a_{s} + 1)(\tau - \frac{1}{2}) + a_{s} - a_{s}D + 1 = - (a_{s} + 1)(\tau + \frac{1}{2}) + a_{s} + a_{s}D + 1} \\
{(a_{s} + 1)\tau - \frac{1}{2}(a_{s} + 1) + a_{s} - a_{s}D + 1 = - (a_{s} + 1)\tau - \frac{1}{2}(a_{s} + 1) + a_{s} + a_{s}D + 1} \\
{2(a_{s} + 1)\tau = 2a_{s}D} \\
{\tau = \frac{a_{s}D}{a_{s} + 1}} \\
\end{array}$$
Moreover, the $\tau$ values for the cases, shown in [Figure 8](#sensors-19-00293-f008){ref-type="fig"}b,c, can be derived in a similar manner as follows:$$\begin{array}{l}
{y_{s1} = y_{1} + y_{s2}} \\
{a_{s}(\tau - \frac{1}{2}) + a_{s} - a_{s}d = ( - a_{s} + 1)(\tau + \frac{1}{2}) + a_{s} + a_{s}d + 1} \\
{\tau = \frac{2a_{s}d + \frac{3}{2}}{2a_{s} - 1}} \\
\end{array}$$ $$\begin{array}{l}
{y_{s1} = y_{s2}} \\
{a_{s}(\tau - \frac{1}{2}) + a_{s} - a_{s}D = - a_{s}(\tau + \frac{1}{2}) + a_{s} + a_{s}D} \\
{\tau = D} \\
\end{array}$$
[Figure 9](#sensors-19-00293-f009){ref-type="fig"} shows the summary of Equations (8) to (10). For any ACF, shown in [Figure 8](#sensors-19-00293-f008){ref-type="fig"}, $\tau$ can be estimated using $a_{s}$ and *D* when the spoofing sweep velocity is very low or when the bandwidth is considerable. Moreover, it is possible to calculate *D* corresponding to the different equations of $\tau$ [Figure 10](#sensors-19-00293-f010){ref-type="fig"} shows the ACF change with respect to the spoofing signal when the spoofing signal strength is lower than the authentic signal strength. We can derive an equation to calculate $\tau$ in the same manner as above. The $\tau$ value for the cases, shown in [Figure 10](#sensors-19-00293-f010){ref-type="fig"}a,b,c can be derived as follows:$$\begin{array}{l}
{y_{1} + y_{s1} = y_{2} + y_{s2}} \\
{(a_{s} + 1)(\tau - \frac{1}{2}) + a_{s} - a_{s}D + 1 = - (a_{s} + 1)(\tau + \frac{1}{2}) + a_{s} + a_{s}D + 1} \\
{\tau = \frac{a_{s}D}{a_{s} + 1}} \\
\end{array}$$ $$\begin{array}{l}
{y_{1} + y_{s2} = y_{2}} \\
{( - a_{s} + 1)(\tau - \frac{1}{2}) + a_{s} + a_{s}D + 1 = - (\tau + \frac{1}{2}) + 1} \\
{\tau = \frac{\frac{3}{2}a_{s} + a_{s}D}{a_{s} - 2}} \\
\end{array}$$ $$\begin{array}{l}
{y_{1} = y_{2}} \\
{(\tau - \frac{1}{2}) + 1 = - (\tau + \frac{1}{2}) + 1} \\
{\tau = 0} \\
\end{array}$$
[Figure 11](#sensors-19-00293-f011){ref-type="fig"} shows the summary of Equations (11) to (13). [Figure 12](#sensors-19-00293-f012){ref-type="fig"} shows a graphical representation of Equations (8) to (13). The blue lines indicate spoofing attack success, whereas the red lines indicate spoofing attack failure. If $a_{s}$ is greater than 1, $\tau$ follows the blue line, and if $a_{s}$ is lower than 1, it follows the red line. When $a_{s}$ is 1, *D* at the time of transition, from (b) to (c) in [Figure 8](#sensors-19-00293-f008){ref-type="fig"}, is −1, and the absolute value of $\tau$ becomes 0.5. In case that the spoofing sweep velocity is infinitely slow or the DLL bandwidth is infinitely large, the condition for a successful spoofing attack is that the power of the spoofing signal sets in greater than that of the authentic signal. If the spoofing signal power is a little greater than the authentic signal power, the replica code phase is lower than −0.5 at D is −1. If the spoofing signal power is a little smaller than the authentic signal power, the replica code phase is higher than −0.5 at D is −1. Thus, we could regard that the $\tau$ value is a boundary value when D is −1. This implies that the absolute value of $\tau$ should exceed 0.5 chip before D approaches −1 for a successful spoofing attack. [Figure 13](#sensors-19-00293-f013){ref-type="fig"}a shows the $\tau$ histories with respect the spoofing signal power. $\varepsilon$ indicates a very small positive value. Slope of each line is differently changed after D exceeds −1. [Figure 13](#sensors-19-00293-f013){ref-type="fig"}b shows the $\tau$ value change with respect the spoofing parameters in the real scenario.
4.4. Derivation of SPE {#sec4dot4-sensors-19-00293}
----------------------
Assuming that the spoofing signal shifts from right to left, the spoofing signal affects the DLL discriminator when *D* approaches within 1.5 chip. Moreover, to determine the spoofing results, we only need to calculate $\tau$ until *D* reaches −1. Therefore, if CI is 0.125 chip, it is possible to determine the spoofing attack results by a total of 19 calculations. In Equation (4), all the previous *τ* values and ACF are required to calculate $\tau\lbrack n\rbrack$. The SPE can be used to calculate *τ* at the point where *D* is −1 to determine whether the spoofing attack is a successful one or not by only one calculation. When setting the CI to 0.125, the ACF variation could be known. However, it is not possible to specify the previous *τ* values. This is because *τ* value at a certain D changes according to the spoofing parameters. However, the range of *τ* can be defined for each D value for *τ* to be close to −0.5 chip when D is −1. The spoofing attack results can be determined by checking whether the absolute value of *τ* at D = −1 exceeds 0.5 chip or not. In our case, *τ*\[19\] is the final *τ* value. For *τ*\[19\] to be close to −0.5, *τ*\[18\] must be in a specific range. Moreover, *τ*\[17\] must be in a specific range for *τ*\[18\] to be in the defined range. Thus, we can define each range according to the D value of the entire process. [Table 4](#sensors-19-00293-t004){ref-type="table"} lists the range of *τ*\[*i*\] for each *D*\[*i*\] where *i* denotes each CI index. There are ACF models of *XE* and *XL* for each D. If each *τ*\[*i*\] is within the defined range for each *D*\[*i*\], *τ*\[19\] will be calculated close to −0.5. The details of the SPE derivation are given in appendix A. Finally, SPE has the following form like:$$\tau\lbrack 19\rbrack = f(a_{s},Vs,B).$$
The inputs to the SPE are the spoofing signal strength, spoofing sweep velocity and DLL bandwidth. When the calculation of SPE is performed, $\tau\lbrack 19\rbrack$ is calculated for D = −1 by just one calculation. The success or failure of the spoofing attack is determined by the absolute value of $\tau$.
5. Analysis of SPE Simulation Results {#sec5-sensors-19-00293}
=====================================
5.1. SPE Performance Analysis {#sec5dot1-sensors-19-00293}
-----------------------------
To verify the performance of the SPE, we compared the estimated SPE results with the original DLL results. [Table 5](#sensors-19-00293-t005){ref-type="table"} presents the various spoofing signal parameters and $\tau$ results in the cases of using the original DLL and SPE at *D* = *−*1.
$\tau_{1ms}$ indicates the estimated replica code phase obtained using the original DLL, the integration time of which is *1ms*. $\tau_{SPE}$ is the estimated replica code phase obtained using the SPE. The calculation time required by the SPE is significantly lower than that required by the original DLL, because $\tau_{SPE}$ can be calculated in just one calculation using the SPE. We can see that the replica code phase values estimated using the two methods are very similar. [Figure 14](#sensors-19-00293-f014){ref-type="fig"} shows the $\tau_{SPE}$ errors with respect to CI. The $\tau_{SPE}$ errors decrease with the decrease in CI. Thus, the SPE error is due to the reduction in the number of DLL calculations during the spoofing attack process.
[Figure 15](#sensors-19-00293-f015){ref-type="fig"} shows the error distribution of the SPE with respect to the spoofing signal strength and sweep velocity on a fixed bandwidth. At the yellow point, the values of the spoofing parameters, namely the signal strength offset, sweep velocity, and bandwidth, are 2 dB, 50 m/s, and 2 Hz, respectively. When $\tau_{SPE}$ is calculated for the above set of spoofing parameter values using the SPE, the error in $\tau_{SPE}$ is the Z axis value corresponding to the yellow point. The SPE performance is the best around the boundary line. The success and failure of the spoofing attack can be divided based on the boundary line.
In other words, $\tau_{SPE}$ of the spoofing parameters on the boundary line is *−*0.5. $\tau_{SPE}$ error increases as the distance from the boundary value and spoofing parameter increases. A large error indicates that the previous $\tau$ values are outside the defined range in the $\tau_{SPE}$ calculation process. We define the range of previous replica code phase range at every *D\[i\]* like [Table 4](#sensors-19-00293-t004){ref-type="table"} in order that the final code phase is calculated around the boundary line at *D is* −1. If the previous code phases, $\left. \tau\lbrack 1\rbrack \right.\sim\tau\lbrack 18\rbrack$, are deviated from the defined range, SPE would provide inaccurate result.
Also, it could be explained that the ACF functions used to calculate *XE* and *XL* vary with respect to the already defined *XE* and *XL*. [Figure 16](#sensors-19-00293-f016){ref-type="fig"} shows the SPE error distribution in two dimensions. The SPE error is lowest around the boundary line. The spoofing parameters are divided using different colors with respect to the SPE error size.
5.2. Determination of Boundary Line and Surface Using SPE {#sec5dot2-sensors-19-00293}
---------------------------------------------------------
The boundary line and surface that divide the spoofing attack success and failure can be estimated using the SPE. The input parameters of the SPE are the spoofing signal strength, spoofing signal sweep velocity, and DLL bandwidth. In Equation (15), if we set each variable as follows:$$- 0.5 = f(a_{s},40,2),$$
Only one variable, i.e., as, remains, and the SPE becomes an equation to calculate as. We use MATLAB solver to obtain ${\widetilde{a}}_{s}$ which is an estimated value of $a_{s}$ obtained using Equation (15). This means that the SPE result of the spoofing parameters, (${\widetilde{a}}_{s}$, 40, 2), becomes −0.5. Therefore, the spoofing parameter, (${\widetilde{a}}_{s} + \varepsilon$, 40, 2), will result in a successful spoofing attack. The other spoofing parameter, (${\widetilde{a}}_{s} - \varepsilon$, 40, 2), will result in a failed spoofing attack. $\varepsilon$ is a small positive value. [Figure 17](#sensors-19-00293-f017){ref-type="fig"}a shows the boundary line dividing the spoofing attack success and failure zones. We obtain the spoofing signal strength values by fixing the other spoofing parameters and $\tau$. [Table 6](#sensors-19-00293-t006){ref-type="table"} presents the estimated ${\widetilde{a}}_{s}$ values with respect to the spoofing parameters. The red line in [Figure 17](#sensors-19-00293-f017){ref-type="fig"}a indicates the boundary line. The boundary can be estimated using the spoofing parameters listed in [Table 6](#sensors-19-00293-t006){ref-type="table"} as follows:$$\begin{array}{l}
{V_{s} = f_{bl}(a_{s})} \\
{\text{ } = \, p_{1} \cdot a_{s}^{3} + p_{2} \cdot a_{s}^{2} + p_{3} \cdot a_{s} + p_{4}} \\
\end{array}$$ where $f_{bl}$ is the function of the boundary line for the DLL bandwidth of 2 Hz. $p_{1}$, $p_{2}$, $p_{3}$, and $p_{4}$ are the coefficients at the boundary line. Moreover, this line expresses the correlation between two parameters for a successful spoofing attack. From Equation (16), we find that as the spoofing signal strength increases, the spoofing attack becomes successful even with a higher sweep velocity.
The spoofing attack success and failure zone is divided based on the boundary line, as shown in [Figure 17](#sensors-19-00293-f017){ref-type="fig"}b. The zone above the boundary line indicates spoofing attack failure, whereas the zone below indicates spoofing attack success. [Figure 18](#sensors-19-00293-f018){ref-type="fig"}a shows the boundary lines for various DLL bandwidths. We find that the receiver becomes more vulnerable to spoofing attacks as its DLL bandwidth increases. With the increase in the DLL bandwidth, the spoofing attack becomes successful even for a low spoofing signal strength when using a fixed spoofing sweep velocity. [Figure 18](#sensors-19-00293-f018){ref-type="fig"}b shows the boundary lines in three dimensions.
The boundary surface can be estimated using the boundary lines, as shown in [Figure 19](#sensors-19-00293-f019){ref-type="fig"}. The boundary surface can be expressed as follows:$$\begin{array}{l}
{B = f_{sf}(a_{s},V_{s})} \\
{\text{\,\, \,\,} = c_{1} + c_{2}a_{s} + c_{3}V_{s} + c_{4}a_{s}^{2} + c_{5}a_{s}^{2}V_{s} + c_{6}a_{s}V_{s}^{2} + c_{7}V_{s}^{3} + c_{8}a_{s}^{3}V_{s} + c_{9}a_{s}^{2}V_{s}^{2} + c_{10}a_{s}V_{s}^{3} + c_{11}V_{s}^{4}} \\
\end{array}$$ where $f_{sf}$ indicates the function of the boundary surface. $\left. c_{1} \right.\sim c_{11}$ are coefficients of $f_{sf}$. For specific spoofing parameters, the spoofing attack results can be determined using $f_{sf}$. Equation (18) is the case for spoofing attack failure, whereas Equation (19) is the case for spoofing attack success:$$B > f_{sf}(a_{s},V_{s}),$$ $$B < f_{sf}(a_{s},V_{s}).$$
[Table 7](#sensors-19-00293-t007){ref-type="table"} presents the computational complexities of the conventional DLL and SPE in terms of the number of iteration and computational time with respect to the spoofing signal velocity. We can see that the proposed method has much lower computational complexity than that of the conventional DLL. The simulation were performed on a personal computer with Intel Core i7-4790k. In case of the conventional DLL, as the spoofing signal velocity decreases, the computational load increases. In contrast, a consistent computational load is required in case of SPE.
6. Discussion {#sec6-sensors-19-00293}
=============
- The SPE can be derived in the same manner regardless of the DLL order. The details of the same are given in [Appendix B](#app2-sensors-19-00293){ref-type="app"}.
- In this paper, we analyzed the effect of the spoofing signal on the local replica code phase using the SPE. However, for a completely successful spoofing attack, the point of FLL tracking should be moved from the authentic signal to the spoofing signal. In the future, we will focus on spoofing process analysis in the frequency domain.
- Our simulation is conducted without any noise. If noise is added to our simulation, the probability distribution around the boundary line can be obtained using the SPE. The probability of spoofing attack success or failure on the boundary line would be 50%.
7. Conclusions {#sec7-sensors-19-00293}
==============
Analyzing the replica code phase variation due to the reception of the spoofing signal is important for developing spoofing attack or anti-spoofing techniques. In this paper, we propose an SPE that can be used to calculate the replica code phase following a spoofing attack and determine whether the spoofing attack is successful using the SPE output. The advantage of the SPE is that it could theoretically create a minimal spoofing signal condition for a successful spoofing attack. The boundary surface dividing the spoofing attack success or failure is obtained using the SPE. The boundary surface shows the correlation of how each spoofing parameter affects the code tracking results. This study is meaningful in that it presents a detailed study about the variation in the replica code phase during a spoofing attack process. We expect that the research results would aid the development of spoofing attack or anti-spoofing techniques.
This work has been supported by the program 'Satellite Navigation Augmentation to Improve Navigation Technology' of Agency for Defense Development, contracted through the SNU-IAMD. This research was supported (in part) by the Institute of Advanced Aerospace Technology at Seoul National University. The Institute of Engineering Research at Seoul National University provided research facilities for this work.
This research was carried out in collaborations with all authors. B.S., M.P., S.J. and C.K. designed the proposed method. B.S. and M.P. performed the simulation. S.J., H.S, and G.K. verified the proposed method and analyzed the simulation results. B.S. and M.P. drafted the manuscript. C.K. corrected the whole manuscript. All authors contributed and improved the manuscript.
The authors declare no conflicts of interest.
{#sensors-19-00293-f0A1}
The details of SPE derivation are as follows. [Figure A1](#sensors-19-00293-f0A1){ref-type="fig"} shows the ACF snapshots for *i* ranging from 1 to 4. If $\tau\lbrack i\rbrack - 0.5$ and $\tau\lbrack i\rbrack + 0.5$ are in the defined range, *XE* and *XL* can be calculated using $y_{1}(\tau\lbrack i\rbrack - 0.5)$ and $y_{2}(\tau\lbrack i\rbrack + 0.5) + y_{s2}(\tau\lbrack i\rbrack + 0.5)$, respectively. Thus, first-order DLL could be expressed until $i \leq 5$ like:$$\tau\lbrack i\rbrack = \tau\lbrack i - 1\rbrack + k\{(a - 2)\tau\lbrack i - 1\rbrack + \frac{3}{2}a_{s} - ad\lbrack i - 1\rbrack\}.$$
Also, $\tau\lbrack 1\rbrack$, $\tau\lbrack 2\rbrack$, $\tau\lbrack 3\rbrack$, and $\tau\lbrack 4\rbrack$ can be expressed as follows:$$\begin{array}{l}
{\tau\lbrack 1\rbrack = - k \cdot \{ R_{1}(\tau\lbrack 1\rbrack - \frac{1}{2}) - R_{1}(\tau\lbrack 1\rbrack + \frac{1}{2})\}} \\
{\text{ } = - k \cdot \{ y_{1}( - \frac{1}{2}) - y_{2}(\frac{1}{2}) - y_{s1}(\frac{1}{2})\}} \\
{\text{ } = k(\frac{3}{2}a_{s} - D\lbrack 1\rbrack)} \\
\end{array},$$
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After $i > 5$, the first-order DLL could be expressed as follows:$$\tau\lbrack i\rbrack = \tau\lbrack i - 1\rbrack + k\{ - (2a_{s} + 2)\tau\lbrack i - 1\rbrack + 2a_{s}d\lbrack i - 1\rbrack\}.$$
If $\tau$ is developed until $i = n$, SPE is complete. SPE has the following generalized form like:$$\tau\lbrack n\rbrack = {\sum\limits_{i = 1}^{n}{g_{i,n}(a_{s})k^{i}}},$$ $$g_{n,n}(a_{s}) = {( - 1)}^{m}{}_{m}C_{m}{(2a_{s} + 2)}^{m}{(a_{s} - 2)}^{l - 1}(\frac{3}{2}a_{s} - ad\lbrack 1\rbrack),$$ $$\begin{array}{l}
{g_{n - 1,n}(a_{s}) = {( - 1)}^{m - 1}{}_{m}C_{m - 1}{(2a_{s} + 2)}^{m - 1}{(a_{s} - 2)}^{l - 1}(\frac{3}{2}a_{s} - a_{s}d\lbrack 1\rbrack)\,} \\
{+ {( - 1)}^{m}3_{m}C_{m}{(2a_{s} + 2)}^{m}{(a_{s} - 2)}^{l - 2}(\frac{3}{2}a_{s} - a_{s}d\lbrack 1\rbrack)} \\
{+ {( - 1)}^{m}3_{m}C_{m}{(2a_{s} + 2)}^{m}{(a_{s} - 2)}^{l - 2}\{ 2\frac{3}{2}a - a(d\lbrack 1\rbrack + d\lbrack 2\rbrack)\}} \\
\end{array},$$ $$\begin{array}{l}
{g_{n - 2,n}(a_{s}) = {( - 1)}^{m - 2}{}_{m}C_{m - 2}{(2a_{s} + 2)}^{m - 2}{(a - 2)}^{l - 1}(\frac{3}{2}a_{s} - a_{s}d\lbrack 1\rbrack)} \\
{\, + {( - 1)}^{m - 1}3_{m}C_{m - 1}{(2a_{s} + 2)}^{m - 1}{(a_{s} - 2)}^{m - 2}(\frac{3}{2}a_{s} - a_{s}d\lbrack 1\rbrack)} \\
{\, + {( - 1)}^{m - 1}2_{m}C_{m - 1}{(2a_{s} + 2)}^{m - 1}{(a_{s} - 2)}^{l - 2}\{ 2\frac{3}{2}a_{s} - a_{s}(d\lbrack 1\rbrack + d\lbrack 2\rbrack)\}} \\
{\, + {( - 1)}^{m}3_{m}C_{m}{(2a_{s} + 2)}^{m}{(a_{s} - 2)}^{l - 3}(\frac{3}{2}a_{s} - a_{s}d\lbrack 1\rbrack) + {( - 1)}^{m}2_{m}C_{m}{(2a_{s} + 2)}^{m}{(a_{s} - 2)}^{l - 3}\{ 2\frac{3}{2}a_{s} - a_{s}(d\lbrack 1\rbrack + d\lbrack 2\rbrack)\}} \\
{\, + {( - 1)}^{m}{}_{m}C_{m}{(2a_{s} + 2)}^{m}{(a_{s} - 2)}^{l - 3}\{ 3\frac{3}{2}a - a(d\lbrack 1\rbrack + d\lbrack 2\rbrack + d\lbrack 3\rbrack)\}} \\
\end{array}.$$
To derive the SPE, each DLL order could be expressed as follows:$$\begin{array}{l}
{\tau\lbrack n + 1\rbrack = \tau\lbrack n\rbrack - \omega_{0} \cdot T \cdot \Delta\tau\lbrack n\rbrack} \\
{\text{ \quad\quad\,} = \tau\lbrack n\rbrack - K_{1} \cdot \Delta\tau\lbrack n\rbrack} \\
\text{ } \\
\end{array}.$$ $$\begin{array}{l}
{\tau\lbrack n + 1\rbrack = \tau\lbrack n\rbrack - \{{(\omega_{1} \cdot T)}^{2} \cdot \Delta\tau\lbrack n\rbrack + a_{2} \cdot \omega_{1} \cdot T \cdot \Delta\tau\lbrack n\rbrack\} + \tau_{1}\lbrack n\rbrack \cdot T} \\
{\text{ \quad\quad\,} = \tau\lbrack n\rbrack - (k_{2} \cdot \Delta\tau\lbrack n\rbrack + k_{3} \cdot \Delta\tau\lbrack n\rbrack) + \tau_{1}\lbrack n\rbrack \cdot T} \\
{\text{ \quad\quad\,} = \tau\lbrack n\rbrack - (k_{2} + k_{3})\Delta\tau\lbrack n\rbrack + \tau_{1}\lbrack n\rbrack \cdot T} \\
{\text{ \quad\quad\,} = \tau\lbrack n\rbrack - K_{2} \cdot \Delta\tau\lbrack n\rbrack + \tau_{1}\lbrack n\rbrack \cdot T} \\
\text{ } \\
\end{array}$$ $$\begin{array}{l}
{\tau\lbrack n + 1\rbrack = \tau\lbrack n\rbrack - \{{(\omega_{2} \cdot T)}^{3} \cdot \Delta\tau\lbrack n\rbrack + {(a_{3} \cdot \omega_{2} \cdot T)}^{2}\Delta\tau\lbrack n\rbrack} \\
{\text{ \quad\quad\, } + (b_{3} \cdot \omega_{2} \cdot T) \cdot \Delta\tau\lbrack n\rbrack\} + \tau_{2}\lbrack n\rbrack \cdot T^{2} + \tau_{3}\lbrack n\rbrack \cdot T} \\
{\text{ \quad\quad\,} = \tau\lbrack n\rbrack - (k_{4}\Delta\tau\lbrack n\rbrack + k_{5}\Delta\tau\lbrack n\rbrack + k_{6}\Delta\tau\lbrack n\rbrack) + \tau_{2}\lbrack n\rbrack \cdot T^{2} + \tau_{3}\lbrack n\rbrack \cdot T} \\
{\text{ \quad\quad\,} = \tau\lbrack n\rbrack - (k_{4} + k_{5} + k_{6})\Delta\tau\lbrack n\rbrack + \tau_{2}\lbrack n\rbrack \cdot T^{2} + \tau_{3}\lbrack n\rbrack \cdot T} \\
{\text{ \quad\quad\,} = \tau\lbrack n\rbrack - K_{3} \cdot \Delta\tau\lbrack n\rbrack + \tau_{2}\lbrack n\rbrack \cdot T^{2} + \tau_{3}\lbrack n\rbrack \cdot T} \\
\text{ } \\
\end{array}$$
Equations (A11) to (A13) are the DLL in case of 1st, 2nd, and 3rd order, respectively. $\omega_{0}$ is the 1st order filter value. $\omega_{1}$ and $a_{2}$ are the 2nd order filter values. $\omega_{2}$, $a_{3}$ and $b_{3}$ are the 3rd order filter values. $K_{1}$, $K_{2}$ and $K_{3}$ are the SPE coefficients with respect to the DLL order, respectively. SPE coefficients are determined through the filter setting. Also, $\tau_{1}\lbrack n\rbrack$ in Equation (A12) can be derived using Equation (A11). And $\tau_{2}\lbrack n\rbrack$ and $\tau_{3}\lbrack n\rbrack$ in Equation (A13) can be derived using Equation (A11) and Equation (A12), respectively. This derivation shows that the SPE could be derived from any DLL order.
{#sensors-19-00293-f001}
{#sensors-19-00293-f002}
{#sensors-19-00293-f003}
{#sensors-19-00293-f004}
{#sensors-19-00293-f005}
{#sensors-19-00293-f006}
{#sensors-19-00293-f007}
{#sensors-19-00293-f008}
{#sensors-19-00293-f009}
{#sensors-19-00293-f010}
{#sensors-19-00293-f011}
{#sensors-19-00293-f012}
{#sensors-19-00293-f013}
{#sensors-19-00293-f014}
{#sensors-19-00293-f015}
{#sensors-19-00293-f016}
{#sensors-19-00293-f017}
{#sensors-19-00293-f018}
{#sensors-19-00293-f019}
sensors-19-00293-t001_Table 1
######
Relationship between spoofing parameters and spoofing results.
Parameters Spoofing Attack Success Probability
-------------------------------------------- -------------------------------------
Increase in the spoofing signal strength Increase
Increase in spoofing signal sweep velocity Decrease
Increase in the DLL bandwidth Increase
sensors-19-00293-t002_Table 2
######
Time interval calculation according to CI.
---------------------------------
Chip Interval\ Time Interval\
(chip) (second)
---------------- ----------------
0.005 0.0183
0.053 0.1941
0.125 0.4578
0.160 0.5860
0.213 0.7801
0.266 0.9742
0.320 1.1720
0.426 1.5602
0.533 1.9521
---------------------------------
sensors-19-00293-t003_Table 3
######
Integration time calculation according to CI.
------------------------------------------------------------------------------------------------------------------------------
Case Spoofing Signal\ Sweep Velocity (m/s) Bandwidth Spoofing Results $\mathbf{\left| \mathbf{\tau} \right|}$\
Strength Offset (dB) at *D* = −1
------ ---------------------- ---------------------- ----------- ------------------ ------------------------------------------
1 1.5 50 3 Success 0.5013
2 1.5 50 5 Success 0.5287
3 1.5 70 3 Failure 0.4362
4 1.5 70 5 Failure 0.4774
5 2 50 3 Success 0.5357
6 2 50 5 Success 0.5741
7 2 70 3 Failure 0.4761
8 2 70 5 Success 0.5104
------------------------------------------------------------------------------------------------------------------------------
sensors-19-00293-t004_Table 4
######
Range of $\tau\lbrack i\rbrack$ and ACF model of *XE* and *XL* according to the *D\[i\]*.
----------------------------------------------------------------------------------------------------------------------------------------------------------------
*i* *D\[i\]* Range of\ Range of\ ACF model\ ACF model\
$\mathbf{\mathbf{\tau}\lbrack\mathbf{i}\rbrack - 0.5}$ $\mathbf{\mathbf{\tau}\lbrack\mathbf{i}\rbrack + 0.5}$ of *XE* of *XL*
----- ---------- -------------------------------------------------------- -------------------------------------------------------- -------------- --------------
1 1.375 −1\~0 0.375\~1 y~1~ y~2~ + y~s1~
2 1.25 −1\~0 0.25\~1 y~1~ y~2~ + y~s1~
3 1.125 −1\~0 0.125\~1 y~1~ y~2~ + y~s1~
4 1 −1\~0 0\~1 y~1~ y~2~ + y~s1~
5 0.875 −1\~−0.25 −0.125\~0.875 y~1~ y~2~ + y~s1~
6 0.75 −0.25\~0 0.75\~1 y~1~ + y~s1~ y~2~ + y~s2~
7 0.675 −0.375\~0 0.625\~1 y~1~ + y~s1~ y~2~ + y~s2~
8 0.5 −0.5\~0 0.5\~1 y~1~ + y~s1~ y~2~ + y~s2~
9 0.375 −0.625\~0 0.375\~1 y~1~ + y~s1~ y~2~ + y~s2~
10 0.25 −0.75\~0 0.25\~1 y~1~ + y~s1~ y~2~ + y~s2~
11 0.125 −0.875\~0 0.125\~1 y~1~ + y~s1~ y~2~ + y~s2~
12 0 −1\~0 0\~1 y~1~ + y~s1~ y~2~ + y~s2~
13 −0.125 −1\~−0.125 0\~0.875 y~1~ + y~s1~ y~2~ + y~s2~
14 −0.25 −1\~−0.25 0\~0.75 y~1~ + y~s1~ y~2~ + y~s2~
15 −0.375 −1\~0.375 0\~0.625 y~1~ + y~s1~ y~2~ + y~s2~
16 −0.5 −1\~−0.5 0\~0.5 y~1~ + y~s1~ y~2~ + y~s2~
17 −0.625 −1\~0.625 0\~0.375 y~1~ + y~s1~ y~2~ + y~s2~
18 −0.75 −1\~−0.75 0\~0.25 y~1~ + y~s1~ y~2~ + y~s2~
19 −0.875 −1\~0.875 0\~0.125 y~1~ + y~s1~ y~2~ + y~s2~
----------------------------------------------------------------------------------------------------------------------------------------------------------------
sensors-19-00293-t005_Table 5
######
Various spoofing parameters and $\tau$ results in case of using original DLL and SPE.
Case Spoofing Signal Strength Offset (dB) Sweep Velocity (m/s) Bandwidth Reference Proposed Error
------ -------------------------------------- ---------------------- ----------- ----------- ---------- --------
1 1.5 55 3 −0.4895 −0.4903 0.0008
2 1.5 60 3 −0.4742 −0.4777 0.0035
3 1.5 60 5 −0.5043 −0.503 0.0013
4 1.5 65 5 −0.4927 −0.4931 0.0004
5 2 60 3 −0.5072 −0.507 0.0002
6 2 65 3 −0.4934 −0.4937 0.0003
7 2 65 5 −0.5257 −0.5217 0.004
8 2 70 3 −0.477 −0.4797 0.0027
9 2 70 5 −0.5112 −0.5104 0.0008
10 2.5 65 3 −0.5253 −0.5231 0.0022
11 2.5 80 3 −0.4759 −0.4787 0.0028
12 2.5 80 5 −0.5147 −0.5136 0.0011
sensors-19-00293-t006_Table 6
######
Estimated ${\widetilde{a}}_{s}$ values according to the spoofing parameters.
------------------------------------------------------------------------------------------------
Number Sweep Velocity (m/s) Bandwidth\ $\mathbf{{\widetilde{\mathbf{a}}}_{\mathbf{s}}}$\
(Hz) (Hz)
-------- ---------------------- ------------ ---------------------------------------------------
1 40 2 1.46
2 45 2 1.69
3 50 2 1.92
4 55 2 2.17
5 60 2 2.42
6 65 2 2.69
7 70 2 2.97
8 75 2 3.26
9 80 2 3.56
10 85 2 3.87
11 90 2 4.20
12 95 2 4.54
13 100 2 4.09
------------------------------------------------------------------------------------------------
sensors-19-00293-t007_Table 7
######
Computational complexities of conventional DLL and SPE.
Sweep Velocity (m/s) Conventional DLL SPE
---------------------- ------------------ ------- ---- ------
40 18312 10.34 19 0.24
50 14650 8.26 19 0.24
60 12208 6.86 19 0.24
70 10464 5.89 19 0.24
80 9156 5.17 19 0.24
| {
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INTRODUCTION
============
Cutaneous neoplasms with two or more distinct cell populations are rare but well documented, and may pose a diagnostic challenge to clinicians and pathologists. They often mimic other cutaneous tumors or present as nonspecific neoplasms. Diagnosis of a collision tumor may be an accidental and unexpected histological finding. Many combinations of collision tumors have been described, with the most frequently reported being the combination of nevus and basal cell carcinoma (BCC). A melanocytic tumor in collision with a benign or malignant epithelial neoplasm is less commonly documented. Some collision tumors involve melanomas, most frequently in combination with a BCC. ^[@r1]-[@r3]^ Even more exceptional is the involvement of an adnexal tumor in a collision tumor. Syringomas are relatively common benign tumors that originate in the sweat glands. They present clinically as 1 to 5 mm yellowish, rounded or flat-topped dermal papules. They are generally multiple, and are distributed symmetrically on the chest, neck, or face, and, especially, the lower eyelids.^[@r4]^ We report an unusual case of a collision tumor on the nose involving a melanoma *in situ* and a syringoma. To the best of our knowledge, this is the first report of this combination.
CASE REPORT
===========
An 82-year-old Caucasian man with a history of atopic dermatitis and osteoarthritis presented with a brown-colored asymptomatic macular lesion on the left nasal root. The lesion had grown progressively for approximately two years and was untreated. Examination revealed an ill-defined 15 mm x 7 mm macule with an irregular shape and asymmetrical distribution of the pigment network ([Figure 1](#f1){ref-type="fig"}). The clinical impression was of lentigo maligna and a punch biopsy was performed to confirm the diagnosis.
Figure 1Irregular, asymmetric pigmented lesion on the left nasal root
Microscopic study of the biopsy revealed lentiginous melanocytic dysplasia, which involved the hair follicles and was positive for Melan-A, HMB45 and S-100 stains. Subsequently, the lesion was completely excised by surgery. Histopathologically, the surgical specimen revealed two distinct histological components. In the basal layer, there were melanocytic dysplasia and nucleocytoplasmic abnormalities with an irregular distribution and involving hair follicles. In the upper and middle dermis there were clusters of small ducts, occasionally with comma-shaped extensions, lined by a double-layered epithelium typical of a syringoma ([Figure 2](#f2){ref-type="fig"}). Immunohistochemical staining confirmed that melanoma was positive for Melan-A and HMB45 and that syringoma was positive for the carcinoembryonic antigen (CEA) ([Figure 3](#f3){ref-type="fig"}).
Figure 2**A:** Hematoxylin & eosin staining of melanoma *in situ* with follicular involvement and syringoma structures in the middle dermis (X2). **B:** Detail of the collision between the melanoma in situ and the syringoma (X200)
Figure 3**A:** Melan-A staining showing the melanoma *in situ* with follicular involvement (x200). Duct structures do not stain. **B:** CEA staining showing the ducts of the syringoma (X100)
DISCUSSION
==========
In recent years, various terms have been used to describe two lesions occurring in one site, resulting in increasing terminological confusion. Satter *et al.* proposed a classification to simplify the nomenclature of these lesions, classifying them as collision, combined, colonized, or biphenotypic tumors.^[@r5]^ Collision tumors are defined as two distinct neoplasms that occur within close proximity of each other but maintain sharply distinct boundaries. Neoplasms consisting of two phenotypically different, yet imperceptibly intertwined populations of malignant cells are referred to as combined tumors, and immunohistochemical stains are often required to appreciate the two tumor cell populations. Colonization describes a situation where large dendritic melanocytes populate another neoplasm, e.g., a melanoma *in situ* colonizing a BCC. Biphenotypic tumors are exceptionally rare neoplasms that arise from a common stem cell precursor that undergoes divergent differentiation.
In our patient, the syringoma was not clinically apparent and there were no syringomas in other facial areas. Melanoma and syringoma were close but separated, indicating a collision tumor; this impression was supported by the immunohistochemical data, which clearly differed and did not overlap. Immunostaining with CEA revealed an epithelial syringomatous population, while HMB45 positivity in the melanocytic population confirmed the presence of a melanoma *in situ*, thus drawing a clear boundary between the two tumors. This, together with the fact that the initial biopsy only showed the melanocytic component and not the syringoma, confirmed that this was a collision tumor.
Reactive eccrine gland ductal proliferation has been reported in a variety of inflammatory skin diseases (e.g. scarring alopecia) and benign and malignant neoplasms. Guitart *et al.* proposed the term of 'syringomatous dermatitis' for those cases of reactive hyperplastic response of the eccrine duct resulting from an inflammatory skin reaction.^[@r6]^ In our case we found sclerotic dermal changes in the area of the syringoma, the ducts were located in the middle dermis and some presented with typical comma-shaped structures. These findings, along with the absence of a previous inflammatory cutaneous process, are distinguishable from reactive eccrine gland ductal proliferation and confirm the diagnosis of a syringoma.
To date, a few cases of collision tumors involving a syringoma have been reported in association with intradermal nevi, BCCs, epidermal cyst and Spitz nevus.^[@r3],[@r4],[@r7]-[@r10]^ Whether the coexistence of a syringoma with these conditions occurs by chance alone remains unclear, but it may be that the silent form of this tumor occurs more frequently than suspected.^[@r4]^ Syringomas are common lesions found on the face, and therefore may occasionally be found in biopsies or excisions of facial lesions performed for unrelated pathologies, just like solar lentigines, seborrheic keratosis or actinic keratosis.
We report the first case of a collision tumor involving a syringoma and a melanoma *in situ*. Syringomas are common, but their association with other types of tumor is rare. Histopathology and immunohistochemical staining were very helpful in distinguishing between the two components. Complete excision biopsies are necessary to establish the diagnosis of collision tumors, and long-term follow up is recommended.
Conflict of Interests: None.
Study conducted at the Hospital Universitari Sagrat Cor de Barcelona - Barcelona, Spain.
Financial Support: None.
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Introduction {#S1}
============
Sleep disorder is associated with various diseases' development and progression, such as obesity, type II diabetes ([@B29]) and cardiovascular diseases ([@B22]). Insomnia is the most prevalent sleep disorder, including sleep apnea, restless legs syndrome (RLS) and narcolepsy, and affects a large proportion of the population on a situational, recurrent or chronic basis and is also one of the most common complaints in medical practice ([@B39]). The signature of insomnia is that patients have difficulty falling asleep, or staying awake, despite plenty of opportunity to sleep. Like many other psychiatric disorders, insomnia is a multifactorial disorder, though the detailed pathological aspects of insomnia remain unclear. Thus, a better understanding of the pathophysiology of insomnia may provide additional therapeutic strategies.
The gut microbiome, a key component of the intestinal environment, has been implicated as an essential modulator for human health ([@B59]). Microbial homeostasis is critical to the host development and health. Dysbiosis perturbs the host immune system and metabolism balance, which leads to the development of various kinds of diseases ([@B17]; [@B41]; [@B57]). Microbial dysbiosis may also contribute to the development of neurological disorders and psychiatric disorders, such as autism spectrum disorder, anxiety disorder, depression and Alzheimer's disease ([@B40]; [@B45]; [@B15]; [@B32]). In addition, several studies have provided preliminary evidence for the involvement of the gut microbiota in sleep disorders of murine models and human patients. It was reported that after 4 weeks of sleep fragmentation in experimental mice, gut flora were dominated by Lachnospiraceae and Ruminococcaceae, with a gradually reduced relative abundance of Lactobacillaceae ([@B46]). In another study with partial sleep deprivation in normal-weighted young individuals, the composition of the gut microbiota was subtly affected with an increased ratio of Firmicutes/Bacteroidetes ([@B7]). However, either sleep fragmentation or sleep deprivation refers to curtailed sleep length due to an externally imposed restriction of the opportunity to sleep, while insomnia refers to the inability to fall asleep adequately, either in length or quality. Considering the significant difference in definition between sleep fragmentation/deprivation and insomnia, to date, study investigating the relationship between insomnia and gut flora remains unexplored.
Thus, in the present investigation we combined 16S rDNA amplicon sequencing and innovative bioinformatic analysis to examine the pathological and physiological significance of the gut microbiota between healthy participants and patients suffering insomnia disorder. Leveraging these innovative analyses, such as redundancy analysis, co-occurrence analysis, PICRUSt, random forest and artificial neural networks (ANN), we demonstrated that the gut taxa composition, signaling pathways, and metabolic functions are perturbed in patients with insomnia disorder. Artificial neural networks were also incorporated by utilizing the relative abundance of the gut microbiota to establish a prediction model for an unbiased evaluation of insomnia. This study is the first to combine high-throughput sequencing and bioinformatic analysis, especially machine learning, to systemically understand the biological effect of the gut microbiota on insomnia. Comprehensive analysis indicated that gut microbiota homeostasis is a strong determinant, which is closely associated with insomnia disorder. Overall, the aforementioned study provides a comprehensive understanding of the link between gut microbiota and insomnia disorder. By utilizing the machine learning approach, we identified the signature gut microbiota, which could be utilized as novel and unbiased prediction targets, which in other aspects could provide additional interventions for clinical application.
Materials and Methods {#S2}
=====================
Volunteer Enrollment {#S2.SS1}
--------------------
The experiment was approved by the Ethics Committee of Jinan University (Approval \#: GNU-20180306).
The volunteers were recruited from the public and The First Affiliated Hospital of Jinan University in Guangzhou, China. After being informed on the rights and obligation, all participants understood the benefits and risks of the experiment totally and signed an informed consent document. In compliance with strict standards for inclusion and exclusion criteria (Detailed in [Supplementary Materials](#FS1){ref-type="supplementary-material"}), all participants were assessed by two psychiatrists. In the event of any dispute or difference of judgment, the participant would be excluded. All participants accepted polysomnography treatment at the Sleep Medicine Center of Jinan University. Finally, twenty qualified volunteers were enrolled and separated into two groups (Insomnia group and Normal Control group). Their fecal samples were collected by sterilized instruments in the morning upon polysomnography treatment, and then stored in a freezer at −80°C for 16S rDNA sequencing.
16S rDNA Amplicon Sequencing {#S2.SS2}
----------------------------
Bacterial DNA from patients' feces was extracted by utilizing the ZR Fecal DNA Kit (Zymo Research, United States). A multiplexed amplicon library covering the V3-V4 region of 16S rDNA gene was PCR-amplified with the optimized primer sets for the Illumina HiSeq 2500 sequencing instrument. A total of 1,534,966 high-quality reads were obtained, with an average of 76,748 reads (range 66,570--84,443) per sample. All chimera sequences were removed by VSEARCH ([@B47]). Chimera-free sequences were processed using a standard QIIME 1.91 pipeline ([@B12]) and clustered into operational taxonomic units (OTUs) at a 97% similarity threshold using an "Open-Reference" approach. Taxonomy was assigned using the RDP classifier against the Greengenes database (May 2013 release) ([@B38]). The raw Illumina pair-end read data for all samples have been deposited in the Short Read Archive under the Bioproject: [PRJNA527914](PRJNA527914).
Bioinformatics Analysis {#S2.SS3}
-----------------------
Alpha rarefaction was analyzed by the Faith's phylogenetic diversity ([@B23]), Chao1 ([@B13]), Shannon and Simpson index ([@B14]). β-diversity was estimated by computing weighted and unweighted UniFrac distance. Principal Coordinates Analysis (PCoA), Redundancy Analysis (RDA) and heatmap of correlation were plotted by "ggplot2," "vegan," and "corrplot" packages of R (version 3.5.1). Manhattan Plot was plotted by "edgeR," "dplyr" and "ggplot2" to present the differential relative abundance between groups. These results were tested by Monte Carlo permutation and Student's *t*-test. Organism-level microbiome phenotype prediction was obtained by BugBase software ([@B49]). To decipher the difference of microbiota structure between groups, LEfSe (linear discriminant analysis effect size) was performed and the cladogram was graphed with default parameter (*p* \< 0.05 and LDA score \> 2.0) ([@B24]). To probe the microbial metabolism and predict metagenome functional content from the marker gene, PICRUSt was utilized to explore differences of the KEGG pathway between groups ([@B34]). To decipher the gut microbiota ecology, co-occurrence analysis was performed with the "igraph" package ([@B42]) of R with data filtered at species level considering only those relative abundance present in at least 70% of the samples in each group. The edges were estimated by Spearman confident index (abs(*r*) \> 0.6, *p* \< 0.05). Communities inside two networks were determined by the fast-greedy modularity optimization algorithm ([@B20]), which was one of the approaches to determine the dense subgraph in Graph Theory. The circle bar was plotted according to the eigenvector centrality scores (ECS) to estimate the importance and betweenness of each node ([@B50]). To identify the key signature microbiota, five-fold cross validation together with Random Forest analysis were performed to compute importance scores (mean decrease accuracy, MDA) to estimate the importance of variables by utilizing the "randomForest" v.4.6-14 package ([@B9]) in R. At species level, in order to establish a prediction model to predict the sleep-related physiological parameter, the ANN was performed on python 3.6.1 with the pyTorch, sklearn, pandas, and numpy packages. The optimized parameters, including learning rate, activation function, layers, number of neurons and dropout, were selected by grid search and cross-validation.
Results {#S3}
=======
Insomnia Disorder Leads to Significant Structural and Functional Changes of Gut Microbiota {#S3.SS1}
------------------------------------------------------------------------------------------
Among the twenty qualified enrolled volunteers, basic personal information including height, weight and BMI presented no significant difference between groups except for age (insomnia: 33.00 ± 6.90; normal: 26.10 ± 1.85) ([Supplementary Figure S1](#FS1){ref-type="supplementary-material"}). Considering previous research demonstrated the gut microbiota differed little in adults based on more than 1,000 very healthy Chinese individuals ([@B8]), only 7 mean-years of difference between groups could be tolerated. All the volunteers were accepted according to inclusion and exclusion criteria (detailed in [Supplementary Materials](#FS1){ref-type="supplementary-material"}). All fecal samples from participants were collected for high-throughput sequencing. 16S rDNA V3-V4 region amplicon sequencing generated 1,534,966 high-quality reads, with an average of 76,748 reads (range 66,570--84,443) per sample. All raw data were filtered by VSEARCH and processed using a standard QIIME 1.91 pipeline against the Greengenes database (May 2013 release). Rarefaction measurement of Shannon and Simpson index, Goods_Coverage, and species accumulation curve (SAC) indicated that sequencing depth was enough to capture all bacterial species and sufficient for downstream analysis ([Supplementary Figure S2](#FS2){ref-type="supplementary-material"}). Rarefaction analysis of chao1 (*p* = 0.007) and PD whole tree (*p* = 0.001) index showed significant difference between the healthy and insomnia groups, suggesting that insomnia disorder may result in alteration of gut microbiota diversity ([Figure 1A](#F1){ref-type="fig"}). Furthermore, β-diversity calculated with the Unweighted UniFrac (*p* = 0.0006) and Weighted UniFrac (*p* = 0.0032) algorithms indicated that the insomnia and normal groups had significant structural difference by the first dimension of space distance ([Figure 1B](#F1){ref-type="fig"}). To confirm the composition of difference between two groups, a Manhattan plot was used to represent the fold change of insomnia/normal group and revealed a significant difference, especially the Firmicutes and Bacteroidetes phylum, which was confirmed by Linear Discriminant Analysis Effect Size (LEfSe) analysis and identified 87 biomarkers ([Figures 1C,D](#F1){ref-type="fig"}). Meanwhile, BugBase algorithm-based prediction suggested that the insomnia group preferentially enriched with the gram-negative and potential pathogenic taxa compared with the normal group ([Figure 1E](#F1){ref-type="fig"} and [Supplementary Figures S3A--F](#FS3){ref-type="supplementary-material"}). Other than the composition and diversity of gut microbiota, PICRUSt algorithm was performed to assess the functional difference by plotting the differential pathways against KEGG database. We identified pathways such as steroid hormone biosynthesis (ko00360), Retinol metabolism (ko00830), Vitamin B6 metabolism (ko00750), Folate biosynthesis (ko00790), Citrate cycle TCA cycle (ko00020) that were predicted to be enriched in the insomnia group (Kruskal test *p* \< 0.05), while Arachidonic acid metabolism (ko00590), Pantothenate and CoA biosynthesis (ko00770), Lysine biosynthesis (ko00300), and Glycerolipid metabolism (ko00561) associated pathways were downregulated (Kruskal test *p* \< 0.05) ([Figure 1F](#F1){ref-type="fig"}).
{#F1}
Insomnia Disorder Disturbs the Gut Flora Interaction {#S3.SS2}
----------------------------------------------------
Whether insomnia disorder is associated with the gut microbiota community network and the network complexity, the graph theory algorithm and Co-occurrence analysis were performed to estimate the gut microbiota ecology between groups. The radar plot computed by the graph theory analysis including the transitivity, graph density, degree centralization, number of vertices and number of edges showed that insomnia disorder did not significantly change the systemic complexity of gut bacteria, indicating that the gut microbiota in insomnia patients had already developed a mature network. With this, based on species data whose relative abundance presented at least 70% of the samples in each group, Co-occurrence analysis was used to further explore the gut microbiota interaction and sub-groups in both the normal and insomnia groups ([Supplementary Figure S4](#FS4){ref-type="supplementary-material"} and [Supplementary Table S1](#TS1){ref-type="supplementary-material"}). The gut flora interaction network was significantly altered for patients under insomnia disorder compared with that of the normal group. Furthermore, the gut microbiota was sub-divided into five and four sub-groups for the normal and insomnia groups, respectively ([Figure 2](#F2){ref-type="fig"}).
{#F2}
Gut Microbiota Alteration Strongly Associated With Insomnia Disorder {#S3.SS3}
--------------------------------------------------------------------
As demonstrated above, significant structure, composition and function of the gut microbiota as well as the bacterial interaction network were significantly changed between the normal and insomnia groups. To further prove whether the insomnia-associated clinical sleep parameter directly contributes to the alteration of the gut microbiota, we performed the redundancy analysis (RDA) to link the insomnia parameter with the relative abundance of gut microbiota at phylum level ([Figure 3](#F3){ref-type="fig"}). These clinical sleep parameters from polysomnography (PSG) and the psychological scale include the Pittsburgh Sleep Quality index (PSQ), Hamilton Anxiety Scale (HAMA), Hamilton Depression Scale (HAMD), Epworth Sleepiness Scale (ESS) and Insomnia Severity Index (ISI). Here, we demonstrated that 67.13% of the variance could be interpreted by twelve environmental factors (in other words: clinical sleep parameter), which means that insomnia disorder could significantly alter the population of the gut microbiota at phylum level and samples from two groups were obviously separated. In particular, according to the Monte Carlo permutation test, some clinical sleep parameters, e.g., Pittsburgh sleep quality index (PSQ, *r^2^* = 0.6074, *p* = 0.002) and rapid eye movement sleep (REM, *r^2^* = 0.2663, *p* = 0.045), play a pivotal role in clustering the distribution of flora between groups. Meanwhile, ANOSIM based on the Bray Curtis distance also confirmed the observation from RDA analysis that the difference between groups was more significant than that within groups (statistic *R*: 0.1944, *p* = 0.015) ([Supplementary Figure S5](#FS5){ref-type="supplementary-material"}). Both RDA and ANOISM analysis clearly suggested that clinical sleep parameters associated with insomnia disorder directly contribute to the separation and clustering of the gut microbiota between groups.
{#F3}
Identification of the Signature Gut Microbiota Associated With Insomnia Disorder by Random Forest {#S3.SS4}
-------------------------------------------------------------------------------------------------
The traditional approaches such as LEfSe by comparing the difference of relative abundance of gut flora between groups resulted in the identification of 87 biomarkers. It is difficult to utilize these markers to establish a prediction model for disease diagnosis. To improve the biomarker identification, we incorporated a robust statistical analysis and applied five-fold cross-validation together with random forest to generate ∼2 million decision trees ([Supplementary Figure S6](#FS6){ref-type="supplementary-material"}), leading to identification of three optimal species biomarkers with consideration of lowest error rate plus standard deviation. With further analysis to identify V68 (g\_\_Prevotella) as an outlier, we thus selected V45 (g\_\_Bacteroides) and V124 (o\_\_Clostridiales) as the most important biomarkers to distinguish the insomnia patients from healthy individuals with a ROC curve at AUC = 0.87 ([Figures 4A--D](#F4){ref-type="fig"}, [Supplementary Figure S7](#FS7){ref-type="supplementary-material"}, and [Supplementary Table S1](#TS1){ref-type="supplementary-material"}). Moreover, V45 was highly correlated with HAMD (*r* = 0.70, *p* \< 0.001), HAMA (*r* = 0.62, *p* \< 0.01), ISI (*r* = 0.62, *p* \< 0.01), sleep efficiency (*r* = −0.56, *p* \< 0.05), PSQ (*r* = 0.63, *p* \< 0.01) and sleep latency (*r* = 0.66, *p* \< 0.01) while V124 correlated with ESS (*r* = −0.45, *p* \< 0.05), ISI (*r* = −0.48, *p* \< 0.05), REM latency (*r* = −0.49, *p* \< 0.05), and PSQ (*r* = −0.51, *p* \< 0.05) ([Figure 4E](#F4){ref-type="fig"} and [Supplementary Figure S8](#FS8){ref-type="supplementary-material"}). Even in the Co-occurrence plot, these two key microbiotas both occupied hub-like positions with high betweenness (Insomnia: V45 0.702499227 V124 0.034447479; Normal: V45 0.467046396 V124 0.044448542) ([Supplementary Figure S9](#FS9){ref-type="supplementary-material"}). All above results strongly demonstrated that the key microbiota we identified via the robust statistical approaches led to the development of an optimal and robust prediction model for insomnia diagnosis.
{#F4}
Relative Abundance of Gut Microbiota-Based Prediction on the Clinical Sleep Parameter {#S3.SS5}
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Given that the gut microbiota tightly was correlated with the clinical sleep parameter, we sought to establish a mathematical model to utilize the relative abundance of the gut microbiota to predict the sleep-related physiological parameter. Here, we utilized a well-established regression model, LASSO regression to link the relative abundance of gut microbiota and clinical sleep parameter resulted in a poor correlation ([Supplementary Figure S10](#FS10){ref-type="supplementary-material"}). To overcome the shortcoming of the traditional machine learning model, we integrated an even more powerful deep learning model, called an ANN, which is considered to be able to imitate biological neural networks. By integrating the clinical sleep parameter into the ANN model, this model could result in a high coefficient of determination respective for WASO number: *r^2^* = 0.14, MAE = 4.80; WASO time: *r^2^* = 0.6, MAE = 15.77; Sleep efficiency: *r^2^* = 0.52, MAE = 5.45; ESS: *r^2^* = 0.54, MAE = 2.41; HAMA: *r^2^* = 0.66, MAE = 1.81; HAMD: *r^2^* = 0.55, MAE = 1.83; ISI: *r^2^* = 0.66, MAE = 2.96; N1: *r^2^* = 0.43, MAE = 2.88; N2 *r^2^* = 0.58, MAE = 3.28; N3: *r^2^* = 0.34, MAE = 4.97; PSQ: *r^2^* = 0.73, MAE = 1.75; REM *r^2^* = 0.40, MAE = 3.59; REM latency: *r^2^* = 0.41, MAE = 38.5; Sleep latency: *r^2^* = 0.42, MAE = 6.12; Total sleep time: *r^2^* = 0.37, MAE = 45.18 ([Figure 5](#F5){ref-type="fig"}).
{#F5}
Discussion {#S4}
==========
Insomnia disorder as a common clinical symptom is a critical part of sleep disorder ([@B44]). It is often accompanied by excessive arousal and sleep debt, which always lead to adverse impacts such as mental or physical fatigue. From the statistics of more than 50 epidemiological studies, the prevalence of insomnia symptoms was estimated at 10∼48% ([@B43]). Insomnia disorder is functionally linked to cardiovascular and nervous system diseases ([@B27]; [@B60]). The classic hypothesis is Spielman's 3P Model including predisposing, precipitating and perpetuating factors ([@B53]). Recently, it has been reported that the hypothalamic--pituitary--adrenal axis (HPA) may contribute to the incidence of insomnia ([@B35]). Moreover, in 2017 a genome-wide association study (GWAS) identified risk genomic loci and genes that are associated with the incidence of insomnia, and suggested that insomnia is highly polygenic ([@B26]). However, none of these studies provided a mechanistic interpretation of the causes or even objective approaches for insomnia diagnosis. Here, our study is first to comprehensively compare the gut microbiota between insomnia patients and healthy individuals. With these data, we established a robust statistical prediction model to utilize the relative abundance of the gut microbiota to distinguish insomnia patients from the normal population and to estimate levels of sleep quality through the novel bioinformatics technology and machine learning algorithm.
The unhealthy shift of gut microbiota, also called dysbiosis, is associated with various metabolic diseases such as obesity, type II diabetes, hypertension and cardiovascular diseases ([@B19]; [@B31]; [@B1]; [@B54]). In this study, we demonstrated that α- and β-diversity of the gut microbiota in insomnia patients is significantly altered. Meanwhile, by comparing the difference of the relative abundance between insomnia and healthy individuals, we identified that Bacteroidetes are the dominant taxa in the insomnia group, while Firmicutes and Proteobacteria were enriched in the normal group, resulting in a decreased ratio of Firmicutes/Bacteroidetes. Our results are different from observations made in previous studies in individuals with sleep deprivation or restriction. In their studies, the F/B ratio shows either no change or is increased after partial sleep deprivation ([@B7]; [@B11]). This discrepancy with respect to the change over the F/B ratio may be due to the difference regarding the clinical definition between sleep restriction/deprivation and insomnia. Sleep deprivation or restriction is not considered to be a specific disease, but rather a result of a wide range of interruption from external environmental factors. It is worth mentioning that although subjects in previous study followed strict experimental protocol, they not only had *ad libitum* access to food/drink throughout the experiment, but also were allowed to read, play video or board games, watch television, and interact with laboratory staff to help remain awake ([@B11]). These environmental factors may contribute to variation in the gut microbiota, which leads to difficulty in interpreting the results. Compared to those with sleep deprivation, patients with insomnia who do not have externally imposed restrictions on the opportunity to sleep still have trouble falling asleep, staying asleep, or waking too early, resulting in daytime impairment ([@B10]). Our study demonstrated that although insomnia and sleep deprivation may result in similar reductions on sleep length in most cases, they may lead to different consequences regarding the dysbiosis of the gut microbiota. In addition, this ratio change is also reported in different life stages and pathological circumstances. A study looks into the ratio of F/B between adults and elders, suggesting that a higher ratio in the adult gut is observed, while it starts to decrease in individuals undergoing aging ([@B21]). Alteration of the F/B ratio is also observed in those with metabolic diseases, such as obesity ([@B61]; [@B30]) and type II diabetes ([@B63]). Furthermore, our BugBase-based phenotypical prediction also demonstrated that the insomnia group was enriched with bacterial taxa to be potentially pathogenic. This may link the insomnia-related sleep disorder population with high potential disease development and progression, as more evidence has proved that chronic sleep disorder is associated with a multitude of health conditions and even systemic metabolic disorder ([@B62]).
The biological and physiological function of gut microbiota could be defined from multiple aspects, such as the taxonomic composition and diversity, which are poorly conserved across individuals while the genetic composition and functional capacity are evolutionally conserved across individuals. Thus, to decipher the metabolic switch of gut bacteria, the PICRUSt algorithm was utilized to map the bacterial genetic pathway against the KEGG database. Compared to normal group, a wide range of pathways was altered obviously in our study. It is interesting to note that vitamin B-related pathways were significantly induced in the insomnia group, while the level of vitamins is highly associated with the clinical practice of insomnia ([@B36]). In our insomnia patients, the analysis suggested vitamin B6 catabolism (ko00750) in the gut microbiota is significantly enhanced, resulting in vitamin B6 deficiency for the host. It was reported that vitamin B6 is administered as a common therapeutic practice for insomnia disorder and its deficiency results in fatigue and depression ([@B5]). Thus, additional vitamin B6 supplementation could ameliorate insomnia symptoms ([@B5]). Moreover, the folate (also called vitamin B9) biosynthesis-related pathway (ko00790) was also increased in the insomnia group. Previous study of serum nutritional biomarkers and dietary supplementation of folate demonstrated that folate acid has a high correlation with the development of sleep disorder ([@B51]; [@B65]). In addition, endogenously synthesized arachidonic acid significantly facilitates the release of GABA in the striatum ([@B16]), while GABA could enhance the catabolism of serotonin into N-acetylserotonin (the precursor of melatonin) in rat ([@B6]). It has long been speculated that GABA is associated with the synthesis of melatonin and thus might exert regulatory effects on sleep functions. In our study, our bioinformatic analysis demonstrates that arachidonic acid biosynthesis was lower in the insomnia group, indicating that lower production of arachidonic acid from gut microbiota may be associated with a high incidence of insomnia. However, whether arachidonic acid supplementation may improve insomnia symptoms requires further clinical investigation. These results provide a link that gut microbiota and their metabolites maybe a mediator with respect to the development of insomnia. With this information, novel therapeutic and intervention approaches could be developed for people suffering from insomnia disorder in the future.
In our study, insomnia disorder leads to the alteration of the gut microbiota composition and diversity. However, whether insomnia disorder directly contributes to the dysbiosis of the gut microbiota is still unknown. Here, our RDA analysis and ANOSIM provide strong evidence to support the role of clinical sleep parameters of insomnia individuals in dysbiosis of gut microbiota, especially PSQ (*r*^2^ = 0.6074, *p* = 0.002) and REM (*r*^2^ = 0.2663, *p* = 0.045), based on a Monte Carlo permutation test. Both results strongly pinpointed the importance of insomnia disorder as a key factor in separating gut microbiota from two groups. Upon establishment of the link between gut microbiota and clinical sleep parameter, taking advantage of the differential test and LEfSe algorithm, we identified 87 differential biomarkers from the normal and insomnia groups. Among the biomarkers, to further classify their importance, the machine learning approach is incorporated, such as random forest. This robust statistical method could identify the signature biomarkers with higher prediction accuracy and coefficiency, especially for the gut microbiota-based diseases prediction and diagnosis ([@B48]; [@B64]). Here, our random forest model together with the cross-validation model identified two key bacterial taxa (g\_\_Bacteroides; o\_\_Clostridiales), which are not only tightly associated with clinical data, but also play a pivotal role in network of gut ecology and could be used as two critical biomarkers to identify patients with insomnia.
Classic diagnosis for insomnia disorder relies on either subjective or objective assessment, including the most common clinical sleep parameters such as PSQ, ESS, ISI, HAMD, and HAMA. However, most of these results are often affected by the subjectivity of individuals, especially for some patients with insomnia disorder ([@B2]; [@B33]). On the other hand, PSG, as the first choice for objective assessment, is the golden standard for insomnia diagnosis worldwide. This is restricted by the cost, equipment and space. Furthermore, the adaptation of the first-night sleep may affect the PSG results because of the temporary change of sleep environment ([@B55]). Thus, a convenient approach is necessary for the diagnosis of insomnia. Given the tight correlation between microbiota and disease incidence, whether there is a method to establish a regression model to predict clinical sleep parameter remains unclear. So, we introduced a LASSO regression model, which is widely used in gut microbiota-based clinical study and has been shown to effectively utilize the relative abundance to predict cancer development and progression, such as irritable bowel diseases and colorectal cancer ([@B56]; [@B25]). Moreover, LASSO could overcome the multicollinearity problem caused by the interaction between microbes ([@B58]), while regression models were limited in microbiology study ([@B3]). However, in our insomnia case, the LASSO model could not collect enough fitness for the current study ([Supplementary Figure S10](#FS10){ref-type="supplementary-material"}). To overcome the limitation of LASSO regression, we introduced ANN, which was originally developed to imitate the biological neural networks of the brain ([@B37]). ANN is not only an algorithm, but also a frame for different machine learning algorithms to incorporate and work together to process complex data. As it works in the same way as the human brain, compared to traditional machining learning such as LASSO, ANN brought out stronger and more robust ability to deal with complex data, offered a good prediction model with high fitness, and thus was applied to various areas, especially in quantum chemistry ([@B4]), general game playing ([@B52]), 3D reconstruction ([@B18]), and medical diagnosis ([@B28]). In these areas, ANN like LASSO regression could also effectively and practically address the multicollinearity problem. Thus, we incorporated an ANN prediction model to assess sleep quality based on the relative abundance of the gut microbiota. Although based on few samples, this model could still obtain good fitness. With this, we are able to utilize the relative abundance of the gut microbiota to provide an alternative and accurate approach for insomnia diagnosis.
Conclusion {#S5}
==========
The model proposed in the current study utilizes the cutting edge bioinformatic algorithm to not only underpin the difference between insomnia and normal health, but also take advantage of ANN to establish the prediction model for insomnia diagnosis and sleep quality evaluation based on the relative abundance of the gut microbiota. Although all methods above are only based on bioinformatics and mathematics, we believe these approaches could validate the results and further prove that even with a small sample size. With this, we could still be able to draw a solid conclusion. Of course, more cases will be collected to provide further evidence in our future work. This will open another gate and a new perspective for the development of novel therapeutic strategies by taking advantage of the information from the gut microbiota.
Data Availability {#S6}
=================
The generated datasets for this study can be found on BioProject accession number [PRJNA527914](PRJNA527914).
Ethics Statement {#S7}
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The experiment was proved by the Ethics Committee of Jinan University and recruited volunteers in public and The First Affiliated Hospital of Jinan University in Guangzhou, China (Approval \#: GNU-20180306).
Author Contributions {#S8}
====================
LX, LL, and JP designed the experiments. LX and JP collected the grant support. BL, SC, and ZL performed the data analysis. WL, TX, GX, YlY, and YfY collected participants' feces. BL and LX drafted the manuscript.
Conflict of Interest Statement {#conf1}
==============================
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
**Funding.** This work was supported by the National Natural Science Foundation of China (Grant No. 8187050617), "GDAS" Project of Science and Technology Development (Grant No. 2019GDASYL-0402001), Natural Science Foundation of Guangdong Province (Grant No. 2017A030313136) and Collaborative Innovation of Industry, University and Research in Guangzhou (Grant No. 201802030013).
Supplementary Material {#S10}
======================
The Supplementary Material for this article can be found online at: <https://www.frontiersin.org/articles/10.3389/fmicb.2019.01770/full#supplementary-material>
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The general information including BMI **(A)**, Height **(B)**, Weight **(C)**, and Age **(D)** between insomnia and normal group.
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Rarefaction measurement of Shannon **(A)** and Simpson **(B)** index presented a saturate platform indicated sequencing depth was enough to capture all bacterial species while Good's coverage index **(C)** and species accumulation curve **(D)** confirmed the sampling was sufficient for the experiment design on OTU taxa.
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BugBase algorithm was used to predict microbiome phenotypes including Gram positive **(A)** or negative **(B)**, aerobic **(C)** or anaerobic **(D)**, Potential_Pathogenic **(E)**, and Forms_Biofilms **(F)** with Mann-Whitney *U* test.
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Radar plot on transitivity, number of edges, number of vertices, degree of centralization, and graph density indicated the gut microbiota in each group developed a mature network with almost same complexity.
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Analysis of similarity (ANOSIM) revealed the difference between groups was more significant than that within groups (statistic *R*: 0.1944, *p* = 0.015).
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The detailed results of random forest in ten different random seed are presented.
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Based on two key bacterial taxa (V45, V124), the random forest prediction obtained an accurate rate with the ROC curve at AUC = 0.87.
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The detailed results of correlation analysis, these key taxa and clinical sleep parameter were mapped.
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Co-occurrence network in each group plotted with "auto layout" parameter in "igraph" packages shows V45 and V124 occupied hub-like position in each network.
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LASSO regression model to utilize the relative abundance of bacterial taxa to predict clinical sleep parameter.
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[^1]: Edited by: Enrica Pessione, University of Turin, Italy
[^2]: Reviewed by: Zongxin Ling, Zhejiang University, China; Giuliana Banche, University of Turin, Italy
[^3]: ^†^These authors have contributed equally to this work
[^4]: This article was submitted to Systems Microbiology, a section of the journal Frontiers in Microbiology
| {
"pile_set_name": "PubMed Central"
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Introduction {#Sec1}
============
Mendelian randomisation (MR) is now widely used to infer the causal influence of one trait (the exposure) on another (the outcome)^[@CR1],[@CR2]^. It generally uses genetic instruments for an exposure, obtained from genome-wide association studies (GWAS). If the instruments are valid, in that they are unconfounded and influence the outcome only through the exposure (vertical pleiotropy), then they will each provide an independent, unbiased estimate of the causal effect of the exposure on the outcome^[@CR3]^. Meta-analysing these estimates can provide a more precise estimate of the effect of the exposure on the outcome^[@CR4],[@CR5]^. If, however, some of the instruments are invalid, particularly because they additionally influence the outcome through pathways that bypass the exposure (horizontal pleiotropy)^[@CR3]^, then the effect estimate is liable to be biased. To date, MR method development has viewed horizontal pleiotropy as a nuisance that needs to be factored out of the analysis^[@CR6]--[@CR9]^. Departing from this viewpoint, here we exploit horizontal pleiotropy as an opportunity to identify alternative traits that putatively influence the outcome. We also explore how this knowledge can improve the original exposure--outcome estimates.
A crucial feature of MR is that it can be performed using only GWAS summary data, where the effect estimate can be obtained solely from the association results of the instrumental single-nucleotide polymorphisms (SNPs) on the exposure and on the outcome^[@CR5]^. This means that causal inference between two traits can be made even if they have never been measured together in the same sample of individuals. Complete GWAS summary results have now been collected from thousands of complex trait and common diseases^[@CR10]^, meaning that one can search the database for candidate traits that might be influenced by SNPs exhibiting possible pleiotropic effects (outliers). In turn, the causal influence of each of those candidate traits on the outcome can be estimated using MR by identifying their instruments (which are independent of the original outlier). Should any of these candidate traits putatively influence the outcome then this goes some way towards explaining the horizontal pleiotropic effect that was exhibited by the outlier SNP in the initial exposure--outcome analyses.
Several methods exist for identifying outliers in MR, each likely to be sensitive to different patterns of horizontal pleiotropy. Cook's distance can be used to measure the influence of a particular SNP on the combined estimate from all SNPs^[@CR11]^, identifying SNPs with large influences as outliers. Steiger filtering removes those SNPs that do not explain substantially more of the variance in the exposure trait than in the outcome, attempting to guard against using SNPs as instruments that are likely to be associated with the outcome through a pathway other than the exposure^[@CR12]^. Finally, meta-analysis tools can be used to evaluate if a particular SNP contributes disproportionately to the heterogeneity between the estimates obtained from the set of instruments, and this has been adapted recently to detect outliers in MR analysis^[@CR13]--[@CR15]^. A potential limitation of heterogeneity-based outlier removal is that this practice could constitute a form of cherry picking^[@CR9],[@CR16]^. While outlier removal can certainly improve power by reducing noise in estimation, it could also potentially induce higher type 1 error rates, which we go on to explore through simulations.
Recent large-scale MR scans have indicated that horizontal pleiotropy is widespread based on systematic analysis of heterogeneity^[@CR14],[@CR17]^. This suggests that many SNPs used as instruments are likely to associate with other traits, which in turn might associate with the original outcome of interest---hence giving rise to heterogeneity. As such we have an opportunity to identify previously unreported pathways by making use of outlying instruments in an MR analysis. Equipped with automated MR analysis software^[@CR10]^, outlier detection methods and a database of complete GWAS summary datasets, we develop MR-TRYX (from the phrase 'TReasure Your eXceptions'^[@CR18]^, popularised by William Bateson, an early pioneer in genetics). This framework is designed to identify putative causal factors when performing a simple exposure--outcome analysis.
In this paper, we describe how MR-TRYX can be implemented in MR analyses and how to interpret its results. A wide range of simulations is performed to show how knowledge of horizontal pleiotropic pathways can be used to improve the power and reliability of the original exposure--outcome association analysis. Our simulations also show that outlier removal methods can induce bias or increase type 1 error rates, but adjustment for detected pleiotropic pathway can improve estimates by reducing heterogeneity without sacrificing study power. We apply MR-TRYX to four exemplar analyses to demonstrate its potential utility, showing that horizontal pleiotropic pathways can be used to discover putative causal factors for an outcome of interest.
Results {#Sec2}
=======
Overview of MR-TRYX {#Sec3}
-------------------
Figure [1](#Fig1){ref-type="fig"} shows an overview of the approach. MR-TRYX is applied to an exposure--outcome analysis in a two-sample MR framework and it has two objectives. The first is to use outliers in the original exposure--outcome analysis to identify putative factors that influence the outcome independently of the exposure. The second is to re-estimate the original exposure--outcome association by adjusting outlier SNPs for the horizontal pleiotropic pathways that might arise through the putative associations. This outlier-adjustment method should be treated as a new approach to be used in conjunction with other methods that already exist in the MR sensitivity analysis toolkit. We provide extensive discussion on the context, advantages and potential pitfalls that come with trying to use a data-driven approach to adjust for horizontal pleiotropy at the end of the paper.Fig. 1Conceptual framework of the study.Illustration of identifying putative factors that influence the original observations. **a** Where (*gx*) is the SNP--exposure effect, (*xy*) is the exposure--outcome effect as estimated through MR analysis from the non-outlier SNPs, (*gp*) is the SNP--candidate trait effect and (*py*) is the causal effect of the candidate trait on the outcome. **b** The open circles represent valid instruments and the slope of the dotted line represents the causal effect estimate of the exposure on the outcome. The closed red circle represents an outlier SNP which influences the outcome through two independent pathways, P and X. **c** One way in which the red SNP can exhibit a larger influence on the outcome than expected given its effect on the exposure is if it influences the outcome additionally through another pathway (horizontal pleiotropy). **d** Using the MR-Base database of GWAS summary data for hundreds of traits we can search for 'candidate traits' with which the outlier SNP has an association. **e** Instruments excluding the original outlier SNP are obtained for each candidate trait, LASSO-based multivariable MR is used to prune the candidate traits to avoid redundancy, and the causal influence of each of those independent candidate traits on the outcome can subsequently be estimated. This allows us to identify alternative traits that putatively influence the outcome and adjust the SNP--outcome associations for pleiotropic pathways in the original exposure--outcome model.
Adjustment of pleiotropic pathways improves MR performance {#Sec4}
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We performed a wide range of simulations (Fig. [2](#Fig2){ref-type="fig"}, Supplementary Data [2](#MOESM3){ref-type="media"}) to evaluate how a variety of methods designed to deal with pleiotropy fare under a set of different scenarios that violate the exclusion restriction principle. Perhaps the most striking result from these simulations is that no method is always reliable, and several methods have similar overall reliability while performing very differently from each other between specific scenarios. Across 47 simulation scenarios, adjusting for detected outliers using the MR-TRYX framework had the highest average rank, and simply performing inverse-variance weighted (IVW) random effects was most often the best performing method, whereas removing detected outliers had the lowest average rank. We note that generally we do not know which of the scenarios are of relevance for any particular empirical analysis and so the metric used to evaluate performance here reflects the methods that are most generally performant. We found that as the proportion of instruments exhibiting pleiotropic effects increased, all methods typically worsened in their performance though there were notable examples in which increasingly widespread pleiotropy does not have an adverse effect. For example, widespread balanced horizontal pleiotropy or mediated pleiotropy does not have a drastic adverse influence on IVW, and MVMR and outlier adjustment is relatively impervious to confounding pleiotropy.Fig. 2Simulations comparing methods across different scenarios.We evaluated three scenarios: confounding pleiotropy, horizontal pleiotropy and mediated pleiotropy (columns of graphs, with DAGs illustrating the scenarios. See Methods for full details). The *x-*axis of each graph represents the proportion of variants used to instrument x that were similated to exhibit pleiotropic effects. Typically, 30 instruments were simulated directly for x but this varies across scenarios where necessary. The *y-*axis of the first row of graphs represents the proportion of simulations that lead to unbiased effect estimates of x on y. The *y-*axis of the second row of graphs represents the sensitivity and specificity of the analysis across the simulations, where the area under the receiving operating curve (AUROC) represents the ability of the method to distinguish between simulations in which the causal effect of x on y is either null or not null. For all graphs, higher *y-*axis values are better. Seven methods are evaluated at each simulation. Raw = IVW random effects estimates applied to all detected instruments; Removed = either all outliers are removed, or only outliers detected to associate with a candidate trait; MVMR = multivariable MR using either candidate traits detected to associate with any instrument or using only candidate traits associated with outlier instruments; Adjusted = adjusting SNP--outcome associations for candidate traits applied either only to variants detected to be outliers, or all variants regardless of outlier status.
It is an obvious conceptual disadvantage in these simulations for IVW and outlier removal, which use only the exposure and outcome data, when compared against MVMR and MR-TRYX which draw on information from other sources. However, we note that the MR-TRYX adjustment approach depends on detecting candidate traits that explain the pleiotropic effect and if the relevant candidate traits are not available, there is no adjustment and the method becomes identical to random effects IVW which generally performs better than outlier removal. We also note that if we use association with candidate traits to determine whether or not to remove an outlier, then improvements can be made over simple outlier removal. What we observe here is intuitive because the potential drawback of outlier removal is that the outliers could be the only valid instruments, or false discovery rates increase due to overly precise confidence intervals. Thus, adding an extra barrier to the removal of outliers can mitigate these problems.
Multivariable MR targets a different estimand than univariable MR---it is estimating the direct effect rather than the total effect of the exposure on the outcome. This strategy performs generally well across the range of simulations except in the case when the candidate trait is a mediator of the x--y association in which case there is a strongly attenuated direct effect. The problem here is that it is hard for MVMR to distinguish between a model where the exposure's influence on the outcome is mediated by a candidate trait (the exposure is causal), vs. where the exposure's apparent effect on the outcome is simply due to pleiotropy through the candidate trait (the exposure is not causal)^[@CR19]^. Here, MVMR performs worse than other methods when the candidate trait is a mediator, as MVMR estimates the direct effect of x on y adjusting for the entirety of x\'s signal on y. Adjusting for outliers escapes this problem to some extent because it only adjusts some proportion of the instruments for x that are most likely to be pleiotropic, allowing some signal of x on y to persist due to the unadjusted variants.
Empirical MR-TRYX analyses {#Sec5}
--------------------------
To examine the performance of MR-TRYX analysis, we tested four independent exposure--outcome hypotheses: (i) systolic blood pressure (SBP) and coronary heart disease (CHD); (ii) urate and CHD; (iii) sleep duration and schizophrenia; and (iv) education level (years of schooling) and body mass index (BMI). For each analysis we: (a) obtain MR estimates of the exposure--outcome causal relationship and detect outlier instruments; (b) identify putative causal influences (candidate traits) on the outcome trait based on their associations with outlier variants (Table [1](#Tab1){ref-type="table"}, Supplementary Data [1](#MOESM2){ref-type="media"}); (c) adjust the original SNP--outcome estimates for the putative influences operating through the candidate traits (Table [2](#Tab2){ref-type="table"}); and (d) compare the changes in heterogeneity in the MR estimates of the adjusted SNP--outcome effects to standard outlier removal methods.Table 1Candidate traits associated with both exposure and outcome.Outlier SNPsNearest geneCategoryPhenotypes^a^*N* SNPs^b^Beta (95% CI)^c^**Empirical analysis 1: systolic blood pressure (mmHg) on coronary heart disease (Odds ratio)**[rs3184504](https://www.ncbi.nlm.nih.gov/snp/?term=rs3184504)*SH2B3*Early developmentBirth weight of first child40−0.312 (−0.498, −0.126)Anthropometric measuresStanding height577−0.208 (−0.264, −0.152)LipidLDL cholesterol780.393 (0.290, 0.497)HDL cholesterol86−0.172 (−0.288, −0.055)Total cholesterol860.378 (0.271, 0.484)[rs9349379](https://www.ncbi.nlm.nih.gov/snp/?term=rs9349379)*PHACTR*MedicationsSelf-reported status of ibuprofen intake2−16.726 (−37.262, −3.811)**Empirical analysis 2: urate (mg/dl) and coronary heart disease (Odds ratio)**rs653178*ATXN2*Early developmentBirth weight of first child310.347 (0.065, 0.628)Birth weight40−0.312 (−0.498, −0.126)Anthropometric measuresComparative height size at age 10357−0.248 (−0.342, −0.154)Hip circumference2750.131 (0.030, 0.231)Impedance of arm (left)305−0.263 (−0.380, −0.145)Standing height577−0.208 (−0.264, −0.152)LipidHDL cholesterol780.393 (0.290, 0.497)LDL cholesterol86−0.172 (−0.288, −0.055)Total cholesterol860.378 (0.271, 0.484)Diseasehypothyroidism/myxoedema (Self-reported)770.847 (0.211, 1.483)SmokingPast tobacco smoking41−0.265 (−0.500, −0.029)MedicationsTreatment/medication: levothyroxine sodium511.231 (0.270, 2.191)rs642803*OVOL1*Anthropometric measuresWaist circumference2180.458 (0.352, 0.563)**Empirical analysis 3: sleep duration (hour/night) and schizophrenia (Odds ratio)**[rs7764984](https://www.ncbi.nlm.nih.gov/snp/?term=rs7764984)*HIST1H2BJ*DiseaseMalabsorption/coeliac disease (self-reported)11−8.401 (−12.842, −3.961)rs13107325*SLC39A8*Anthropometric measuresImpedance of leg (left)2820.179 (0.047, 0.311)MemoryProspective memory result24.493 (1.851, 7.135)**Empirical analysis 4: years of schooling (years) and body mass index (kg/m**^**2**^**)**[rs6882046](https://www.ncbi.nlm.nih.gov/snp/?term=rs6882046)*LINC00461*DrinkingAlcohol intake frequency310.347 (0.065, 0.628)[rs4800490](https://www.ncbi.nlm.nih.gov/snp/?term=rs4800490)*NPC1*DrinkingAlcohol intake frequency310.347 (0.065, 0.628)ExerciseUsual walking pace22−1.595 (−2.364, −0.825)[rs8049439](https://www.ncbi.nlm.nih.gov/snp/?term=rs8049439)*ATXN2L*DrinkingAlcohol intake frequency310.347 (0.065, 0.628)*SNP* single-nucleotide polymorphism, *VLDL* very low-density lipoprotein, *HDLC* high-density lipoprotein cholesterol, *LDLC* low-density lipoprotein cholesterol, *N SNPs* number of SNPs, *CI* confidence interval.^a^Candidate traits that are associated with outliers (*p* \< 5 × 10^−8^) and both exposure and outcome are listed. The listed traits were used in the adjusted model to investigate whether they are associated with the hypothesised outcome.^b^The number of SNPs used for two-sample MR analysis of candidate traits on the outcome.^c^The results were presented as IVW beta coefficient (95% CI), derived from two-sample MR analyses. Empirical analysis 1: systolic blood pressure (mmHg) and coronary heart disease (log odds); Empirical analysis 2: urate (mg/dl) and coronary heart disease (log odds); Empirical analysis 3: sleep duration (hour/night) and schizophrenia (log odds); Empirical analysis 4: years of schooling (years) and body mass index (kg/m^2^).Table 2Results of empirical analyses with different IV estimators derived from various MR methods.MethodsAll variantsEstimates (95% CIs)Removing outliersRemoving candidate outliersAdjustment for candidate outliers**Empirical analysis 1: systolic blood pressure (mmHg) on coronary heart disease (Odds ratio) ** Heterogeneity (*Q*)^a^682.7 (*N* SNPs = 157)312.1 (*N* SNPs = 150)448.7 (*N* SNPs = 155)567.6 (*N* SNPs = 157)IVW random effects1.761 (1.474, 2.104)1.876 (1.655, 2.125)1.797 (0.558, 5.789)1.706 (1.449, 2.008)Egger random effects2.641 (1.490, 4.679)2.951 (1.970, 4.419)2.206 (0.314, 15.472)--Intercept0.980 (0.969, 0.992)0.990 (0.982, 0.998)0.996 (0.988, 1.004)--Weighted median1.770 (1.528, 2.050)1.782 (1.539, 2.065)1.765 (0.576, 5.403)--Weighted mode1.770 (1.264, 2.479)1.726 (1.218, 2.447)1.740 (0.600, 5.043)--**Empirical analysis 2: urate (mg/dl) and coronary heart disease (Odds ratio)**Heterogeneity (*Q*)81.6 (*N* SNPs = 24)20.7 (*N* SNPs = 21)33.4 (*N* SNPs = 22)44.1 (*N* SNPs = 24)IVW random effects1.081 (0.996, 1.174)1.054 (1.008, 1.103)1.062 (1.057, 1.122)1.070 (0.992, 1.155)Egger random effects0.952 (0.846, 1.071)1.008 (0.937, 1.084)0.990 (0.910, 1.077)--Intercept1.015 (1.003, 1.027)1.006 (0.998, 1.014)0.992 (0.984, 1.000)--Weighted median1.019 (0.961, 1.081)1.016 (0.958, 1.078)1.017 (0.961, 1.077)--Weighted mode1.028 (0.975, 1.084)1.022 (0.966, 1.082)1.025 (0.970, 1.083)--**Empirical analysis 3: sleep duration (hour/night) and schizophrenia (Odds ratio)**Heterogeneity (*Q*)204.8 (*N* SNPs = 36)54.1 (*N* SNPs = 30)121.4 (*N* SNPs = 34)147.7 (*N* SNPs = 36)IVW random effects1.184 (0.573, 2.445)1.289 (0.828, 2.008)1.215 (0.674, 2.192)1.181 (0.634, 2.197)Egger random effects0.866 (0.056, 13.383)2.428 (0.485, 12.158)2.363 (0.254, 21.955)--Intercept1.004 (0.968, 1.042)0.991 (0.969, 1.013)0.991 (0.963, 1.020)--Weighted median1.276 (0.774, 2.104)1.249 (0.746, 2.090)1.250 (0.761, 2.052)--Weighted mode1.327 (0.679, 2.593)1.504 (0.728, 3.105)1.428 (0.702, 2.904)--**Empirical analysis 4: years of schooling (years) and body mass index (kg/m**^**2**^**)**Heterogeneity (*Q*)211.9 (*N* SNPs = 59)101.9 (*N* SNPs = 56)101.9 (*N* SNPs = 56)197.8 (*N* SNPs = 59)IVW random effects−0.272 (−0.386, −0.158)−0.232 (−0.314, −0.150)−0.232 (−0.314, −0.150)−0.265 (−0.377, −0.153)Egger random effects0.013 (−0.677, 0.703)−0.404 (−0.910, 0.102)−0.404 (−0.910, 0.102)--Intercept−0.005 (−0.017, 0.007)0.003 (−0.005, 0.011)0.003 (−0.005, 0.011)--Weighted median−0.209 (−0.307, −0.111)−0.217 (−0.315, −0.119)−0.217 (−0.315, −0.119)--Weighted mode−0.141 (−0.413, 0.131)−0.127 (−0.405, 0.151)−0.127 (−0.405, 0.151)--*N SNPs* number of single nucleotide polymorphisms, *95% CIs* 95% confidence intervals, *IVW* inverse variance weighted. Empirical analysis 1: systolic blood pressure (mmHg) and coronary heart disease (log odds); Empirical analysis 2: urate (mg/dl) and coronary heart disease (log odds); Empirical analysis 3: sleep duration (hour/night) and schizophrenia (log odds); Empirical analysis 4: years of schooling (years) and body mass index (kg/m^2^).^a^Heterogeneity amongst the estimates were assessed based on contribution of individual variant to Cochran's statistic.
Example 1: Systolic blood pressure and coronary heart disease: Blood pressure is a well-established risk factor for CHD. Random effects IVW estimates indicated that higher SBP is causally associated with higher risk of CHD (odds ratio \[OR\] per 1 SD: 1.76; 95% CI: 1.47, 2.10). While there was substantial heterogeneity in this estimate (*Q* = 682.7 on 157 SNPs, *p* = 5.74 × 10^−67^), the estimates from MR-Egger, weighted median and weighted mode methods were consistent (Table [2](#Tab2){ref-type="table"}). Seven of the 157 SNPs were detected as strong outliers based on *Q* statistics. We identified 69 candidate traits that were associated with these outliers (*p* \< 5 × 10^−8^). We manually removed redundant traits and traits that are similar to the exposure and the outcome (e.g. hypertension). Among the remaining candidate traits, 15 were putatively causal for CHD (Fig. [3a](#Fig3){ref-type="fig"}). After we applied LASSO regression, six traits remained (Table [1](#Tab1){ref-type="table"}): anthropometric measures (e.g. height), lipid levels (e.g. cholesterol level) and self-reported ibuprofen use were among the candidate traits that associated with CHD, which were all uncovered due to two outliers ([rs3184504](https://www.ncbi.nlm.nih.gov/snp/?term=rs3184504) near *SH2B3* and [rs9349279](https://www.ncbi.nlm.nih.gov/snp/?term=rs9349279) near *PHACTR*).Fig. 3Causal associations between candidate exposures and hypothesised outcome.Each candidate trait related to an outlier from an analysis is represented by a point in these plots. Along the *x-*axis, different phenotype groups are shown in different colours. The *y-*axis presents log transformed *P* value for each trait, multipled by the sign of the causal effect estimate on the outcome. Filled circles in each category indicate the evidence of association between candidate traits and exposure or outcome (using an FDR \< 0.05 threshold; see Methods for discussion of this). **a** Empirical analysis 1: systolic blood pressure (mmHg) and coronary heart disease (log odds). **b** Empirical analysis 2: urate (mg/dl) and coronary heart disease (log odds). **c** Empirical analysis 3: sleep duration (hour/night) and schizophrenia (log odds). **d** Empirical analysis 4: years of schooling (years) and body mass index (kg/m^2^).
We next adjusted the exposure--outcome association for the detected pleiotropic pathways and obtained an adjusted IVW estimate. The total heterogeneity, based on adjusting only these two of 157 SNP effects, was reduced by 17% (*Q* = 567.6). The effect estimate remained consistent with the original estimate, as did the IVW estimates when removing all outliers, or just outliers known to associate with candidate traits that associated with the outcome (Fig. [4a](#Fig4){ref-type="fig"}). However, the width of the confidence interval was substantially larger (including the null) after removing outliers known to associate with candidate traits (1 OR per SD: 1.80; 95% CI: 0.56, 5.79).Fig. 4Exposure--outcome association adjusting the SNP effects on the candidate traits.Radial plots of MR associations. The *x*-axis represents the weight (w) that each SNP contributes to the overall estimate, and the *y*-axis represents the product of the causal effect and weight of each SNP. The slopes represent causal effect estimates from different models (linetype). The arrows in this radial scatter plot indicates changes in the SNPʼs contribution to the overall causal effect estimate after conditioning on the effect of candidate traits on the outcome. The candidate traits that influence the association of the original exposure and the original outcome were listed in the box. **a** Empirical analysis 1: systolic blood pressure (mmHg) and coronary heart disease (log odds). **b** Empirical analysis 2: urate (mg/dl) and coronary heart disease (log odds). **c** Empirical analysis 3: sleep duration (hour/night) and schizophrenia (log odds). **d** Empirical analysis 4: years of schooling (years) and body mass index (kg/m^2^). Note that we use radial plots here as they explicitly show that one consequence of SNP-outcome effect adjustment is that the standard errors get larger (lower values on the *x*-axis). This leads to the adjusted variant contributing less weight to the causal effect and heterogeneity estimates, a process that acts in concert with the intention of attenuating the pleiotropic effect.
Example 2: Urate and coronary heart disease: Here we show an example with mixed findings from previous studies. The influence of circulating urate levels on risk of coronary heart disease has been under debate. Several MR studies have investigated the inflated effect of urate on CHD, which appeared to be influenced by pleiotropy^[@CR20],[@CR21]^ . We re-estimated the associations here using a range of MR methods. As has been previously reported the estimate from IVW suggested a weak association between urate and the risk of CHD using all variants (OR per 1 SD: 1.08; 95% CI: 1.00, 1.17), while there was a large intercept in the MR-Egger analysis (intercept = 1.02; 95% CI: 1.00, 1.03) with a much-attenuated causal effect estimate (Table [2](#Tab2){ref-type="table"}). The median and mode-based estimates were also consistent with the MR-Egger estimate, indicating weak support for urate having a causal influence on CHD. Here, three variants were detected as outliers, which associated with 61 candidate traits (*p* \< 5 × 10^−8^). Among those outliers, rs653178 and rs642803 were associated with 14 traits that had conditionally independent influences on the outcome (Fig. [3b](#Fig3){ref-type="fig"}), including anthropometric measures (e.g. hip circumference), cholesterol levels, diagnosis of thyroid disease and smoking status.
Removing the outliers in the IVW analysis led to a more precise (though slightly attenuated) estimate of the influence of higher urate levels on CHD risk (OR per 1 SD: 1.05; 95% CI: 1.01, 1.10 and OR per 1 SD: 1.06; 95% CIs: 1.06, 1.12, respectively, Table [2](#Tab2){ref-type="table"}). The adjustment model indicated an attenuated IVW estimate in comparison to the 'raw' approach, with confidence intervals spanning the null (OR per 1 SD: 1.07; 95% CI: 0.99, 1.16) while the degree of heterogeneity was reduced by half by accounting for the pleiotropic pathways through two outlier SNPs. The adjusted scatter plot showed that outliers moved towards the fitted line after controlling for the SNP effect on the candidate traits (Fig. [4b](#Fig4){ref-type="fig"}). The results in this analysis suggest that it is unlikely that urate has a strong causal influence on CHD. Here, outlier removal appears to strengthen evidence that may lead to a wrong conclusion.
Example 3: Sleep duration and schizophrenia: previous studies have shown that sleep disorder is associated with schizophrenia^[@CR22]^. However, none of them confirmed the causality between sleep disorder and schizophrenia. We observed weak evidence for any association between sleep duration and schizophrenia (OR per 1 SD: 1.18; 95% CIs: 0.57, 2.45), but there was substantial heterogeneity when all SNPs were used (*Q* = 204.8; *p* = 6.9 × 10^−26^). Six outlier instruments were detected, which associated with 46 candidate traits (*p* \< 5 × 10^−8^). Among those outliers, the SNPs [rs7764984](https://www.ncbi.nlm.nih.gov/snp/?term=rs7764984) (near *HIST1H2BJ)* and rs13107325 (near *SLC39A8)* were associated with three traits that putatively influenced the outcome: self-reported coeliac disease, body composition (impedance of leg) and memory function (Fig. [4c](#Fig4){ref-type="fig"}).
We re-estimated the original association accounting for the detected outliers. The degree of heterogeneity was reduced by 74% (*Q* = 54.1) when removing all six outliers and by 46% (*Q* = 147.7) when adjusting for the two SNP effects that had putative pleiotropic pathways. Both methods of outlier removal and adjustment provide similar estimates in terms of direction, while the magnitude of estimates differed. After removing outliers, MR-Egger causal estimates were substantially larger (OR per 1 SD = 2.43; 95% CI: 0.49, 12.16 and OR per 1 SD = 2.36; 95% CI: 0.25, 21.96, respectively) than those from the method using all variants. IVW causal estimates from the adjustment method were virtually identical with the original estimates, with narrower CIs (OR per 1 SD = 1.18; 95% CI: 0.63, 2.20). While all methods indicate that sleep duration is unlikely to be a major causal risk factor for schizophrenia, pursuing outliers in the analysis provided putative indications that coeliac disease and memory function may be risk factors for schizophrenia (Fig. [4d](#Fig4){ref-type="fig"}).
Example 4: Years of schooling and body mass index: The association of education and health outcome is well established in social science^[@CR23]^. Higher socioeconomic position is generally thought to lead to a lower risk of obesity in high-income countries^[@CR24],[@CR25]^. We used 59 independent genetic instruments^[@CR26]^ to estimate the influence of years of schooling on BMI^[@CR27]^ (Table [2](#Tab2){ref-type="table"}). All MR methods indicated that years of schooling has a causal beneficial effect on BMI (e.g. IVW beta: −0.27; 95% CI: −0.39, −0.16), except the estimate from MR Egger which had a very imprecise estimate (beta: 0.01; 95% CI: −0.67, 0.70), but the degree of heterogeneity was large (*Q* = 211.9 on 59 SNPs; *p* = 2.20 × 10^−8^). Three outliers ([rs6882046](https://www.ncbi.nlm.nih.gov/snp/?term=rs6882046) near *LINC00461*, [rs4800490](https://www.ncbi.nlm.nih.gov/snp/?term=rs4800490) near *NPC1*, [rs8049439](https://www.ncbi.nlm.nih.gov/snp/?term=rs8049439) near *ATXN2L*) were identified as contributors to heterogeneity, and they showed associations (*p* \< 5 × 10^−8^) with 48 candidate traits. Among those candidate traits, two were associated with BMI (Fig. [3b](#Fig3){ref-type="fig"}): alcohol intake frequency (which associated with all three outliers) and usual walking pace.
We next re-estimated the influence of years of schooling on BMI by accounting for outliers. Adjusting the outliers for candidate trait pathways such as alcohol intake and usual walking pace reduced heterogeneity by 15% and had a small reduction in the confidence intervals while the point estimate remained consistent (Table [1](#Tab1){ref-type="table"}). By contrast, there was a 48% reduction in heterogeneity when removing outliers. Point estimates remained largely consistent across all outlier removal methods. However, we note that Fig. [4b](#Fig4){ref-type="fig"} shows that one of the outliers ([rs4800490](https://www.ncbi.nlm.nih.gov/snp/?term=rs4800490), near gene *NPC1*) on the scatter plot moved away from the fitted line after adjusting for the pleiotropic pathway, indicating that if this outlier is due to a pleiotropic pathway we have estimated its indirect effect inaccurately or partially (e.g. where GWAS summary statistics are not available to identify other influential pleiotropic pathways).
Discussion {#Sec6}
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The problem of instrumental variables being invalid due to horizontal pleiotropy has received much attention in MR analysis. Detecting and excluding such invalid instruments, based on whether they appear to be outliers in the analysis, is now a common strategy that exists in various forms^[@CR7],[@CR8],[@CR14],[@CR15],[@CR28]^. We have shown here that outlier removal could, in some circumstances, compound rather than reduce bias, and misses an opportunity to better understand the traits under study. We developed the MR-TRYX framework, which utilises the MR-Base database^[@CR10]^ of GWAS summary data to identify potential explanations for outlying SNP instruments, and to improve estimates by accounting for the pleiotropic pathways that give rise to them. We have also demonstrated the use and interpretation of MR-TRYX in four sets of empirical analyses.
To be effective, MR-TRYX depends upon the performance of three methodological components: (i) detecting instruments that exhibit horizontal pleiotropy; (ii) identifying the candidate traits on the alternative pathways from the variant to the outcome; and (iii) adequately estimating the effects of the candidate traits on the outcome. Each of these components present difficult problems, but they are all modular and build upon existing methods and resources, and the MR-TRYX framework will naturally improve as those methods and resources themselves improve. We will now discuss the consequences of underperformance of each of these components on the TRYX analysis.
First it is important to notice that a major motivation for development of MR is that observational associations are often deemed unreliable because it is impossible to prove that there is no residual or unmeasured confounding biasing the effect estimate. But somewhat ironically, we find ourselves in a situation now where horizontal pleiotropy poses a similar challenge, in that proving that it is either absent or perfectly balanced is impossible. While several 'pleiotropy-robust' methods attempt to model out pleiotropic effects by assuming a particular model of genetic architecture, another strategy is to adjust for horizontal pleiotropy, by including in the same model the genetic effects on one or more traits that are hypothesised to mediate the horizontal pleiotropic pathways (e.g. MVMR^[@CR29]^). The adjustment approach depends upon those pathways being identified, which leaves it in a similar predicament to observational associations in that we cannot easily prove that all biasing pathways have been included in the model. The MR-TRYX approach falls within this category also, but we note that as fewer and fewer of the biasing pathways are identified and available to the adjustment model, the adjusted estimate will tend towards the IVW random effects estimate, which our simulations indicate can have good performance compared to, e.g., outlier removal methods. So, while clearly not a panacea for causal inference analysis, it is a valuable method within the MR toolkit, and its efficacy has been demonstrated. There is also an important contrast between outlier adjustment and multivariable MR in that the formulation of the latter is to estimate the direct effect of each exposure conditional on the others, whereas the former is to obtain an unbiased estimate of the total effect. MVMR may fail to distinguish between a pleiotropic model where the exposure (X) does not influence the outcome (Y) but has instruments that associate with another trait (A) which does influence Y, vs. a causal model in which trait A mediates the causal effect of X on Y. In both situations X will be deemed to be non-causal, despite it being indirectly causal in the latter case. This issue is discussed in detail elsewhere^[@CR19]^. Here, outlier adjustment improves on the matter because MVMR will nullify all instruments for the exposure after adjusting for the mediator, leading to the exposure being dropped. When only the outlier variants are adjusted, the risk of erroneously removing the entire exposure signal is replaced by the lesser risk of incorrectly nullifying the effects of the outliers only. This will introduce heterogeneity and slight bias but is unlikely to remove the exposure's entire signal.
The classification of an outlier in MR analysis can be based on the statistical estimates of how a SNP being included as an instrument is due to being reverse causal (Steiger filtering)^[@CR12],[@CR17]^, the extent to which a single SNP disproportionately influences the overall result (e.g. Cook's distance), or most commonly the extent to which an SNP contributes to heterogeneity (e.g. Cochran's *Q* statistic, MR-PRESSO, and implicitly in median- and mode-based estimators)^[@CR7],[@CR8],[@CR14],[@CR15]^. The philosophy of the latter two approaches is that proving horizontal pleiotropy is impossible, but that it should lead to outliers^[@CR9]^. While a useful approximation, these approaches have two main limitations. First, determining whether a SNP is an outlier depends on the use of arbitrary thresholds, and this entails a trade-off between specificity and sensitivity. Second, if most variants are pleiotropic, then it is possible that the outlier SNPs are the valid instruments. Such a scenario can arise for complex traits such as gene expression or protein levels that have a few large effects and many small effects. For example, for C-reactive protein (CRP) levels, the SNP in the *CRP* gene region is likely the only valid instrument in some analyses^[@CR30]^. In this context, bias due to horizontal pleiotropy cannot be avoided by selection of instruments since this approach may generate more bias^[@CR31]^. This is supported by our simulation which demonstrates that in the presence of extensive pleiotropy removing outliers increased FDR and bias.
MR-TRYX should, in principle, avoid the problem of outlier removal because instead of removing outliers in their entirety, it attempts to eliminate the component of the SNP--outcome effect that is due to horizontal pleiotropy. Hence, we avoid implicitly cherry picking from among the SNPs to be used in the analysis, and if we have low sensitivity (i.e. a more relaxed threshold for outlier detection) it does not mean that there will be an unnecessary loss of power in the overall analysis. Previous work has adjusted for the effect of pleiotropic phenotypes, but they treated pleiotropic phenotypes as exogenous variables that are not associated with the causal pathways of interest^[@CR32]^. In MR-TRYX, candidate traits are treated as endogenous variables to account for the effect of the traits on the original association. Moreover, our method is applicable in the two-sample context, whereas the previous method requires individual level data. The problem of outlier detection which remains in MR-TRYX could be sidestepped by applying the adjustment approach to all SNPs irrespective of their contributions to heterogeneity.
Upon identification of potentially pleiotropic SNPs, MR-TRYX can only account for these if the pathways through which pleiotropy is acting can be identified. Detecting the pathways depends on the density and coverage of the human phenome available for the analysis. We use the MR-Base database of GWAS summary results, which comprises several hundred independent traits (we selected 605 traits from UK Biobank and 342 other complex traits and diseases obtained from previous GWA studies). While being the largest available resource, it is certainly not covering the whole human phenome. Therefore, even if a pleiotropic variant is detected correctly, it may not be possible to adjust it away if the phenotype associated with the variant cannot be identified. In the empirical analyses, often fewer than half of the candidate traits were inferred to be associated with the outcome. Yet, as we illustrated, MR-TRYX allows for an informative analysis that could routinely be applied in MR analyses. Broadening phenotype coverage is an on-going pursuit that will continually improve MR-TRYX analysis^[@CR33]^. It is also important to note that in estimating the adjusted effect, the SNP--outcome standard error is liable to increase, which is one avenue through which heterogeneity is reduced as its outlying contribution will be down-weighted in the subsequent IVW analysis. We used radial MR plots to illustrate this explicitly in Fig. [4](#Fig4){ref-type="fig"}.
MR-TRYX is an automated framework, and this comes with several limitations in addition to those discussed already. First, our LASSO extension to multivariable MR is used to automate the selection of exposures that will be used for adjustment. A shrinkage step of LASSO may increase the SNP--exposure effect heterogeneity, which is necessary to assess the power of multivariable MR^[@CR34]^. Multivariable MR is adept at establishing conditionally independent exposures but the reason that some exposures have attenuated effects in comparison to their total effects could be because (a) their total effects were biased by pleiotropy or (b) they are mediated by the exposures that are included in the model. Interpretations of (a) and (b) are very different, because in the case of mediation the exposure is a causal factor for the outcome. Second, we were primarily using the multivariable approach for practical purposes to avoid having multiple highly related exposures taken forward to the adjustment step (e.g. multiple different measures of body composition such as body weight and BMI). This approach worked effectively, although a problem remains unsolved in automating the removal of traits that are similar to the outcome. For example, if a trait similar to the outcome CHD associates with an outlier and is included in the multivariable analysis of multiple exposures against CHD, then all the other putative exposures will be dropped from the model. In the analyses presented we manually removed traits that came up as candidate pleiotropic pathways but were, in fact, synonymous with or closely related to the outcome. Third, we note that heterogeneity does not necessarily arise only because of pleiotropy, for example the non-collapsibility of odds ratios will introduce heterogeneity automatically which cannot be adjusted away through the TRYX approach. Many other mechanisms exist that can lead to bias in MR, as has been described in detail elsewhere. Fourth, SNPs can appear to be outliers not through being pleiotropic, but through other mechanisms, such as population stratification (association of alleles with phenotypes being confounded by ancestral population), canalisation (developmental compensation to a genetic change)^[@CR2],[@CR35]^, or the influence on phenotype being changeable across the life course^[@CR36]^. Fifth, since MR-TRYX uses the resource from MR-Base, it is recommended that the user acknowledge the limitation and restriction of MR-Base^[@CR10]^. For example, the population should be the same for the exposure (or the candidate traits) and the outcome traits to avoid mis-estimation of the magnitude of the association. Also, sample overlap should be recognised between the GWAS studies for the SNP--exposure and SNP--outcome association to prevent effect estimates being biased^[@CR37]^. Users should consider modifying their analyses when the limitations indicated above are avoidable. Sixth, in the case of a binary outcome, there may be parametric restrictions on the conditional causal odds ratio in our multivariable MR model where the exposure effect is linear in the exposure on the log odds ratio scale^[@CR38]^. However, the two-stage estimator with a logistic second-stage model still yields a valid test of the causal null hypothesis^[@CR38]^. Finally, it is necessary for the effects through the identified pleiotropic pathways to be accurately estimated. This is a recursive problem---MR-TRYX adjusts the SNP--outcome effects based on the pleiotropic effect through the outlier SNP, but it does this by introducing more SNPs into the analysis that instrument the candidate traits. These new SNPs may themselves exhibit pleiotropic effects that could lead to bias in the estimates of the candidate traits on the outcome, requiring a second round of TRYX-style candidate trait searches, and so on. In the example of education level and BMI, adjustment for the pleiotropic pathway failed to substantially reduce the degree of heterogeneity. Further developments could involve recursively analysing alternative pathways. For example, Steiger filtering could be applied at all stages of MR estimation to attempt to automatically remove reverse causal instruments or those that arise due to confounding pleiotropy^[@CR17]^.
In this study, we demonstrated the use of MR-TRYX through four examples of identifying putative pathways. In the first empirical example (SBP on CHD), we illustrated the validity of MR-TRYX to detect the traits that possibly influence the disease outcome. Apart from SBP, MR-TRYX also detected well-established risk factors for CHD including adiposity, cholesterol levels, and standing height. An interesting finding from this example is that headache-related traits (e.g. experience of pain due to headache and self-reported status of ibuprofen intake) were identified as candidate traits, which may influence the original association. In support of the putative finding for self-reported ibuprofen use associating with CHD, we also found that pain experienced in the last month (headache) and self-reported migraine were associated with lower risk of CHD (OR per 1 SD: 0.33; 95% CI: 0.12, 0.89 and beta = 0.02; 95% CI: 0.0004, 0.65, respectively). A previous study reported shared genetic risk between headache (migraine) and CHD, suggesting a potential role of migraine in vascular mechanisms^[@CR39]^. An alternative mechanism that could give rise to this association is that the effect of pain on lower CHD risk is mediated through the use of medications such as aspirin that have known protective effects on CHD.
The example of urate and CHD demonstrated the benefit of the adjustment method showing that the noise due to pleiotropy was substantially reduced after correcting for the effect of candidate traits. The presence of hypothyroidism and self-reported levothyroxine sodium intake status were identified as putative risk factors for risk of CHD, which is consistent with previous clinical trials: thyroid dysfunction is associated with overall coronary risk^[@CR40]^, which can be reversed by levothyroxine therapy^[@CR41]^. In the education--BMI example, we showed that increased alcohol intake and slower usual walking pace may influence obesity. These identified traits have been reported as possible risk factors for higher BMI and obesity^[@CR42],[@CR43]^. Additionally, the example of sleep duration and risk of schizophrenia suggested coeliac disease and body composition as putative risk factors for schizophrenia. A number of observational studies suggested that schizophrenia is linked with body composition^[@CR44]^ and coeliac disease^[@CR45]^. MR of binary exposures is often difficult to interpret because the instrument effects are on liability to disease, not the presence or absence of the disease. Hence, the association between coeliac disease and schizophrenia may be better interpreted as an indication of shared disease aetiology. Nevertheless, this is a valuable finding since the causal effect of those putative risk factors on risk of schizophrenia has not been investigated using an MR approach. Therefore, our example illustrates how outliers can be used to identify alternative pathways, opening the door for hypothesis-free MR approaches and a network-based approach to disease.
In conclusion, we have introduced a framework to deal with the bias from horizontal pleiotropy, and to identify putative risk factors for outcomes in a more directed manner than typical hypothesis-free analyses, by exploiting outliers. Heterogeneity is widespread across MR analyses and so we are tapping into a potential new reservoir of information for understanding the aetiology of disease. The strategy is a departure from previous ones dealing with pleiotropy---enlarging the problem by searching across all traits for a better understanding of a specific exposure--outcome hypothesis can be fruitful.
Methods {#Sec7}
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Outlier detection {#Sec8}
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Several outlier detection methods now exist that are based on the contribution of each SNP to overall heterogeneity in an IVW meta-analysis^[@CR46]^. In order to estimate heterogeneity accurately, it is important to appropriately weight the contribution of each SNP to the overall estimate. We used the approach implemented in the RadialMR R package (<https://github.com/WSpiller/RadialMR>) to detect outliers. Full details are provided elsewhere^[@CR15]^, but briefly, we used the so-called 'modified 2^nd^ order weighting' approach to estimate total Cochran's *Q* statistic as a measure of heterogeneity, as well as the individual contributions of each SNP, *q*~*i*~^[@CR15]^. This has been shown to be comparable to the simulation-based approach in MR-PRESSO, providing a well-calibrated test statistic for outlier status whilst being computationally more efficient^[@CR14],[@CR47]^. The probability of a SNP being an outlier is calculated based on *q*~*i*~ being chi-square distributed with one degree of freedom. For demonstration purposes we adopted a *p* value threshold that was Bonferroni corrected for the number of SNPs tested in analysis (*p* \< 0.05/number of SNPs). We are not, however, suggesting that this arbitrary threshold will necessarily be optimal for identifying outliers, and users can apply other approaches or thresholds through the MR-TRYX software.
Candidate trait detection {#Sec9}
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Traits associated with the detected outliers could causally influence the outcome. MR-TRYX searches the MR-Base database to identify the traits that have associations with the detected outliers. By default, we limit the search to traits for which the GWAS results registered at MR-Base have more than 500,000 SNPs and sample sizes exceeding 5000. Traits that have an association with outlier SNPs at genome-wide *p* value threshold (*p* \< 5 × 10^−8^; in keeping with traditional GWAS thresholds used for instrument selection) are regarded as potential risk factors for the outcome and defined as candidate traits. Each candidate trait is tested for its influence on the original exposure (X) and outcome (Y) traits (Fig. [1](#Fig1){ref-type="fig"}) using the IVW random effects model. We take forward putative associations based on false discovery rate (FDR) \< 0.05, where the null hypothesis is true, but we note that the use of arbitrary thresholds is problematic^[@CR48],[@CR49]^, and we use them here to make high dimensional investigations more manageable.
Assessing effect of the candidate traits on the outcome {#Sec10}
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Once candidate traits are detected, we can identify instruments specifically for the candidate traits and model how the exposure and candidate traits together associate with the outcome. This involves the following process, which we go on to describe in full detail below:Identify instruments for the candidate traits.Estimate the influence of the candidate traits on *y* conditioning on *x* using multivariable MR.
Suppose we have *g*~0~, *g*~*x*1~,...,*g*~*xE*~ instruments for the exposure *x* where *g*~0~ is an outlier in the *x*--*y* MR analysis due to an association with candidate trait *P*, and where *E* indicates the number of genetic variants for the exposure. Also, *P* has *g*~0~, *g*~*P*1~,...,*g*~*PM*~ genetic instruments, where *M* is the number of genetic variants for *P*. To obtain the estimate of (*py*) uncontaminated by shared genetic effects between *P* and *x* (Fig. [1a](#Fig1){ref-type="fig"}), we perform multivariable MR analysis^[@CR34]^. We generate a combined list of instruments for both *x* and *P* and clump them to obtain a set of independent SNPs. The original outlier is removed from amongst these SNPs. We then obtain the genetic effects of each of these SNPs on the exposure (*gx*), candidate trait (*gp*), and outcome (*gy*). Finally, we estimate the causal influence of *P* on *y* conditioning on *x* by regressing (*gy*) \~ (*gx*) + (*gp*) weighted by the inverse of the variance of the (*gy*) estimates. The whole process is automated within the TwoSampleMR R package which connects to the MR-Base database.
In the case of an outlier SNP associating with many candidate traits we first apply a modified form of multivariable MR, involving LASSO regression of (*gy*) \~ (*gx*) + (*gp*~i~)+...+(*gp*~*p*~) and use cross-validation to obtain the shrinkage parameter that minimises the mean squared error. We retain only the candidate traits that are putatively associated with the outcome and have non-zero effects after shrinkage. Then we apply remaining traits in a multivariable model with *x* against the outcome, as described above^[@CR34]^. We perform the LASSO step because many traits in the MR-Base database have considerable overlap and redundancy, and the statistical power of multivariable analysis depends on the heterogeneity between the genetic effects on the exposure variables^[@CR34]^. Using LASSO therefore automates the removal of redundant traits (Supplementary Fig. [1](#MOESM1){ref-type="media"}, Supplementary Tables [2](#MOESM1){ref-type="media"} and [3](#MOESM1){ref-type="media"}). We then obtain estimates of (*py*) that are conditionally independent of *x* and jointly estimated using all remaining *P* traits by combining them in a multivariable analysis on the outcome *y*. A detailed discussion of dealing with multiple candidate traits per outlier SNP is presented in Supplementary Note [1](#MOESM1){ref-type="media"}.
Adjusting causal estimates for candidate-trait associations {#Sec11}
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An illustration of how outliers arise in MR analyses is shown in Fig. [1](#Fig1){ref-type="fig"}. If a SNP *g* has some influence on exposure *x*, and *x* has some influence on outcome *y*, the SNP effect on *y* is expected to be (*gy*) = (*gx*)(*xy*), where (*gx*) is the SNP effect on *x* and (*xy*) is the causal effect of *x* on *y*. Any substantive difference between (*gy*) and (*gx*)(*xy*) could be due to an additional influence on *y* arising from the SNP's effect through an alternative pathway.
If a SNP influences a 'candidate trait', *P*, which in turn influences the outcome (or the exposure and the outcome), then the SNP's influence on the exposure and the outcome will be a combination of its direct effects through *x* and indirect effects through *P*^[@CR34]^. If we have estimates of how the candidate trait influences the outcome, then we can adjust the original SNP--outcome estimate to the effect that it would have exhibited had it not been influencing the candidate trait. In other words, we can obtain an adjusted SNP--outcome effect conditional on the 'candidate-trait--exposure' and 'candidate-trait--outcome' effects. If the SNP influences *P* independent candidate traits (as selected from the LASSO step), then the expected effect of the SNP on *y* is$$\documentclass[12pt]{minimal}
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\begin{document}$$\left( {gy} \right) = \left( {gx} \right)\widehat {\left( {xy} \right)} + \mathop {\sum}\limits_{i = 1}^P {\left( {gp_i} \right)\widehat {\left( {p_iy} \right)}}.$$\end{document}$$
Hence, the effect of the SNP on the outcome adjusted for alternative pathways *p*~1~,..., *p*~*p*~ is$$\documentclass[12pt]{minimal}
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\begin{document}$$\left( {gy} \right) ^\ast = \widehat {\left( {gy} \right)} - \mathop {\sum}\limits_{i = 1}^P {\left( {gp_i} \right)\left( {p_iy} \right)}.$$\end{document}$$
We use parametric bootstraps to estimate the standard error of the $\documentclass[12pt]{minimal}
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\begin{document}$${\left( {gy} \right)}^\ast$$\end{document}$ estimate, where 1000 resamples of (*gy*), (*gp*), and (*py*) are obtained based on their respective standard errors and the standard deviation of the resultant $\documentclass[12pt]{minimal}
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\begin{document}$$\widehat {\left( {gy} \right)}^\ast$$\end{document}$ estimate represents its standard error. Finally, an adjusted effect estimate of (*xy*) due to SNP *g* is obtained through the Wald ratio.$$\documentclass[12pt]{minimal}
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\begin{document}$$\widehat {\left( {xy} \right)} = \frac{{\left( {gy} \right) ^\ast }}{{(gx)}}.$$\end{document}$$
Occasionally it might be possible that a candidate trait *P* is a redundant trait for *y*, for example if the outcome is coronary heart disease, the outliers might detect traits such as 'medication for heart disease' as a potential candidate trait. It would make no sense to attempt to adjust the SNP--outcome association for a trait that is essentially the same as the outcome, it would just nullify the association. We have not yet developed an automated method to remove such traits, but we recommend manually checking any traits that are selected for automated outlier adjustment.
Simulations {#Sec12}
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IVW effect estimates are liable to be biased when at least some of the instrumenting SNPs exhibit horizontal pleiotropy, and those SNPs tend to contribute disproportionately towards the heterogeneity in the effect estimate. We conducted simulations to evaluate how different methods perform at estimating the causal effect of *x* on *y* under different circumstances. The simulations are principally designed to evaluate the potential value of adjusting outliers for putative explanatory pathways. Other aspects of the MR-TRYX framework, for example, dealing with redundant traits in the GWAS database are dealt with separately (Supplementary Note [1](#MOESM1){ref-type="media"}). In all circumstances there are 30 independent genetic effects on *x* (*Gx*), and *x* either has no direct influence on *y*, or has a direct effect of 0.1 on *y*. For all simulations, we used 10,000 individuals, and repeated each circumstance 1000 times. We summarised each scenario in two ways: (a) We estimated the proportion of simulations that gave a biased estimate of the causal effect of *x* on *y* (*b*~*xy*~). For each simulation we calculated the probability of the effect estimate being substantially different from the true simulated effect based on whether the true effect fell outside the 95% confidence interval of the estimate. Then for the set of 1000 simulations, we calculated the proportion of estimates that were 'unbiased'. (b) We summarised the power and FDR by estimating the area under the receiver operator curve, characterising the sensitivity and specificity of each method at determining whether the true causal effect estimate was null or non-null. Each simulation is conducted by first simulating data to satisfy the parameters described below. We then search for instruments for *x* across all simulated genetic variants and retain those that are significant after Bonferroni correction, and applying the summary data-based methods based on the genetic associations for the instruments on *x* and *y*. All genetic variants are simulated to be Hardy Weinberg equilibrium with an allele frequency of 0.5.
We investigated three scenarios that could give rise to invalid instruments (Fig. [2](#Fig2){ref-type="fig"}).
In the confounding pleiotropy scenario, there are instruments detected for *x* that primarily influence a confounder variable (e.g. *u*~1~ that influences both *x* and *y*). Therefore, the term 'confounding pleiotropy' indicates that the instrument's horizontal pleiotropic effect arises because it primarily influences a confounder of *x* and *y*. See Fig. [2](#Fig2){ref-type="fig"} (column 1) for a DAG describing the model. The confounder *u*~1~ has a set of independent genetic influences, *G*~*u*1~, which may be detected as instruments for *x*.$$\documentclass[12pt]{minimal}
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\begin{document}$$u_1 = \mathop {\sum }\limits_j^{m_{u1}} G_{u1,j}b_{gu1,j} + e_{u1},$$\end{document}$$$$\documentclass[12pt]{minimal}
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\begin{document}$$y = u_1b_{u1y} + xb_{xy} + e_y.$$\end{document}$$
Parameters: *b*~*gu*1,*j*~ values are sampled for each SNP *m*~*u*1~ from a normal distribution such that they explain 60% of the variance in *u*~1~. The value of *b*~*u*1*x*~ is chosen such that *u*~1~ explains 60% of the variance in *x* and 40% of the variance in *y*. The values of *b*~*gx*~,~*j*~ are sampled from a normal distribution for each of *m*~*x*~ SNPs such that they explain 20% of the variance in *x*. The causal effect *b*~*xy*~ is set to either 0, or some value such that *x* explains 10% of the variance in *y*. Values for *e*~*u*1~,*e*~*x*~, and *e*~*y*~ are sampled from normal distributions with mean 0 and variances that are scaled to satisfy the variances of all other parameters described for the model. Different sets of simulations are run with different proportions of invalid instruments by simulating different numbers of genetic variants directly influencing *u*~1~ or *x*:$$\documentclass[12pt]{minimal}
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In the case of horizontal pleiotropy, at least some of the instruments for *x* have an independent effect on *y* that is mediated through some other pathway that does not include *x*. In these simulations, the pleiotropic influence of each instrument, *G*~*x,i*~, is mediated by a different trait, *u*~2,*i*~$$\documentclass[12pt]{minimal}
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\begin{document}$$u_{2,i} = \mathop {\sum }\limits_j^{m_{u2,i}} G_{u2,i,j}b_{gu2,i,j} + G_{x,i}b_{plei,i} + e_{u2,i},$$\end{document}$$$$\documentclass[12pt]{minimal}
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\begin{document}$$m_{\mathrm{{plei}}} \in \{ 5,10,15,20,25,30\}$$\end{document}$ of 30 G~*x*~ instruments for *x* are selected to have pleiotropic effects, such that they influence *y* each mediated by an independent trait *u*~2,*i*~ which itself has its own set of 30 direct genetic influences *G*~*u*2~,*i*. The *b*~*gu*2,*i*,*j*~ values for the genetic effects on *u*~2,*i*~ are sampled from a normal distribution such that they explain 20% of the variance in *u*~2,*i*~. Each pleiotropic *G*~*x,i*~ instrument has an influence on *u*~2,*i*~ that explains 20% of its variance (*b*~plei,*i*~). The influence of each *u*~2,*i*~ on *y* is such that *b*~*u*2,*i*,*y*~ is normally distributed with mean 0 and variance 0.4. The outcome *y* is also influenced by *x* where the causal effect *b*~*xy*~ is set to either 0, or some value such that *x* explains 10% of the variance in *y*. Values for *e*~*u*2,*i*~, *e*~*x*~, and *e*~*y*~ are sampled from normal distributions with mean 0 and variances that are scaled to satisfy the variances of all other parameters described for the model.
Mediation pleiotropy is treated as in 'confounding pleiotropy', except the pleiotropic relationships arise due to a trait that is mediating the path from *x* to *y*, rather than confounding it (Fig. [2](#Fig2){ref-type="fig"}, column 3). Specifically, the influence of *x* on *y* is at least partially mediated by another trait *u*~3~, and at least some of the instruments for *x* have an independent pleiotropic influence on *u*~3~.$$\documentclass[12pt]{minimal}
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\begin{document}$$u_3 = \mathop {\sum }\limits_j^{m_{u3}} G_{u3}b_{gu3,j} + \mathop {\sum }\limits_i^{m_{\mathrm{{plei}}}} G_{x,i}b_{\mathrm{{plei}},i} + xb_{x,u3} + e_{u3},$$\end{document}$$$$\documentclass[12pt]{minimal}
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\begin{document}$$m_{\mathrm{{plei}}} \in \{ 5,10,15,20,25,30\}$$\end{document}$ of 30 *Gx* instruments for *x* are selected to have pleiotropic effects, such that they influence *u*~*3*~ which itself mediates an effect from *x* to *y*, and has its own set of 30 direct genetic influences *G*~*u*3~. The *b*~*gu*3,*j*~ values for the genetic effects on *u*~3~ are sampled from a normal distribution such that they explain 20% of the variance in *u*~3~. Each pleiotropic *Gx,i* instrument has an influence on *u*~3~ such that *b*~plei*,i*~ are sampled from a normal distribution explaining 20% of the variance in *u3* in total. The indirect influence of *x* on *y* is generated such that *x* explains 30% of the variance in *u*~3~, and *u*~3~ explains 40% of the variance of *y*. The outcome *y* may also be influenced directly by *x* where the causal effect *b*~*xy*~ is set to either 0, or some value such that *x* explains 10% of the variance in *y*. Values for *e*~*u*3~, *e*~*x*~, and *e*~*y*~ are sampled from normal distributions with mean 0 and variances that are scaled to satisfy the variances of all other parameters described for the model.
In these simulations we ask: if we can identify the pathway through which an outlier SNP has a horizontal pleiotropic effect, can adjustment for that pathway improve the original exposure--outcome analysis? We assess the performance of the following methods for each simulation.Raw, where all detected instruments are used in a standard IVW random effects analysis.Adjusted SNP--outcome effectsWhere outlier SNPs are tested for association with all candidate traits and adjusted for the effect of the candidate trait on the outcome using MR-TRYX.Where attempts are made to adjust all detected instruments regardless of outlier status.Removed instrumentsWhere all detected outliers are removed.Where only outliers that are found to influence a candidate trait are removed.Multivariable MR (MVMR)Where the traits selected to be included in the model are the candidate traits associated with outliers.Where the traits selected to be included in the model are the candidate traits associated with any of the detected instruments regardless of outlier status.
Empirical analyses {#Sec13}
------------------
As applied examples, we chose two robust findings and two controversial findings that are potentially biased due to pleiotropy: (i) systolic blood pressure (SBP) and coronary heart disease (CHD); (ii) urate and CHD; (iii) sleep duration and schizophrenia; and (iv) education level (years of schooling) and body mass index (BMI). Those examples were chosen based on previous findings^[@CR20],[@CR22],[@CR50],[@CR51]^ to illustrate how pleiotropic variants can be used to identify other pathways and adjusted to estimate the causal effect of the original exposure on the outcome independent of pleiotropic bias.
Summary statistics (β-coefficients and SEs) for the associations of the SNPs with each exposure were obtained from the publicly available GWAS database (Supplementary Table [1](#MOESM1){ref-type="media"}). Selected SNPs were harmonised for the analysis, excluding palindromic SNPs and pruning for linkage disequilibrium (*r*^2^ \< 0.001). We primarily used the two-sample MR IVW method to obtain causal estimates between exposures and outcomes allowing each SNP to have a different mean effect (random effects model). A number of sensitivity analyses were applied to evaluate the consistency of causal effect estimates under different models of pleiotropy among the SNPs, including the MR-Egger^[@CR6]^, weighted median, and weighted mode approaches^[@CR7],[@CR8]^.
Outliers were detected among the instruments for each exposure (*p* \< 0.05/the number of SNPs). We searched the MR-Base database to identify the candidate traits that are associated with outliers (*p* \< 5 × 10^−8^). We then performed multivariable MR analysis to test which candidate trait can explain the heterogeneity in the original exposure--outcome association. To perform multivariable MR, more SNPs that instrument the candidate traits were introduced into the analysis.
Subsequently we re-estimated the association of the original exposure and the original outcome using different sets of instruments: (a) all SNPs (corresponding to the raw method in our simulation), (b) outliers adjusted, (c) all outlier removed, and (c) candidate outliers removed.
All analyses were conducted with the TwoSampleMR package (<https://github.com/MRCIEU/TwoSampleMR>) and the MR-TRYX package (<https://github.com/explodecomputer/tryx>) in R statistical software (ver 3.4.1). Detailed information are provided in Supplementary Note [1](#MOESM1){ref-type="media"} and the scripts used for the simulations and empirical analyses can be found here <https://github.com/explodecomputer/tryx-analysis>.
Reporting summary {#Sec14}
-----------------
Further information on research design is available in the [Nature Research Reporting Summary](#MOESM5){ref-type="media"} linked to this article.
Supplementary information
=========================
{#Sec15}
Supplementary Information Supplementary Data 1 Supplementary Data 2 Peer Review File Reporting Summary Description of Additional Supplementary Files
**Peer review information** *Nature Communications* thanks Hans van Kippersluis, Kostas Tsilidis and Marie Verbanck for their contribution to the peer review of this work. Peer reviewer reports are available.
**Publisher's note** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
=========================
**Supplementary information** is available for this paper at 10.1038/s41467-020-14452-4.
This study was supported by the UK Medical Research Council (MC_UU_00011/1; MC_UU_00011/4), which founds Integrative Epidemiology Unit at the University of Bristol where Y.C., P.C.H., E.S., T.R.G., J.Z., G.D.S. and G.H. work. G.H. was supported by the Wellcome Trust and Royal Society \[208806/Z/17/Z\].
Y.C., G.D.S. and G.H. conceived the study and developed the statistical analysis plan. Y.C. and G.H. developed the model and methods. Y.C., G.D.S. and G.H. prepared the first draft of manuscript. Y.C., P.C.H., E.S., T.R.G., J.Z., A.P.M., G.D.S., and G.H. contributed to the writing of the manuscript. All authors reviewed and agreed on the manuscript.
The data that support the findings of this study are available from IEU GWAS database (<https://gwas.mrcieu.ac.uk/>).
A copy of the code used in this analysis is available at <https://github.com/explodecomputer/tryx> and <https://github.com/explodecomputer/tryx-analysis>.
The authors declare no competing interests.
| {
"pile_set_name": "PubMed Central"
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Most pancreatic tumors are primary, and the majority are adenocarcinomas of ductal origin \[[@b1-jptm-2020-03-04],[@b2-jptm-2020-03-04]\]. Although direct invasion of the pancreas by adjacent aggressive tumors is often observed, pancreatic metastasis from distant primary sites is very rare \[[@b1-jptm-2020-03-04],[@b3-jptm-2020-03-04]\]. Metastatic pancreatic cancer accounts for less than 2% of all pancreatic malignancies \[[@b4-jptm-2020-03-04],[@b5-jptm-2020-03-04]\]. Renal cell carcinoma is the most common primary tumor to metastasize to the pancreas \[[@b4-jptm-2020-03-04],[@b5-jptm-2020-03-04]\]. Other common tumors are colorectal cancer, melanoma, breast cancer, lung cancer, and sarcoma \[[@b4-jptm-2020-03-04]\]. Among these, cases of metastatic sarcoma to the pancreas are limited \[[@b4-jptm-2020-03-04]\]. Metastatic sarcoma to the pancreas may present a diagnostic and therapeutic challenge due to its rarity and the difficulty in distinguishing between primary and metastatic tumors based on their radiologic features, especially for solitary mass \[[@b3-jptm-2020-03-04],[@b6-jptm-2020-03-04]\]. Furthermore, tumors located in the body or tail of the pancreas often present with no symptoms \[[@b3-jptm-2020-03-04],[@b7-jptm-2020-03-04]\]. Therapeutic approaches can vary greatly between primary pancreatic cancer and metastatic sarcoma \[[@b3-jptm-2020-03-04],[@b4-jptm-2020-03-04],[@b8-jptm-2020-03-04]-[@b10-jptm-2020-03-04]\]. With continued improvements in treatment and survival for sarcoma patients, the frequency of detection of metastasis to unusual sites is increasing \[[@b11-jptm-2020-03-04]\]. However, the index of suspicion for metastatic sarcoma in the pancreas is still very low, and a standard management regimen for metastatic pancreatic sarcoma has not been established. Prolonged survival after surgery for isolated or resectable pancreatic metastatic sarcoma has been reported \[[@b10-jptm-2020-03-04],[@b12-jptm-2020-03-04]-[@b16-jptm-2020-03-04]\].
In this paper, we present 12 cases of metastatic sarcoma to the pancreas and report their clinical and histologic features.
MATERIALS AND METHODS
=====================
One hundred twenty-four cases of metastasis to the pancreas diagnosed by biopsy (n = 49, 39.5%) or surgical resection (n = 75, 60.5%) at Asan Medical Center between 2000 and 2017 were reviewed. Only distant metastatic tumors to the pancreas were included; adjacent tumors directly extending to the pancreas were excluded. Patient medical records were retrospectively reviewed to evaluate clinical presentation, treatment, and patient status. Hematoxylin and eosin-stained slides of both primary tumors and pancreatic metastases were reviewed by two pathologists (M.L. and K.J.C.).
Ethics statement
----------------
This study was approved by the appropriate institutional review board (2020-0048), and the informed consent was waived.
RESULTS
=======
The primary tumors of 124 patients with pancreatic metastases included 111 carcinomas (89.5%), 12 sarcomas (9.6%), and one melanoma (0.8%). The metastatic carcinomas (n = 111) were mainly renal cell carcinomas (n = 50, 40.3%), followed by lung cancer (n = 18; eight small-cell carcinomas, five adenocarcinomas, three squamous cell carcinomas, and two sarcomatoid carcinomas), colorectal cancer (n = 12, all adenocarcinomas), gastric cancer (n = 12; six tubular adenocarcinomas, three poorly cohesive carcinomas, two mucinous adenocarcinomas, and one hepatoid adenocarcinoma), ovarian or fallopian tube cancers (n = 7; six serous carcinomas and one clear-cell carcinoma), hepatocellular carcinoma (n = 4), and other cancers (n = 8).
Of the 12 sarcoma patients, nine were female and three were male, with ages ranging from 20--70 years ([Table 1](#t1-jptm-2020-03-04){ref-type="table"}). Among the primary lesions, the most common site was bone (n = 4); followed by brain, lung, soft tissue (n = 2 for each), uterus, and pulmonary vein (n = 1 each) ([Table 1](#t1-jptm-2020-03-04){ref-type="table"}). Histologically, the sarcomas included two World Health Organization (WHO) grade III solitary fibrous tumors/hemangiopericytomas, and one case each of synovial sarcoma, malignant solitary fibrous tumor, undifferentiated pleomorphic sarcoma, osteosarcoma, mesenchymal chondrosarcoma, intimal sarcoma, myxofibrosarcoma, myxoid liposarcoma, rhabdomyosarcoma, subtype uncertain, and high-grade spindle-cell sarcoma of uncertain type ([Table 1](#t1-jptm-2020-03-04){ref-type="table"}). In the case of synovial sarcoma, the presence of an SYT-SSX fusion product was confirmed by reverse transcription polymerase chain reaction.
The primary tumor of case no. 8 arose in the medullary cavity of the metaphysis of the distal femur. The tumor destroyed the cortex and extended into the adjacent soft tissue. Clinically, osteosarcoma was highly suspected, but neoplastic bone formation, which is the essential diagnostic feature of osteosarcoma, was not observed in either primary or metastatic tumor. Thus, we diagnosed it as a high-grade spindle-cell sarcoma of uncertain type.
The median interval between primary sarcoma diagnosis and detection of pancreatic metastasis was 28.5 months. In one exceptional case, a pancreatic metastasis of an osteosarcoma manifested 15 months prior to detection of the osteosarcoma in the femur ([Table 1](#t1-jptm-2020-03-04){ref-type="table"}). This patient first presented with abdominal pain, and abdominal computed tomography revealed a 2.5-cm mass-like lesion in the pancreatic tail. Other radiologic workup, such as imaging of the extremities, was not performed at that time. A 2.7-cm, ovoid, solid mass was identified during distal pancreatectomy ([Fig. 1A](#f1-jptm-2020-03-04){ref-type="fig"}). Microscopy revealed proliferation of pleomorphic spindle cells, numerous giant cells with infiltrative margins ([Fig. 1B](#f1-jptm-2020-03-04){ref-type="fig"}), high mitotic activity, and focal osteoid and bone formation ([Fig. 1C](#f1-jptm-2020-03-04){ref-type="fig"}). Vascular invasion was also identified ([Fig. 1D](#f1-jptm-2020-03-04){ref-type="fig"}). The tumor was diagnosed as a sarcomatoid carcinoma of the pancreas. The patient was hospitalized with left leg pain 15 months after distal pancreatectomy. Magnetic resonance imaging revealed a 9-cm mass in the left femur with fracture and adjacent soft-tissue extension ([Fig. 1E](#f1-jptm-2020-03-04){ref-type="fig"}). A biopsy was performed, revealing histologic features similar to those of the pancreatic mass ([Fig. 1F](#f1-jptm-2020-03-04){ref-type="fig"}). We concluded that the osteosarcoma of the femur had metastasized to the pancreas, and the original diagnosis of sarcomatoid carcinoma of the pancreas was incorrect.
Metastatic tumors were located in the body or tail of the pancreas (n = 8), the head of the pancreas (n = 3), and throughout the entire pancreas (n = 1) ([Table 1](#t1-jptm-2020-03-04){ref-type="table"}). Nine cases were single masses, and three cases showed multiple masses. Pancreatic tumor size ranged from 2.5--19 cm (median, 2.7 cm) ([Table 1](#t1-jptm-2020-03-04){ref-type="table"}). Primary lesions and pancreatic metastatic lesions ([Fig. 2](#f2-jptm-2020-03-04){ref-type="fig"}) showed similar histologic features except in one case in which about 30% of the round-cell components were present in the primary myxoid liposarcoma but not in the metastasis.
Primary tumors from 12 patients had undergone surgical resection: two patients with distant metastasis synchronously received neoadjuvant chemotherapy, six received postoperative or preoperative radiation therapy, and four received adjuvant chemotherapy ([Table 1](#t1-jptm-2020-03-04){ref-type="table"}). The chemotherapy regimens varied, and several combinations of methotrexate, ifosfamide, doxorubicin, cisplatin, and etoposide were used. In five patients, local recurrence of the primary lesions occurred prior to detection of pancreatic metastasis ([Table 1](#t1-jptm-2020-03-04){ref-type="table"}).
After diagnosis of metastatic pancreatic lesions, nine of 12 patients underwent surgical resection with or without preoperative chemotherapy or radiation therapy. Five cases received postoperative radiation therapy or chemotherapy ([Table 1](#t1-jptm-2020-03-04){ref-type="table"}). Lymphnode dissection was performed in eight cases, and none of the cases showed metastasis to the lymph nodes. After detection of pancreatic metastasis, eight patients died between 5--48 months, and four patients remained alive from 4--92 months ([Table 1](#t1-jptm-2020-03-04){ref-type="table"}).
DISCUSSION
==========
Sarcomas preferentially metastasize via the blood rather than the lymphatic system, and lung and bone tissue are the most frequent sites of metastatic sarcoma \[[@b17-jptm-2020-03-04]\]. Lymph node metastasis is uncommon, but some histologic sarcoma types exhibit high metastasis rates via lymphatic drainage, including rhabdomyosarcoma, angiosarcoma, clear-cell sarcoma, epithelioid sarcoma, and myxoid liposarcoma \[[@b18-jptm-2020-03-04],[@b19-jptm-2020-03-04]\]. In our study, the absence of lymph node metastasis in all eight lymph node dissection cases suggests that metastatic sarcoma to the pancreas is more likely to occur via the blood than the lymphatic system. The connective interspaces and cavities such as the peritoneum or pleural space, in which tumor cells can become entrapped, are also possible routes of metastatic dissemination \[[@b18-jptm-2020-03-04]\].
Synovial sarcoma metastasizes in approximately 50% of cases, and common metastatic sites are the lung and bone \[[@b12-jptm-2020-03-04]\]. Unlike other sarcomas, spread to the lymph nodes is not infrequent (3%--23%) \[[@b12-jptm-2020-03-04]\]. For disseminated disease, chemotherapy based on adriamycin and ifosfamide is usually attempted. Makino et al. \[[@b12-jptm-2020-03-04]\] suggested that pancreatic metastasis from synovial sarcoma can be successfully treated by surgical resection in cases of solitary pancreatic lesions with no extra-pancreatic metastasis and a more than 3-year interval between diagnosis of the primary tumor and pancreatic metastasis. Only four cases of metastatic synovial sarcoma to the pancreas have been reported in the literature \[[@b5-jptm-2020-03-04],[@b10-jptm-2020-03-04],[@b12-jptm-2020-03-04],[@b20-jptm-2020-03-04]\], making our case the fifth. The patient reported here was also treated by surgical resection. Our patient and the two patients described by Makino et al. \[[@b12-jptm-2020-03-04]\] are doing well, without recurrence or distant metastasis for 92 months, 40 months, and 30 months after surgical resection, respectively.
Solitary fibrous tumors/hemangiopericytomas of the central nervous system (CNS) show two distinct histologic phenotypes, a solitary fibrous tumor phenotype and a hemangiopericytoma phenotype \[[@b21-jptm-2020-03-04]\]. The hemangiopericytoma phenotype has a high recurrence rate (75%--90%) and a high rate of distant metastasis (20%--33%), especially to the liver, lung, and bone \[[@b14-jptm-2020-03-04],[@b21-jptm-2020-03-04]-[@b24-jptm-2020-03-04]\]. Both our cases showed the hemangiopericytoma phenotype and were grade III. The metastatic sites of these cases were the lung, bone, breast, and pancreas. A total of 18 cases of metastasis from the brain to the pancreas have been reported to date \[[@b14-jptm-2020-03-04],[@b25-jptm-2020-03-04]\]. Interestingly, the interval between diagnosis of the primary tumor and development of pancreas metastasis was long (5.3--24 years, with a median of 10.13 years) \[[@b14-jptm-2020-03-04],[@b25-jptm-2020-03-04]\]. After detection of pancreatic metastasis, eight patients died between 2.6--16 years, and seven patients lived for 3.4--25 years.
Outside the CNS, such as in the soft tissue, pleura, and other visceral sites, malignant solitary fibrous tumors showed increased cellularity and mitotic activity (\> 4 per 10 high-powered fields). Metastatic malignant solitary fibrous tumors to the pancreas have been reported in two cases, one originating in the chest wall \[[@b13-jptm-2020-03-04]\] and one originating in the kidney \[[@b26-jptm-2020-03-04]\]. Our case originated in the lung.
Osteosarcoma has a high potential for metastasis \[[@b27-jptm-2020-03-04]\]. The high incidence of metastasis is related to its strong propensity for early hematogenous spread \[[@b15-jptm-2020-03-04],[@b16-jptm-2020-03-04],[@b28-jptm-2020-03-04]\]. Prolonged survival is possible with treatment including multi-centric chemotherapy and resection of the primary tumor \[[@b3-jptm-2020-03-04],[@b11-jptm-2020-03-04],[@b15-jptm-2020-03-04],[@b28-jptm-2020-03-04]\]. This result is attributed to the delayed appearance of metastasis and increased metastasis incidence at unusual sites \[[@b11-jptm-2020-03-04]\]. The common metastasis sites are the lung, bone, pleura, and liver \[[@b3-jptm-2020-03-04],[@b27-jptm-2020-03-04],[@b29-jptm-2020-03-04]\]. Twelve cases of metastatic pancreatic osteosarcoma have been reported \[[@b3-jptm-2020-03-04],[@b6-jptm-2020-03-04],[@b11-jptm-2020-03-04],[@b15-jptm-2020-03-04],[@b16-jptm-2020-03-04],[@b27-jptm-2020-03-04]-[@b32-jptm-2020-03-04]\], making our case the thirteenth. The median interval between primary osteosarcoma diagnosis and development of pancreas metastasis was three years (range, -1.25 to 11 years), including our case, which was the first reported case with a pancreatic metastasis diagnosis prior to primary osteosarcoma diagnosis. Among the eight patients with available follow-up information, four who had undergone surgical resection of the pancreatic metastasis were alive for 7--13 months. Three patients died two weeks, three months, and 18 months after pancreatic metastasis development, respectively \[[@b3-jptm-2020-03-04],[@b28-jptm-2020-03-04],[@b29-jptm-2020-03-04],[@b32-jptm-2020-03-04]\].
Mesenchymal chondrosarcoma mainly metastasizes to the lungs \[[@b33-jptm-2020-03-04]\]. Metastasis to the pancreas is extremely rare and has only been reported in six cases, making our case the seventh \[[@b1-jptm-2020-03-04],[@b10-jptm-2020-03-04],[@b33-jptm-2020-03-04]-[@b36-jptm-2020-03-04]\]. The primary site was extra-skeletal soft tissue (thigh, brain, buttock, or femoral vein) in five cases and bone (skull) in two cases. Among the four patients with available follow-up information, two were alive 6 years and 10 years after surgery for pancreatic metastasis, and two patients died 2 and 4 years after surgery.
Myxoid liposarcoma generally has a favorable prognosis. The round-cell content is a poor prognostic factor in this disease. The most common metastatic site is the abdomen, including the retroperitoneum, abdominal wall, and abdominal cavity, followed by bone \[[@b37-jptm-2020-03-04],[@b38-jptm-2020-03-04]\]. Until now, metastasis to the pancreas has not been reported. Our case showed metastasis from the thigh to the bone and the pancreas. Microscopy showed round-cell content of approximately 30% in the primary lesion, but not in the metastatic lesion.
Myxofibrosarcoma was first described in 1977 as a myxoid variant of malignant fibrous histiocytoma and was reclassified as myxofibrosarcoma in 2013 by the WHO \[[@b39-jptm-2020-03-04]\]. The most common metastatic site is the lung, followed by the pleura, lymph node, bone, and retroperitoneum \[[@b40-jptm-2020-03-04],[@b41-jptm-2020-03-04]\]. In this report, a case of myxofibrosarcoma in the knee showed metastasis to the pancreas. Only two cases of pancreatic metastasis of a myxofibrosarcoma have been previously reported, although there are some papers that present cases of metastatic myxofibrosarcoma without describing the specific metastatic sites \[[@b40-jptm-2020-03-04]\]. Some studies show that adjuvant or neoadjuvant radiation therapy may improve recurrence and metastasis in this indication.
In this study, we present several rare cases of metastatic sarcomas to the pancreas. This is the largest reported case series of pancreatic metastatic sarcomas to date. Metastatic sarcoma comprised 10% of pancreatic metastases, which is high given the overall sarcoma incidence. Furthermore, pancreatic metastasis may manifest as a primary solitary lesion before detection of the primary tumor. Intensive whole-body imaging screening is necessary in sarcoma patients, and metastasis should remain a differential diagnosis in pancreatic tumors with sarcomatoid features. Recently, the efficacy of surgical resection of pancreatic metastases was shown for sarcoma \[[@b10-jptm-2020-03-04],[@b12-jptm-2020-03-04]-[@b16-jptm-2020-03-04]\] as well as renal cell carcinoma \[[@b4-jptm-2020-03-04]\]. Clinicians caring for patients with sarcoma should be aware of the potential for increased survival following surgical resection of the metastasis. This study will contribute to a better understanding of this unusual clinical circumstance and, thus, may lead to improved diagnostic accuracy and treatment.
**Author Contributions**
Conceptualization: KJC.
Data curation: ML, KJC.
Formal analysis: ML, KJC.
Funding acquisition: KJC, SMH.
Investigation: ML, KJC.
Methodology: ML, KJC.
Project administration: ML.
Resources: JSS, SMH, SJJ, JK, KBS, JHL, KJC.
Supervision: KJC.
Validation: ML, KJC.
Visualization: ML.
Writing---original draft: ML, KJC.
Writing---review & editing: ML, KJC.
**Conflicts of Interest**
The authors declare that they have no potential conflicts of interest.
**Funding**
No funding to declare.
{#f1-jptm-2020-03-04}
{#f2-jptm-2020-03-04}
######
Clinicopathologic characteristics of primary sarcoma and pancreatic metastasis in 12 patients
Case No. Sex/age at pancreatic metastasis (yr) Primary tumor Pathologic diagnosis Interval between primary tumor detection and pancreatic metastasis (mo) Pancreatic metastasis Associated metastases Survival after pancreatic metastasis (mo)
---------- --------------------------------------- --------------------- ---------------------- ------------------------------------------------------------------------- ----------------------- ----------------------------------------------------------------- ------------------------------------------- ------------------ ---------- ----- ------------------ -------- --------------------------------------------------------- ---- ----
1 F/62 Lung 7.5 OP No Synovial sarcoma, FNCLCC grade 3 40 Body Single 2.7 NeoCTx + OP DP No \- 92
2 M/45 Lung 7 OP Yes Malignant solitary fibrous tumor, FNCLCC grade 3 11 Tail Single 2.6 OP + RTx and CTx T Umbilicus, adrenal gland 24 \-
3 F/48 Brain 7.5 OP + RTx Yes Solitary fibrous tumor/ hemangiopericytoma, WHO grade III 64 Body, tail Multiple 3.5 RTx + OP DP Bone (femur, tibia), breast, lung 31 \-
4 F/70 Brain NA OP + RTx Yes Solitary fibrous tumor/ hemangiopericytoma, WHO grade III 135 Head Single 2.6 OP + RTx PPPD Bone (humerus), lung, \- 41
5 F/58 Bone (tibia) 6 NeoCTx + OP + CTx No Undifferentiated pleomorphic sarcoma, FNCLCC grade 3 88 Head Single 3.5 RTx + CTx Biopsy Bone (pelvis) \- 10
6 F/57 Bone (femur) 9 OP + RTx + CTx No Osteosarcoma, FNCLCC grade 3 15 mo prior Tail Single 2.7 OP + CTx DP Skin (scalp), bone (rib, ilium, sternum, pelvis), brain 18 \-
7 F/32 Bone (skull) 4 OP + RTx No Mesenchymal chondrosarcoma, FNCLCC grade 3 65 Tail Single 19 OP DP Lung, mesentery, bone (femur) 48 \-
8 F/28 Bone (femur) 11.7 NeoCTx + OP + CTx No High-grade spindle-cell sarcoma, type uncertain, FNCLCC grade 3 20 Body Single 4 OP DP Bone (pelvis) 11 \-
9 F/35 Pulmonary vein 4.6 RTx + OP Yes Intimal sarcoma, FNCLCC grade 2 14 Tail Single 2.6 CTx Biopsy Retroperitoneal LN, bone (left humerus), lung 29 \-
10 M/52 Soft tissue (knee) 8.2 OP + RTx + CTx No Myxofibrosarcoma, FNCLCC grade 2 26 Head Single 2.5 OP PPPD No \- 4
11 M/35 Soft tissue (thigh) 22 OP No Myxoid liposarcoma, FNCLCC grade 2 0 Tail Multiple 11 OP + CTx DP Bone (humerus) 16 \-
12 F/60 Uterus 8.5 OP Yes (vaginal stump) Rhabdomyosarcoma, subtype uncertain, FNCLCC grade 3 8 Head, body, tail Multiple NA CTx Biopsy No 5 \-
F, female; OP, operation; FNCLCC, Fédération Nationale des Centres de Lutte Contre le Cancer; NeoCTx, neoadjuvant chemotherapy; DP, distal pancreatectomy; M, male; RTx, radiation therapy; CTx, adjuvant chemotherapy; T, tumorectomy; WHO, World Health Organization; NA, not available; PPPD, pylorus-preserving pancreaticoduodenectomy; LN, lymph node.
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Dolly, Polly, and friends proved that somatic cells are potentially totipotent, but the reprogramming that a somatic cell nucleus must undergo during cloning remains an error-prone black box. James Byrne, John Gurdon, and colleagues (University of Cambridge, UK) have now shown that the biochemically tractable frog oocyte system can be used to model reprogramming. A modified version of their protocol might allow the isolation of elusive reprogramming factors and, eventually, the reprogramming of somatic human cells for self-transplantation of stem cells.
FigureA frog oocyte germinal vesicle (center) can reprogram injected nuclei (white).Gurdon/Elsevier
The Cambridge group chose frog oocytes because, unlike most eggs, oocytes are not at all active in replication but very strongly so in transcription. To see if this transcriptional activity extended to reprogramming, Gurdon microinjected the oocytes with various cells: first mouse fetal fibroblasts, then mouse adult thymic cells, and finally human lymphocytes. All cell types eventually showed robust expression of *oct4*, whose expression is specific to and preserves the fate of stem cells.
Transcriptional activity not associated with stem cells, such as that of *β-actin* and the thymus marker *thy-1*, was reduced or extinguished by the transfer. But the extent of the transformation is not yet clear. "It\'s possible that what we are doing is turning everything into an oocyte," says Gurdon. But he believes that *oct4* expression is a good sign that the cells are at least headed toward becoming stem cells.
Stem cell characteristics may develop only through sequential inductive events. But the optimists are hoping that there is a single extract that will do the entire conversion. The success of the current experiments, says Gurdon, is "one of the more compelling reasons for believing that to be true." ▪
Reference:
Byrne, J.A., et al. 2003. Curr. Biol. 13:1206--1213. 12867031
[^1]: <[email protected]>
| {
"pile_set_name": "PubMed Central"
} |
Introduction
============
The articles in this Special Topic cover a range of issues concerning long-distance projecting cortical GABAergic neurons, in the context of interneuron diversity. As several authors report, these neurons are attracting renewed attention spurred by new techniques and markers which show great potential for deciphering their role in cortical organization and microcircuitry. Other authors have emphasized developmental origins of particular subpopulations and their roles in early cortical circuitry. Notable recurring themes are species-specific features and probable implications for normal and pathological cortical functioning. A corollary theme, evident in many of these articles, concerns nomenclature. Several terms are almost interchangeably used, but nevertheless distinct; that is: subplate, layer 7, layer VIB, pioneer and interstitial neuron (see comments to follow Clancy et al., below, among others). In this article the main conclusions, and some of what the host editors (Kathleen Rockland and Javier DeFelipe) consider the most interesting remarks, have been extracted from each of the individual articles. These commentaries are not necessarily directly derived from the original work of the authors, and may be the result of the collective work of several different laboratories. This is followed by a section dedicated to more general comments and a discussion of the issues raised. The authors who have participated in this article are listed in alphabetical order.
A commentary on
Cross-species analyses of the cortical GABAergic and subplate neural populations
by Clancy, B., Teague-Ross, T. J., and Nagarajan, R. (2009). Front. Neuroanat. 3:20. doi:10.3389/neuro.05.020.2009.
Remarks and Main Conclusions
============================
1. Cortical GABAergic neurons can be parceled into a number of subgroups based on variations in morphology, birthplace, mature locations, colocalized peptides, and electrophysiological parameters. Despite such diversity, conventional models of cortical function include GABAergic neurons as participators only in local connectivity, such that the designation of "interneuron" is often used interchangeably with GABAergic to describe cortical neurons that play an inhibitory role. However, cortical GABAergic categories recently were extended to include a subset of phylogenetically conserved neurons that project axons across long distances -- the newly identified long-range interneurons, perhaps more precisely called cortical GABAergic projection neurons.
2. One intriguing aspect of the latest subgroup is that the majority of the long-range GABAergic projections extend from neurons located in cortical layer I, cortical white matter, and the subgriseal region of the cortex (subjacent to cortical layer VI). This prompted the suggestion that the GABAergic projection neurons might be a subset of the little-studied cells that persist in the adult brain from the developmental subplate.
3. Early in development, the cells of the future layer I, as well as the future subplate/white matter neurons, are merged as the preplate before neurons of the developing cortical plate split the preplate into a superficial region (later called layer 1) and a subgriseal region (later called the subplate). The exact percentage of subplate cells that survive into adulthood is somewhat difficult to identify, but it is well-documented that some survive an early wave of cell death to remain in the mature white matter in human and non-human primate cortex and in carnivores.
4. GABAergic neurons account for 15--25% of all the neurons in the region depicted as the persisting subplate, similar to their percentage in the cortex overall.
5. Similar to the sometimes confusing GABAergic nomenclature recently addressed by the Pettilla committee, surviving subplate cells have been assigned a variety of different names in mature cortex, including border neurons, white matter neurons, subgriseal neurons, layer VII, layer VIb, deep layer VI, upper subplate neurons, and the deep cortical band. In this study we will use the term "persisting subplate neurons" for those resilient cells that remain from the developmental subplate in and above the white matter across maturation, where they continue to participate in cortical function, and apparently include the GABAergic subset that sends projections for long distances.
6. Both GABAergic and subplate populations include numerous and diverse morphological subsets that are different from the more prevalent cortical pyramidal neurons (although each population may include cells with pyramidal morphology), and both populations contain a subset whose projections may travel long distances, sometimes crossing areal boundaries, as well as a subset that focuses projections on cortical layer I. Both populations are similarly heterogeneous in their electrophysiological properties and in the numerous signaling chemicals they sequester. Moreover, subsets may share a common non-cortical birthplace in the ganglionic eminences, raising the possibility that some may descend from similar sets of precursors. Supporting this notion, both populations use somewhat similar molecular modes of migration, different from the mechanism used by pyramidal cells.
7. Although the contribution of the GABAergic interneurons to cortical function is undisputed, and the critical role of the subplate in cortical development is well-accepted, conventional models of mature cognitive function do not yet incorporate contributions of either the projection GABAergic or the persisting subplate neurons. When numbers are reduced compared to other neural populations, there may be a tendency to simply dismiss those that persist as "sparse," "remnants," or "relics". Unfortunately, such terminology implies a fairly unessential function, and it seems important to avoid such categorization until additional information on their function is available.
8. The mathematical principles underlying small-world networks suggest that sparse connectivity is a plausible design underlying important cognitive function. Long-range inhibition, even from relatively sparse connections, can be a potent network component. In small-world networks, clusters of cells link to their nearest neighbors, while some connect to distant clusters. This pattern can serve as the basis for a surprisingly strong communication network, especially when it is amplified by local input, as is likely the case for both the long-range GABAergic and the persisting subplate populations.
9. In a series of previous studies, mathematical models have been used to successfully identify both similarities and relative differences in the timing of neural "events" when comparing primate and nonprimate development. For the purpose of this review, "neural events" are defined as milestones pertaining to neural development such as the post conception (PC) date when neurons destined for the various cortical layers are generated. Mathematical approaches are valid because despite species differences, including differences in the duration of development, the size of most brain regions scales similarly across species. Central to this meta analysis, the timing of events that occur in most neural regions is remarkably conserved.
10. The most pragmatic application of statistical modeling is that neural events empirically derived in one species can be compared and successfully applied to another. Therefore, given the potentially important contributions of the long distance GABAergic and persisting subplate populations, we reasoned any additional information about these two populations, including comparative cross-species data, is likely to be useful. At this time, no empirical developmental data are yet available specifically for the cortical GABAergic projection neurons. However developmental data are available for both general populations that include them, the GABAergic and subplate populations. We assembled a database of GABAergic and subplate developmental events (e.g. the PC day subplate neurogenesis begins, the day GABAergic cells are first found in the subplate). We then applied cross-species statistical modeling, and tested if species similarities and differences might be indicated by statistical analysis of the developmental sequences.
11. Similarities between the two rodent populations (rat and mouse) are striking, permitting us to identify developmental dates for GABAergic and subplate neural events in rats that were previously identified only in mice, as well as the timing in mouse development for events previously identified in rats. Primate comparative data are also compelling, although slight variability in statistical error measurement indicates differences in primate GABAergic and subplate events when compared to rodents. Although human extrapolations are challenging because fewer empirical data points are available, and because human data display more variability, we also produce estimates of dates for GABAergic and subplate neural events that have not yet been, or cannot be, determined empirically in humans.
12. The pragmatic value of the cross species translations -- in direct savings of time and resources when intervals for studies might be narrowed -- is a compelling reason to add this type of analysis to the growing array of modern neuroanatomical tools. In some case, it might eliminate the necessity of repeating a study already accomplished in one species (as our data suggest is possible when translating from mice to rats, or rats to mice), or at the least contribute to a narrowing of time intervals (as our data suggest is possible for humans). And although correlations between data available in the empirical literature and data produced by the model were already significant, we expect future predictions will become more accurate as additional data points are added to our database.
13. On the other hand, we certainly do not suggest that there are no differences in the brain development of diverse mammalian species. Mathematical modeling can successfully adjust for some rodent/primate differences, such that comparisons and predictions are not overly distorted by differences in brain sizes, or what might seem to be a relatively prolonged time window for neurogenesis in primates when compared to rats. Yet there are questions that analysis of our database does not yet permit us to address, such as the possibility of an effect on differences in cell cycle mechanisms, or the effect of variability in the location of GABAergic proliferation when comparing rodents to humans.
14. However, it is clear that the timing of neurogenesis alone is an important factor in development, given the evidence that timing might predict such properties as laminar position, electrophysiology responses, and neuronal morphology, including projection patterns. As datapoints are added to our database, we hope to test if a neuron\'s role is more closely related to the location of its birthplace, or the timing of its birth date. One additional limitation we should point out arises because early postnatal days in rodent brain development correspond to *in utero* timing in humans. The result is that the effects of a perinatal wave of synaptogenesis, the onslaught of experience surrounding birth, and the mother/offspring interaction are not included, as we have not yet identified data points associated with these events that fit into statistical models.
General Comments and Discussion
===============================
Espinosa and fairén
-------------------
Comment on point 2.
\(a\) It would be of interest to confirm by birth-dating the developmental association of GABA projection neurons with the preplate and its derivatives, marginal zone and subplate.
\(b\) What is the proportion of GABA projecting neurons among the total GABA neurons persisting from the subplate? Re: comments by DeFelipe and Rockland to Higo et al. (below).
Rockland
--------
Comment on point 3.
If, as seems increasingly possible, subpopulations can be reliably identified by distinctive markers, a next step can be rigorous quantification across species, areas, and developmental epochs. In this regard, the relatively small size of the long-distance projecting GABAergic population in particular is a further advantage.
Clancy
------
Reply to Rockland point 3.
This seems a wonderful next step (or rather several wonderful next steps)!
Kanold
------
The identification of subpopulations is the necessary next step. But the comparison of different species here is almost even more important as well.
Rockland
--------
Comment on point 5.
re Nomenclature, see DeFelipe comment, following Kanold article.
Clancy
------
Reply to Rockland point 5.
There would be value in establishing consistent terminology for persisting subplate cells. Those that remain from development in the white matter fibers have had some nomenclature stability after Cajal named them white matter interstitial cells in 1911 (although often abbreviated as interstitial neurons, or white matter neurons); prior to this they also had been called subcortical cells, extracortical (non plate) cells, and outlying cells (Okhotin and Kalinichenko, [@B28]). Assigning a distinct appropriate name for the persisting subplate cells that remain in a more superficial position has proved more difficult. As noted, they are referred to as layer VII (Reep, [@B30]), the deep cortical band (Kristt, [@B22]), upper subplate neurons (Marin-Padilla and Marin-Padilla, [@B23]), deep layer VI (McDonald and Burkhalter, [@B24]), layer VIb (Gomez-Pinilla and Cotman, [@B13]), subgriseal neurons (Clancy and Cauller, [@B8]), and border neurons (Hogan and Berman, [@B16]).
Espinosa and fairén
-------------------
Comment on points 5--8.
re Nomenclature. Although the group of "persisting subplate neurons" includes both glutamatergic and long-range GABAergic neurons, we would suggest some problems of definition in point 8: "...both the long-range GABAergic and the persisting subplate populations".
Clancy
------
Reply to Espinosa and Fairén.
In addition to the nomenclature problems, it seems persisting subplate cells are sometimes dismissed as simply a remnant population, likely because many die across development, or because they were once considered a rodent specialization (Reep and Goodwin, [@B31]). Yet because so many theories of human cognition are based on studies accomplished in rodents, more information on this subset of cells would be valuable even if they were specific to rodents. However, the "rodent specialization" assignment has evolved (Reep, [@B30]), and it seems unlikely that persisting subplate cells are mere remnants in adult cortex, such that their connectivity serves no definable purpose in mature cognitive function. For a subset of neural cells to persist as a remnant population would be an expensive metabolic strategy, so it is more likely that even if they began as remnants, they would eventually have been incorporated into some functional role. (It may also be worth noting that large numbers of cells in the spinal cord also die prior to maturity, but those that remain are not considered vestigial.)
Kanold
------
Reply to Clancy.
As far as "rodent specialization" goes, it would be very useful if one could compare the subtypes of subplate neurons (surviving or not), their associated circuits, and survival between rodents and at least one carnivore species (ferret, cat, etc).
I agree that remaining subplate neurons probably play some functional role, although it could be a very different role from their role in early development.
Luhmann
-------
Comment on point 8.
It has been recently demonstrated in hippocampal slices from immature rodents that a subpopulation of GABAergic interneurons with long axonal arborizations play a central role in synchronizing spontaneous network activity ("hub" neurons) (Bonifazi et al., [@B2]). These cells were preferentially located in the stratum oriens and lucidum at the two borders with the pyramidal cell layer. Two different morphological types of hub neurons were identified following intracellular labelling and reconstruction: (i) cells displaying a long axon spanning regions with sparse collaterals, and (ii) basketlike neurons with a dense but more local arborization pattern. Subplate cells may serve as hub neurons in the developing cerebral cortex (Voigt et al., [@B39]; Hanganu et al., [@B15]).
A commentary on
Subtypes of GABAergic neurons project axons in the neocortex
by Higo, S., Akashi, K., Sakimura, K., and Tamamaki, N. (2009). Front. Neuroanat. 3:25. doi:10.3389/neuro.05.025.2009.
Remarks and Main Conclusions
============================
1. GABAergic neurons with axons projecting in the ipsilateral hemisphere seem to have similar chemical features and often exhibit somatostatin (SS)-immunoreactivity (IR) (91%), neuropeptide Y (NPY)-IR (82%), and neuronal nitric oxide synthase (nNOS)-IR (71%). Considering these observations, the fact that most nNOS-positive neurons are a subpopulation of SS- and NPY-IR neurons, and nNOS-, NPY-, and SS-triple-positive cells are less than 0.5% of GABAergic neurons, it was speculated that the nNOS-positive GABAergic projection neurons compose a very small subpopulation in the neocortical GABAergic neurons.
2. Nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d)-positive somata in the white matter originate thick NADPH-d-positive fibers in the white matter of the cat neocortex. These emanate with other non-labeled arcuate fibers, without originating any thick branches, and without bending toward the gray matter of the neocortex. These features may indicate that the total length of NADPH-d-positive fibers in the white matter is long enough to be called as projection fibers. We regarded GABAergic neuron extending an axon longer than 1.5 mm from the soma as the GABAergic projection neuron in the mouse.
3. NADPH-d reaction labeled axon fibers of nNOS-positive GABAergic neurons in cats and mice, while GFP-immunohistochemistry in GAD67-Cre/GFP Cre-reporter mouse labeled both projection fibers originating in nNOS-positive GABAergic neurons and those in the other subtypes of GABAergic neurons. However, the distribution pattern of the labeled axon fragments was similar in both preparations. NADPH-d reactive axons and GFP-IR axons were found to be very sparse in the corpus callosum, sparse in the anterior commissure, and more numerous in the fimbria. Labeled fibers in the fimbria will include afferent- and efferent-fibers from and to the subcortical nuclei, in addition to the commissural fibers through the ventral hippocampal commissure. Excluding subcortical afferent and efferent fibers, we speculate that the GABAergic commissural fibers reciprocally interconnect archi- and paleo-cortex.
4. One of the important results elucidated in this study is that a large number of GABAergic neurons are also involved in the cortico-fugal, cortico-cortical, and callosal projections. Each subtype of GABAergic projection neuron may contribute to information processing in the neocortex in different ways.
General Comments and Discussion
===============================
DeFelipe
--------
Comment on point 1.
Are parvalbumin expressing neurons not part of the subpopulation of GABA projecting cells?
Rockland
--------
Reply to DeFelipe.
Tomioka et al. ([@B34]) (mouse) and Tomioka and Rockland ([@B35]) (primate) also reported an absence of PV+ neurons. However, see Jinno, below, about PV+ GABAergic projection neurons in the hippocampus.
Tamamaki
--------
Reply to DeFelipe.
While parvalbumin expressing GABAergic neurons in the septum project axons to the neocortex, we did not find any of those in the neocortex project axons cortico-cortically. Less than 0.5% of GABAergic neurons (nNOS, NYP, and somatostatin-expressing GABAergic neurons) seemed to project axons cortico-cortically. However, we also agree that there will be other subtypes of GABAergic neurons projecting cortico-cortically in the neocortex. They would be generated in embryonic stage and maintained in adult rodent, feline, primate, and maybe, human brain. They may be found in very specific areas such as boundary between area 17 and 18. Although they may be very weak in GAD67 promoter activity and we could not describe them well in GAD67-GFP and GAD67-Cre/GFP-reporter mice, we would like to continue our observation of GABAergic projection neurons in the GAD67-GFP and GAD67-Cre knock-in mice.
DeFelipe
--------
In Figures 2A,B of Higo et al. (2009), GFP-positive axons in the fimbria and anterior commissure of the GAD67-Cre/GFP Cre-reporter mouse brain have numerous varicosities. I am wondering if they establish synapses. Is there any information regarding this issue?
DeFelipe
--------
Comment on points 1 and 4.
There is an apparent discrepancy between the claim of Higo and colleagues that a large number of GABAergic neurons are involved in the cortico-fugal, cortico-cortical and callosal projections (point 4), since it was estimated that the proportion of GABAergic neurons that project over long distances represent less than 0.5% (point 1). Thus, I am wondering whether 0.5% is an underestimation.
Rockland
--------
Continuing DeFelipe.
Peters et al. ([@B29]) demonstrated transcallosally projecting non-pyramidal neurons in the supragranular cortical layers of the area 17/18 border region in the cat. These were estimated as 10--32% of the total non-pyramidal cell population.
DeFelipe
--------
Is the proportion of GABA projecting neurons similar in mouse, rat, cat and monkeys? Are these neurons more common in juvenile than in adult animals?
Espinosa and fairén
-------------------
It seems that not all projection GABAergic neurons are associated to subplate or marginal zones? In neocortex there are exceptions (see Rockland comment on A. Peters). GABA projection neurons in the hippocampus, however, may be situated in every layer including for instance the stratum radiatum (see Jinno paper, this issue) and might not be related to the hippocampal preplate. Nevertheless, in general, hippocampal GABA neurons are among the earliest generated (Soriano et al., [@B32], [@B33]).
A commentary on
Structural organization of long-range GABAergic projection system of the hippocampus
by Jinno, S. (2009). Front. Neuroanat. 3:13. doi:10.3389/neuro.05.013.2009.
Remarks and Main Conclusions
============================
1. Using anatomical, molecular and electrophysiological approaches, several types of GABAergic projection neurons have been shown to exist in the hippocampus. The target areas of these cells are the subiculum and other retrohippocampal areas, the medial septum and the contralateral dentate gyrus. The long range GABAergic projection system of the hippocampus may serve to coordinate precisely the multiple activity patterns of widespread cortical cell assemblies in different brain states and among multiple functionally related areas.
2. The most studied remote target of the hippocampal GABAergic projection neurons is the medial septum. Using intraseptal injection of horseradish peroxidase as the retrograde tracer, it was found that a subset of GABAergic neurons of the hippocampus innervate the medial septum (H-MS cells) in the rat brain. The H-MS cells were found in all regions of the hippocampus, but they were distributed in a layer specific manner: in the CA1 region, they were mainly located in the stratum oriens; in the CA3 region, they were scattered throughout all the layers; and in the dentate gyrus (DG), they were exclusively located in the hilar area. The morphological characteristics of H-MS cells were examined using fixed slice preparations of the rat hippocampus, and the following cells were identified: stellate cells in the hilus, horizontal basket cells in the stratum oriens of CA1 and CA3, stellate cells in the stratum radiatum of CA3 and pyramid-like cells in the stratum radiatum of CA1. The main postsynaptic targets of H-MS cells in the medial septum were parvalbumin (PV)- expressing GABAergic neurons and, to a lesser extent, cholinergic neurons.
3. Several studies, employing different methods, have demonstrated conflicting results with regard to the local targets of H-MS cells. In juvenile rats, the local axons of CA1 H-MS cells recorded *in vitro* were reported to innervate predominantly hippocampal GABAergic neurons. By contrast, the main local targets of *in vivo* recorded or retrogradely labeled GABAergic cells projecting to the medial septum and subiculum were pyramidal neurons in the CA1 area of the adult rats. Although it is difficult to explain the discrepancy, differences in ages of animals and labeling methods might be related to the inconsistent results in local postsynaptic targets of H-MS cells.
4. The hippocampus also receives GABAergic inputs from the medial septum, and thus the medial septum and the hippocampus are connected reciprocally. The two major components of the septo-hippocampal projection are cholinergic and GABAergic neurons. The cholinergic projection terminates on all types of hippocampal cells, whereas septal GABAergic neurons specifically innervate hippocampal GABAergic neurons. Recently, Takács and coworkers demonstrated direct reciprocity by using combined retrograde and anterograde tracing; that is, H-MS cells are the postsynaptic targets of GABAergic septo-hippocampal axons. On the other hand, GABAergic terminals of H-MS cells in the medial septum were shown to innervate septo-hippocampal neurons retrogradely labeled from the ventral hippocampus. This reciprocal loop between the hippocampus and the medial septum via GABAergic neurons is considered to play a critical role for generating the rhythmic activity and synchronization.
5. Recent studies have demonstrated the existence of hippocampal GABAergic cells projecting to the subiculum (H-Sub cells). Using retrograde labeling, the distributions of H-Sub cells were estimated in the rat brain. Differently from the H-MS cells, the retrogradely labeled H-Sub cells were mainly found in the CA1 region, and only a few cells were detected in the CA3 region and the dentate hilus. In the CA1 region, H-Sub cells were scattered throughout all the layers. The majority of H-Sub cells in the stratum oriens were large-sized horizontal cells, while those in the strata radiatum and lacunosum-moleculare were small to medium-sized bipolar and multipolar cells. The postsynaptic targets of long-range axons in the subiculum were assumed to be pyramidal neurons, although the ratios were not determined due to technical limitations. The major local targets of seven H-Sub cells were the dendritic shafts of pyramidal neurons. But, one H-Sub cell expressing enkephalin (ENK) innervated dendritic shafts of GABAergic and pyramidal neurons. The targets of ENK-expressing H-Sub cells were associated with the location of axonal arbors, i.e., interneurons were the main targets in the alveus, both interneurons and pyramidal cell dendrites were innervated in the other layers, and interneurons were the exclusive targets in the subiculum.
6. It should be noted that four *in vivo* recorded cells projected from the CA1 stratum oriens to both the subiculum and the medial septum. Thus, there might be three groups of projection neurons in the CA1 region: those sending axons exclusively to the medial septum, those innervating both the medial septum and the subicular area, and those exclusively sending axons to the subicular/retrosplenial cortex. Although the cells exclusively projecting to the medial septum have not yet been identified, their existence has been suggested by the numerical data showing that the numbers of retrogradely labeled cells after injection into the medial septum are much larger than those after injection into the subiculum.
7. A few GABAergic neurons in the CA1 region are identified by injections of retrograde tracers into the retrosplenial cortex. The majority (about 65%) of hippocampal GABAergic neurons projecting to the granular retrosplenial cortex (H-Rsp cells) were detected at the border between strata radiatum and lacunosum-moleculare of the CA1 region, and a smaller population was located in the stratum radiatum. Many fewer cells (\<10%) were found in the stratum oriens or stratum pyramidale of the CA1 region. In the CA3 and DG, virtually no cells were retrogradely labeled after the injection of tracer into the granular retrosplenial cortex.
8. A small number of GABAergic neurons in the hilus of the DG have axonal projections to the contralateral DG through the hippocampal commissure. Although the targets of these neurons are not strictly extrahippocampal, hilar GABAergic neurons with commissural projection should logically be included as long-range GABAergic projection neurons. The postsynaptic targets of GABAergic commissural projections are thought to be dendrites of granule cells. It has not been clearly proven whether the hilar cells innervating the medial septum simultaneously send commissural axons to the contralateral DG.
9. Several species differences have been reported in the functional organization of the hippocampus. Most notably, glutamatergic hilar mossy cells showed neurochemical discrepancies between mice and rats. The calcium-binding protein calretinin is expressed in mossy cells in the mouse ventral hilus, but not in the rat hilus. On the contrary, calcitonin gene-related peptide is localized in the rat mossy cells, but not in the mouse mossy cells. However, interestingly enough, the morphofunctional similarities in hippocampal GABAergic neurons have been repeatedly reported in mice and rats. The numerical densities of chemically defined subpopulations of GABAergic neurons in the mouse hippocampus were comparable to those in the rat hippocampus. Taken together, it is possible to hypothesize that the chemical characteristics of GABAergic projection neurons in the rat and mouse hippocampus are rather similar to each other.
10. Somatostatin (SOM) is one of the key molecules of H-MS cells. The vast majority of the H-MS cells express SOM in the mouse hippocampus. In contrast, one half of SOM-positive neuron in the CA3 region and DG, and one fourth in the CA1 region projected to the medial septum. Similar results were obtained from the rat hippocampus. In the CA1 region, one half of H-Sub cells were SOM-positive, while no H-Sub cells were immunoreactive for SOM in the strata radiatum and lacunosum-moleculare. In the CA3 strata radiatum/lacunosum-moleculare and the dentate hilus, SOM was detected in less than half and one-fourths of H-Sub cells, respectively. In the rat CA1 region, SOM was not detected in H-Rsp cells. On the other hand, the vast majority of SOM-positive cells project to the contralateral hippocampus via the commissural pathway in the rat dentate hilus.
11. In the Ammon\'s horn, less than half of H-MS cells were positive for neuropeptide Y (NPY), whereas virtually all H-MS cells in the DG contained NPY. The expression ratios of NPY in H-Sub cells were generally similar to those in H-MS cells.
12. Another key molecule of H-MS cells is calbindin D28K (CB). It has been reported that the majority of H-MS cells are CB-positive in the rat hippocampus. However, immunofluorescent multiple labeling showed that less than half of retrogradely identified H-MS cells were CB-positive in the mouse hippocampus. Approximately half of H-MS cells in the Ammon\'s horn expressed CB, while none of them were positive for CB in the DG. Similar results were shown in the rat hippocampus. Expression ratios of CB in retrogradely labeled H-Sub cells were generally lower than those in H-MS cells.
13. Parvalbumin (PV) is expressed in a small population of H-MS cells and H-Sub cells in the rat hippocampus. On the other hand, no H-Rsp cells contained PV. In the DG, the majority of PV-positive neurons commissurally project to the contralateral hippocampus.
14. It is well known that calretinin (CR) is expressed in one of the populations of GABAergic neurons called interneuron-specific (IS) cells, i.e., those exclusively innervating other GABAergic neurons. In addition, CR is also expressed in approximately one-fourths of H-MS cells of the rat hippocampus. But, neither H-Sub cells nor H-Rsp cells were positive for CR. In the DG, one third of CR-positive GABAergic neurons projected commissurally.
15. Muscarinic Acetylcholine (ACh) receptor type 2 (M2R) is commonly expressed in a considerable population (approximately 40%) of H-MS cells and H-Sub cells in the rat hippocampus. In contrast, a smaller subset (15%) of H-Rsp cells was positive for M2R.
16. In the Ammon\'s horn, the majority of H-MS cells expressed mGluR1α (69% in the CA1, and 84% in the CA3 region), whereas only 15% of H-MS were mGluR1α-positive in the DG. H-Sub cells also expressed mGluR1α, but the expression ratios were lower (40% in the CA1 region) than those of H-MS cells.
17. In the CA1 area, enkephalin (ENK) has been detected in a population of IS cells. In addition, a recent retrograde labeling study showed that ENK was expressed in approximately 10% of H-Sub cells. The ENK-positive H-Sub cells were located in the stratum radiatum. All tested ENK-positive H-Sub cells were co-labeled for the orphan nuclear receptor chicken ovalbumin upstream promoter-transcription factor II, but none for CR.
18. Interestingly, different types of cells innervating specific extrahippocampal targets fire with distinct spike timing during network oscillation. The diversity of GABAergic projection cell classes in the hippocampus may result from the need to coordinate precisely the multiple activities of distributed neural circuits in different brain states and among multiple functionally related brain areas.
19. There is a growing body of evidence suggesting that the spatially and temporally organized synaptic inhibition mediated by specific subclasses of GABAergic neurons is impaired in mental illness. Along these lines, it has also been postulated that cognitive and affective impairments in psychiatric disorders may be related to a failure to integrate the activity of widely spread neural circuits. Interestingly, recent imaging data suggest that functional connectivity between remote regions is impaired in individuals with mental illness, such as schizophrenia. Because GABAergic neurons are considered to play a critical role in the long-range fast synchronization of neural activities across brain regions, these findings suggest that defects of long-range GABAergic projection system might be associated with the neurobiology of psychiatric disorders.
General Comments and Discussion
===============================
DeFelipe
--------
Comment on point 12.
Regarding the expression of CB in H-MS, there seems to be some discrepancy between the studies of Tóth and Freund ([@B37]), Jinno and Kosaka ([@B18]) and Jinno et al. ([@B17]) (the majority versus half or less than half). Thus, it is not clear to me whether CB is also a key molecule in H-MS cells as the authors claim.
Jinno
-----
Reply to DeFelipe point 12.
At present, it is difficult explain the reason of the discrepancy between the studies of Tóth and Freund ([@B37]), Jinno and Kosaka ([@B18]) and Jinno et al. ([@B17]). It might be attributed to specificity differences in antibodies against CB. Even so, the expression ratios of CB in GABAergic projection cells are generally high, and I believe CB is one of the key molecules of projection neurons.
Rockland
--------
Comment on PV+ subpopulation.
As I comment above (Higo et al.), the occurrence of PV-positive GABAergic projecting neurons in the hippocampal regions seems to be a specialization not reported so far for cortico-cortical GABAergic projections. However, Jinno and Kosaka ([@B19]) describe PV-positive GABAergic corticostrial neurons in mouse. These were demonstrated from both retrosplenial and somatosensory cortices, and estimated to comprise 5% and 3%, respectively, of the total inhibitory neuron population.
DeFelipe
--------
Comment on point 14.
CR is expressed in IS cells and while approximately one quarter of H-MS cells are also CR-positive, the vast majority of the H-MS cells express SOM (point 10). Thus, it follows that a subpopulation of H-MS represent IS cells that express both CR and SOM. Is this correct?
Jinno
-----
Reply to DeFelipe point 14.
Yes. A subpopulation of H-MS cells represent IS cells, and they are considered to express both CR and SOM (please see Gulyas et al., [@B14]).
A commentary on
Subplate neurons: crucial regulators of cortical development and plasticity
by Kanold, P. (2009). Front. Neuroanat. 3:16. doi:10.3389/neuro.05.016.2009.
Remarks and Main Conclusions
============================
1. Subplate neurons are among the earliest generated neurons in the cerebral cortex of mammals and are located in the developing white matter of all cortical regions. In humans subplate neurons comprise up to 50% of the cortical neurons in the second trimester and are present in the first few years of life (depending on cortical area). In rodents some subplate neurons can remain into adulthood forming layer 6b. Subplate neurons thus comprise additional cortical circuits that are only present during cortical development, and these circuits appear to play a major role in development and early cortical function, but are only beginning to be characterized.
2. Subplate neurons receive glutamatergic input from the thalamus before these thalamic axons grow to their targets in layer 4. Subplate axons mainly project to cortical layer 4, thus there is a time period when subplate neurons are in a key position to relay thalamic input to layer 4. After thalamic axons grow into layer 4, thalamocortical synapses and GABAergic circuits in layer 4 undergo refinement and maturation and over this time are particularly influenced by sensory experience (defining the "critical period"). During this time subplate neurons are still present, receive direct thalamic input, and project to layer 4. The majority of subplate neurons are gradually eliminated postnatally by programmed cell death and remaining neurons are retained as interstitial neurons.
3. Subplate neurons can be selectively ablated in early development by excitotoxic kainic acid injections. Ablation of subplate neurons before thalamic axons invade layer 4 causes these axons to bypass the ablated area and grow into layer 4 at areas that contain subplate neurons. Thus subplate neurons seem to provide a guidance role in targeting thalamic axons to layer 4. Since subplate neurons project radially to layer 4 they might provide a scaffold that enables thalamic axons, which travel tangentially below their eventual target layer, to find their targets.
4. After thalamic axons grow into layer 4 they make synaptic connections with layer 4 neurons and build up these connections over time to adult strength. The strengthening of the thalamocortical synapses from an initially weak state occurs while there is already strong input from subplate neurons and possibly intracortical connections. Recent experiments indicate that subplate neurons play a major role in the developmental strengthening of thalamocortical projections. Subplate ablation after thalamic afferents have grown into layer 4 but before these afferents have made a strong synapse with layer 4 neurons prevents the strengthening of thalamocortical connections. In addition, the frequency -- but not amplitude -- of spontaneous excitatory synaptic events in layer 4 cells is increased, which is consistent with a lack of functional refinement of cortical connections. Together these data indicated that without subplate neurons, there is a failure of appropriate synapses to strengthen and others to weaken, and the visual cortex becomes functionally decoupled from its thalamic inputs.
5. The maturation of intracortical inhibition is central to normal cortical function. In addition GABAergic activity is thought to be involved in the maturation of glutamatergic circuits. Despite this importance of inhibition, the cells and circuits that control inhibitory development are unknown. Key processes of inhibitory maturation occur postsynaptically by changes in the subunit composition of the GABA~A~ receptor and the intracellular Cl^−^-concentration (which affects the ion flow through the GABA~A~ receptor). The Cl^−^-reversal potential (E~Cl~) controls if GABA~A~-ergic activity is depolarizing or hyperpolarizing. E~Cl~ is mediated by Cl^−^ transporters such as KCC2 and NKCC1 that control Cl^−^ levels in the cytosol. KCC2 levels are low (E~Cl~ high) in early development, thus GABA can be depolarizing. Depending on the amount of depolarization, depolarizing GABA can be excitatory or have a shunting inhibitory influence. Over development, KCC2 levels increase (decreasing E~Cl~), rendering GABA inhibitory. The strengthening of both excitatory and inhibitory circuits while maintaining "appropriate" activity levels might be achieved by wiring up GABAergic circuits first and then utilizing depolarizing GABA to aid in maturing glutamatergic connections.
6. The maturation of inhibition depends on normal sensory experience. Sensory deprivations (i.e. dark rearing, deafness, whisker trimming) prevent inhibitory maturation and high expression levels of BNDF, which is involved in inhibitory maturation. Because subplate neurons form a crucial relay of sensory information, and because subplate neurons provide excitation to developing circuits, subplate neurons are in a key position to regulate the maturation of cortical GABAergic inhibition. In particular since subplate neurons are driven by thalamic afferents, strong synaptic inputs between subplate neurons and cortical neurons might amplify the action of sensory inputs.
7. Removal of subplate neurons at early ages, when inhibition is immature, prevents both the developmental increase in KCC2 expression and the expression of a mature complement of GABA~A~ receptor subunits. Consistent with these molecular abnormalities, electrophysiological recordings showed that GABAergic circuits remain depolarizing. How then is KCC2 regulated, and how are subplate neurons involved? Recent experiments have led to the hypothesis that KCC2 expression can be regulated by GABAergic depolarization, while others report no influence of GABAergic signaling on KCC2 expression. However, there are other sources for depolarization. Blocking glutamatergic signaling during early ages *in vivo* is sufficient to prevent the developmental increase in KCC2. Thus early glutamatergic activity might be required for GABAergic maturation in layer 4.
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1. There are three sources of glutamatergic inputs to cortical layer 4: intracortical, thalamus and subplate neurons. Subplate removal by itself prevents inhibitory development despite the presence of intracortical and thalamic input. Thus, together these data suggest that glutamatergic excitation from subplate neurons is needed for inhibitory maturation. Such a role of subplate neurons in inhibitory maturation would require that subplate neurons depolarize layer 4 neurons, which can be achieved either by exciting GABAergic neurons and increasing early depolarizing GABAergic activity or by exciting the targets of GABAergic neurons directly. Thus by providing feed-forward excitation to the developing cortical circuits subplate neurons can regulate both the maturation of glutamatergic thalamocortical and GABAergic intracortical synapses. By controlling cortical inhibition, subplate neurons might also play a role in regulating cortical activity levels after GABAergic circuits have matured. Since subplate ablation prevents inhibitory maturation, maybe by directly activating GABAergic circuits, subplate activation is likely able to dampen cortical activity levels. Thus even temporary depression of subplate activity could lead to cortical hyperexcitability, which might underlie pathophysiological conditions.
2. Lesioning subplate at a time when thalamocortical axons are present in layer 4, but before these projections have refined into a mature pattern, revealed a role for subplate neurons in thalamocortical patterning. The organizational pattern observed in the visual cortex is that of the ocular dominance columns (ODCs). These columns are formed by the segregation of thalamic afferents innervated by either eye into alternating bands of left eye or right eye dominance. In cat ODCs form during the postnatal period from an initially non-segregated state. Analysis of the ODCs following ablation of subplate neurons in V1 shows that subplate ablation prevents the formation of ODCs. This deficit in thalamocortical patterning is present even though both thalamic axons and their target neurons are present in layer 4. Thus subplate neurons are necessary for the patterned organization of the cerebral cortex.
3. The function of inhibitory circuits is crucial for critical period plasticity in the visual cortex. Sensory manipulations that alter inhibition also result in impaired synaptic plasticity mechanisms that underlie critical period plasticity. Thus, there is a co-regulation of inhibition and critical period plasticity. Because subplate neurons play a crucial role in maturation of inhibition, it is likely that they also mediate cortical plasticity during the critical period.
4. The disappearance of subplate neurons might ensure that certain processes, such as critical period plasticity, occur only once. While critical period plasticity might be distinct from adult plasticity by its extent and transience, the underlying mechanisms might be similar to processes underlying adult learning via attention based mechanisms. This attentional modulation of cortical circuits develops postnatally, thus subplate neurons could provide a circuit that enables large-scale cortical plasticity mechanisms before attention is functioning.
5. Subplate activity can influence layer 4 synapses and enable thalamocortical strengthening to occur. In particular, subplate input to layer 4 can strongly depolarize layer 4 cells. Since subplate neurons are driven by thalamic activity, this subplate mediated depolarization of layer 4 cells occurs at the same time as direct thalamocortical input to layer 4 and may lead to a strengthening of thalamocortical synapses by associative long-term potentiation. The strength of the subplate to layer 4 connection is evidenced by the evoked disynaptic sinks in layer 4 during white matter stimulation. Therefore, subplate neurons can act somewhat like a "teacher" entraining layer 4 neurons to respond to appropriate thalamic inputs.
6. Given the central role of subplate neurons in the maturation of cortical circuits, damage to subplate neurons at any point during development could lead to neurological diseases. Subplate neurons are particularly prone to injury (especially hypoxic-ischemic injuries) during development and are especially vulnerable at time points when injuries are associated with many neurodevelopmental disorders (second trimester). The enhanced vulnerability of subplate neurons may be due to their early maturation and therefore higher metabolic requirements. This vulnerability of subplate neurons might be more pronounced in infants born prematurely that are at a higher risk for neurodevelopmental disorders disorders as a large period of development occur *ex utero*. In animals, neonatal hypoxia damages subplate neurons and prevents normal critical period plasticity, supporting the idea that such injuries damage circuits needed for the development of normal tuning and plasticity. In humans, such hypoxic-ischemic injuries, especially in the second trimester are associated with various neurodevelopmental disorders such as cerebral palsy and epilepsy.
7. Subplate ablation in animals is followed by a period of seizures indicating hyperactivity. These seizures develop a couple days after the time of ablation and thus are likely reflecting adjustments of the cortical network. The origin of these seizures is unclear. Seizures could be generating by depolarizing GABAergic activity (Kanold and Shatz, [@B20]) or alternatively be generated by glutamatergic activity that is not balanced appropriately by inhibitory GABAergic circuits. The different possible origins of seizures after subplate lesion are of clinical relevance. GABA~A~ agonists can be used to treat seizures if they decrease firing probability. However, a GABAergic origin of seizures would also indicate that GABA~A~ agonists would increase seizures instead of preventing them.
8. Many neurodevelopmental disorders are characterized by abnormal neuronal activity, hyperexcitability, and learning impairments due to impaired inhibition, suggesting that altered inhibitory development underlies these disorders. Thus, a common outcome for early injuries and deprivations is that both alter inhibitory development, which in turn might alter critical period plasticity and normal development.
9. In addition to subplate lesions, the activity of subplate neurons can be altered by neuromodulators such as GABA, acetylcholine and glycine. The subplate is also innervated by serotonergic fibers and subplate neurons selectively express progestin receptor. Thus, maternal or neonatal exposure to drugs (ranging from nicotine to sedatives and antidepressants) or hormones might alter subplate activity and thereby potentially disrupt cortical development. Therefore monitoring the status of subplate neurons in human infants is of high clinical relevance.
10. Subplate neurons are present in the cerebral cortex of all mammals. Subplate neurons are more prominent in species with increased radial and tangential cortical connectivity such as cat, monkey and human, suggesting that subplate neurons might be needed for the establishment of more complex processing capabilities. The disappearance of subplate neurons over development suggests that their role is purely developmental. As discussed above, subplate neurons enable the functional maturation of cortical circuits. However, other areas in the brain (such as subcortical areas) seem mature without neurons equivalent to subplate neurons. Thus one can speculate that the role of subplate neurons might have to do with unique properties of the cerebral cortex. One hallmark of the cerebral cortex is complex interconnectivity and its ability to adjust its connectivity during early development in response to altered patterns of spontaneous and sensory inputs. This capability for rewiring of the cerebral cortex is greatly diminished after the critical period. Thus removing these enabling (or "teacher") circuits is one way to ensure that plasticity occurs only once and only during early development and might allow the development of higher cognitive processes at later stages of cortical processing at later ages.
General Comments and Discussion
===============================
DeFelipe
--------
Comment on points 1 and 18.
There is apparently a discrepancy between Kanold\'s article and that of Clancy and colleagues. Kanold states that subplate neurons contribute to cortical circuits that are only present during cortical development. However, as stated in the *Remarks and main conclusions* of the article by Clancy et al., and that of Luhmann et al., it is well-documented that some subplate cells persist in the white matter of the mature human and non-human cerebral cortex.
Espinosa and fairén
-------------------
Comment on points 1 and 18.
Indeed, white matter neurons (carnivores and primates) and layer VIb neurons (rodents) are neither a small nor a residual population. Moreover, during development, some GABA neurons in the subplate are migratory neurons on their way to the cortical plate.
Clancy
------
Continuing DeFelipe, Clancy, Espinosa and Fairén.
Connections from the persisting subplate cells in adult brains extend throughout the white matter and to all cortical layers (see [Figure](#F1){ref-type="fig"}), including a stable projection that focuses specifically on cortical layer 1 (Clancy and Cauller, [@B8]; Clancy et al., [@B9]), evidence they retain (or reform) the earliest cortical developmental circuit. In layer 1 they converge with connections from non-specific thalamic and "top down" cortical projections, as well as callosal, brain stem nuclei, claustrum, zona incerta and nucleus basalis on the dendrites of cells in layers II/III and V. As a consequence, they may factor into one of the few neural correlates of primate conscious perception able to be isolated in a laboratory setting, the behaviorally relevant N1, an EEG deflection recorded following evoked sensory stimuli. Changes in the N1 component of a ERP accurately predict sensory perception in primates, a response that is initiated in cortical layer I (Cauller and Kulics, [@B6]; Cauller et al., [@B5]). Thus these connections, first formed in the developing subplate, are implicated as components of an interactive strategy for mature cognitive processing, within which sensory information from the environment may be primed, guided and interpreted (Koch and Davis, [@B21]; Cauller, [@B4]).
![**Proposed persisting subplate/white matter connectivity circuits in mature mammalian cortex (Clancy and Cauller, [@B8]; Clancy et al., [@B9]; Friedlander and Torres-Reveron, [@B12])**.](fnana-04-007-g001){#F1}
Kanold
------
Continuing DeFelipe, Clancy, Espinosa and Fairén.
While there are surviving subplate neurons in many species, the fractions of subplate neurons surviving is different between species, especially considering that there are more subplate neurons in higher species. A careful comparative accounting has to be done to see what is the percentage of subplate neurons and more importantly, which subplate neurons are eliminated/survive in different species. One key issue is the use of proper markers to identify subplate neurons at different ages that also do not label migrating neurons. For example, we can see and patch many subplate neurons at early ages that do not stain well with common markers (Zhao et al. [@B40]). Birth- dating might give the best estimate.
In addition, given the functional changes in the subplate (see Zhao et al., [@B40]) the role of the subplate in development can diminish (or change) simply based on the maturation of intrinsic membrane characteristics of subplate neurons and the maturation of the thalamocortical synapse.
As for subplate projections (figure above), functionally we find in young rodents excitatory subplate input to layer 4 and not layer 2/3 (Zhao et al., [@B40]) consistent with earlier work in cat by Friauf and Shatz ([@B11]). Thus while subplate axons are present in other layers, their functional role might be limited. In addition the projection patterns might be different at different stages ofof subplate neurons might be changing over development.
A commentary on
Subplate cells: amplifiers of neuronal activity in the developing cerebral cortex
by Luhmann, H. J., Kilb, W., and Hanganu-Opatz, I. L. (2009). Subplate cells: amplifiers of neuronal activity in the developing cerebral cortex. Front. Neuroanat. 3:19. doi:10.3389/neuro.05.019.2009.
Remarks and Main Conclusions
============================
1. The subplate forms a transient layer in the developing cerebral cortex and consists of migratory and postmigratory neurons, dendrites, axons, growth cones, synapses and glial cells. The subplate is located between the intermediate zone and the cortical plate, which during further development differentiates into the neocortical layers II to VI.
2. Kostovic and Molliver were the first who identified the subplate as a distinct layer in the embryonic human cerebral cortex. Subsequently Rakic described this layer in the monkey neocortex. A subplate can be identified in all mammals although its relative thickness, developmental profile and persistence in adulthood vary among species. Anatomical data indicate an evolutionary difference in the ontogenetic fate of the subplate. In the rat and other rodents many subplate cells survive into adulthood forming layer VIb or VII.
3. Prominent species differences also exist in the relative thickness of the subplate, which increased during evolution. In humans the subplate develops to approximately six times the thickness of the cortical plate around 29 weeks of gestation (Mrzljak et al., [@B27]), whereas in rodents it remains a relatively thin layer during development (Uylings et al., [@B38]).
4. A substantial proportion of subplate cells are not born in the ventricular neuroepithelium, but instead originate in the medial ganglionic eminence and follow a tangential migratory route to their positions in the developing cortex.
5. Subplate cells represent a rather heterogeneous neuronal population according to their morphology, neurotransmitter identity and connectivity.
6. Subplate cells play important roles in the structural and functional organization of the cerebral cortex and in early necortical plasticity. Axons arising from subplate neurons pioneer the cortico-fugal pathway and have been proposed to form a cellular scaffold for guiding thalamocortical axons. Subplate neurons receive a transient synaptic input from "waiting" thalamic axons and early deletion of subplate neurons in kitten visual cortex prevents the segregation of thalamocortical axons within layer IV and the formation of ocular dominance columns. Furthermore, subplate cells regulate the maturation of GABAergic synaptic transmission and establish the balance between excitation and inhibition in the developing neocortical network.
7. Subplate neurons reveal a large variety of morphologies. Inverted pyramidal-like and horizontal cells as well as polymorphic neurons with different shapes and spiny or smooth dendrites have been classified as subplate neurons. Due to their earlier generation and more mature developmental stage, subplate neurons show a relatively extensive dendritic tree when compared to the more immature pyramidal neurons of the cortical plate. Descending dendrites from subplate neurons may invade the underlying intermediate zone and ascending dendrites may extend into the cortical plate. This morphological heterogeneity is accompanied by a large variation in immunoreactivity. Subplate neurons reveal markers for GABA or glutamate and may co-express various peptides. The morphological and neurochemical heterogeneity also explains, why various attempts failed to identify a specific marker for subplate neurons. Recently Hoerder-Suabedissen and coworkers succeeded in identifying a number of novel markers for murine subplate cells, which may soon allow the definition of different subpopulations of subplate neurons.
8. Subplate cells participate in local and long-distance axonal connections indicating that these neurons may function as local circuit as well as projection neurons. They show a dense axonal arborization within the subplate, but also project to the marginal zone/layer I and to the cortical plate, where they form axonal collaterals within layer IV.
9. In ferrets and cats, the majority of the subplate neurons projecting into the cortical plate reside in the upper half of the subplate and provide a glutamatergic synaptic input to the developing cortical plate, including the layer IV neurons. Long-distance axons from subplate neurons invade the thalamus during early stages of corticogenesis and form an axonal scaffold for the establishment of cortical efferent and afferent projections. Beside these local and long-distance projections arising from glutamatergic subplate neurons, GABAergic subplate cells also project to both neighbouring and more distant neocortical regions and form a cortico-cortical synaptic network. Since GABA may act as an excitatory neurotransmitter during early cortical development, the postsynaptic action of GABAergic subplate neurons may be also depolarizing.
10. Ultrastructural studies of subplate cells in various species have demonstrated symmetrical as well as asymmetrical synapses with relatively mature properties, indicating that subplate neurons receive GABAergic as well as glutamatergic synaptic inputs. As suggested by Kostovic and Rakic, glutamatergic inputs onto subplate neurons may arise from the thalamus and other neocortical areas, whereas GABAergic synaptic inputs may originate from GABAergic interneurons in the subplate. Thalamocortical synaptic contacts with spines and shafts of subplate neuron dendrites have been demonstrated in the neonatal ferret and a dense network of cortico-cortical fibers have been reported in the subplate of the embryonic mouse.
11. *N*-Methyl-d-aspartate (NMDA), α-amino-3-hydroxy 5 methylisoxazole-4-propionic acid (AMPA) and kainate receptors and the essential subunits for their receptor function have been demonstrated in the subplate of various species, suggesting the presence of functional glutamatergic synapses in subplate neurons. The expression of benzodiazepine binding sites, GABAA receptors and GABAA receptor subunits in the subplate indicate that functional GABAergic synaptic inputs should also be present in subplate neurons. These morphological, ultrastructural and immunohistochemical data are complemented by functional studies on the properties of the subplate and single subplate cells in different mammalian species.
12. In contrast to the heterogeneity in morphological and chemical appearance, electrophysiological recordings from single subplate neurons in rodents demonstrate rather homogeneous functional properties. Subplate neurons exhibit relatively uniform passive membrane properties.
13. In comparison to other neurons in the immature cerebral cortex, subplate cells also reveal the most mature properties in action potential characteristics and in the biophysical properties of voltage-dependent sodium and calcium currents. These observations have been made in developing rodent as well as in human cerebral cortex, indicating that these relatively mature functional properties enable subplate cells to transmit afferent neuronal activity faithfully to the developing cortical plate.
14. Intracellular labeling of single subplate cells with fluorescent dyes or biocytin revealed an extensive neuronal network of dye-coupled neurons in the subplate and cortical plate. In newborn rats, on average about nine neurons are dye-coupled to a single subplate cell and these gap junction coupled networks are often organized in a columnar manner.
15. Subplate cells are strongly coupled via electrical synapses and form a functional columnar syncytium with neurons located in the cortical plate. It is tempting to speculate that this early columnar organization results from the radial, column-like neuronal migration of newly-generated neurons into the developing neocortex, which is also controlled by gap junctional coupling.
16. Electrophysiological and optical imaging recordings further support the hypothesis that subplate neurons are well integrated in the developing cerebral cortex. *In vitro* intracellular recordings and current-source density analyses in late embryonic and early postnatal kitten visual cortex demonstrated that subplate neurons receive functional excitatory synaptic inputs from axons that course in the developing white matter. Subplate cells in newborn rat somatosensory cortical slices reveal a substantial amount of spontaneous postsynaptic currents (sPSCs) with different kinetics and pharmacological profile demonstrating that subplate neurons receive functional synaptic inputs mediated by AMPA, NMDA and GABA~A~ receptors.
17. Beside a glutamatergic thalamocortical input with relatively mature functional properties (short delay, fast kinetics, reliable responses), subplate cells in newborn rodents receive additional intracortical synaptic inputs from various presynaptic sources. Local glutamatergic synaptic inputs arise from pyramidal neurons in the cortical plate and from glutamatergic subplate cells, which both activate postsynaptic AMPA/kainite and NMDA receptors. This intra-subplate glutamatergic input differs from the thalamocortical input. Anatomical studies in rodent and human immature cerebral cortex indicate that glutamatergic synaptic inputs most likely also arise from other neocortical sources via cortico-cortical connections, but the functional properties of these long-distance synaptic inputs onto subplate cells are currently unknown.
18. Subplate cells in rodents receive a GABAergic synaptic input from neighbouring GABAergic neurons located in the subplate and probably also in the cortical plate. However, as in other immature brain structures, this GABAergic input most likely has a pure excitatory postsynaptic effect.
19. Subplate neurons are not only well integrated in the developing cortical circuit, but during certain developmental periods they also receive a very selective input from neuromodulatory brain structures. Both in primates as well as in rodents the subplate is specifically innervated by cholinergic fibers arising from the basal forebrain and from monoaminergic inputs. The activation of postsynaptic nicotinic acetylcholine receptors elicits in subplate cells a marked depolarization which is largely mediated by the activation of α4/β2 receptors. The responsiveness to activation of muscarinic acetylcholine receptors (mAChR) is more complex and reveals a remarkable oscillatory discharge mode of subplate cells.
20. Gap junctional coupling, depolarizing GABA actions and a tonic non-synaptic GABA release contribute to the generation and maintenance of the cholinergic network oscillations.
21. Electrophysiological and calcium imaging recordings in acute neocortical slices from newborn rodents as well as calcium imaging data obtained in neocortical cell cultures indicate that GABAergic subplate cells play a central role in generating synchronous neuronal network activity. Voigt and coworkers estimated that a minimal number of two GABAergic subplate neurons per square millimeter are required for the occurrence of synchronous network activity. The unique structural and functional properties enable subplate cells to receive, synchronize and amplify the afferent and intrinsic synaptic inputs.
22. A large proportion of newly-generated GABAergic interneurons arising from the medial ganglionic eminence and pyramidal neurons from the ventricular zone must migrate through the subplate on their way to the developing cortical plate. Therefore the subplate may also have a profound influence on the migration pattern. GABAergic and glutamatergic subplate neurons partly release their neurotransmitter in a paracrine, non-synaptic manner, thereby regulating the neuronal migration pattern. Neurotransmitter release in the subplate may rise substantially during oscillatory network activity, thereby activating low affinity or extrasynaptic receptors causing alterations in neuronal migration.
23. On the basis of the currently available data we suggest the following model: During early cortical development, in most mammals before birth, subplate cells with relatively mature structural properties (elaborated dendritic tree, complex axonal projections, mature symmetrical and asymmetrical synapses) receive a functional glutamatergic synaptic input from specific thalamic nuclei and a selective input from neuromodulatory systems (e.g. cholinergic inputs from the basal forebrain). In rodents, subplate neurons are densely interconnected via electrical and chemical synapses and upon activation of muscarinic (and probably also other metabotropic) receptors discharge in repetitive ∼20-Hz bursts. GABAergic subplate neurons releasing GABA in a synaptic and tonic non-synaptic manner facilitate the generation of oscillatory network activity. Intra-subplate connections arising from glutamatergic subplate cells may boost the subplate activity in the 10--40-Hz frequency range. Subplate cells are dye-coupled to cortical plate neurons in a columnar manner and faithfully transmit synchronized 10--20-Hz network oscillations generated and amplified in the subplate to the cortical plate and marginal zone.
24. Elimination or interruption of the subplate prevents the generation of the subplate-driven synchronized activity patterns and disturbs the maturation of the columnar architecture. Under normal conditions and with further development, subplate cells disappear by apoptosis or transform into white matter interstitial cells or layer VIb (layer VII) neurons. In the normal mature cerebral cortex, subplate neurons have lost their capabilities to receive and amplify incoming neuronal activity and to generate synchronized network oscillations. However, as already suggested by Jones, disturbances in the pattern of programmed cell death in the subplate may cause a failure to establish normal patterns of connections in the overlying cerebral cortex, leading to long-term neurological deficits such as schizophrenia. Abnormal placement of (surviving subplate?) neurons in the white matter and atypical circuits have been observed in the prefrontal cortex of schizophrenic patients. It has been further suggested that subplate-like neurons may persist in cortical dysplasia and contribute to the manifestation of pharmacoresistant epilepsy in adults. Interestingly in the resectioned human tissue GABA application induced depolarizing postsynaptic responses and spontaneous GABAergic synaptic potentials even elicited action potentials.
General Comments and Discussion
===============================
DeFelipe
--------
Comment on point 24.
It seems that most, if not all, interstitial neurons of the adult neocortical white matter are derived from the population of subplate neurons (e.g., Chun and Shatz, [@B7]). According to Luhmann and colleagues, these neurons have lost their capacity to receive and amplify incoming neuronal activity, and to generate synchronized network oscillations. This implies that during the transformation of subplate neurons to interstitial cells, the latter do not receive afferent inputs? Are there other anatomical, molecular and electrophysiological differences between interstitial neurons and subplate neurons? Are there any clues about what may be the function of interstitial cells?
Luhmann
-------
Reply.
Torres-Reveron and Friedlander (Torres-Reveron and Friedlander, [@B36]) demonstrated in adolescent (P10--P20) rat visual cortex that surviving interstitial cells in the white matter and also subplate cells (layer VIb or VII) receive glutamatergic and GABAergic synaptic inputs. Studies in mature (\>2-months rodents) cortex are lacking and it is unkown whether interstitial cells or subplate neurons in adolescent or adult cortex can synchronize network oscillations.
Interstitial cells and subplate neurons in adolescent rat cortex reveal similar passive and active electrophysiological properties (Torres-Reveron and Friedlander, [@B36]).
The exact function of interstitial cells is unknown, but it is tempting to speculate that surviving interstitial cells may play a pathophysiological role in subcortical or white matter heterotopia (Ackman et al., [@B1]; Croquelois et al., [@B10]; Meroni et al., [@B25]).
Rockland
--------
Comment on points 8 and 9.
From this and other articles, information is emerging on the circuitry of long-distance projection neurons. A key point remains the identification of the principal postsynaptic targets. An interesting observation, emphasized in this article, is that GABAergic projecting neurons can have both local and long-distance targets.
Espinosa and fairén
-------------------
Comment on points 4, 7, 10 and 8.
These cells coming from the medial ganglionic eminence are GABA neurons. Some of these cells in the subplate are just interneurons en route to the cortical plate (see point 22), and this should be taken into account for interpreting peptide expression in subplate neurons. Some GABA neurons may have neuronal interactions in subplate neuronal circuits, or may become persisting GABA neurons in the white matter or layer VIb.
Rockland
--------
Comment on point 21.
It\'s fascinating that as little as two GABAergic subplate neurons per square millimeter can be effective for synchronous network activity.
Kanold
------
Continuing Rockland on point 21.
It is interesting that relatively small gap junction conductances can drive cortical oscillations. In addition it is very interesting that the morphological diversity in subplate neurons so far is not reflected in obvious physiological diversity.
Luhmann
-------
Reply to Rockland point 21.
Yes and please see also my comment to point 8 of Clancy et al.
A commentary on
Primate-specific origins and migration of cortical GABAergic neurons
by Petanjek, Z., Kostovic, I., and Esclapez, M. (2009). Primate specific origins and migration of cortical GABAergic neurons. Front. Neuroanat. 3:26. doi:10.3389/neuro.05.026.2009.
Remarks and Main Conclusions
============================
1. Up to this date most of our knowledge regarding GABAergic neuron development is based on studies performed in rodents. However, differences in cortical GABAergic circuitry exist between rodents and primates. Although many types of cortical interneurons appear to be common to all species, some types in primates, like the double bouquet cells, or interneurons of cortical layer I, display more elaborate features. These types may represent evolutionary specializations. In addition, several studies suggest an increased proportion of cortical GABAergic neurons between rodents and primates. Such increases in the number and diversity of GABAergic neurons have been suggested to be closely related to the tremendous increased brain complexity that occurs during mammalian evolution.
2. Whereas there is a consensus for all species examined that principal glutamatergic neurons originate in the local ventricular (VZ) and subventricular zones (SVZ) of the dorsal telencephalon (pallium) and migrate along radial glia to their target cortical layer, this seems not to be the case for GABAergic neurons. In rodents, there are compelling evidences that cortical GABAergic neurons are not generated in these cortical (pallial) proliferative zones. They are produced in proliferative zones of the ventral (basal) telencephalon (subpallium), the ganglionic eminence (GE). From this region, newly born GABAergic neurons migrate tangentially into the cortex. Surprisingly, data obtained in the human and monkey cortex point out significant evolutionary changes with respect to the origin of cortical GABAergic neurons.
3. In rodents GABAergic neurons originate exclusively from the ganglionic eminence and migrate tangentially to the cortex. These migrating neurons follow several routes to reach their target regions. The major stream of tangentially migrating GABAergic neurons is present at the border of IZ and SVZ (lower part of IZ and upper part of SVZ). In addition, smaller streams of migrating cells are present in the subplate (SP) and the upper part of MZ.
4. There are compelling evidences that different subdomains within the GE generate different populations of cortical GABAergic neurons. The GE displays two major divisions: the medial and lateral GE. Recent findings demonstrated the existence of two additional components, the caudal and septal divisions. The vast majority of cortical GABAergic neurons are provided by the medial GE and to a lesser extent by the caudal GE. However, significant, although smaller numbers of cortical GABAergic neurons originate from the lateral GE, especially at later stages. Among the different subpopulations of GABAergic neurons, most of somatostatin-expressing neurons and all parvalbumin-containing cells derive from the medial GE whereas the majority of calretinin-containing cells and half of the NPY-expressing neurons arise from the caudal GE. Furthermore, somatostatin cells are primarily generated within the dorsal part of the medial GE whereas parvalbumin neurons are provided by the ventral part of the medial GE. Therefore, the GE is organized in molecularly different subdomains that produce different subpopulations of cortical GABAergic neurons.
5. In addition to spatial diversity in the origin of GABAergic neuron subpopulations, the generation of these different subgroups may occur at specific time windows. However, the precise spatio-temporal pattern of production, for the different GABAergic subtypes, has yet to be established.
6. In human and non-human primates, cortical GABAergic neurons are produced not only by the GE (as in rodents), but also massively by the proliferative zones of the dorsal telencephalon.
7. Both in human and macaque, neurogenesis of GABAergic neurons within the ventral and dorsal telencephalon occurs with distinct temporal profiles. Whereas at early stages of primate fetal development, GE is an exclusive site of origin for cortical GABAergic neurons, later on the dorsal telencephalic proliferative layers is the major source for these neurons.
8. The dorsal and ventral sites of neurogenesis produce different populations of cortical GABAergic neurons. A recent study from Fertuzinhos and coworkers investigate the proportion of different subpopulations of GABAergic neurons in brains of human fetuses or infants affected by holoprosencephaly. In holoprosencephaly, the numbers of cortical GABAergic neurons expressing nitric oxide synthase, neuropeptide Y, or somatostatin were significantly reduced in comparison to healthy infants. In contrast, calretinin-containing neurons were present in normal numbers as well as principal neurons. These findings show that, in human, nitric oxide synthase-, neuropeptide Y- and somatostatin-containing neurons originate from the GE whereas calretinin neurons are generated by the dorsal telencephalon.
9. The population of calretinin neurons, generated mainly in the dorsal telencephalon in human, corresponds to the population of GABAergic neurons whose proportion increases dramatically in primates and which displays primate-specific subtypes such as double bouquet cells. Altogether, these studies favor the hypothesis that dorsal production occurs principally as an answer to an increased evolutionary need for specific classes of cortical GABAergic neurons.
10. During tangential migration, prospective GABAergic neurons move parallel to the surface of the brain. They pass by a complex route between numerous growing axon bundles and cross regional boundaries. The length of their trajectory is greater than that of radially migrating neurons. Therefore, the mechanisms of cellular interactions enabling proper positioning and specification of GABAergic neurons are more complex than for radially migrating neurons. One can suggest that, dorsal production of cortical GABAergic neurons in primates might occur in order to facilitate migration routes through an expanding neocortex. An exclusive ventral telencephalic origin of cortical GABAergic neurons in primates would imply extremely long and complex migratory routes for such neurons. In keeping with this hypothesis, it might be expected that the percentage of GABAergic neurons produced dorsally will increase in larger brains in order to keep migratory routes shorter and simpler. However, the data obtained in ferret do not support this hypothesis. That is, the ferret brain displays a convoluted neocortex significantly larger compared to rodents, but shows limited cortical GABAergic neurons generated by the dorsal telencephalon such as described in mouse. Extensive dorsal production of GABAergic neurons in primates can be related to an increased need in number and/or specific types for GABAergic neurons in brains with more complex cortical circuitries.
11. The very massive dorsal telencephalic origin of cortical GABAergic neurons suggests distinct properties of dorsal telencephalic progenitors in primates compared to rodents. Interestingly, it was demonstrated that the proliferative behavior of cortical neuronal precursors during neurogenesis differs between rodents and primates. This leads to significant differences in morphology and function of their dorsal proliferative zones.
12. In the human embryo, in comparison to the rodent embryos, there is a dramatic increase in surface area and thickness of the VZ at the earliest stage of proliferation. In comparison to other species, the primate SVZ is larger and more complex. It displays a different type of cellular organization. There is a new, outer compartment within the SVZ, which displays a number of unique features, and exists much longer during development. So, it is not unreasonable to suggest that the increase in complexity, or even the specific cellular and laminar organization of dorsal proliferative zones in primates are significantly connected to the massive production of GABAergic neurons. However, no data to prove this hypothesis are currently available.
General Comments and Discussion
===============================
Rockland
--------
It will be interesting to establish whether common spatial-temporal origins denote share functional characteristics.
Espinosa and fairén
-------------------
The finding of interneuron precursors in the dorsal cortex by combination of Ki67 and GAD65 labeling is fascinating and significant.
A commentary on
Two separate subtypes of early non-subplate projection neurons in the developing cerebral cortex of rodents
by Espinosa, A., Gil-Sanz, C., Yanagawa, Y., and Fairén, A. (2009). Two separate subtypes of early non-subplate projection neurons in the developing cerebral cortex of rodents. Front. Neuroanat. 3:27. doi:10.3389/neuro.05.027.2009.
Remarks and Main Conclusions
============================
1. The preplate and its derivatives contain projection neurons, collectively named pioneer neurons, whose axonal arborizations establish the earliest cortico-fugal projection systems during cortical development. These neurons have traditionally been associated with the subplate. However, certain preplate neurons never associate with the subplate but instead with the marginal zone.
2. Proteins that are strongly expressed in pioneer neurons include the calcium-binding proteins calbindin (CB) and calretinin (CR) in rats. CB and CR co-label a single population of preplate pioneer neurons that end up in the marginal zone. In the preplate, another population is made up of CB^+^ cells interspersed among the CB+/CR+ cells. Most of these CB^+^-only cells end up in the subplate.
3. Surprisingly, neither of these two calcium-binding proteins labels early projecting neurons in mice. In mice, CB is a marker for most migrating GABA interneurons at the earliest stages of cortical histogenesis, including early migrating GAD67-GFP^+^ interneurons that arrived to the preplate before its partition. We found that in rats as well, CB and CR decorated putative migrating interneurons.
4. Thus, we searched for alternative immunohistochemical markers to try to identify the homologues in mice of the calcium-binding protein- expressing pioneer neurons of rats. We used antibodies to the neural cell adhesion and recognition molecules L1 and TAG-1/cntn2/axonin1, found previously to label pioneer cells in mice, in embryos of the two rodent species.
5. We have identified two subtypes of non-subplate pioneer neurons in rats: the cells of the first subtype co-express CB, CR and L1 and are situated in the upper part of the preplate before its partition. Cells of the second subtype are characterized by the expression of TAG-1 and are located slightly deeper than the previous population in the preplate. After the preplate partition, the neurons co-expressing CB, CR and L1 remain in the marginal zone whereas those expressing TAG-1 become localized in the upper cortical plate. In mice, we identified two subtypes of non-subplate pioneer neurons that express either L1 or cntn2 (TAG-1). We propose that these two subtypes of mouse cells are homologues of the two subtypes of non-subplate pioneer neurons of the rat. The anatomical distribution of these neuron populations is identical in rat and mouse.
6. The T-box transcription factor Tbr1 is expressed early after the differentiation of cortical progenitors, and is functionally important for corticogenesis. Tbr1 is expressed by Cajal-Retzius cells, subplate cells and glutamatergic neurons, but not by GABAergic cells. Since we characterized the CB^+^/CR^+^ cells of the rat preplate and marginal zone as projecting neurons, we wished to confirm that these cells co-expressed the T-box transcription factor Tbr1. This was indeed the case, and we additionally observed that Tbr1 was expressed in the mouse preplate by pioneer neurons both of the L1 and the TAG-1 subtypes.
7. Apparent differences in anatomy and tempo of deployment of early axonal projections of subplate vs. non-subplate neurons may result from technical factors; that is, the different advantages and pitfalls of the axonal tract-tracing methods utilized in different studies. Here, we decided to trace axonal projections by taking advantage of the chemical properties of the early neurons, as detected by immunohistochemistry. In fortunate cases, this approach produced selective staining of different neuronal populations together with high resolution anatomical detail of their projecting axons.
8. The different populations of non-subplate pioneer neurons differ in their axonal projections. Non-subplate pioneer neurons of the L1 subtype project to the ganglionic eminences and the anterior preoptic area, but avoid following the internal capsule that leads to the dorsal thalamus. Thus, we can conclude there is no projection of neurons of L1 subtype to the dorsal thalamus. The projections of the neurons of the TAG-1 subtype end up at the lateral parts of the ganglionic eminences. A corticothalamic TAG-1^+^ projection becomes apparent much later, in postnatal mice.
9. A major discrepancy among the various studies of the early cortico-fugal connections concerns whether or not subplate axons are considered as reaching the dorsal thalamus, or, according to more recent studies, these subplate axons (and axons deriving from the marginal zone) are considered as entering only the part of the internal capsule that traverses the ganglionic eminences. However, subplate axons seem not to project to the dorsal thalamus at the initial steps of corticogenesis. If this is so, the subcortical distributions of subplate axons are similar to that of the non-subplate axons described here.
10. A caveat: both adhesion molecules TAG-1/cntn2 and L1 are simultaneously expressed by different neurons of the marginal zone and the subplate. This makes it hard to distinguish which descending cortico-fugal projections originate from neurons located in each one of these two transitory cortical compartments, or from neurons residing in both places. Unfortunately, this seems to somewhat limit the usefulness of most neurochemical markers of the marginal zone and the subplate described so far.
11. The diversified group of early cortical neurons with their diversified axonal projections that we have considered here has not yet been identified in species other than rodents. However, further study is highly relevant to advancing our understanding of cerebral cortex development. Not only would this support the development of functional murine experimental models, but there is the promise of extending these observations to non-human primates and humans. To emphasize the importance of such a comparative approach, it may suffice to consider two examples; namely, the *pioneer plate* described in humans by Meyer et al. ([@B26]) and the *predecessor* *neurons* described in the human preplate by Bystron et al. ([@B3]).
12. We have described here a close anatomical relationship of pioneer L1^+^ and TAG-1^+^ axons with migrating GABA interneurons. Medial L1^+^ axons coincide with a massive stream of migrating interneurons during their exit from the ganglionic eminences to the pallium, along the intermediate zone of the cortex. Other less abundant interneurons migrate in a compartment located more laterally, where they acquire spatial relationships with L1^+^ and TAG-1^+^ axons. TAG-1^+^ axons also accumulate below the subplate, and are accompanied by migrating interneurons in this territory.
13. Our observations strongly suggest that L1^+^ and TAG1^+^ cortico-fugal axons could be a substrate for migration of interneurons along the cortical intermediate zone, even though a role for L1 in the migration of cortical interneurons has been excluded in *in vitro* perturbation experiments. The role of TAG-1 in interneuron migration is controversial, although this molecule is active in the control of tangential migration of other neuron cohorts.
General Comments and Discussion
===============================
Rockland
--------
Comment on point 5.
Do you think that L1 and TAG-1 are actively involved in the spatial sorting of these subtypes; that is, neurons co-expressing CB, CR, and L1 in the marginal zone vs. a deeper location, in the upper cortical plate, for TAG-1 expressing neurons?
Espinosa and fairén
-------------------
Reply.
This is an attractive idea, and of course something of that sort could be expected on the grounds of the exquisite specificity of the two markers for distinct projection neuron populations. Our work in progress seems to indicate that the absence of L1 in knockout mice embryos affects the distribution of early Tbr1^+^ neurons in the cortical anlage.
Espinosa and fairén
-------------------
Comment on point 11.
We need novel data about molecular markers of pioneer cells or predecessor cells described in humans, in order to determine (1) if they are unique to the human brain or may have homologues in other mammals and (2), how the patterns of early cortico-fugal projections are shared in mammalian evolution.
Espinosa and fairén
-------------------
Comment on point 13.
The observation that migrating GABAergic interneurons contact certain sets of cortico-fugal axons does not directly imply a function for either L1 or TAG-1 in the control of these migrations, because experimental proof is still lacking. It is the authors' opinion, however, that the issue of axonophilic migration of certain sets of GABAergic interneurons merits further analyses. Interneurons migrating along the marginal zone do not seem to use any axonal system as scaffold.
A commentary on
Doublecortin-expressing cells persist in the associative cerebral cortex and amygdala in aged non-human primates
by Zhang, X., Cai, Y., Chu, Y., Chen, E., Feng, J., Luo, X., Xiong, K., Struble, R. G., Clough, R. W., Patrylo, P. R., Kordower, J. H., and Yan, X. (2009). Front. Neuroanat. 3:17. doi:10.3389/neuro.05.017.2009.
Remarks and Main Conclusions
============================
1. One of the most fundamental features of the brain is its plasticity, a constant interplay between neural structure, function and experience. The scope and complexity of neuroplasticity appear to increase during evolution in parallel with the enlargement of brain through encephalization, corticalization and gyrification. Thus, plasticity is likely the foundation of the so-called "higher brain functions" (e.g., learning, memory, decision making, or intelligence) that are mostly sophisticated in humans.
2. In general, the associative cortical areas together with the amygdala and hippocampal formation, which are greatly expanded during late mammalian evolution, are the anatomic niches for many high-level cognitive functions. Greater neuroplasticity is likely inherent with higher vulnerability to environmental and internal insults. In line with this notion, cellular deficits and structural changes in the associative cortical and limbic areas are associated with some neurological or neuropsychiatric disorders (e.g., mode disorders, schizophrenia, epilepsy, Alzheimer\'s disease) in the adolescent, adult and aged human populations.
3. Neuroplasticity is a complex process involving structural modulations at synaptic, neuronal and circuitry/pathway levels. Of particular interest, formation of new neurons in the adult brain has been recently recognized as a key substrate for neuroplasticity and cognition. For instance, adult neurogenesis in the forebrain subventricular and subgranular zones (SVZ, SGZ) appears to be essential for olfaction and hippocampus-dependent learning and memory in rodents.
4. Newly-generated neurons in the SVZ and SGZ express a set of immature neuronal markers including doublecortin (DCX+) and polysialylated neural cell adhesion molecule (PSA-NCAM). Concurrent with morphological development, these new neurons differentiate through a correlated process of downregulation of the above-mentioned immature markers and upregulation of common (e.g., neuron-specific nuclear protein, NeuN) or specific terminal neuronal markers, thus eventually become fully integrated into functional neuronal circuitries in the adult brain.
5. Besides the SVZ/SGZ, a novel population of DCX+ cells coexpressing other immature neuronal markers has been recently characterized in the cerebral cortex of guinea pigs, rabbits, cats and primates from young to mid-age adulthood. These DCX+ cells are more common in the associative relative to primary cortical areas, and appear to develop into interneuron subgroups in guinea pigs and cats. We show here in rhesus monkeys that DCX+ cells persist into advanced ages in the associative frontal and temporal lobe cortex and amygdala, even in very old animals with substantial cerebral amyloid pathology. In contrast, DCX+ cells are rare in the hippocampus by midage.
6. Recent studies by our group and others have gathered substantial information on cortical DCX+ cells or alike in various mammalian species, which now allows a comparison between these novel cortical cells and the conventional adult-born neuronal populations in SVZ and SGZ. Taking the newly-generated SGZ immature neuronal population as an example, we attempted to update relevant features of cortical and hippocampal DCX+ cells. Similar properties of these two populations include that both groups: (1) express the very same set of immature neuronal markers including DCX, PSA-NCAM, neuron-specific β-tubulin-III (TuJ1) and Hu; (2) exhibit a diverse variation in somal size and shape, nuclear appearance in bisbenzimide stain and complexity of neuritic processes, all suggesting cell growth and morphological maturation; (3) display a pattern of transient expression of immature neuronal markers that overlap partially with the emergence of mature neuronal markers but correlate with morphological development; (4) may arrange as tightly-apposed cell clusters and/or as migratory chains, suggestive of certain neuroblastlike behavior or property; (5) reduce in number with age in all studied mammals; (6) might be modulated by some antidepressants at least according to rodent studies.
7. The most confusing issue regarding cortical DCX+ cells is whether they are generated prenatally, postnatally or even throughout adult life. Several groups report a low rate of BrdU incorporation into DCX+ cells in the piriform or temporal lobe cortex in small laboratory rodents and primates. In contrast, others suggest that this population of cortical cells is produced prenatally. With this regard, the current finding of persistent occurrence of DCX+ cells in the cortex and amygdale virtually to the end of life in non-human primates is very puzzling.
8. The arrangement of cortical DCX+ cells as extended tangential migratory chains and expanded/distorted migratory apparatus in layers I/II in mid-age and aged primates is of interest. Cortical DCX+ cells in young adult cats and monkeys appear to mostly migrate from layer II to deeper layers. Thus, the increased tendency of tangential cell migration in older animals might implicate certain type of age-related alteration in the distribution of cortical DCX+ cells.
9. Previous studies suggest that the radial migration of newly-generated hippocampal neurons might be impaired in aged rodents, dogs and monkeys. Thus, DCX+ cells in aged dentate gyrus tend to retain at the SGZ with their long somal diameter parallel to the GCL. In contract, dentate DCX+ cells in younger animals migrate across the GCL during their morphological maturation. Thus, the extended tangential migration and distorted clusterization of DCX+ cells around layers I and II in aged monkeys could potentially reflect certain deficit of migration or dispersion/descending of these cells in the cortex.
10. Little is currently known about the functional implication for a life-long presence of putative immature and developing neurons in mammalian associative cerebral cortex and amygdala. Giving their potential GABAergic fate, it seems plausible that these novel cells might be involved in interneuron plasticity under physiological conditions, and perhaps interneuron dysfunction under certain disease conditions. Recent studies suggest that interneuron deficits might relate to the etiology of certain neuropsychiatric diseases. It appears that bipolar disorder, major depression and schizophrenia are associated with reduced GABAergic interneurons and/or altered inhibitory neurotransmission in the prefrontal cortex.
11. A role for impaired neurogenesis in cognitive decline during aging and in Alzheimer\'s disease has been lately proposed according to studies of adult-born neuronal populations in the hippocampus and SVZ. We observe diminished DCX+ subgranular cells in mid-age and aged monkeys, consistent with other reports that hippocampal neurogenesis is dramatically reduced around mid-age in most mammals. Our current study however reveals a remarkably intriguing fact that putative immature cortical neurons persist into very old age in non-human primates, in sharp contrast to a great loss of hippocampal neurogenesis well before the onset of senescence. Thus, there exists an extended time window for a potential involvement of cortical and amygdalar DCX+ cells in aging-related changes in neuronal plasticity and perhaps cognitive decline in non-human primates.
General Comments and Discussion
===============================
Rockland
--------
Comment on point 5.
Can you make any finer demarcation of where in the "associative frontal and temporal lobe cortex" the DCX+ cells can be found? In particular, both lobes have neocortical and limboid components (respectively, dorsolateral frontal and lateral temporal vs. orbitofrontal and entorhinal areas). Any correlation or trend?
Yan
---
Reply.
We did not correlate the pattern of DCX+ cells with Brodmann areas in part because there are age-related changes in area distribution. In addition, the distribution appeared to be more regional than area-specific. In general, we found that labeled cells are more numerous ventrally across the anterior-posterior cerebral dimension, which is particularly clear in young adults. In aged monkeys, DCX+ cells remain fairly numerous in temporal lobe areas, both the paleo- and neo-cortical areas. In addition, a small number of these cells also persist in the medial and ventral prefrontal cortex including the medial and lateral orbital gyri, as well as around the ventral portion of the occipital lobe cortex.
| {
"pile_set_name": "PubMed Central"
} |
1. Introduction {#sec1}
===============
Although gastric cancer has been decreasing in terms of both incidence and mortality in most developed countries in recent decades, it still causes substantial disease burden \[[@B1]\]. Over 90% of people with early stage gastric cancer survive for five years or longer after surgical treatment \[[@B2], [@B3]\], whereas the prognosis of those with advanced gastric cancer is poor, with a 5-year survival rate of about 20% in stage III and 7% in stage IV patients, respectively \[[@B4]\].
Risk stratification is important in informing treatment decision, resource allocation, and patient recruitment in clinical trials \[[@B5]\]. The tumor-node-metastasis (TNM) staging system is widely used for risk stratification \[[@B4]\]. Nevertheless, even patients with the same TNM stage may have significantly different responses to treatment and clinical outcomes \[[@B6]\], suggesting that more accurate stratification could be beneficial \[[@B7]\]. Previous studies have identified numerous other prognostic factors for gastric cancer, which can be broadly divided into four categories: patient-, tumor status-, biomarker-, and treatment-related factors \[[@B8]--[@B10]\]. As no single prognostic factor suffices for satisfactory risk stratification, much interest has been raised in developing multivariable prognostic models, which quantitatively combine two or more prognostic factors \[[@B11], [@B12]\].
The American Joint Committee on Cancer has increasingly recognized the importance of incorporating prognostic models into practice to achieve personalized cancer management \[[@B13]\]. However, despite plenty of prognostic models published in the literature, very few have been adopted in clinical use. We carried out this systematic review to summarize the characteristics of existing models for predicting overall survival of patients with primary gastric cancer, with an emphasis on identifying potential problems in model development and validation and informing future research.
2. Methods {#sec2}
==========
This systematic review was conducted following the *CHARMS checklist* \[[@B14]\], which was developed to guide data extraction and critical appraisal in systematic reviews of prediction model studies.
2.1. Eligibility Criteria {#sec2.1}
-------------------------
We included primary studies that reported the development and/or validation of prognostic models predicting overall survival of patients with primary gastric cancer. A prognostic model was defined as a combination of at least two prognostic factors, based on multivariable analysis, to estimate individual risk of a specific outcome, presented as regression formula, nomogram, or in a simplified form, such as risk score \[[@B15]--[@B17]\]. We only included prognostic models for predicting overall survival or all-cause death, excluding other outcomes, such as progression-free survival after treatment or disease-specific survival.
We excluded studies if (1) they enrolled patients with other types of cancer and the information on gastric cancer model could not be separately extracted; (2) they used short-term mortality (for example, death within 30 days after surgery) as the outcome; or (3) they validated prognostic models that were not initially developed for gastric cancer patients.
2.2. Literature Search {#sec2.2}
----------------------
We searched MEDLINE and EMBASE to identify all relevant studies from their inceptions through 30 May 2018, using the following three groups of terms: (1) *gastric tumor^∗^* OR *gastric tumour^∗^* OR *gastric cancer^∗^* OR *gastric neoplasm^∗^* OR *gastric carcinoma^∗^* OR *stomach tumor^∗^* OR *stomach tumour^∗^* OR *stomach cancer^∗^* OR *stomach neoplasm^∗^* OR *stomach carcinoma^∗^* OR *Siewert* OR *esophagogastric* OR *EGJ*, and (2) *prognos^∗^* OR *survival* OR *death* OR *mortality*, and (3) *scor^∗^* OR *model^∗^* OR *index^∗^* OR *nomogram^∗^* OR *rule^∗^* OR *predict^∗^* OR *indices* OR *formula^∗^* OR *equation^∗^* OR *algorithm*^∗^ \[[@B18], [@B19]\]. The search terms were limited to title and abstract. We also manually checked the reference lists of eligible studies to identify extra relevant studies.
2.3. Study Selection {#sec2.3}
--------------------
After excluding duplicates, we screened all titles and abstracts to identify potentially eligible studies and then retrieved their full texts for further examination. Final eligibility was confirmed by two authors (QF and ZYY). Discrepancy was resolved by discussion with a third author (JLT).
2.4. Data Extraction and Quality Assessment {#sec2.4}
-------------------------------------------
The data extraction form was designed according to the *CHARMS checklist* \[[@B14]\], supplemented with other items obtained from methodological guidance studies and previous systematic reviews \[[@B15], [@B16]\]. Briefly, the following information was extracted for each model development and external validation: publication year, country, data source, patient characteristics, length of follow-up, outcome, candidate predictors, training sample size, number of deaths, missing data, model development/validation methods, final predictors, predictive performance, and presentation formats.
In this study, candidate predictor refers to the potential predictors (and their functional forms, if any) that are selected to be examined in multivariable analysis but might or might not be included in final model. Final predictor refers to the predictors that are included in final models. Event per variable (EPV) is the ratio between the number of events and the number of candidate predictors, which is a rule of thumb to empirically evaluate the power of regression analysis, with a value of 10 or higher recommended to avoid potential overfitting \[[@B20], [@B21]\]. If one study included multiple model developments and validations, we extracted relevant information for each model development and validation separately. Data extraction was undertaken by two authors (QF and ZYY), and any uncertainty in data extraction was resolved by discussion with a third author (JLT).
We used a preliminary version of the *Prediction Model Study Risk Of Bias Assessment Tool* (PROBAST) to evaluate the methodological quality of each model development and validation \[[@B22]\]. This tool evaluated the levels of risk of bias in five domains: participant selection, definition and measurement of predictors, definition and measurement of outcome, sample size and participant flow, and statistical analysis. Each domain was rated as high, low, or unclear risk of bias. Overall judgment of risk of bias was derived from the judgments on all domains: low risk if all domains had low risk of bias, high risk if any domain had high risk of bias, otherwise unclear risk.
2.5. Statistical Analysis {#sec2.5}
-------------------------
We mainly used descriptive statistics to summarize the characteristics of model developments and validations. All final predictors were assigned into one of the four categories: patient, tumor status, biomarker, and treatment. We counted the frequency of each final predictor being included in models. We compared models that have been externally validated with those that have not, regarding their characteristics (training sample size, number of events, number of final predictors, EPV, c statistic, etc.).
3. Results {#sec3}
==========
In total, 16334 citations were identified and 99 eligible publications (Supplementary [](#supplementary-material-1){ref-type="supplementary-material"}) were included in this review ([Figure 1](#fig1){ref-type="fig"}). Of the 99 publications, 75 performed model development only, 9 performed external validation only, and 15 performed both model development and external validation. One-hundred and one distinct models were extracted from 90 studies. Thirty-two external validations for 20 models were extracted from 24 studies.
3.1. Model Development {#sec3.1}
----------------------
### 3.1.1. Basic Characteristics of Model Development {#sec3.1.1}
Characteristics of the 101 model developments are summarized in [Table 1](#tab1){ref-type="table"}. Three models were published before 2000, and the number has rapidly risen by 2- and 30-fold during 2001--2010 and 2011--2018, respectively ([Figure 2](#fig2){ref-type="fig"}). Most models (76/101) originated from East Asian populations, which was not surprising, given the fact that this region has the highest incidence and prevalence of gastric cancer.
Patient characteristics varied substantially across studies in terms of age, sex, tumor status, and treatment ([Table 1](#tab1){ref-type="table"}). The median proportion of male patients was 67.8% (range 30.9% to 80.3%), and the median age was 60 years (range 51 to 70). Thirty-six models were developed from gastric cancer patients at TNM stages I--III only and 17 models from TNM stage IV only. Most models (71/101) recruited only patients who had received surgery.
### 3.1.2. Summary of Model Development Methods {#sec3.1.2}
Most models (91/101) were developed by retrospective cohort studies based on routine clinical data, which were not collected for the purpose of model development. To deal with missing data, which is a common problem with routine clinical data, seven models adopted the multiple imputation approach, while the remaining 94 models conducted complete-case analysis. The medians of total sample size and number of events included in analysis were 360 (range 29 to 15320) and 193 (range 14 to 9560), respectively. The starting point of follow-up for overall survival varied across models. Seven studies did not report their candidate predictors clearly. EPV can be estimated in 83 model developments, with the median of 25.1 (range 0.2 to 1481.3). A favored EPV (\>10) was achieved in 64 model developments.
As for selection of candidate predictors, 63 models used univariable analysis, 30 models prespecified candidate predictors based on clinical knowledge, five models employed a combination of the two, and the other three models did not specify this issue clearly. Various statistical models were used for prognostic model development, with Cox proportional hazard model being the most popular one (used in 90 models). Sixty-eight models used a stepwise approach in multivariable analyses to select final predictors. Statistical assumptions of the methods were examined and reported in only nine studies.
The median number of final predictors was 5 (range 2 to 10). In total, 180 different predictors were included, of which 21 were patient-related, 34 tumor-related, 116 biomarkers, and 9 treatment-related ([Table 2](#tab2){ref-type="table"}; more details in the Supplementary [](#supplementary-material-1){ref-type="supplementary-material"}). The most consistent predictors for overall survival (included by more than 10 models) were age at diagnosis, sex, lymph node involvement, metastasis, invasion depth, TNM stage, tumor size, tumor site, differentiation, and histologic type, all of which were patient- and tumor-related.
The models were mostly presented in simplified forms, such as risk score (35/101) and nomogram (47/101). For model performance, 33 and 55 models did not report discrimination and calibration, respectively. Among the studies reporting relevant information, the median c statistic for discrimination was 0.748 (range 0.627 to 0.961). Forty-two models were compared with TNM stage alone regarding c statistic value, and all models outperformed TNM stage, with a median increase in c statistic value of 0.050 (range 0.015 to 0.180).
3.2. External Validation {#sec3.2}
------------------------
There were 32 external validations for 20 distinct models, with 22 of them reporting in the same study as the model development. The majority (81/101) of models developed have not been externally validated. Five models were externally validated more than once, and two models \[[@B23], [@B24]\] more than five times. The characteristics of training datasets and validation datasets were compared in 19 external validations. Five validations did not assess discrimination, and 24 did not assess calibration ([Table 3](#tab3){ref-type="table"}). The median (range) of c statistic for discrimination was 0.770 (0.576 to 0.868). The difference in c statistic between development and validation ranged from −0.044 to 0.290 with a median of 0.029.
3.3. Quality Assessment {#sec3.3}
-----------------------
The model developments had either high (97/101) or unclear (4/101) risk of bias, and all model validations had high risk of bias. Ninety-one developments and 31 validations had high risk of bias in participant selection, mainly due to retrospective data collection. Forty-six developments and six validations had high risk of bias in sample size and participant flow, mainly due to small sample size and inappropriate method of dealing with missing data. Eighty-three developments and 13 validations had high risk of bias in analysis, mainly due to inappropriate method dealing with continuous variable, lack of statistical assumption examination, lack of overfitting detection, and insufficient reporting of model performance (Supplementary [](#supplementary-material-1){ref-type="supplementary-material"}).
3.4. Comparison of Externally Validated Models with Not-Validated Models {#sec3.4}
------------------------------------------------------------------------
When comparing development characteristics of externally validated models with not-validated models, we found that the validated models tended to have larger training sample size, bigger number of events, higher EPV, older age, and higher c statistic value, while the differences in number of final predictors seemed to be insignificant ([Table 4](#tab4){ref-type="table"}). Multivariable logistic regression showed that models were more likely to be externally validated if they were developed with bigger training sample and higher c statistic.
4. Discussion {#sec4}
=============
This systematic review identified 101 models predicting overall survival of gastric cancer patients, with 20 of them externally validated.
van den Boorn et al. published a systematic review \[[@B25]\] summarizing prediction models for esophageal and/or gastric cancer patients, but the present study substantially differed from it and has its own value. Firstly, this study focused on prognostic models designed for primary gastric cancer patients only, whereas van den Boorn et al. included models for both gastric and esophageal cancers. Secondly, we identified 40 more newly published models and provided a more comprehensive picture of their characteristics. Thirdly, van den Boorn et al. focused on models\' performance and clinical application, but our study emphasized more on the methodology of model development and validation.
We observed substantial heterogeneity regarding patient types in model development. Many studies developed prognostic models for specific subgroups of gastric cancer patients (e.g., those with a certain tumor stage and those receiving certain treatment) to make their models unique from those developed by others. This strategy of patient restriction may limit the model\'s generalizability, increasing uncertainty when applying it to other types of patients. In addition, an underlying assumption of restricting a model development to specific patient subgroups is that there exists effect modification or interaction between the restriction variable(s) and the main prognostic factors of interest. However, most studies did not check this assumption.
We also identified common statistical shortcomings that may cause bias in model development. Firstly, most models were developed from routinely collected clinical data, in which missing data was common. Most models simply performed complete-case analysis by excluding the patients with missing data. However, complete-case analysis works well only when missing data occurs completely at random, which is rare in reality \[[@B26]\]. To address this issue, multiple imputation has been recommended \[[@B27], [@B28]\]. This method has been employed in prediction model studies of other diseases \[[@B29], [@B30]\] but has not been applied in gastric cancer until 2017 \[[@B31]--[@B33]\].
Secondly, univariable analysis was commonly used to select candidate predictors. However, this data-driven method has high risk of wrongly excluding a potentially significant variable or including a potentially insignificant variable when its association with the outcome is confounded by others \[[@B34], [@B35]\]. The bootstrap resampling method could be used to increase the stability of variable selection, by selecting variables with high inclusion frequency across multiple bootstrapping samples \[[@B36]\]. Moreover, variable selection should take into account clinical or biological knowledge and combine results of multivariable analysis with sensitivity analysis for cautious conclusion \[[@B34], [@B35]\].
Thirdly, the majority of studies did not examine the assumptions of the statistical models, such as hazard-proportionality for Cox regression and linearity assumption for continuous variables. The results of examination are important in selecting appropriate statistical models and determining predictors\' functional forms \[[@B37], [@B38]\]. Cox regression is the most commonly used model for survival data, but the underlying proportional-hazard assumption was often violated, in which case parametric survival models could be considered \[[@B39], [@B40]\]. In addition, algorithms from machine learning (e.g., neural network) are less strict with assumptions and have been used more and more in prognostic model development.
Fourthly, detection of model overfitting was neglected in most studies. Overfitting is more likely to occur in studies with small sample size and many predictors, resulting in overestimation of risk in high-risk patients and underestimation low-risk ones \[[@B41]\]. This issue can be detected by internal validation with cross-validation or bootstrap resampling and handled with statistical methods, such as shrinkage and penalized regression \[[@B42], [@B43]\].
Underreporting is another common problem. Outcome definitions, variable selection method, assessment of discrimination, and calibration measures were not reported in 34%, 23%, 33%, and 55% of model developments, respectively. Because there is no standard method for model development and multiple feasible options exist at each step in model development, underreporting of methodological details may cause difficulty in assessing a model\'s internal and external validity. Future studies are suggested to follow relevant reporting guidelines such as the *TRIPOD* \[[@B44], [@B45]\].
In this study, we found 55 predictors that were included more than twice in models, 10 of which (age at diagnosis, sex, lymph node involvement, metastasis, invasion depth, TNM stage, tumor size, tumor site, differentiation, and histologic type) were included more than 10 times. This can be regarded as indirect evidence for their predictive power in gastric cancer prognosis. Direct evidence, i.e., magnitude of their association with the outcome, such as hazard ratio, can be found in previous systematic reviews and meta-analyses \[[@B46], [@B47]\]. Therefore, we suggest that future model development, if necessary, to build upon these existing evidence. Though a large number of biomarkers were studied, their frequency of being included in final models was very low (mostly once or twice).
Prognostic models can be used to inform patients of their prognosis and assist clinical decision-making. However, despite the much effort devoted into model development so far, very few prediction models other than the TNM stage system have been adopted in clinical practice. Apart from the problems discussed above, other reasons may include the complexity of those models as compared with TNM system and lack of external validation and clinical impact studies \[[@B48]\]. External validation evaluates a model\'s predictive performance in local setting and updates model if necessary. An impact study evaluates the effects of a prognostic model on clinical decision-making, behavioral change, subsequent health outcomes of individuals, and the cost-effectiveness of applying the model, with the optimal design being a clustered randomized controlled trial \[[@B49], [@B50]\]. The gap between prognostic model and clinical decision rule is another big concern. Prognostic models compute the probability of an event on a continuous scale or risk scores on an ordinal scale, whereas clinical decision is a binary choice regarding whether to use an intervention or not. Unfortunately, the translation of risk estimates derived from existing prognostic models to clinical decisions is much less investigated \[[@B50]\].
Therefore, future research should try to avoid repeatedly developing new models for similar predictive purposes with small sample size and high risk of bias. Instead, more emphasis should be put on improving methodological quality of model development, validating and updating models for use within their own setting \[[@B51]\], translating model prediction into clinical decision rules \[[@B50]\], and assessing the models\' clinical impact \[[@B52], [@B53]\].
5. Conclusion {#sec5}
=============
This systematic review identified 101 prognostic models for predicting overall survival of patients with gastric cancer, which were limited by high risk of bias, methodological shortcomings, insufficient reporting, and lack of external validation and clinical impact assessment. Future prognostic model research should pay more attention to their methodological and reporting quality, and more importantly, emphasized more on external validation and impact studies to assess the models\' effectiveness in improving clinical outcomes.
Conflicts of Interest
=====================
The authors declare that they have no conflicts of interest.
Authors\' Contributions
=======================
JLT and ZYY conceived the study. QF and ZYY did literature search and data extraction. QF analyzed the data. QF, ZYY, MTM, and SI interpreted the results. QF wrote the draft manuscript. All authors critically reviewed the manuscript.
Supplementary Materials {#supplementary-material-1}
=======================
######
Supplementary file 1: full list of included studies. Supplementary file 2: model presentation and the predictors included in final models. Supplementary file 3: quality assessment of model development and validation.
######
Click here for additional data file.
{#fig1}
{#fig2}
######
Characteristics of 101 model developments.
Model developments (*n* = 101)
------------------------------------------------ --------------------------------
*Study characteristics*
Publication year
Before 2000 3
2001--2010 7
2011--2018 91
Study location
East Asia (China/Japan/Korea) 76
Non-Asian 25
Data source
Clinical data/retrospective cohort 91
Prospective cohort 7
Randomized controlled trial 3
*Patient characteristics*
Male% (4/101 missing) 67.6 (30.9, 80.3)^a^
Age (5/101 missing)
Median (min, max) of mean 60.0 (51.0, 70.0)^a^
Tumor TNM stage
All 46
I--III 36
IV 17
No information 2
Gastrectomy
No restriction 28
Only patients with gastrectomy 71
Only patients without gastrectomy 2
*Model development*
Sample size (training set) (14/101 missing) 360 (29, 15320)^a^
Number of events 193 (14, 9560)^a^
Event per variable (18/101 missing) 25.1 (0.2, 1481.3)^a^
Length of follow-up (month) (53/101 missing) 44.0 (6.7, 111.6)^a^
Start of outcome follow-up
From diagnosis 3
From surgery 49
From other time points^b^ 15
Unclear 34
Candidate selection methods
Prespecification 30
Univariable analysis 63
Prespecification + univariable analysis 5
Unclear 3
Statistical model
Cox proportional hazard regression 90
Others^c^ 11
Final predictor selection
Full model 10
Stepwise (including forward and backward) 68
Unclear 23
Statistical assumptions ever checked 9
Number of final predictors 5 (2, 53)^a^
Formats of presentations
Score 35
Nomogram 47
Equation 9
Others (decision tree and neural network) 4
No 6
Predictive performance
Discrimination
AUC/c statistic 67
Others 1
No 33
Calibration
Calibration plot 45
Hosmer--Lemeshow test 3
No 55
Model validation
Internal 30
External 21
No 54
^a^Median (min, max). ^b^Initiation of chemotherapy (*n* = 10), metastasis (*n* = 3), and randomization (*n* = 2). ^c^CART, Cox Lasso, discrimination analysis, Weibull model, neural network, and logistic model. AUC: area under curve.
######
Final predictors included in the models.
Category Number of predictors Number of predictors selected multiple times Predictors selected multiple times^a^
---------------- ---------------------- ---------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Patient 21 9 Age, sex, ethnicity, performance score, year of diagnosis, family history, smoking, residency, and addiction to opium
Disease status 34 21 T stage, N stage, TNM stage, tumor site, tumor size, differentiation, metastasis, histologic type, Lauren type, LN ratio, lymphovascular invasion, bone metastasis, Borrmann type, liver metastasis, number of metastasis sites, lung metastasis, number of examined LN, metastasis LN, perineural invasion, LODDS, and TTP after chemotherapy
Biomarker 116 19 CEA, NLR, ALP, albumin, bilirubin, CA199, Hb, CES1, IS, LDH, LNR:ART, lymphocyte count, MGAT5, mGPS, NPTM, platelet, sodium, TNFRSF11A, and WBC
Treatment 9 6 Chemotherapy, gastrectomy, lymphedenectomy, resection margin, extent of resection, and radiotherapy
^a^The table lists only the predictors that have been included more than once. LN: lymph node. LODDS: log odds of positive LN. CEA: carcinoembryonic antigen. NLR: neutrophil/lymphocyte ratio. ALP: alkaline phosphatase. Hb: hemoglobin. MGAT5: *β*1, 6-N-acetylglucosaminyltransferase-5. mGPS: modified Glasgow Prognostic Score. CA199: cancer antigen 199. NPTM: number of positive tumor markers (cancer antigen 125, CA199, CEA). WBC: white blood cell. TTP: time to progression.
######
Characteristics of model external validations.
External validations (*n* = 32)
---------------------------------------------- ---------------------------------
Data source
Clinical 27
Prospective cohort 3
Randomized controlled trial 2
Validated in
The original development study 22
Independent study 10
Sample size for validation 610 (71, 26019)^a^
Discrimination
AUC/c statistic 25
Others 2
No 5
Calibration
Calibration plot 6
Hosmer--Lemeshow test 2
Calibration in large 1
No 24
Compared validation set with development set 19
^a^Median (min, max). AUC: area under curve.
######
Characteristics of models with external validation and those without.
Externally validated models (*n* = 20) mean (SD) Not externally validated models (*n* = 81) mean (SD) *P* value
-------------------------------- -------------------------------------------------- ------------------------------------------------------ -----------
Training sample size 3902.55 (5777.62) 634.17 (926.30) 0.021
Number of events 2825.12 (4069.04) 344.75 (613.35) 0.028
Number of candidate predictors 75.80 (204.53) 12.83 (28.26) 0.185
EPV 364.21 (542.04) 44.70 (82.97) 0.033
Number of final predictors 6.65 (3.44) 5.94 (6.08) 0.490
Length of follow-up (month) 64.24 (29.65) 43.76 (19.15) 0.122
Age 63.00 (4.99) 59.87 (3.39) 0.034
Male% 64.92 (4.10) 67.29 (6.54) 0.053
c statistic 0.80 (0.06) 0.75 (0.07) 0.042
EPV: event per variable.
[^1]: Academic Editor: Tsutomu Nishida
| {
"pile_set_name": "PubMed Central"
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Introduction
============
The cereal prolamins are broadly classified into cysteine (Cys)-poor and Cys-rich species ([@B33]). In wheat and barley, most prolamins are Cys-rich species (gliadins and glutenins in wheat and hordeins in barley) and are deposited in protein storage vacuoles (PSVs) in the form of very large aggregates ([@B2], [@B20]). In contrast, maize endosperm contains relatively large amounts of Cys-poor prolamins, α-zeins, which form single spherical inclusion granules surrounded by the endoplasmic reticulum (ER) membrane ([@B19]). These two major cereals exemplify the two modes of intracellular deposition of seed storage proteins.
The rice prolamins account for about 18--20% of the total seed protein content ([@B26], [@B21]). They consist of 60% Cys-rich prolamins and 40% Cys-poor prolamins ([@B26], [@B12]). The prolamin fraction of the *japonica* rice variety Kinmaze consists of 10, 13 (indicated as 13b in [@B26]), 14 (indicated as 13a in [@B26]) and 16 kDa molecular species. [@B26]) demonstrated that the 10, 14 and 16 kDa prolamins are Cys-rich species, while the 13 kDa prolamin is a Cys-poor species.
Based on the primary sequences derived from cDNA sequences, the four prolamins are encoded by three distinct classes of genes ([@B10], [@B11], [@B23], [@B22], [@B35], [@B37], [@B36], [@B38]). The Cys-poor 13 kDa (CysP13) and Cys-rich 14 kDa (CysR14) and 16 kDa prolamins (CysR16) share considerable homology (∼70%) and differ only in that the former species lack cysteine residues. The 10 kDa prolamins (CysR10) share minimal sequence homology with the other two classes and are characterized by their high content of methionine (20%) and cysteine (10%) residues ([@B23]). Both Cys-rich prolamin classes contain the three A, B and C cysteine motifs which are typically observed in cereal Cys-rich prolamins ([@B32]).
Two types of protein bodies (PBs), called PB-I and PB-II, are observed in rice ([@B1], [@B40]). Prolamins are accumulated in PB-Is as intracisternal protein granules, while glutelins are accumulated in PB-IIs derived from the PSV ([@B40], [@B26]). PB-I is spherical with a diameter of 1--2 μm and surrounded by rough ER membranes with attached polysomes ([@B1], [@B40], [@B25], [@B24]). When viewed by electron microscopy, the structure of PB-I consists of an electron-dense center core surrounded by electron-lucent layers, which are interspersed with concentric rings of varying electron density ([@B1], [@B40], [@B13], [@B26]). Similar PB structures are also observed in *Sorghum* ([@B34]) and *Setaria* ([@B30]). It is not known how the electron-dense core structure is formed and how prolamin polypeptides assemble to form a tightly compact, spherical intracisternal inclusion granule within the ER.
As first observed for the maize zeins, the rice prolamins are synthesized on rough ER membranes and are co-translationally translocated into the ER lumen ([@B44]). In maize, the various zein classes are not randomly distributed within the PBs; the Cys-rich β-zeins and γ-zeins are localized to the PB periphery, which surrounds the centrally located Cys-poor α-zeins and Cys-rich δ-zeins ([@B19], [@B5]). PB formation is initiated by the accumulation of Cys-rich γ-zeins and β-zeins to give a small electron-dense granule, whereupon accumulation of Cys-poor α-zeins displaces the β- and γ-zeins from the center to the periphery ([@B19]). These cytochemical results suggest that Cys-rich β- and γ-zeins play an important role for initiation of PB formation and the sequestration of α-zeins within the PBs in maize endosperm.
Kumamaru et al. ([@B15], [@B16]) characterized rice mutants for storage protein and isolated three prolamin mutant classes. The varied prolamin polypeptide composition was reflected in the morphology of their prolamin PBs ([@B27]). Endosperm storage protein mutants *esp1* and *Esp4* are characterized by low levels of CysP13, with the latter also containing elevated levels of Cys-rich prolamins. On the other hands, the *esp3* mutant contains low levels of CysR10, CysR14 and CysR16 ([@B15], [@B16], [@B27]).
In order to identify the processes responsible for PB-I formation, we evaluated the temporal accumulation of CysR10 and CysP13 during rice seed development and their spatial distributions within the PB using immunocytochemical and immunofluorescence analysis of the wild type, genetic mutants for prolamin accumulation and CysR10 RNA interference (RNAi) lines. Our results indicate that these prolamin species are asymmetrically distributed within PB-I and that CysR10 is essential for formation of the tightly compact, spherical shaped structure of PB-I.
Results
=======
The distribution of CysR10 and CysP13 in PB-I
---------------------------------------------
PB-I exhibits a non-uniform pattern of electron density when stained with lead acetate and uranyl acetate ([@B40]). The intracisternal inclusion granule contains a dark staining center core surrounded by one or more layers of increasing lighter density extending from the core to the periphery ([Fig. 1](#F1){ref-type="fig"}A, C). In many instances the electron-lucent peripheral region is interspersed with non-continuous, electron-dense concentric rings. Fig. 1Distribution of CysR10 and CysP13 in PB-Is of developing rice endosperm at 3 WAF. (A) Electron micrographs of PB-Is. (B, C) Immunoelectron microscopy of PB-Is which were labelled with anti-CysR10 antibody (15 nm gold particles) and anti-CysP13 antibody (5 nm gold particles). (C) Enlarged image of (B). Arrows in C show the 5 nm gold particles (CysP13). The double arrow in C denotes the middle layer containing CysR10 and CysP13. Bars in (A, B): 1 μm, bars in (C): 0.5 μm. (D--F) Immunofluorescence microscopy of PB-Is from developing rice endosperm at 3 WAF. (D) The distribution of CysR10 as visualized using anti-CysR10 and rhodamine-conjugated secondary antibodies. (E) The localization of CysP13 using anti-CysP13 and FITC-conjugated secondary antibodies. (F) Merged image of D and E. Bars in (D--F): 10 μm.
To investigate the spatial relationship of prolamin polypeptides within PB-I, immunoelectron microscopy was carried out using thin sections taken from developing rice endosperm at 3 weeks after flowering (WAF). [Fig. 1](#F1){ref-type="fig"}B and [C](#F1){ref-type="fig"} show the distribution of CysR10 labeled with secondary antibodies conjugated with 15 nm gold particles and CysP13 visualized with 5 nm gold particles. The CysR10 was concentrated at the interior while the CysP13 was distributed to the outer electron-lucent peripheral layer ([Fig. 1](#F1){ref-type="fig"}B, C). Additionally, a middle layer containing both CysR10 and the CysP13 was evident ([Fig. 1](#F1){ref-type="fig"}C, double arrow).
The spatial distribution of these prolamins within the PBs was confirmed by immunofluorescence light microscopy. CysR10 was visualized as small particles when labeled by anti-CysR10 followed by rhodamine-conjugated secondary antibodies ([Fig. 1](#F1){ref-type="fig"}D). The distribution of CysR10 co-localized with CysP13, which was visualized with fluorescein isothiocyanate (FITC)-conjugated secondary antibodies ([Fig. 1](#F1){ref-type="fig"}E, F arrows). CysR10 was observed at the center of the PBs, whereas the distribution of CysP13 was more varied. In some PBs, CysP13 appeared to be distributed uniformly throughout the PB, while in others, CysP13 was found either at lower levels or was entirely absent from the center core region ([Fig. 1](#F1){ref-type="fig"}E, F).
To examine the spatial relationship between the prolamins in more detail, serial thin sections from developing endosperm at 3 WAF were obtained and analyzed by immunofluorescence microscopy. In [Fig. 2](#F2){ref-type="fig"} image 1, a PB sectioned near its periphery is labeled with the CysP13 antibody but not by the CysR10 antibody (arrow). The succeeding series of images 2--4 show cross-sections of this same PB taken from the periphery towards the center. In image 2, the CysP13 was distributed mainly around the periphery with the center of the PB showing a trace of the CysR10. The next image ([Fig. 2](#F2){ref-type="fig"} image 3), which depicts a section near the center of the PB, shows a non-uniformed distribution of CysP13 with lower levels at the center region, which coincided with the location of CysR10, and higher amounts at the peripheral areas. Image 4 shows a section at the center of the PB where the center contains an 'apparent cavity' when viewed for CysP13 but an area filled with CysR10. These observations are consistent with the distribution of these prolamin polypeptides in PB-I as viewed by immunocytochemistry at the electron microscope level. PB-I is composed of a center core of CysR10, surrounded by a middle layer of a mixture of CysR10 and CysP13, and then followed by a peripheral layer containing CysP13 devoid of CysR10. Fig. 2Immunofluorescence microscopy of serial sections of PB-Is obtained from developing rice endosperm at 3 WAF. The numbers at the left of the image denote the order of the sections. The PBs in each section were labeled with antibodies against CysR10 and CysP13. The CysR10--antibody interactions were visualized with rhodamine-conjugated secondary antibodies (A--D), while those for CysP13s were visualized with FITC-conjugated secondary antibodies (E--H). (I--L) Merged images. The arrow indicates the location of the same PB in the serial sections. Bars: 4 μm.
Developmental changes in the prolamin composition of the interior PB-I
----------------------------------------------------------------------
The temporal accumulation patterns of the various prolamin classes were examined by SDS--PAGE followed by immunoblot analysis. The accumulation of the CysR10 polypeptide was first detected at 4 days after flowering (DAF) and attained maximum steady-state levels (on a seed mg basis) at 6 DAF ([Fig. 3](#F3){ref-type="fig"}, [Supplementary Fig. S1](http://pcp.oxfordjournals.org/cgi/content/full/pcr053/DC1)). Significant levels of CysR14 and CysR16 were initially detected at 6 DAF and steadily increased as the seed developed ([Fig. 3](#F3){ref-type="fig"}, [Supplementary Fig. S1](http://pcp.oxfordjournals.org/cgi/content/full/pcr053/DC1)). Only small amounts of CysP13 were detected in developing seed at 6--8 DAF although their levels increased prominently from 9 to 14 DAF. These results show that the rice prolamins are differentially accumulated during seed development. CysR10 was initially detected as early as 4 DAF, followed closely by the accumulation of CysR14 and CysR16 polypeptides. In turn, CysP13 was the last prolamin to be accumulated during seed development. The results of shorter and longer exposures of the membranes in all the immunoblotting analyses ([Supplementary Fig. S1](http://pcp.oxfordjournals.org/cgi/content/full/pcr053/DC1)) were essentially the same as those shown in [Fig. 3](#F3){ref-type="fig"}. Fig. 3Immunoblot analysis of the four prolamin species during rice seed development. DAF are shown in the top of the figure, and the illustrations depict the shape of the developing seeds at the corresponding DAF. Asterisk shows a non-specific band in CysR16 prolamin. M, mature seed.
The steady-state RNA levels for CysR10 and CysR16 as assessed by reverse transcription--PCR (RT--PCR) ([Supplementary Fig. S2](http://pcp.oxfordjournals.org/cgi/content/full/pcr053/DC1)) are consistent with their respective polypeptide levels during seed development. CysR10 RNAs were readily detected at 2 DAF and attain their maximum levels at 3 DAF, while significant levels of CysR16 were evident at 4 DAF and reach their highest levels at 8 DAF. Interestingly, steady-state RNA levels for CysR14 and CysP13 were very similar during early seed development, a pattern which contrasts with the differential temporal accumulation patterns of their polypeptides ([Fig. 3](#F3){ref-type="fig"}). As CysR14 was readily observed at 6 DAF whereas a significantly high level of CysP13 was only detected 3 days later ([Fig. 3](#F3){ref-type="fig"}), CysR14 RNAs appear to be preferentially translated compared with CysP13 transcripts during early seed development.
To examine further the development and maturation of PB-I formation, double indirect immunofluorescence studies using anti-CysR10 and anti-CysP13 antibodies were carried out using developing seeds harvested at 1, 2 and 3 WAF. In developing seeds at 1 WAF, PB-Is were observed as small reddish particles with a diameter of 0.5--2.1 μm containing mainly CysR10 ([Fig. 4](#F4){ref-type="fig"}A). Small amounts of CysP13 were also seen as small locules embedded in the matrix of larger PBs composed of CysR10 ([Fig. 4](#F4){ref-type="fig"}D). As the developing seed matures (2 WAF), CysP13 was detected in all of the PBs ([Fig. 4](#F4){ref-type="fig"}B), while CysR10 was detected at the center in some PBs with a diameter of 0.7--3.0 μm ([Fig. 4](#F4){ref-type="fig"}B, E). Interestingly in the very large PB depicted in [Fig. 4](#F4){ref-type="fig"}E, CysP13 was not uniformly distributed in the region surrounding the CysR10 central core. Instead a narrow layer devoid of CysR10 and CysP13 was evident. The basis for this layer is probably due to the presence of other prolamins (see Discussion). At 3 WAF, PBs with a diameter of 1.0--3.4 μm contained the CysR10 core (reddish center) surrounded by a mixture of CysR10 and CysP13 (yellowish layer) which, in turn, was surrounded by CysP13 (greenish layer) ([Fig. 4](#F4){ref-type="fig"}C, F). Fig. 4Double indirect immunofluorescence microscopy of PB-Is in rice endosperm during seed development. Endosperm sections were labeled with the antibodies against CysR10 and CysP13 and visualized by rhodamine- and FITC-conjugated antibodies, respectively. (D--F) Enlarged images of (A--C), respectively. Bars in (A--C): 10 μm. Bars in (D--F): 5 μm.
To obtain a more detailed structure of PB-I, we analyzed its ultrastructure during different stages of seed development by immunoelectron microscopy ([Fig. 5](#F5){ref-type="fig"}). In developing seeds at 5 DAF (see seed illustration in [Fig. 3](#F3){ref-type="fig"}), PB-I was seen as a small particle with an average diameter of about 0.3 μm with attached polysomes ([Fig. 5](#F5){ref-type="fig"}A). These small particles were readily labeled with antibodies to CysR10 but not with those to CysP13 ([Fig. 5](#F5){ref-type="fig"}A, E). At 1 WAF, the PB was spherical with an average diameter of about 0.9 μm ([Fig. 5](#F5){ref-type="fig"}B). CysR10 was located in the electron-dense center region which accounted for the bulk of the PB, with a small amount of the CysP13 localized to the peripheral region ([Fig. 5](#F5){ref-type="fig"}B, F). These results are consistent with those observed by immunofluorescence microscopy ([Fig. 3](#F3){ref-type="fig"}). At 2 WAF, the observed spherical PB was about 1.3 μm in average diameter ([Fig. 5](#F5){ref-type="fig"}C). Several electron-dense ring structures, which surrounded the electron-dense core structure, were observed ([Fig. 5](#F5){ref-type="fig"}C). These ring structures reacted with the CysR10 antibody, indicating that CysR10 is one of the polypeptides that comprise this structure ([Fig. 5](#F5){ref-type="fig"}G). CysP13 was evident in the electron-lucent peripheral layer and the electron-lucent matrix between the electron-dense ring and core structures in PB-I ([Fig. 5](#F5){ref-type="fig"}G). At 3 WAF, the PBs, which increased to an average size of about 1.5 μm in diameter, were devoid of the middle ring structures observed in PBs at 2 WAF ([Fig. 5](#F5){ref-type="fig"}D). The middle concentric electron-dense matrix appeared to merge with the center region, with its outer layer gradually being more electron lucent towards the periphery ([Fig. 5](#F5){ref-type="fig"}H). The distribution of CysR10 gradually decreased in this middle region, while that for CysP13 increased toward the periphery ([Fig. 5](#F5){ref-type="fig"}H). Fig. 5Immunoelectron microscopy of PB-I structure during rice endosperm development. PB-I was labeled with antibodies against CysR10 (5 nm gold particles) and CysP13 antibodies (15 nm gold particles). (E--H) Enlarged images of (A--D), respectively. (I) Schematic images of A--D. Bars in (A--D): 400 nm. Bars in (E--H): 200 nm.
Changes in the structure of PB-I in rice mutants
------------------------------------------------
Various seed mutants which show altered levels of rice prolamins were previously identified and studied ([@B15], [@B16], [@B27]). Mature *esp3* seeds contain low amounts of CysR10 as well as reduced levels of CysR14 and CysR16. The *esp1* mutant showed low levels of CysP13 while the *Esp4* mutant showed low levels of CysP13 as well as high levels of Cys-rich prolamins ([@B27]). To determine how these changes in the rice prolamin levels affect PB formation, the distribution of CysR10 and CysP13 in the PBs from these rice prolamin mutant endosperms was analyzed.
Immunofluorescence microscopy of *esp1* at 1 WAF indicated that there were many small PBs up to 1.8 μm in diameter containing CysR10 (red) and very little, if any, CysP13 (green) ([Fig. 6](#F6){ref-type="fig"}B). Similarly, *Esp4* endosperm at 1 WAF contained many small PBs up to 1.5 μm in diameter containing CysR10 and very little, if any, CysP13 ([Fig. 6](#F6){ref-type="fig"}D). The structure of PBs in both mutants was similar to that of the wild-type PB at 1 WAF ([Fig. 6](#F6){ref-type="fig"}A). At 2 WAF, PBs of *esp1* and *Esp4* were labeled almost entirely with the CysR10 antibody and had an average size similar to that of the PBs in wild-type endosperm ([Fig. 6](#F6){ref-type="fig"}E, F, H). The peripheral layer containing CysP13 typically observed in wild-type PBs was not observed in *Esp4* ([Fig. 6](#F6){ref-type="fig"}H). At 3 WAF, the PBs of *esp1* increased in size, with an average size of about 2.6 μm ([Fig. 6](#F6){ref-type="fig"}J), and the PBs of *Esp4* increased only slightly in size, with an average size of about 1.6 μm ([Fig. 6](#F6){ref-type="fig"}L). While the level of CysP13 was lower in *Esp4* endosperm, this was compensated by increases in levels of all of the Cys-rich prolamins ([@B16], [@B27]). This result, together with the cytological findings reported here, suggests that the reduction in size of *Esp4* PB-Is is due to the tighter packaging of Cys-rich prolamins and loss of CysP13, which accumulates in the peripheral region of PBs in the wild type. Fig. 6The structure of PB-I in the wild type, *esp1*, *esp3* and *Esp4* as viewed by double indirect immunofluorescence microscopy. Endosperm sections from the wild type and the three mutant lines were labeled with antibodies against CysR10 and CysP13, followed by labeling with secondary antibodies conjugated with rhodamine and FITC, respectively. Note that the small pinpoint red dots in (A--E) are non-specific background, which is observed occasionally in immunofluorescence microscopy. Bars: 10 μm.
Immunofluorescence microscopy of *esp3* showed that only PBs labeled by the CysP13 antibody were observed at all stages of seed development ([Fig. 6](#F6){ref-type="fig"}). At 1 WAF, PBs labeled by the CysP13 antibody were about 1.6 μm in diameter ([Fig. 6](#F6){ref-type="fig"}C), similar in size to those from the wild type ([Figs. 6](#F6){ref-type="fig"}A, [4D](#F4){ref-type="fig"}). At 2 WAF, the PBs, which were wholly recognized by anti-CysP13 antibody, were significantly larger (about 1.8--2.8 μm in diameter) than those from the wild type ([Figs. 6](#F6){ref-type="fig"}G, [4B](#F4){ref-type="fig"}). At 3 WAF, larger PBs about 2.8--4.2 μm in diameter were observed ([Fig. 6](#F6){ref-type="fig"}K). Though individual PBs were spherical in shape, many were clustered and appeared to be fused together ([Fig. 6](#F6){ref-type="fig"}K, arrow).
To analyze the PB structures in these mutants in more detail, immunoelectron microscopy was performed. At 1 WAF, the *esp1* and *Esp4* PBs exhibited a homogeneous electron density, with CysR10 (15 nm gold particles) distributed nearly uniformly over the whole PB ([Fig. 7](#F7){ref-type="fig"}F, J). A few gold particles (5 nm) denoting the presence of CysP13 polypeptide were evident in several PBs ([Fig. 7](#F7){ref-type="fig"}F, J, arrows). At 3 WAF, the localization of both prolamins was randomly distributed within clearly spherical PB-Is, and the size was slightly larger than at 1 WAF ([Fig. 7](#F7){ref-type="fig"}H, L). Fig. 7The structure of PB-I in the wild type, *esp1*, *esp3* and *Esp4* as viewed by immunocytochemistry at the electron microscopy level. Endosperm sections, except for (K, L and Q, R), were incubated with anti-CysR10 and anti-CysP13 antibodies followed by treatment with secondary antibodies which were conjugated with 15 and 5 nm gold particles, respectively. The sections in (K, L and Q, R) were labeled with anti-CysR10 antibody (5 nm gold particles) and anti-CysP13 antibody (15 nm gold particles). (A--D) The wild type (Kinmaze). (E--H) *esp1*. (I--L) *Esp4.* (M--T) *esp3*. White arrows in (F, H, J and L) show the 5 nm gold particles (CysP13). The left two tiers show the PB-I at 1 WAF and the right two tiers show the PB-I at 3 WAF. (B, D, F, H, J, L, N, P, R and T) Enlarged images of each left panel. Arrows in (M, N, Q and R) show the cracks in the PBs. Open arrows in T show the boundary of PB-I compressed by PB-II. Asterisks in (S, T) show the matrix in protein storage vacuoles (PSVs). Am, amyloplast. Bars in (A--R): 200 nm, Bars in (S, T): 500 nm.
The morphology of the PBs in the *esp3* endosperm at 1 WAF was significantly different from that seen in wild-type endosperm ([Fig. 7](#F7){ref-type="fig"}A, B). The PBs were oval in shape, were electron lucent, and displayed a wrinkled surface ([Fig. 7](#F7){ref-type="fig"}M, N). At 1 WAF, CysP13 (5 nm gold particles) was uniformly distributed throughout the whole PB ([Fig. 7](#F7){ref-type="fig"}M, N). In some instances, small amounts of CysR10 (15 nm gold particles) were evident and distributed as a ring ([Fig. 7](#F7){ref-type="fig"}M, N). At 3 WAF, the PBs were labeled with only the CysP13 antibody ([Fig. 7](#F7){ref-type="fig"}O, P). The electron-dense core and ring structures observed in the wild type ([Fig. 7](#F7){ref-type="fig"}C, D) were not observed in *esp3* PBs, which were electron lucent and of uniform electron density ([Fig. 7](#F7){ref-type="fig"}O, P and [Q, R](#F7){ref-type="fig"}). The PBs were elongated ([Fig. 7](#F7){ref-type="fig"}Q) or irregularly shaped, and appeared to be less rigid as their shape was compressed by other organelles ([Fig. 7](#F7){ref-type="fig"}S, and arrowheads in [Fig. 7](#F7){ref-type="fig"}T). These irregular-shaped PBs contained cracks and cavities, spaces presumably free of prolamins ([Fig. 7](#F7){ref-type="fig"}Q, R, arrows). These cavities were detected in PBs at 1 WAF ([Fig. 7](#F7){ref-type="fig"}M, N, arrow). The larger PB size but lower levels of the Cys-rich prolamins ([@B16], [@B27]) indicate that prolamins are packaged much less compactly in *esp3* compared with normal conditions. These results indicate that the decrease in the levels of the Cys-rich prolamins results in the formation of abnormal PB-Is. The degeneration of the electron-dense core containing CysR10 is especially remarkable. Although the levels of the other Cys-rich prolamins were reduced in *esp3*, we hypothesized that the reduction of CysR10 and, in turn, the absence of the core were responsible for the deformation of PB-I.
To determine whether CysR10 is required for normal PB-I formation, an RNAi cassette for CysR10 was transferred into rice by *Agrobacterium*-mediated transformation, resulting in the generation of 10 independent transformant lines. The strongest silenced line, which showed significantly reduced amounts of CysR10 but normal levels of the other Cys-rich prolamins, was selected for further study ([Fig. 8](#F8){ref-type="fig"}A, B). This RNAi line also contained lower amounts of CysP13 ([Fig. 8](#F8){ref-type="fig"}A, B, and [Supplementary Fig. S3](http://pcp.oxfordjournals.org/cgi/content/full/pcr053/DC1)). The PB-Is in seeds at 2 WAF from the CysR10-repressed transformant and wild type were stained with rhodamine and examined by confocal laser microscopy. PB-Is from the wild type are seen as ring-like structures as the hydrophobic character of the prolamins causes them to bind to this vital stain ([@B3], [@B28]) ([Fig. 8](#F8){ref-type="fig"}C). Although the PB-I organelles could also be detected by rhodamine staining in developing endosperm of the CysR10 RNAi plant, their structures were significantly altered. The PB-Is in the RNAi line were non-spherical ([Fig. 8](#F8){ref-type="fig"}D). The PB-I in the CysR10 RNAi plant was observed by immunocytochemistry at the electron microscope level ([Fig. 9](#F9){ref-type="fig"}). The ribosome-attached membrane was observed around the non-spherical PBs ([Fig. 9](#F9){ref-type="fig"}C, D), indicating that the PBs were derived from the ER. Except for a change in size of the PBs, the deformed PBs from the CysR10 RNAi plant were similar to the irregular-shaped PBs in *esp3* which contained reduced levels of CysR10, CysR14 and CysR16 ([Figs. 6](#F6){ref-type="fig"}K, [7M, Q](#F7){ref-type="fig"}). These results support the view that the compact size and spherical morphology of PB-I is dependent on the presence of CysR10. Fig. 8Down-regulation of CysR10 by RNAi. (A) SDS--PAGE analysis of the prolamin/glutelin fraction extracted from mature seed. Lane 1, wild-type, Kinmaze; lane 2, *esp3* mutant derived from Kinmaze; lane 3, CysR10-repressed transformant derived from Yukihikari; lane 4, wild-type, Yukihikari. (B) Immunoblot analysis of the prolamin/glutelin fraction with anti-prolamin antibodies. (C) Confocal laser scanning microscopy of rhodamine-stained wild-type endosperm cells. (D) Confocal laser scanning microscopy of rhodamine-stained endosperm cells from the CysR10-repressed transformant. Bars in (C, D): 5 μm. Fig. 9The effect of reduced levels of CysR10 on PB-I formation as viewed by immunoelectron microscopy of the CysR10-RNAi transformant. PB-I was labeled with anti-CysR10 (15 nm gold particles) and anti-CysP13 (5 nm gold particles) antibodies (A--D). (A) Wild-type PB-I at 3 WAF. (B--D) CysR10-repressed transformants at 3 WAF. (D) Enlarged image of the PB-I (open arrowhead) shown in B. Asterisks in (A) and (B) denote PB-I. Arrows in (C) and (D) indicate the rough ER. Am, amyloplast; CW, cell wall; PSV, protein storage vacuole. Bars: 500 nm.
Discussion
==========
The results in this study demonstrate that the formation of the rice prolamin-containing PB (PB-I) is initiated by the accumulation of CysR10. We suggest that CysR10 is needed for the initial assembly of prolamins to form a small focus which ultimately becomes the core of PB-I and that CysP13 is synthesized and layered over the core in two separate concentric rings. We found that the inner ring immediately surrounded the central core and contained both CysR10 and CysP13, whereas the outer ring contained only the latter species ([Supplementary Figs. S4, S5](http://pcp.oxfordjournals.org/cgi/content/full/pcr053/DC1)). Interestingly, in the very large PB ([Fig. 4](#F4){ref-type="fig"}E) at 2 WAF, there was a thin ring between the CysP13 peripheral layer and the CysR10 core which was not recognized by either antibody ([Fig. 4](#F4){ref-type="fig"}E). This clear ring most probably contains other prolamin classes such as CysR14 and CysR16. We indeed found that the distribution of CysR16 within PB-I was very defined and appeared as two concentric rings ([Supplementary Fig. S4B](http://pcp.oxfordjournals.org/cgi/content/full/pcr053/DC1), panel c). It is thus possible that the inner CysR16 ring may correspond to the thin ring lacking both CysR10 and CysP13 in the PB depicted in [Fig. 4](#F4){ref-type="fig"}E. The distribution of CysR14 in PB-I was also non-uniform. Immunofluorescence studies showed that CysR14 was distributed in a donut pattern where it was excluded from the central core region ([Supplementary Fig. S5](http://pcp.oxfordjournals.org/cgi/content/full/pcr053/DC1)). Such an arrangement was verified by immunoelectron microscopy where CysR14 was located around the central core and distributed in a pattern similar to that seen for CysP13 ([Supplementary Fig. S5E--H](http://pcp.oxfordjournals.org/cgi/content/full/pcr053/DC1)).
The prolamin-containing PB-Is were non-spherical in the CysR10-repressed endosperm and *esp3* mutant. Additionally, the PBs in *esp3* were significantly larger. Unlike the case in *esp3* where the levels of CysR14 and CysR16 were also reduced, their amounts are not significantly different in the CysR10-repressed line. These observations strongly suggest that CysR10 is required for forming PB-Is into the spherical shape and for the compact size, and that it not only forms the central core but also interacts with other Cys-rich prolamins to assemble the concentric ring structure within the PB-I.
Although the PBs in the CysR10-repressed line and *esp3* mutant appear similar in being non-spherical, there are notable differences. PB-Is in *esp3* appear to be more elastic as their irregular shape could be further misshapen by other organelles including starch-containing amyloplasts and glutelin-containing PSVs ([Fig. 7](#F7){ref-type="fig"}T). In contrast, the irregular shape of PB-Is in the CysR10-repressed line is independent of other organelles and appears to be due to the less ordered arrangement of prolamin polypeptides ([Fig. 9](#F9){ref-type="fig"}C, D and [Supplementary Fig. S6](http://pcp.oxfordjournals.org/cgi/content/full/pcr053/DC1)). It is inferred from the structural difference in PB-I between CysR10-RNAi transformant and *esp3* that the deformed shape of PB-I in the CysR10-RNAi transformant is induced by the presence of CysR14 and CysR16.
Interestingly, RNAi suppression of CysR10 also slightly reduced the expression level of CysP13. The molecular basis for this reduction in the levels of CysP13 is unclear, but the interaction of CysR10 with the other Cys-rich prolamins around the core region may be required to stabilize the accumulation of CysP13. It should be pointed out that the decline in CysP13 is not the reason for the irregular shape of PB-Is in the CysR10-repressed line as PB-Is in CysP13-deficient *esp1* and *Esp4* are spherical ([Fig. 7](#F7){ref-type="fig"}F, J; [@B16], [@B27]).
The smaller size of PB-I in *esp1* and *Esp4* mutant lines is consistent with the results of a recent study ([@B8]), in which the expression of 13 kDa prolamins is repressed. However, unlike this study where large, abnormally shaped PB-I structures were seen in our CysR10-deficient RNAi line, smaller spherical PB-Is were observed when CysR10 was down-regulated ([@B8]). The reason for the discrepancy is not clear, but we note that the CysR10 RNAi plants in this study and that of [@B8]) had significant differences in the quantities of the other prolamin species. Seeds of our CysR10 RNAi line contained normal or slightly higher amounts of CysR16 and CysR14 and lower amounts of CysP13 ([Fig. 8](#F8){ref-type="fig"}A). Although lower amounts of CysP13 (RM2 and RM4) prolamins were also observed by [@B8]), their 10 kDa prolamin-suppressed plant accumulated substantially higher levels (about three times compared with the wild type) of CysR14 (RM1 and RM9) and CysR16 (RP16) prolamins than normal. Hence, the elevated levels of CysR14 and CysR16 may compensate for the loss of CysR10 and contribute to form a rigid normal sized PB-I. This view is supported by *esp3* which contains reduced levels of CysR16, CysR14 and CysR10 and displays abnormal large PBIs.
Alternatively, differences in the methods for inducing RNAi may, in part, account for the different PB-I structures. In the study of [@B8]), the target protein genes are down-regulated by expressing a modified human *Glucagon-like peptide-1* (*GLP-1*). Although highly effective in repressing target genes, the mechanism remains unclear ([@B45]). In the current study, we employed a conventional RNAi method, in which a DNA fragment containing a part of the CysR10 open reading frame in an inverted manner is designed to be transcribed under the control of an endosperm-specific promoter, as reported previously ([@B28], [@B29]). The two approaches are likely to be responsible for the differences in the expression of CysR14 and CysR16 between the two CysR10 RNAi lines.
The stratified distribution of Cys-rich (CysR10) and Cys-poor (CysP13) prolamins in PB-I differs substantially from that observed for the maize PBs. Cys-rich β- and γ-zeins in maize initially form a small PB, but they are subsequently displaced to the peripheral region by the accumulation of the Cys-poor δ-zein and Cys-rich δ-zein in the core ([@B19], [@B5]). The exact reason for the different arrangements of prolamin species in the PBs of rice and maize is not known, but it has been suggested that the overall differences in hydrophobicity of maize prolamins may play a role in the formation of the respective PBs ([@B9]). Analysis of the zein primary amino acid sequences indicates that the hydrophobicity of δ- and α-zein is substantially higher than that of the β- and γ-zeins, with δ-zein being the most hydrophobic ([@B9]; [Supplementary Fig. S7](http://pcp.oxfordjournals.org/cgi/content/full/pcr053/DC1)). Segregation of the maize prolamins inside the PB according to their hydrophobicity profiles may establish a more thermodynamically stable arrangement of the prolamins. Interestingly, however, a similar hydropathy analysis of rice prolamins revealed that differences in the overall hydrophobicity profiles between CysR10 (RP10) and CysP13 are not as readily apparent as those seen for the maize prolamins ([Supplementary Fig. S8](http://pcp.oxfordjournals.org/cgi/content/full/pcr053/DC1)). In view of the differences in spatial arrangement of the maize and rice Cys-rich and Cys-poor prolamins and in their overall hydrophobicity, the mechanisms responsible for PB formation are likely to be different in these cereals.
Since neither rice nor maize prolamins contain the typical C-terminal KDEL retrieval signal ([@B23], [@B14]), ER retention of both prolamins is likely to be dependent on their capacity to assemble into an intracisternal granule at a rate greater than their capacity to be exported from the ER. The localization of these RNAs to the PB ER would facilitate the assembly of these proteins to form an inclusion granule by concentrating their proteins within the PB lumen ([@B4], [@B41]). The CysR10 polypeptides are the first prolamin species to be synthesized, followed soon after by the other species. Immunofluorescence microscopy using anti-CysR14 indicated that CysR14 surrounded the center core containing CysR10 ([Supplementary Fig. S4](http://pcp.oxfordjournals.org/cgi/content/full/pcr053/DC1)). This region also contained CysR10 ([Figs. 4](#F4){ref-type="fig"}, [5](#F5){ref-type="fig"}) and as well as CysR16 and CysP13, as the synthesis of these proteins began a few days later. The CysR16, CysR14 and CysR10 species could interact with each other in the layer surrounding the center core by non-covalent as well as covalent bonds via disulfide linkages. Interactions by the latter are likely to be responsible for the electron-dense rings evident in PBs in seeds at mid-development ([Fig. 5](#F5){ref-type="fig"}). Although CysP13, which lacks a cysteine residue, is unable to bind to other prolamins through disulfide bonds, the prolamin shares considerable sequence identity with CysR14 and CysR16 ([@B10], [@B11], [@B23], [@B22], [@B35], [@B37], [@B36], [@B38]), suggesting possible hydrophobic interactions between CysP13 and Cys-rich prolamins. Although direct evidence for protein--protein interactions for formation of the PB-I in the rice endosperm has not yet been obtained, the available evidence supports such a role. The Cys-rich prolamins contain the three conserved A, B and C motifs containing cysteine residues ([@B32]), which have been demonstrated to be involved in interchain disulfide bonding ([@B7]). In a recent study, we have shown that protein disulfide isomerase-like protein (PDIL) 2;3 is localized mainly on the surface of PB-I in the ER lumen, and that PDIL1;1 (Esp2) and PDIL2;3 play distinct roles in the accumulation of CysR10 and CysP13 in PB-I ([@B29]). It is possible that intermolecular disulfide bond formation between CysR molecules facilitated by PDILs may be important for the spatial arrangement of prolamins in PB-I.
Materials and Methods
=====================
Plant materials
---------------
The *esp1*, *Esp4* and *esp3* mutant lines, CM21, PM164 and PM163, respectively, induced by *N*-methyl-*N*-nitrosourea (MNU) treatment, and the wild-type rice cultivar, Kinmaze, were used in this study. *esp1* shows low levels of CysP13, *Esp4* shows low levels of CysP13 and high levels of CysR10, CysR14 and CysR16, while *esp3* shows low levels of the Cys-rich prolamins and high levels of CysP13 ([@B15], [@B16], [@B17]). *esp1*, *Esp4* and *esp3* mutant lines are registered as CM21, CM1834 and CM1675, respectively, in the Rice National Bio Resources Project database, Oryzabase (<http://www.shigen.nig.ac.jp/rice/oryzabase/top/top.jsp>). These rice plants were grown in field plots at the university farm of Kyushu University and the Agricultural Experimental Station of Yamaguchi Prefecture. The RNAi plant was cultivated in the greenhouse at the National Institute of Agrobiological Science. Developing seeds were tagged on the day after flowering and collected at various developmental stages. The seeds for chemical analysis were immediately frozen with liquid nitrogen then store at −80°C before use, while developing seeds for cytochemical analysis were immediately chemically fixed.
SDS--PAGE and immunoblot analysis
---------------------------------
SDS--PAGE of the total rice seed protein extract and immunoblot analysis were performed as described previously ([@B39], [@B18]). The total rice protein was extracted in 20% (v/v) glycerol, 4% (w/v) SDS, 88 M urea, 5% 2-mercaptethanol, 500 mM Tris--HCl, pH 6.8 (30 μl of extraction buffer per 11 mg of developing seeds), and 7 μl of the extracted sample was subjected to SDS--PAGE. The proteins separated by SDS--PAGE were transferred to a polyvinylidene fluoride (PVDF) membrane. Antisera against CysR10, CysP13, CysR14 and CysR16 were diluted 1/4,000, 1/2,000, 1/8,000 and 1/4,000, respectively.
Antibodies
----------
The CysR10, CysR14 and CysR16 proteins were purified by elimination of Cys-poor prolamins from matured rice seed powder by 60% (v/v) *n*-propanol solution and then extraction of Cys-rich prolamins by 60% (v/v) *n*-propanol solution containing 5% (v/v) 2-mercaptoethanol. Proteins from the latter extraction were separated by SDS--PAGE. Each prolamin band was excised and separated again by SDS--PAGE. The purified CysP13 was produced by extraction of the Cys-poor prolamin from matured rice seed powder in 60% (v/v) *n*-propanol solution. The extracted proteins were then separated by isoelectric focusing and the major CysP13 band (pI 6.65) was excised and then re-purified by preparative SDS--PAGE. Antibodies against CysR10 and CysR14 were prepared in rabbits, while antibodies against CysP13 and CysR16 were raised in mice. Antibodies against CysR10 and CysR14 are very specific because prolonged exposures showed no cross-reaction with other prolamins ([Fig. 3](#F3){ref-type="fig"}, [Supplementary Fig. S1](http://pcp.oxfordjournals.org/cgi/content/full/pcr053/DC1)). Antibody against CysP13 showed a weak cross-reaction with unidentified proteins in the total protein containing the albumin or globulin fraction ([Fig. 3](#F3){ref-type="fig"}), but the antibody reacted only with CysP13 in the prolamin fraction ([Fig. 8](#F8){ref-type="fig"}B). Antibodies to CysR16 occasionally reacted faintly with a polypeptide of small size (∼15 kDa) ([Fig. 3](#F3){ref-type="fig"}), but the identity of the polypeptide was not investigated further because the reaction was sporadic ([Fig. 8](#F8){ref-type="fig"}B).
Microscopy analysis
-------------------
For immunoelectron and immunofluorescence microscopic observations, the samples were fixed as described previously ([@B39], [@B31]). Sections were prepared using a microtome (Leica) and then immunolabeled with antisera toward each prolamin. Incubation of the sections with conjugated secondary antibodies for each microscopic technique was performed as described previously ([@B39], [@B31]). Images obtained by fluorescence microscopy were analyzed by Image-Pro plus (Planetron). Immunolabeled sections for electron microscopy were sequentially stained with 0.25% KMnO~4~ and 1% uranyl acetate. Samples for electron microscopy were embedded in epon and sections were stained in Reynolds\'s lead staining solution consisting of 3.44% Pb(NO~3~)~2~, 4.63% Na~2~(H~6~H~5~O~7~)·2H~2~O, 0.21 N NaOH in CO~2~ free distilled water. The diameters (minimum, maximum and average) of PBs at various developing stages of seed development were measured from multiple micrographs using a graphic software package (Canvas X, ACD Systems of America Inc).
Plasmid construction and rice transformation
--------------------------------------------
A full-length cDNA clone for CysR10 (RP10, AK108254) was obtained from the National Institute of Agrobiological Sciences (Tsukuba, Japan). For the CysR10-specific RNAi construction, 4,299 bp (Asp41 to stop codon) of the cDNA clone for CysR10 was used to make inverted repeats. The RNAi cassette was transferred to the binary vector containing the α-globulin promoter and the nopaline synthase (Nos) terminator described previously ([@B43], [@B7]), and the hygromycin phosphotransferase gene. The CysR10 RNAi plasmid was transformed into the rice variety Yukihikari as described previously ([@B6], [@B28]).
Extraction of prolamin/glutelin fraction
----------------------------------------
The powder of an individual rice seed was suspended in 200 mM Tris--HCl (pH 6.8) containing 0.55 M NaCl (60 μl per 11 mg of mature seeds). The suspension was sonicated for 55 min on ice. After centrifugation, the supernatant was discarded. The precipitate was suspended with 0.55 ml of 0.1255 M Tris--HCl (pH 6.8) containing 4% (w/v) SDS, 88 M urea and 5% (v/v) 2-mercaptoethanol. After centrifugation, the supernatant was collected as the prolamin/glutelin fraction. The protein composition of the fraction was analyzed by SDS--PAGE.
Confocal laser scanning microscopy
----------------------------------
Rhodamine labeling of PBs in developing rice seed at 2 WAF was performed as described previously ([@B28]). The fluorescence images of the subaleurone cells were analyzed with a confocal laser scanning microscope with a laser beam of 5,433 nm (TCS SP2 AOBS; Leica).
Supplementary data
==================
[Supplementary data](http://pcp.oxfordjournals.org/cgi/content/full/pcr053/DC1) are available at PCP online.
Funding
=======
This work was supported by the Japan Society for the Promotion of Science \[grant-in-aid for Scientific Research to T.K. (16380009 and 21380008\]; Bio-oriented Technology Research Advanced Institution (BRAIN) \[the program for Promotion of Basic Research Activities for Innovative Biosciences\]; the Ministry of Agriculture, Forestry and Fisheries of Japan \[grant to Y.K. (Genomics for Agricultural Innovation, IPG-0023); United States National Science Foundation \[grants IOS-1021699 and DBI-0605016 to T.W.O.\]. United States Department of Agriculture-Cooperative State Research, Education and Extension-National Research Initiative \[grant 2006-35301-17043 to T.W.O.\].
Supplementary Material
======================
###### Supplementary Data
We thank Mr. Kazuhiko Kaneko, Mr. Hiroshi Kajiwara and Mr. Masayasu Hajima for enabling us to use the transmission electron microscope at the Yamaguchi Prefectural Agricultural Experimental Station.
DAF
: days after flowering
ER
: endoplasmic reticulum
*esp*
: endosperm storage protein mutant
FITC
: fluorescein isothiocyanate
PB
: protein body
PDIL
: protein disulfide isomerase-like protein
PSV
: protein storage vacuole
RNAi
: RNA interference
WAF
: weeks after flowering.
| {
"pile_set_name": "PubMed Central"
} |
Introduction
============
Aging is associated with compromised function, impaired mobility, and loss of independence, resulting in decreased quality of life. Osteoporosis is characterized by decreased mineral mass and mechanical strength of bone leading to fragility fractures in the elderly. Vertebral fractures are one of the commonest fragility fractures. Vertebral fractures can increase mortality, with a reported age-standardized mortality ratio of 1.23 to 1.66 in women and 2.38 in men \[[@b1-asj-2019-0049],[@b2-asj-2019-0049]\].
Muscle mass contributes to bone mineral mass, and the interaction between bone and muscle increases the mechanical strength of bone under loading and maintains normal musculoskeletal function \[[@b3-asj-2019-0049],[@b4-asj-2019-0049]\]. Clinical studies have shown that loss of muscle mass occurs concurrently with osteoporosis \[[@b5-asj-2019-0049]\]. The loss of muscle mass with increasing age associated with either decreased muscle strength or decreased physical performance is defined as sarcopenia \[[@b6-asj-2019-0049]\]. Presarcopenia is the decrease in muscle mass alone, whereas severe sarcopenia is defined as the presence of a decrease in muscle mass, muscle strength, and physical performance. Muscle mass can be measured using various methods like dual-energy X-ray absorptiometry (DEXA), bioimpedance electrical analysis, computed tomography (CT) or magnetic resonance imaging (MRI). MRI is the most accurate method and total psoas muscle cross-sectional area (TPA) measured on MRI is an accepted muscle mass measure which shows atrophy and fat infiltration in sarcopenia \[[@b7-asj-2019-0049]\]. Muscle strength is measured by handgrip strength using a dynamometer, and physical performance by tests such as usual gait speed or short physical performance battery.
Observational studies have shown that sarcopenia, decreased paraspinal muscle cross-sectional area, and fat infiltration of paraspinal muscles are risk factors for vertebral fractures. These factors have been associated with an increased number of vertebral fractures in the elderly \[[@b8-asj-2019-0049]-[@b10-asj-2019-0049]\]. However, some of these studies are limited as they did not account for muscle strength or physical performance, thus, diagnosing presarcopenia rather than sarcopenia. Moreover, some studies have used DEXA to assess muscle mass, which is inferior to MRI.
We hypothesized that sarcopenia, diagnosed by decreased TPA and decreased handgrip strength, is an independent risk factor for fresh vertebral fragility fractures in elderly. We did a comparative analysis between a study group of elderly patients with fresh vertebral fragility fractures and a control group of patients without fractures and analyzed the effect of sarcopenia and other variables. Our objective was to determine whether sarcopenia increased the risk for vertebral fragility fractures among the elderly.
Methods
=======
After obtaining Institutional Review Board approval (IRB approval no., 2017/06/03, dated 28/07/2017 of Ganga Medical Centre and Hospitals Pvt. Ltd., Coimbatore) and informed consent, we included patients presenting to a tertiary spine center during the period between September 2017 and April 2018. Patients ≥50 years of age with acute back pain, history of trivial trauma, and presence of vertebral fracture on radiographs were included in the study group. Patients presenting after 3 weeks of symptoms, those with significant trauma, diagnosed malignancies or neuromuscular disorders, and those who were bedridden for more than a month in the preceding year were excluded. Out of 65 patients fulfilling the inclusion criteria, 14 were excluded (13 incomplete investigations and one lack of consent) and the remaining 51 patients were recruited. A detailed history was taken, including the history of drug intake and that of being bedridden. Anthropological measurements of height, weight, and body mass index were noted. Equal numbers of age and sexmatched patients who presented with backache, but without fresh vertebral fractures, were assigned to the control group. Age was matched to within 1 year. As sarcopenia is known to cause decreased bone mineral density (BMD), BMD was excluded as a matching variable to prevent overmatching.
MRI of the whole spine was performed using a 1.5-Tesla machine (Magnetom Essenza; Siemens Healthineers, Erlangen, Germany) to assess fracture, presence of previous fractures, exclude other pathologies and calculate the TPA. DEXA scan was performed to assess BMD at the femoral neck for evaluating osteoporosis with values of Caucasian females as reference (Lunar Prodigy Advance; GE Healthcare, Chicago, IL, USA). Femoral neck BMD was chosen instead of vertebral BMD as vertebral fractures and osteophytes are known to lead to falsely high values of BMD. Biochemical laboratory analysis of calcium, phosphorus, vitamin D, and parathyroid hormone was also performed.
The primary evaluation included the presence of sarcopenia, diagnosed according to the European Working Group on Sarcopenia in Older People (EWGSOP) criteria \[[@b8-asj-2019-0049]\], which diagnoses sarcopenia by the presence of decreased muscle mass with either decreased muscle strength or physical performance. Although they mention that DEXA is the most commonly used method of assessment of muscle mass, MRI is the gold standard and most appropriate in the research setting. The cut-off values for diagnosis rely on measuring normative data from a reference population of young adults, with separate values for men and women. Muscle mass was assessed using TPA on MRI. We also examined the role of standardized muscle mass based on the psoas lumbar vertebral index (PLVI) to assess its role in addition to that of absolute muscle mass measured by TPA. PLVI is calculated as TPA divided by the vertebral body area at the same axial level. This measure is useful for standardizing muscle mass for stature. Sarcopenia was diagnosed based on both absolute muscle mass and standardized muscle mass for separate analysis. Muscle strength was tested using a hydraulic handheld dynamometer (Jamar Medical Hand Dynamometer; Patterson Medical, Warrenville, IL, USA) which measures peak hand strength in kilograms. Grip strength of the dominant hand was measured thrice, and the average was considered for diagnosis. Cut-off values for low muscle strength were specific for the Asian population (26 kg in males and 18 kg in females) according to the criteria of the Asian Working Group for Sarcopenia (AWGS) \[[@b11-asj-2019-0049]\]. We excluded measurements of physical performance as the presence of an acute fracture and back pain in the patients precluded gait speed measurements.
All measurements were taken by the principal investigator. The TPA at L4 upper endplate was used to measure muscle mass, as described by Shen et al. \[[@b12-asj-2019-0049]\]. All the patients underwent MRI using the same protocol, with disk level parallel section at the upper L4 endplate used for measurements in all cases. TPA and PLVI were measured at the same level from T2-weighted axial images using the hospital picture archiving and communications system (PACS; Medsynapse PACS, ver. 5.0.1.3; Medsynaptic Pvt. Ltd., Pune, India). Outlines of the left and right psoas muscles and vertebral body were drawn manually to define the region of interest. Fat infiltration, seen as hyperintensity on T2-weighted films were excluded from the region of interest, thus measuring the fat-free cross-sectional area. The software calculated the area automatically ([Figs. 1](#f1-asj-2019-0049){ref-type="fig"}, [2](#f2-asj-2019-0049){ref-type="fig"}). Sarcopenia was diagnosed as TPA and PLVI 2 standard deviations (SDs) below the mean of the reference population of normal young adults.
We calculated the reference values by analyzing MRI images of all persons between the ages of 20 and 40 years who had undergone MRI of the lumbar spine between January 2012 and December 2017. From medical records, we identified patients who had come for evaluation of a first episode of acute-onset back pain without any clinical abnormalities and normal imaging. Patients with significant pain, limitation of activity, or abnormal physical findings were excluded. The final list of persons with normal MRI was used to measure the psoas area and PLVI. Mean and SD were calculated separately for men and women. Cut-off values for diagnosing sarcopenia were then determined.
For calculating the reliability of interobserver assessment, measurements of 50 random subjects were taken independently by a senior registrar. For intraobserver reliability, measurements were repeated by the principal investigator after a gap of 3 months. Intraclass correlation coefficient (ICC) estimates and their 95% confidence intervals (CIs) were calculated using IBM SPSS ver. 22.0 (IBM Corp., Armonk, NY, USA). Based on a single-measurement, absolute-agreement, two-way random effects model for interobserver reliability, the ICC for TPA was 0.932 (95% CI, 0.865--0.964) and that for PLVI was 0.788 (95% CI, 0.619--0.881). Based on a single-measurement, absolute-agreement, two-way mixed effects model for intraobserver reliability, the ICC for TPA was 0.944 (95% CI, 0.902--0.968) and that for PLVI was 0.943 (95% CI, 0.886--0.970).
Statistical analysis was performed using IBM SPSS ver. 22.0 (IBM Corp.). The mean and SD for normative values were calculated. Significance level was set at *p*=0.05. Paired t-test and McNemar's test were used to analyze continuous and categorical variables, respectively. Conditional regression analysis was used to analyze variables in multivariate analysis. Correlation between continuous variables within a group was analyzed using Pearson's coefficient.
Results
=======
1. Normative study
------------------
The normative data were calculated from 249 adults (114 men and 135 women). The TPA of the reference population was 1,576 mm^2^ (standard error \[SE\]=25.3, SD=295) for women and 2,723 mm^2^ for men (SE=50.6, SD=541). Based on these, cut-off values for women and men with sarcopenia were 986 mm^2^ and 1,641 mm^2^, respectively. The PLVI of women and men in the reference population was 1.51 (SE=0.026, SD=0.31) and 2.05 (SE=0.04, SD=0.46), with cut-off values of 0.89 and 1.13, respectively ([Table 1](#t1-asj-2019-0049){ref-type="table"}).
2. Case-control study
---------------------
There were 15 men (mean age, 70.05±9.8 years) and 36 women (69.3±9.2 years) each in the case and control groups. Compared with cases, TPA was significantly higher in the control group (1,569 mm^2^ versus 1,278 mm^2^, *p*=0.001). There was no statistically significant difference between women in both groups with respect to handgrip strength (15.8 kg versus 14.3 kg, *p*=0.226). However, men in the control group had significantly higher handgrip strength (30 kg versus 23 kg, *p*=0.016). Twenty-one cases and seven controls had a T-score less than −2.5, with an odds ratio (OR) of 5.67 (*p*=0.004). The mean T-scores were significantly different between the two groups. There was no significant difference between the two groups with respect to biochemical parameters. [Table 2](#t2-asj-2019-0049){ref-type="table"} shows the comparison of continuous variables between cases and controls, with separate analysis for males and females.
Sarcopenia, when diagnosed based on TPA, was present in 15 cases (29.4%) compared to four controls (7.8%) (*p*=0.005). However, when diagnosed based on PLVI, the prevalence of sarcopenia was 23 (45.1%) and 14 (27.4%) among the cases and controls, respectively, and it was not significantly different between groups (*p*=0.064). McNemar's test revealed a significant OR of 12 (*p*=0.006) for sarcopenia diagnosed by TPA, in contrast to the PLVI, where the difference was not significant between groups (OR, 2.29; *p*=0.095) ([Table 3](#t3-asj-2019-0049){ref-type="table"}).
Twenty-nine cases and seven controls had previous vertebral fractures, showing a significantly higher prevalence of previous fractures in the study group (OR, 12; *p*\<0.001). The prevalence of sarcopenia was significantly higher among those with previous fractures (38% versus 7.6%; OR, 7.76; *p*\<0.001). None of the controls without previous fractures had sarcopenia. TPA (1,563 mm^2^ versus 1,168 mm^2^) and handgrip strength (19.6 kg versus 16.3 kg) were significantly higher in those without previous fractures (*p*=0.001 and *p*=0.05). TPA showed a strong positive correlation with handgrip, height, weight, and BMD in all groups.
Univariate analysis of different variables showed that the presence of previous fractures, BMI, T-score, and sarcopenia diagnosed by TPA were significant risk factors for developing fresh fractures. Multivariate analysis was performed using the occurrence of fresh fractures as the dependent variable ([Table 4](#t4-asj-2019-0049){ref-type="table"}). Sarcopenia was not identified as a significant risk factor for fresh fractures in multivariate analysis (*p*=0.4). The presence of previous fractures had a significant OR for developing fresh fractures (OR, 7.049; *p*=0.015). T-score showed a significant value (OR, 0.604; *p*=0.036) indicating a 40% decreased odds for each unit increase in the T-score.
Discussion
==========
Sarcopenia is the age-related decrease in muscle mass, power, and physical performance. According to EWGSOP criteria, its diagnosis requires the presence of both decreased muscle mass and either decreased muscle strength or physical performance. MRI is the most accurate method for determining muscle mass \[[@b13-asj-2019-0049]\]. In all previous studies that diagnosed sarcopenia using CT or MRI, cutoff points were derived arbitrarily, as the lowest tertile or quartile of muscle area \[[@b14-asj-2019-0049]-[@b18-asj-2019-0049]\]. In this study, the reference range of TPA for Indian men and women was calculated from records of clinically and radiologically normal adults. Then, sarcopenia was diagnosed as 2 SDs below the sex-specific population means, which increases the accuracy of the diagnosis. Handgrip strength is an easy, reliable, and reproducible indicator of muscle strength that is commonly used for the screening and diagnosis of sarcopenia. In this study, decreased handgrip strength was based on cut-off values according to the AWGS criteria \[[@b11-asj-2019-0049]\].
In our study, sarcopenia showed a significantly higher prevalence among the elderly with vertebral fragility fractures compared to matched controls. However, multivariate analysis showed that sarcopenia was not an independent risk factor for fresh vertebral fractures. The close relationship between sarcopenia and low BMD may be responsible for the increased prevalence of sarcopenia among the elderly with vertebral fractures. However, this denotes only an association and not causation. Our analysis indicates that sarcopenia alone may not result in vertebral fractures and that low BMD and presence of previous fractures have more important roles in fracture occurrence.
Sarcopenia showed a significantly higher prevalence among the elderly with vertebral fragility fractures compared to matched controls suggesting that absolute muscle mass may have a more important role in preventing fractures than relative muscle mass. Women generally have lower muscle mass than men with a higher prevalence of vertebral fragility fractures, which could be a result of the loss of protective muscle mass \[[@b19-asj-2019-0049]\]. Ignasiak et al. \[[@b20-asj-2019-0049]\] simulated the effect of aging and sarcopenia in a spine model to determine muscle recruitment patterns and spinal loads. They simulated the loss of muscle fibers of back extensors, erector spinae, and multifidus that occurs with normal aging and sarcopenia. In the normal aging model, they found that loss of these muscles was compensated by an increased activity of other muscles, whereas in sarcopenia, the compensatory muscle groups were ineffective.
The presence of previous vertebral fractures was found to be significantly higher in the case group. Increasing age, preexisting vertebral fracture, and osteoporosis are known risk factors for a new vertebral fracture \[[@b21-asj-2019-0049]-[@b23-asj-2019-0049]\]. It is likely that an incident vertebral fracture will lead to sarcopenia, particularly in the case of prolonged immobilization, which can lead to a positive correlation between them.
Previous studies have shown a positive correlation between sarcopenia, osteoporosis, and fragility fractures \[[@b23-asj-2019-0049]-[@b26-asj-2019-0049]\]. Hida et al. \[[@b9-asj-2019-0049]\] reported that sarcopenia, measured as decreased leg skeletal mass index, was a risk factor for vertebral fractures in elderly Japanese women. In a study of Hida et al. \[[@b9-asj-2019-0049]\], the prevalence of sarcopenia was 42% and 25% in fracture and non-fracture groups, respectively. Our study found a prevalence of 29% and 7.8% in the fracture and non-fracture groups, respectively. In a study of Hida et al. \[[@b9-asj-2019-0049]\], they measured only muscle mass and did not consider strength or physical performance. As decreased muscle mass is not always associated with decreased strength, this could result in overestimation of sarcopenia, which may explain the higher prevalence reported in their study. Moreover, they did not consider the presence of previous fractures in their study. In contrast, in our study, we found that the presence of previous fractures was the strongest risk factor for a new fracture, more so even than sarcopenia or osteoporosis alone.
Yu et al. \[[@b27-asj-2019-0049]\] conducted a prospective study in a large cohort of elderly Chinese men and found that sarcopenia at baseline was an independent risk factor for incident fragility fractures, and the addition of sarcopenia to other risk factors, such as BMD, increased the predictive ability of the model. However, the group with sarcopenia had an incidence of vertebral fractures of only 2.7% over an average follow-up period of 11 years. Although they mentioned the proportion of people with preexisting fractures at baseline, they did not analyze the effect of preexisting fractures on the incidence of new fractures.
Not all studies demonstrated sarcopenia as a risk factor for fragility fractures. Trajanoska et al. \[[@b28-asj-2019-0049]\] followed up 5,911 subjects in a Rotterdam study and found that sarcopenia did not carry a higher risk for fracture compared to BMD. Though sarcopenic individuals had a higher prevalence of fractures, this was associated with lower BMD in this group. Sarcopenia did not add to the risk of fracture in osteoporotic individuals. Harris et al. \[[@b29-asj-2019-0049]\] also found a similar result in their prospective study on elderly women and found that sarcopenia was not an additional risk factor for fractures in women with osteoporosis.
Iolascon et al. \[[@b8-asj-2019-0049]\] analyzed 67 women with vertebral fractures, of which 32 had multiple fractures and found that sarcopenia was more prevalent in women with multiple fractures than in those with a single fracture. Our findings were similar, indicating that sarcopenia is strongly associated with preexisting fractures. However, the causal relationship between sarcopenia and previous fractures cannot be determined without a prospective study.
In our sample, there were 20 pairs of cases and controls without preexisting fractures. Among these, five cases had sarcopenia, whereas none of the controls had sarcopenia. Though not statistically significant, this might indicate the contributory effect of sarcopenia for the development of the first episode of fractures. Future studies matched for the presence of previous fractures and osteoporosis may reveal the exact role of sarcopenia in the development of new fractures and its contribution in patients with preexisting fractures.
The small sample size is the biggest limitation of this study. There are longitudinal studies of large populations that examined the relationship between sarcopenia and osteoporotic vertebral fractures. However, in our country, we lack a common data registry and hence, we had to opt for a case-control study instead of a longitudinal study. The study was conducted in a single center, which might have introduced a selection bias. Although we measured muscle mass using MRI and diagnosed sarcopenia based on reference values obtained from a reference population, this method has not been rigorously validated in other studies. Reference values were obtained from a subset of the population that had attended the hospital to tend to a specific complaint rather than from the community, which was not operationally feasible.
Conclusions
===========
Our results showed that sarcopenia is not an independent risk factor for vertebral fractures among the elderly despite its increased prevalence in this population. The presence of preexisting fractures and decreased T-score still remain the most important predictors of new fracture occurrence.
No potential conflict of interest relevant to this article was reported.
**Author Contributions**
AA, APS, KRR, KSSV, RMK, and SR were involved in conception and design. AA, KRR, and KSSV were involved in data acquisition and analysis. AA, APS, and KRR contributed to drafting the manuscript. APS, RMK, and SR provided critical revisions. And APS and SR provided supervision.
{#f1-asj-2019-0049}
{#f2-asj-2019-0049}
######
Results of normative study for TPA
Variable Women (n=135) Men (n=114)
-------------------------------- --------------- -------------
TPA (mm^2^)
Mean±SD 1,576±295 2,723±541
SE 25.3 50.6
PLVI
Mean±SD 1.51±0.31 2.05±0.46
SE 0.026 0.04
Cut-off values for TPA (mm^2^) 986 1,641
Cut-off values for PLVI 0.89 1.13
TPA, total psoas cross-sectional area; SD, standard deviation; SE, standard error of mean; PLVI, psoas lumbar vertebral index.
######
Comparison between cases and controls with respect to continuous variables
Variable Women *p*-value Men *p*-value
------------------------------------------ ------------- ------------- --------- ------------- ------------- -------
Age (yr) 69.4±9.2 69.3±9.2 0.949 70.3±9.9 69.8±9.7 0.883
Body mass index (kg/m^2^) 25.18±4.4 28.5±5.2 0.005 24.87±4.1 27.15±5.89 0.229
Total psoas cross-sectional area (mm^2^) 1,103±297 1,379±323 \<0.005 1,699±354 2,025±408 0.027
Handgrip strength (kg) 14.3±5.6 15.8±4.7 0.223 23.02±9.2 30.22±5.8 0.016
Psoas lumbar vertebral index 0.83±0.26 0.992±0.28 0.015 1.11±0.26 1.32±0.28 0.037
T-score -2.37±1.4 -1.27±1.3 0.001 -1.12±1.2 -0.127±1.2 0.034
Serum calcium in (mg/dL) 9.85±0.77 9.81±0.49 0.796 9.88±0.82 9.68±0.51 0.43
Serum phosphorus (mg/dL) 3.87±0.65 3.96±0.43 0.501 3.76±0.59 3.76±0.39 0.99
Serum vitamin D (ng/mL) 34.82±19.03 30.30±17.53 0.298 33.11±19.57 24.21±10.93 0.14
Serum parathyroid hormone (pg/mL) 39.99±18.16 43.43±13.10 0.369 36.77±19.34 40.11±13.29 0.59
Values are presented as mean±standard deviation.
######
Comparison between cases and controls with respect to categorical variables, McNemar's test was used
Variable Cases (n=51) Controls (n=51) Odds ratio *p*-value
----------------------------------- -------------- ----------------- ------------ -----------
Sarcopenia
Total psoas cross-sectional area 15 4 12 0.005
Psoas lumbar vertebral index 23 14 2.29 0.064
Old fractures 29 7 12 \<0.001
######
Conditional logistic regression analysis: occurrence of fresh fracture is the dependent variable
Variable Wald *p*-value Odds ratio (95% confidence interval)
----------------- ------- ----------- --------------------------------------
Old fracture 5.888 0.015 7.049 (1.456--34.132)
Sarcopenia 0.673 0.412 2.709 (0.250--29.296)
Body mass index 0.212 0.645 0.967 (0.838--1.116)
T-score 4.377 0.036 0.604 (0.377--0.969)
| {
"pile_set_name": "PubMed Central"
} |
Background
==========
Parkinson\'s disease is a neurodegenerative disorder which affects approximately 1% population over the age of 60 \[[@B1]\]. The most motor symptoms of this disease are caused by dysfunction of the nigro-striatal pathway. DAergic neurons in the substantial nigral pars compacta (SNc) project axons to striatum; when PD patients display symptoms, more than half of the DAergic neurons in the SNc are lost. In the last two decades, several different sources of DAergic cells as transplantation therapy have been tried in animal models and in patients with PD \[[@B2]-[@B6]\]. RPE cell transplantation has been applied in experimental and clinical studies for its capability of producing L-dopa as intermediate product of melanin \[[@B7],[@B8]\]. RPE cell transplantation therapy has many advantages: it does not require immune suppression, the cells are relatively easy to obtain, and the procedure has minimal ethic concern, which make this approach attractive \[[@B9]\].
RPE cells are melanin containing cells that constitute a monolayer between the neural retina and the choroid. In RPE cells, tyrosine is catalyzed by tyrosinase to L-dopa that is polymerized to form melanin \[[@B10]\]. It is hypothesized that L-dopa in the RPE cells can be converted into DA in the terminates of nigrostriatal DAergic neurons and provide DA to nigro-striatal system directly after RPE cells are transplanted \[[@B7]\]. However, such assumption has yet to be verified.
RPE cells play a key role in maintaining the normal function of retina and can express several neurotrophic factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and pigment-derived epithelial factor (PEDF) \[[@B11]\], which nourish the neurosensory retina and also probably provide trophic effects on the host DAergic neurons.
In the present study, we attempt to determine whether the neurotrophic effects of RPE cells play a role in restoring the function of nigrostriatal system in the transplanted model of PD, and to examine whether RPE cells have the ability to synthesize and release DA in the cultures. Our works provide the first evidence that RPE cells can secrete the neurotrophic factors GDNF and BDNF, and synthesize DA, which probably contribute to their beneficial effects of RPE cells transplantation in animal model of PD.
Methods
=======
Cell cultures
-------------
Human RPE cells were obtained from the RPE Cell Bank at the Shanghai 1^st^Hospital. The method to collect the RPE cells was similar to the previous description \[[@B12]\]. In brief, human eyes were dissected by a circumferential incision above the ora serrata near the limbus; the anterior segment and lens were separated and discarded. The neural retina was detached and layer of RPE cells were separated from the choroid. The layer of RPE cells was dissociated in 0.25% trypsin (Gibco-Invitrogen, USA), by gentle titration, and the cells were collected by centrifuge at 100 × g for 5 minutes. Then the cells were calculated and seeded at the density of 10^5^per cm^2^. Growing medium consisted of Dulbecco\'s modified Eagle\'s medium (DMEM, Gibco-Invitrogen, USA), 10% fetal bovine serum (FBS, heat-inactivated, Gibco-Invitrogen, USA) and 100 unit/ml penicillin and streptomycin. At confluence, cells were subcultured by trypsinization.
SH-SY5Y cells were cultured on poly-D-lysine (Sigma, USA) precoated dishes in DMEM supplemented with 10% FBS, and the medium was changed every 3 days.
To culture primary ventral mesencephalic (VM) cells, pregnant Sprague-Dawley (SD) rats at gestation day 14 (Experimental Animal Center of Shanghai, China) were anaesthetized with chloral hydrate (400 mg/kg, i.p.) and VM tissues were dissected from embryonic brain and trypsinized into single-cell suspension using sterilized micropipette tips. The cells were resuspended in DMEM and Ham\'s F12 at 1:1 (D-F12), supplemented with 10% FBS and plated at a final density of 5 × 10^5^viable cells/cm^2^in 24-well plates (Nunc, Denmark) precoated with poly-D-lysine. The cells were incubated at 37°C for 12 hours and then switched to the serum-free medium, consisting of D-F12 with 2% B27 supplement (Gibco-Invitrogen, USA). For differentiation of VM cells, the cultured cells were incubated in serum-free medium for 6 days, and the culture medium was changed each 3 days.
Preparation of conditioned medium
---------------------------------
CM by RPE cells (RPE-CM) was collected as previously described \[[@B13]\]. Briefly, RPE cells were incubated with FBS-deprived medium for 3 days, and the medium was collected and centrifuged at 1000 × *g*for 10 minutes at 4°C to remove cells and debris. The supernatant was concentrated 5-fold in an Amicon Ultra tube (Millipore, USA) by centrifugation (4,000 × *g*, 2 hours) at 4°C. The concentrated medium was diluted by fresh DMEM to 1-fold concentration. The proteins which molecular weight is lower than 10 kDa were removed by filtration.
Determine the protective role of RPE cells in vitro
---------------------------------------------------
SH-SY5Y cells were incubated in DMEM supplemented with 10% FBS. Then the culture medium were replaced with three different medium. One group was incubated with the RPE-CM containing rotenone or 6-OHDA. The second group was exposed to the normal medium containing rotenone or 6-OHDA. The third group was cultured in normal medium without toxins. After 24 hours of incubation, 10 μl of the dye 3, \[4,5-dimethylthiazol-2-yl\]-diphenyltetrazolium bromide (MTT) (5 mg/ml) was added to make the final concentration at 0.5 mg/ml, and then the plates were incubated for 4 hours at 37°C. After medium were removed, 100 μl dimethyl sulfoxide per well was added and the plate was incubated at 37°C for 15 minutes. Color intensity was assessed with a microplate reader at the 570 nm wavelength. Each experiment was performed in triplicate independently.
In order to test the protective role of CM against rotenone, the VM cultures were treated under different conditions as following for 8 hours. 1) RPE-CM with 25 nM rotenone; 2) normal medium with 25 nM rotenone; 3) normal medium without rotenone. Then the neurons were immunostained against TH and the number of TH-immunoreactive (TH-ir) neurons was counted in a blind manner by an unrelated investigator. Ten fields per well (113 mm^2^surface area) were counted using a field lens, and the size of field was 4 mm^2^and 10 fields consisted of about 35% of the whole surface of the cultured well.
For 6-OHDA treatment, the VM cultures were treated under different conditions as following for 24 hours: 1) RPE-CM with 40 μM 6-OHDA; 2) normal medium with 40 μM 6-OHDA; 3) normal medium without 6-OHDA.
Measurement of GDNF and BDNF using Enzyme-linked immunosorbent assay (ELISA)
----------------------------------------------------------------------------
After two days culture of 10^6^RPE cells, 2 ml serum-free medium were collected and ultrafiltered using the Amicon Ultra Tube (10 kDa). To determine in vivo neurotrophic factors expression, 15 mg wet tissues with microcarriers-RPE cells and tissues with microcarriers were lysed for ELISA assay. The lysis buffer was prepared according to the manual in a ratio of 1 mg tissue to 10 μl buffer. The concentrations of GDNF and BDNF were determined using Emax ImmunoAssay System (Promega, USA) \[[@B14]\].
High performance liquid chromatography (HPLC) analysis
------------------------------------------------------
10^6^RPE cells were homogenized in 200 μl 0.4 M perchloric acid. Homogenates were centrifuged at 12,000 rpm for 20 minutes at 4°C and the supernatants were collected for HPLC (Eicom HTEC-500, Japan), while the pellet was dissolved in 0.1 M NaOH for BCA protein analysis (Pierce, USA). One liter mobile phage was consisted of 8.84 g citric acid monohydrate, 10 g sodium acetate anhydrate, 220 mg sodium octane sulfonate, 5 mg EDTA-2Na and 200 ml methanol.
To analyze the DA release, the RPE cells was treated with high potassium solution (56 mM K^+^) (84 mM NaCl, 55 mM KCl, 1 mM MgSO~4~, 1.25 mM KH~2~PO~4~, 2 mM CaCl~2~, 16 mM NaHCO~3~, and 10 mM glucose) as previously described \[[@B15]\]. The high potassium solution collected from RPE cells was mixed with 0.4 M perchloric acid in the ratio of 1:1 and was centrifuged before HPLC assay.
Western blot
------------
10^6^RPE cells were lysed in RIPA lysis buffer \[(in mM): Tris-HCl, 50, pH 7.4; NaCl, 150; 0.1% sodium dodecyl sulphate (SDS), EDTA, 1; 1% Triton X-100, 1% sodium deoxycholate, PMSF, 1; 5 μg/ml aprotinin, 5 μg/ml leupeptin\]. Protein concentration was measured and 40 μg of total proteins were loaded to SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred onto polyvinylidene difluoride (PVDF, Millipore, USA) membrane, and incubated with anti-DDC antibody (Proteintech Group, USA) or anti-dopamine transporter (DAT) antibody (Santa Cruz, USA) overnight. After incubation, the membrane was washed and incubated with peroxidase-conjugated goat anti-rabbit IgG (Pierce, USA), and developed with Super Signal West Dura Extended Duration Substrate (Pierce, USA).
Reverse transcription PCR analysis
----------------------------------
Total RNA from RPE cells was prepared using Trizol reagent (Invitrogen, USA) and digested with RNase-free DNase for 30 minutes to remove genomic DNA. 2 μg of RNA were reverse transcribed into cDNA with the Reverse Transcription System (Promega, USA) in 20 μl volume. cDNA was used as template in the following PCR assay. The primers used for PCR assays were as follows: (1) DDC, forward: 5\'-TTACTCATCCGATCAGGCACAC-3\', reverse: 5\'-GGCAGAACAGTCAAAATTCACC-3\'; (2) DAT, forward: 5\'-CGAGGCGTCTGTTTGGAT-3\', reverse: 5\'-CAGGGAGTTGATGGAGGTG-3\'; (3) GAPDH, forward: 5\'-CCATGTTCGTCATGGGTGTGAACCA-3\', reverse: 5\'-GCCAGTAGAGGCAGGGATGATGTTC-3\'. PCR conditions were 95°C for 10 minutes, followed by 35 cycles of 94°C for 45 seconds, 58°C for 45 seconds, 72°C for 1 minute, and a final extension step at 72°C for 5 minutes.
RPE cells-microcarriers attachment
----------------------------------
The microcarriers which were dextran particles coated with gelatin (Cytodex 3, Sigma, USA) were sterilized and hydrated according to the manufacture manual (Sigma, USA). Dry microcarriers were swollen in Ca^2+^, Mg^2+^-free phosphate buffered saline (PBS) (50--100 ml/g Cytodex) for at least 3 hours and the microcarriers were sterilized by autoclaving (120°C, 20 minutes). The microcarriers were rinsed using medium three times before mixed with RPE cells, suspended at 2 × 10^6^density in 1 ml medium and then mixed with 10^5^microcarriers. The mixture was shaken in the rate of 60 rpm for 2 hours at 37°C, and then was cultured for 24 hours \[[@B16]\].
6-OHDA lesion and RPE cell transplantation
------------------------------------------
SD rats were housed pre- and post-surgery in a temperature and humidity controlled room with a 12 hours light-dark cycle. Food and water were freely available.
Experimental rats were anesthetized with chloral hydrate (400 mg/kg) and received brain injection on a stereotaxic frame (Myneurolab, USA). 6-OHDA (6 μg in 3 μl) was dissolved in normal saline containing ascorbic acid (0.2 mg/ml), and injected into the right medial forebrain bundles (MFB; anterior-posterior: -4.2 mm, lateral: -1.5 mm from bregma, dorsal-ventral: -7.7 mm from dura, toothbar set at -2.4 mm) via a 10 μl hamilton syringe with a blunt-tip needle at a flow rate of 1 μl/minute. After injection, the needle was left *in situ*for 10 minutes and then slowly withdrawn. A gelfoam plug was placed on the broken dura and the skin was sutured \[[@B17]\].
Microcarriers with RPE cells were washed three times with Ca^2+^, Mg^2+^-free PBS and were kept at 4°C before transplantation. The cell transplantation was performed by stereotaxic injection into the right side of striatum (anterior-posterior 1.5 mm, lateral -2.0 mm from bregma, dorsal-ventral -5.0 mm from dura, set toothbar at -3.3 mm) as described previously \[[@B18]\]. Some of the rats were transplanted with the microcarriers alone as control.
Behavioural testing
-------------------
SD rats were tested for the AIR behavior two weeks after 6-OHDA lesion and four weeks after transplantation by administration with apomorphine (0.2 mg/kg, i.p.). Only the rats that exhibited a mean rotation toward the healthy side at least 6.0 full body turns per minute were used for transplantation. Four weeks after transplantation, rotation behavior of rats was examined again. Rats transplanted with microcarriers alone were used as control for the behavior test.
Histological procedure and immunostaining
-----------------------------------------
Rats were deeply anesthetized with chloral hydrate and sacrificed by transcardial perfusion with PBS (37°C) for 20 minutes followed by 4% paraformaldehyde (PFA) (4°C) for 10 minutes. Brains were removed, postfixed for 2 hours in PFA and then cryoprotected for 24 hours in PBS with 30% sucrose. Before frozen in -80°C, the brains were embedded in embedding medium compound (Sakura, USA). Coronal sections (10 μm) were made through the striatum containing transplants and mounted to gelatin coated slides. Adjacent sections were processed for hematoxylin-eosin (HE) stain and immunohistochemistry.
The sections were stained against cytokeratin antibody (1:300, Sigma, USA), and the primary VM neurons were stained against TH (1:3000, sigma, USA). A biotinylated secondary rabbit anti-mouse antibody (Vector Laboratories, UK) and peroxidase-coupled avidin-biotin staining kit (ABC kit, Vector Laboratories, UK) were used.
For HE staining, the tissue sections were submerged into the hematoxylin solution (0.5% hematoxylin, 5% aluminium ammonium sulphate, 1% ethanol, 0.1% sodium iodate, 2% acetic acid and 30% glycerol) for 10 minutes and washed by tap water. Place the sections in acid alcohol (0.3% concentrated hydrochloric acid in 70% ethanol) for several seconds and then in eosin solution (0.1% eosin, 0.4% acetic acid in 95% ethanol) for 1 minute. Then the sections were dehydrated and sealed.
Statistics
----------
All data were expressed as means ± SEM. Independent *t*-test followed by *post hoc*Bonferroni tests were used for the analysis of other data via the SPSS 10.0 soft packages (SPSS Inc., USA). The criterion for statistical significance was set at *p*\< 0.05.
Results
=======
RPE-CM protects against rotenone and 6-OHDA toxicity through GDNF and BDNF secretion
------------------------------------------------------------------------------------
The neuroprotective ability of the RPE-CM was determined by adding CM into neurotoxins-treated DAergic cell cultures. SH-SY5Y cultures were challenged by rotenone or 6-OHDA. After exposure to 10 μM rotenone for 24 hours, the cell viability in the cultures was determined by MTT assay. It was found that rotenone treatment resulted in 43.1% decrease in cell viability as compared with control cultures (Fig [1A](#F1){ref-type="fig"}). Incubation with RPE-CM significantly attenuated the rotenone-induced decrease in cell viability by 72.9% (Fig [1A](#F1){ref-type="fig"}). When SH-SY5Y cells were challenged by 50 μM 6-OHDA, RPE-CM showed a similar protective ability on the cells viability. 6-OHDA decreased the cell viability by 65.3%, treatment with RPE-CM protected the cell viability by 56.7% (Fig [1B](#F1){ref-type="fig"}).
{#F1}
BDNF and GDNF are believed to be the most important neurotrophic factors in the survival of DAergic cells \[[@B14],[@B19],[@B20]\]. So we focused on these two neurotrophic factors and measured the level of these two neurotrophic factors by ELISA assay to determine whether the protective effect of RPE-CM is mediated by the secretion of GDNF and BDNF. We found the RPE-CM contained high levels of GDNF (0.019 pg/ml) and BDNF (0.49 pg/ml) (Table [1](#T1){ref-type="table"}). Furthermore, adding antibodies of GDNF and BDNF to abolish their biological effects demonstrated that GDNF and BDNF were key elements in the neurotrophic protection of RPE-CM. RPE-CM with antibodies against GDNF or BDNF (1 μg/ml) was added into the SH-SY5Y cultures in the presence of 10 μM rotenone or 50 μM 6-OHDA. After 24 hours incubation, the cell viability of SH-SH5Y cultures was measured by MTT assay. Application of antibody against GDNF decreased the CM-mediated protection on SH-SY5Y cells by 41.4% and 46.7% against rotenone and 6-OHDA induced injury, respectively (Fig [1C, D](#F1){ref-type="fig"}). While antibody against BDNF could reduce the CM-induced protection on SH-SY5Y cells by 38.7% and 85.9% against rotenone and 6-OHDA induced injury, respectively (Fig [1C, D](#F1){ref-type="fig"}).
######
Neurotrophic factors secreted by RPE cells
Trophic factors BDNF GDNF
--------------------------------- ------------- ---------------
Concentration in medium (pg/ml) 0.49 ± 0.09 0.019 ± 0.005
Serum-free medium was incubated with RPE cells for two days, and subjected to ELISA assay.
To further support our findings, we then tested the neuroprotection of RPE cells in primary VM DA neurons culture. Exposure to 25 nM rotenone for 8 hours resulted in a significant loss of the TH-positive cells by 50.6% as compared with control cultures without rotenone treatment (Fig [2B](#F2){ref-type="fig"}), while incubation with RPE-CM significantly attenuated the rotenone-induced loss of TH-positive cells by 44.3% (Fig [2A](#F2){ref-type="fig"}).
{#F2}
When DAergic neuron cultures were challenged by 6-OHDA at 40 μM, RPE-CM showed a similar protective ability on the TH-positive cells. 6-OHDA treatment caused a 43.2% loss of TH-positive cells as compared with non-toxin control cultures (Fig [2F](#F2){ref-type="fig"}), while RPE-CM attenuated the 6-OHDA-induced TH-positive cell loss by 63.1% (Fig [2E](#F2){ref-type="fig"}).
RPE cells express GDNF and BDNF after transplantation
-----------------------------------------------------
As the role of GDNF and BDNF was demonstrated in the neuroprotection of RPE-CM against 6-OHDA and rotenone neurotoxicity in vitro, we measured the levels of GDNF and BDNF in the RPE cell-grafted striatal tissues. Four weeks after transplantation the striatal tissues with microcarriers-RPE cells were taken out and homogenated, followed by centrifugation at 12000 rpm for 20 minutes. The striatal tissues transplanted with microcarriers were used as control. ELISA assay showed that tissues with microcarriers-RPE cells had 41.2% and 68.1% higher levels of GDNF and BDNF as compared with the control group which contained microcarriers only (Fig [3A, B](#F3){ref-type="fig"}).
{#F3}
RPE cells express DDC and synthesize DA
---------------------------------------
DDC is an enzyme that converts L-dopa to DA; the expression of DDC indicates the RPE cells have the ability to produce DA. To determine whether the RPE cells can synthesize DA, we measured the DDC mRNA by RT-PCR and DDC protein by immunoblot, which showed that the RPE cells could transcribe DDC mRNA and express abundant DDC protein (Fig [4](#F4){ref-type="fig"}). But the mRNA and the protein of DAT which transports DA through the membrane couldn\'t be detected by RT-PCT and immunoblot (Fig [4](#F4){ref-type="fig"}).
{#F4}
Furthermore, we measured the content of DA and its metabolite homovanillic acid (HVA) in RPE cells by HPLC (Fig [5](#F5){ref-type="fig"}), which showed DA (peak time: 5.43 minutes) level as 29.13 ng/mg protein and HVA (peak time: 11.51 minutes) level as 267.89 ng/mg protein (Table [2](#T2){ref-type="table"}). However, DA release into the buffer was not detected after 56 mM potassium chloride treatment in the cultured RPE cells, suggesting that the high potassium-depolarization can not induce DA release from RPE cells (Table [2](#T2){ref-type="table"}). It\'s likely that RPE cells may have other mechanism to transfer DA throughout the membrane.
######
DA and HVA in RPE cells extract and DA release after potassium treatment
DA HVA
----------------------------------- -------------- ----------------
Cells extract (ng/mg protein) 29.13 ± 4.11 267.89 ± 16.10
Release after potassium treatment None None
RPE cells were homogenated and centrifuged. The supernatant was examined by HPLC for DA and its metabolic. To determine the DA release, RPE cells were depolarized with high potassium (56 mM K^+^) and the buffer was subjected to HPLC assay.
{#F5}
Microcarriers-RPE cells survive in the host striatal tissues and significantly improve AIR in 6-OHDA-lesioned rats
------------------------------------------------------------------------------------------------------------------
To demonstrate the RPE cells survival in the host striatum, we performed HE staining and cytokeratin immunostaining. HE staining showed that transplants were accurately placed into the striatum (Fig [6A](#F6){ref-type="fig"}) and RPE cells were attached outside the microcarriers (Fig [6B](#F6){ref-type="fig"}); immunostaining demonstrated that these cells were cytokeratin-immunoreactive, a morphological marker of live RPE cells (Fig [6D](#F6){ref-type="fig"}).
{#F6}
Before transplantation, AIR showed the basal level of rotation in 6-OHDA lesioned rats. We selected the rats that exhibited rotation toward the healthy side at least 6.0 full body turns per minute for transplantation of microcarriers-RPE or microcarriers alone as control. After transplantation, microcarriers-RPE grafted animals displayed a significant reduction in AIR behavior compared to control rats that was transplanted with microcarriers alone (*p*\< 0.05) (Fig [6F](#F6){ref-type="fig"}).
Discussion
==========
Most of the cell-based therapies for PD are focused on two goals: one is to provide a source of neurotrophic factors which may modify disease course and the other is to provide a constant level of DA. RPE cell transplantation is a promising therapy as shown in preliminary clinical trial. In the present study, we attempt to elucidate the mechanisms of this therapy by determining: whether RPE cells exert protective effects on DAergic cells when challenged by neurotoxins; whether RPE cells can produce and release DA. Our results indicate that 1) RPE cells can express and secrete GDNF and BDNF and protect DAergic cells against neurotxoins-induced injury; 2) RPE cells can express DDC and synthesize DA; 3) RPE cells attached to microcarriers can survive in the host striatum and produce high level GDNF and BDNF after transplantation; and 4) RPE cells transplantation produce a statistically significant improvement of AIR.
In the previous transplantation studies, microcarriers were used to increase the survival of grafted cells \[[@B21]\]. Indeed, microcarriers provide a substrate to which the cells can establish a basal lamina and thus create a more favorable microenvironment. Furthermore, cells attached to beads may alter the immunogenic properties of cells, which may prevent the recognition and immunological surveillance \[[@B16]\]. In our experiments, we used cytodex 3 which consists of a thin layer of denatured collagen chemically coupled to a matrix of cross-linked dextran, and these microcarriers facilitate the survival of transplanted RPE cells.
We demonstrate that RPE cells can provide trophic effect on DAergic cells, which may be one of the possible mechanisms underlying RPE cell therapy. Previous studies had showed that RPE cells expressed several neurotrophic factors such as PEDF, PDGF, EGF, and VEGF \[[@B11]\]. Our results elucidate that RPE cells can secrete BDNF and GDNF and these two factors play important role in the neurotrophic effects of RPE cells. Although RPE cells can express PEDF, it accounts for only a portion of the neurotrophic effect \[[@B22]\]. In this study we demonstrate that GDNF and BDNF in RPE-CM contribute for the most part of trophic effect. We also demonstrate that GDNF and BDNF are expressed by grafted RPE cells.
Besides the neurotrophic effect of RPE cells, we document that RPE cells can express DDC and produce DA. L-dopa is a precursor of DA, and can be synthesized by RPE cells as an intermediate product of melanin \[[@B23]\]. DDC, an enzyme to convert L-dopa to DA, is found in the RPE cells in our study. However, the depolarization-induced DA release is not detected in the cells, indicating that the DA release machinery as seen in most excitable cells is not present in the RPE cells. It\'s possible that RPE cells may have other mechanism to transfer DA throughout the membrane. Previous report by Dalpiaz et al \[[@B24]\] showed that DA could permeate the membrane of RPE cells, and this permeation seems to be mediated by organic cation transporter 3 \[[@B25]\]. The ability of DA synthesis in RPE cells suggests RPE cells transplantation may be one of the advantages for the cell replacement therapy to treat advanced PD patients.
Conclusion
==========
RPE cells not only replenish L-dopa as elucidated by previous study, but can also synthesize DA and neurotrophic factors which protect the intrinsic neurons after transplantation. These findings make this cell replacement a more viable and promising therapy for PD.
Competing interests
===================
The authors declare that they have no competing interests.
Authors\' contributions
=======================
The studies were designed by MM and WL and were performed by MM, XL, and XF. Human RPE cells were separated and cultured by QG. DY, LL and SC gave advises on the work and helped in the interpretation of the data. WL supervised all the work and wrote the paper together with MM. All authors read and approved the final manuscript.
Acknowledgements
================
This work was supported by a grant from 973 National Project (NO. 2005CB724302), the National Natural Science Foundation (NO. 30730096), the National Basic Research Program of China from Science and Technology Commission (NO. 2007CB947904) and the Technology Commission (863 project 2007AA02Z460).
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#Sec1}
============
Gastric cancer (GC) is the fifth most common malignancy and the third leading cause of cancer-related death worldwide, and the highest mortality rates are seen in East Asia, including China \[[@CR1], [@CR2]\]. Although overall survival has improved with the implementation of standard D2 lymphadenectomy and the advancement of chemotherapy and targeted treatments \[[@CR3]--[@CR5]\], the survival rate of patients with GC is less than 30% \[[@CR2]\]. Currently, the mechanisms of GC remain unclear. However, recent evidence has indicated that cross-talk between tumor cells and immune or nonimmune stromal cells creates a unique microenvironment that is essential for tumor growth, invasion, and metastasis \[[@CR6], [@CR7]\]. The interaction between tumor cells and stromal cells may polarize stromal cells to favour tumor promotion \[[@CR7]\].
In addition to tumor cells, a variety of immune stromal cells are the main components of the GC environment. Neutrophils, as the most abundant circulating leucocytes, are also one type of mostly infiltrating immune and inflammatory cells in GC \[[@CR8]\]. It has been reported that elevated peripheral blood neutrophils and the neutrophil/lymphocyte ratio predict poor outcomes in many types of cancers, including GC \[[@CR9]--[@CR11]\]. The same has been found in neutrophils that have infiltrated in tumors \[[@CR9]\]. In the tumor microenvironment, tumor-associated neutrophils (TANs) have been proposed to promote cancer initiation, progression, and metastasis \[[@CR12]\]. In addition to contact-dependent mechanisms, neutrophils may influence tumor progression through the paracrine release of cytokines and chemokines with protumor or antitumor functions, depending on the tumor microenvironment \[[@CR13]\]. Moreover, neutrophils can release neutrophil extracellular traps (NETs), which can trap circulating tumor cells in vitro; trapping by NETs is associated with increased formation of micrometastasis in vivo \[[@CR14], [@CR15]\]. These novel aspects of neutrophil biology may contribute to GC progression and metastasis. However, direct evidence that supports a role of neutrophils in the immunopathogenesis of human cancers is scarce.
Epithelial-mesenchymal transition (EMT), a well-characterized embryological process, has been identified to play a critical role in tumor progression, invasion and metastasis, and is a way by which cancer cells gain more aggressive properties. In the process of EMT, epithelial cells undergo a phenotypic switch by losing their cell polarity and expression of epithelial markers (E-cadherin, β-catenin), to become mesenchymal cells through the acquisition of mesenchymal markers (N-cadherin, Vimentin, ZEB1) expression; thus, these transformed epithelial cells acquire fibroblast-like properties and exhibit reduced cell-cell adhesion and increased motility \[[@CR16]--[@CR18]\]. The enhanced motility and invasiveness afforded by EMT is critical in the initiation of metastasis for cancer progression, and the acquisition of a mesenchymal phenotype has also been shown to enhance resistance to chemotherapy and lead to a poor prognosis \[[@CR19], [@CR20]\]. The expression of these EMT markers can be induced by a number of growth factors/cytokines such as transforming growth factor (TGF)-β, interleukin-6 (IL-6), and CXCL12 \[[@CR21]--[@CR23]\], as well as a variety of transcription factors such as STAT3 and hypoxia-inducible factor-1α (HIF-1α) \[[@CR22], [@CR24]\].
Epithelial cell-stromal cell interactions usually regulate EMT, and the factors that induce EMT are often originated from the stromal cells that constitute the tumor microenvironment. Neutrophils are essential components of the tumor stroma and the key players in regulating tumor progression \[[@CR12], [@CR25]\]. It has been observed that TANs actively communicate with tumor cells through growth factors or inflammatory cytokines such as TNFα, CCL2, IL-8, and IL-17a, which can promote tumorigenesis and progression \[[@CR9], [@CR26]\]. Neutrophils are a source of IL-17a in the setting of inflammation and autoimmune diseases \[[@CR27], [@CR28]\]. IL-17a is an immune and inflammatory mediator with multiple biological activities. It is widely found in the inflammatory microenvironment of various tumors, including GC, and is involved in promoting tumor cell migration and invasion, chemotherapy resistance, and immunosuppression, which causes tumor progression and metastasis \[[@CR29]--[@CR31]\]. Clinical studies have suggested that an elevated number of IL-17a-producing cells is an independent marker of adverse survival in cancer \[[@CR32]\]. IL-17a exerts its effects by binding to IL-17Ra, a common cytokine receptor, which leads to activation of the Janus kinase (JAK) family of tyrosine kinases and the signal transducers and activators of transcription (STAT) family, particularly STAT3 \[[@CR33], [@CR34]\]. Activation of the IL-17a/JAK2/STAT3 pathway plays an active role in the progression of a variety of tumors \[[@CR29], [@CR33], [@CR35]\]. Studies have shown that both STAT3 and phosphorylated STAT3 increased in intestinal-type GC compared with normal gastric tissues \[[@CR36]\]. Furthermore, phosphorylated STAT3 was positively associated with poorly differentiated adenocarcinoma, lymph node metastasis, and poor prognosis \[[@CR37]\]. However, the role of TANs and IL-17a in GC has not been well addressed. Therefore, our aim was to determine how TANs promote the migration and invasiveness of GC cells and to reveal the association between TANs and activation of the IL-17a/JAK2/STAT3 pathway in the progression of GC cells.
In this study, we have shown that neutrophils in GC correlate with prognosis. Neutrophils are a source of IL-17a in clinical sample analysis and experimental studies. We tested the hypothesis that TAN-derived IL-17a enhances migration, invasiveness and EMT of GC cells. We show that EMT is induced by GC neutrophils via secretion of IL-17a and activation of JAK2/STAT3 signalling in GC cells. This effect was blocked by application of an IL-17a neutralizing antibody. Thus, an IL-17a neutralizing antibody could serve as a novel therapeutic strategy in GC.
Materials and methods {#Sec2}
=====================
Patients and clinical specimens {#Sec3}
-------------------------------
In all, 327 patients who presented between 2007 and 2009 at the Department of Gastroenterological Surgery of Harbin Medical University Cancer Hospital (Harbin, China) were recruited for this study. In this study, all patients were diagnosed with gastric adenocarcinoma, and no patients were treated with neoadjuvant chemotherapy. Patients with infectious diseases, autoimmune disease or multiple primary cancers were excluded from the study. Paraffin-embedded and formalin-fixed tissues were obtained from 327 patients with GC. The medical data and follow-up information of GC patients were identified from our prospective database. Fresh paired intratumoral and nontumoral (at least 5 cm from the tumor site) tissues were obtained from patients with GC who underwent surgical resection, 10 samples were used for immunofluorescence, 10 samples were used for neutrophils isolation, and 10 samples were used to prepare tumor tissue culture supernatants (TTCS) or non-tumor tissue culture supernatants (NTCS). Peripheral blood from 20 healthy donors was also used for neutrophils isolation.
The tumor size was defined according to the longest diameters of the samples. The eighth edition of the American Joint Committee on Cancer Tumor Node Metastasis (AJCC TNM) staging classification for carcinoma of the stomach was used for tumor staging. The Lauren classification was defined as intestinal type, diffuse type, and mixed type. The histological grade was classified as G1, G2, G3 adenocarcinoma, signet ring cell carcinoma, and mucinous adenocarcinoma. Lymphovascular invasion and perineural invasion were diagnosed by H&E-stained slides. Disease-free survival (DFS) was defined as the date of surgery to the date of identification of disease recurrence, which was either radiological or histological. Disease-specific survival (DSS) was calculated from the date of surgery to the date of death from GC; patients who died of causes unrelated to the disease were censored at the last follow-up. This retrospective study was approved by the ethics committee of Harbin Medical University Cancer Hospital, China.
Immunohistochemistry and immunofluorescence {#Sec4}
-------------------------------------------
Immunohistochemical staining was performed using the avidin-biotin-peroxidase complex method. Briefly, paraffin-embedded and formalin-fixed tissues were cut into 4 μm sections and incubated on slides. The sections were deparaffinized in xylene and rehydrated in graded ethanol solutions. Then the slides were incubated in 3% H~2~O~2~ to block the endogenous peroxidase activity. Antigen retrieval was performed by autoclaving the sections for 2 min in citrate buffer (pH 6.0). Primary antibodies against CD66b (1:400 dilution, 555,723, BD) and IL-17a (1:200 dilution, ARG55256, Arigo) were applied to the slides, which were incubated at 4 °C overnight. The Envision-plus detection system was applied to the sections with anti-mouse polymer (1:500 dilution, ab205719, Abcam) or anti-rabbit polymer (1:500 dilution, ab6721, Abcam) at 37 °C for 30 min. Staining was performed with 3,30-diaminobenzidine tetra hydrochloride and counterstaining was performed with Mayer's haematoxylin. In all assays, we included negative control slides with the primary antibodies omitted. The tissue sections were screened using an inverted research microscope (Nikon, Japan).
For immunofluorescence analysis, frozen sections of human GC tissues were fixed in acetone for 15 min and were permeabilized with 0.1% Triton X-100 for 15 min at room temperature. After blocking with normal nonimmune goat serum for 30 min, tissue sections were incubated with CD66b (1:100 dilution, 555,723, BD) and IL-17a (1:100 dilution, ab9565, Abcam) antibodies. Then, the tissue sections were stained with Alexa Fluor 555-conjugated anti-mouse IgG (A16071, Invitrogen) and Alexa Fluor 488-conjugated anti-rabbit IgG (31,635, Invitrogen) antibodies, Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Negative control staining was performed by omission of the primary antibody. Immunofluorescence images were observed using a fluorescence microscope equipped with the MetaMorph Imaging System (Universal Imaging Corporation).
Tumor cell lines {#Sec5}
----------------
Gastric cell lines (GES-1, MKN45, and MKN74) were purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, and were cultured at 37 °C in a humidified atmosphere of 5% CO~2~ in RPMI-1640 medium containing 10% FBS with 100 U/ml penicillin and 100 U/ml streptomycin. For co-culture studies, 12-well Transwell chambers with 0.4 μm porous polycarbonate membranes (Corning, Union City, CA, USA) were used with 1 × 10^6^ MKN45 or MKN74 cells seeded 24 h before co-culture and 2.5--5 × 10^6^ TANs added to the upper or lower chamber. Humanized anti-IL-17a receptor antibody (Abcam, USA, 10 μg/ml) or human IgG antibody (10 μg/ml) were used in the indicated experiments and added to the selected wells. After 24 to 48 h, GC cells and CD66b + TANs cells were collected.
Neutrophil isolation {#Sec6}
--------------------
### Neutrophils from peripheral blood {#Sec7}
Peripheral blood was collected from healthy donors after written informed consent was obtained. First, 5.0 ml of anti-coagulated whole blood was layered over 5.0 ml of PolymorphPrep (Axis-Shield PoC AS, Oslo, Norway) in a 15 ml centrifuge tube. The samples layered over PolymorphPrep were centrifuged at 500 g for 30 min in a swing-out rotor at room temperature. After centrifugation, two leukocyte bands could be visible. The top band at the sample/medium interface should consist of mononuclear cells, while the lower band should consist of polymorphonuclear neutrophils (PMNs). The cell bands were harvested using a Pasteur pipette. The remnant RBCs were lysed using a hypotonic lysis procedure to obtains a pure PMN population. The morphological examination and cell count were performed to determine the number and purity of the PMNs. Neutrophils were cultured in RPMI-1640 containing 10% FBS with 100 U/ml penicillin and 100 U/ml streptomycin. The purity of the neutrophils was 98% after this procedure. The sorted cells were used unless their viability was determined \> 90% and their purity was determined \> 95%.
### Neutrophil from gastric tissues {#Sec8}
For neutrophil isolation, fresh gastric tissues were sliced into small pieces and digested in RPMI-1640 supplemented with 0.05% collagenase IV (Sigma-Aldrich, St. Louis, MO), 0.002% DNase I (Roche, Indianapolis, IN), and 20% fetal bovine serum at 37 °C for 60 min. We filtered dissociated cells through a 150 mm mesh and then these cells were centrifuged at 2500 rpm for 20 min at a 1 ml cell suspension and 10 ml of Ficoll-Hypaque (Stemcell Technologies, Vancouver, Canada) in a 15 ml tube. Thereafter, the leukocytes were harvested and CD66b + neutrophils were isolated using the EasySep PE Selection Kit (Stemcell Technologies, Vancouver, Canada) according to the manufacturer's protocol. We confirmed purification of neutrophils via fluorescence-activated cell sorting analysis with anti-CD66b antibodies, which showed that their viability was greater than 85% and that their purity was greater than 85% for TANs.
### Preparation of TTCS and NTCS and supernatant-conditioned neutrophils {#Sec9}
Tumor tissue culture supernatants (TTCS) or non-tumor tissue culture supernatants (NTCS) were prepared by plating autologous tumor or non-tumor gastric tissues in 1 ml RPMI-1640 containing 10% FBS with 100 U/ml penicillin and 100 U/ml streptomycin for 24 h. The supernatant was then centrifuged and harvested. To generate supernatant-conditioned neutrophils, neutrophils from healthy donors were first harvested and cultured with 30% TTCS or NTCS at a density of 2.5 × 10^6^ cells per ml for 24 h and were then washed with RPMI-1640 medium for three times. Neutrophils cultured with RPMI-1640 medium were used as controls.
### Enzyme-linked immunosorbent assay (ELISA) {#Sec10}
The concentration of IL-17a in the medium was measured by an ELISA (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions.
### Cell migration and invasion assay {#Sec11}
The cell migration/invasion assay was performed in a 24-well Boyden chamber with an 8-mm pore size polycarbonate membrane (Corning, Union City, CA, USA). An appropriate amount of Matrigel (1:8) was added to the upper chamber of the Transwell plates for the invasion assay, while the plates without Matrigel in the upper chamber were used for the migration assay. The treated cells were incubated in serum-free medium for 24 h and 1.25--2.5 × 10^5^ TANs in 500 μl RPMI-1640 containing 10% FBS were added to the lower chamber before 5 × 10^4^ MKN45 or MKN74 cells in 500 μl in RPMI-1640 containing 10% FBS were added to the upper chamber. The cells were incubated at 37 °C for 24--48 h in a 5% CO2 incubator. Cancer cells remaining on the upper surface of the membrane were removed. The migrated cells on the lower surface of the membrane were rinsed with PBS for 5 min to remove residual neutrophils and were subsequently fixed and stained with crystal violet. Subsequently, at least six randomly selected fields were counted and the average number was presented.
### Quantitative real-time PCR (qRT-PCR) {#Sec12}
Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's manual. RNA (1 μg) was reverse transcribed into cDNA using a Reverse Transcription System (Promega, Madison, WI, USA). To quantify the IL-17a mRNA level, real-time PCR was performed using a Light Cycler 480 SYBRGreen Kit (Roche Applied Science, Mannheim, Germany) according to the manufacturer's instructions. GAPDH served as the endogenous reference. Data were analysed by using the comparative Ct method. The specificity of the resulting PCR products was confirmed by examination of the melting curves. The primers used in this assay were:
IL-17a: 5′-CGGTCCAGTTGCCTTCTCCC-3′ (upper)
and 5′-GAGTGGCTGTCTGTGTGGGG-3′ (lower);
GAPDH: 5′-GGACCTGACCTGCCGTCTAG-3′ (upper)
and 5′-GTAGCCCAGGATGCCCTTGA-3′ (lower).
### Western blot analysis {#Sec13}
Total protein was extracted from cells using RIPA buffer in the presence of protease inhibitor (Sigma, USA) and phosphatase inhibitor cocktail (Sigma, USA). The protein concentration was determined with a BCA Protein Assay Kit (Beyotime, Shanghai, China). Proteins were separated by 10% SDS-PAGE and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, MA, USA). The membranes were blocked with 5% nonfat milk (BD Biosciences, WA, USA) and 0.1% Tween-20 in Tris-buffered saline and immunoblotted overnight with primary antibodies at 4 °C with gentle shaking. Subsequently, the membranes were stained with HRP-conjugated secondary antibody. Proteins were visualized using ECL Western Blotting Substrate or Super Signal West Femto Chemiluminescent Substrate (Pierce, Rockford, IL, USA) followed by exposure to film. Antibodies used in this study are as follows: Anti-GADPH (1:1000 dilution, ARG10112, Arigo), anti-E-cadherin (1:1000 dilution, ARG66195, Arigo), anti-Vimentin (1:1000 dilution, ARG66199, Arigo), anti-ZEB1 (1:500 dilution, ARG57524, Arigo), anti-p-JAK2 (1:500 dilution, ARG57812, Arigo), anti-JAK2 (1:500 dilution, ARG57629, Arigo), anti-p-STAT3(1:500 dilution, ARG51549, Arigo), and anti-STAT3 (1:500 dilution, ARG53604, Arigo). The secondary goat anti-rabbit or goat anti-mouse (1:5000 dilution, Abcam, USA).
### Statistical analyses {#Sec14}
SPSS 19.0 software (Version 19.0, Chicago, IL, USA) and Graphpad prism 5.0 were used for all statistical analyses. The results were expressed as the mean ± S.D. Analyses of variance and Pearson chi-square tests were used to assess any associations between variables. Clinical outcomes were calculated by Kaplan-Meier survival curves, and the groups were compared using the log-rank test. Stepwise multivariate Cox proportional analysis was also performed. The level of significance permitting multivariate analysis inclusion and the statistical significance for all other tests used was set at *P* \< 0.05.
Results {#Sec15}
=======
The distribution of neutrophils in GC and its relationship with clinicopathological features {#Sec16}
--------------------------------------------------------------------------------------------
In total, 327 patients were enrolled in the study. The clinicopathological features of GC patients are shown in Additional file [1](#MOESM1){ref-type="media"}: Table S1. The median DFS and DSS of the GC patients were 25.1 months and 33.9 months, respectively. Moreover, 221 patients (67.7%) experienced postoperative recurrence, and 214 (65.4%) had died of GC by the final follow-up. The median follow-up duration was 45.7 months (range 3.03--112.1 months), and the average age was 57.3 years (range 29--88 years). In addition, 186 of 300 patients received postoperative adjuvant chemotherapy, and the average number of harvested lymph nodes after surgical resection was 37.5 (range 10--69).
Neutrophils were widely distributed in the gastric tissues of patients with GC and were obviously increased in number in GC tissues, especially, at the invasion of the edge. The number of neutrophils at the invasion margin was significantly higher than that at the nontumoral tissues (101.70 ± 3.3 vs 8.46 ± 0.49, *P* \< 0.001) and that at the tumor center (101.70 ± 3.3 vs 59.96 ± 2.22, *P* \< 0.001) (Fig. [1](#Fig1){ref-type="fig"}). No correlation was observed between the number of neutrophils at the nontumoral tissues and the clinicopathological features of GC (all *P* \> 0.05) (Additional file [1](#MOESM1){ref-type="media"}: Table S1). The number of neutrophils at the invasion margin was significantly associated with TNM stage (*P* = 0.016), lymphovascular invasion (*P* = 0.008), and perineural invasion (*P* \< 0.001). However, the number of neutrophils was not associated with age, gender, tumor location, tumor size, Lauren type, or histological grade (Additional file [1](#MOESM1){ref-type="media"}: Table S1). The number of neutrophils at the tumor center was correlated with lymphovascular invasion (*P* \< 0.001), and perineural invasion (*P* \< 0.025), but was not associated with age, gender, tumor location, tumor size, TNM stage, Lauren type, or histological grade (Additional file [1](#MOESM1){ref-type="media"}: Table S1).Fig. 1Representative picture of immunohistochemical staining of CD66b + neutrophils at the nontumoral, invasive margin and tumor center tissues of GC sampers. **a** High CD66b + neutrophils density at the nontumoral gastric tissues. **b** Low CD66b + neutrophils density at the nontumoral gastric tissues. **c** High CD66b + neutrophils density at the invasive margin of GC tissues. **d** Low CD66b + neutrophils density at the invasive margin of GC tissues. **e** High CD66b + neutrophils density at the tumor center of GC tissues. **f** Low CD66b + neutrophils density at the tumor center of GC tissues. Magnifications: × 200
The relationship between neutrophils in GC and prognosis {#Sec17}
--------------------------------------------------------
The univariate analysis indicated that the number of neutrophils at the nontumoral tissues was not associated with DFS or DSS (*P* = 0.981 and *P* = 0.896), but the number of neutrophils at the invasion margin and at the tumor center all demonstrated a statistically significant association with DFS and DSS (*P* = 0.001 and *P* \< 0.001, *P* = 0.001 and *P* \< 0.001, respectively) (Additional file [2](#MOESM2){ref-type="media"}: Table S2 and Additional file [3](#MOESM3){ref-type="media"}: Table S3) (Fig. [2](#Fig2){ref-type="fig"}). In addition, ASA (*P* = 0.002), tumor site (*P* = 0.022), tumor size (*P* \< 0.001), TNM stage (*P* \< 0.001), lymphovascular invasion (*P* = 0.002), perineural invasion (*P* \< 0.001), and postoperative chemotherapy (*P* = 0.032) all demonstrated a statistically significant association with DFS, whereas age, gender, Lauren type, and histological grade had no prognostic significance for DFS (Additional file [2](#MOESM2){ref-type="media"}: Table S2). ASA (*P* = 0.001), tumor site (*P* = 0.029), tumor size (*P* \< 0.001), TNM stage (*P* \< 0.001), lymphovascular invasion (*P* = 0.001), perineural invasion (*P* \< 0.001), and postoperative chemotherapy (*P* = 0.010) were all significantly associated with patients with DSS, whereas age, gender, Lauren type, and histological grade had no prognostic significance for DSS (Additional file [3](#MOESM3){ref-type="media"}: Table S3).Fig. 2Kaplan-Meier curves of DFS and DSS based on the number of neutrophils at the nontumoral, invasive margin and tumor center tissues of patients with GC. **a**, **d** Higher number of neutrophils at the nontumoral tissues were not correlated with prognosis (*P* = 0.981 and *P* = 0.896). **b**, **e** Higher number of neutrophil at the invasive margin of GC tissues were closely correlated with poor DFS and DSS (*P* = 0.001 and *P* = 0.001). **c**, **f** Higher number of neutrophil at the tumor center of GC tissues were closely correlated with poor DFS and DSS (*P* \< 0.001 and *P* \< 0.001)
The multivariate analysis showed that TNM stage (*P* \< 0.001), perineural invasion (*P* = 0.015), postoperative chemotherapy (*P* = 0.006), and CD66bIM (*P* = 0.001) were independent risk factors for DFS in GC (Additional file [2](#MOESM2){ref-type="media"}: Table S2). In addition, TNM stage (*P* \< 0.001), perineural invasion (*P* = 0.013), postoperative chemotherapy (*P* = 0.001), and CD66bIM (*P* \< 0.001) were independent risk factors for DSS in GC (Additional file [3](#MOESM3){ref-type="media"}: Table S3). The results of the comprehensive analysis showed that higher TNM stage, perineural invasion, lack of postoperative chemotherapy, and CD66bIM were independent risk factors for the prognosis of GC.
IL-17a protein is primarily expressed by neutrophils in GC {#Sec18}
----------------------------------------------------------
Neutrophils play an important role in promoting tumor progression in GC. But the role of IL-17a in tumors is still unclear. Although the role of IL-17a in lymphocytes has been widely studied, recent studies have found that IL-17a is also expressed in other immune inflammatory cells, including neutrophils, but the association between neutrophils and IL-17a + cells is unknown. Therefore, we assessed the association between neutrophils and IL-17a + cells in GC, with a specific focus on tissue micro-location of the cells.
Immunohistochemical staining showed that IL-17a + cells were mainly distributed in the peritumoral stroma (Fig. [3](#Fig3){ref-type="fig"}a) and that IL-17a was negatively related to DFS and DSS (*P* \< 0.001 and *P* \< 0.001) (Additional file [4](#MOESM4){ref-type="media"}: Figure S1). Both CD66b + neutrophils and IL-17a + cells were observed in the same area (R^2^ = 0.155, *P* \< 0.001) (Fig. [3](#Fig3){ref-type="fig"}b). Immunofluorescence further found that (61.6 ± 7.5) % of IL-17a protein was expressed by CD66b + neutrophils in the tumor stroma which was significantly higher than that in the nontumoral tissues (15.4 ± 5.8)% (*P* \< 0.001) (Fig. [3](#Fig3){ref-type="fig"}c). Therefore, neutrophils are the primary cells that produce IL-17a.Fig. 3IL-17a protein is primarily expressed by neutriphils in GC. **a**, **b** Association of CD66b + neutriphils and IL-17a + cells at the invasive margin of GC tissues by immunohistochemical staining. Magnifications: × 200. **c** Analysis of IL-17a and CD66b distribution in GC tissues by immunofluorescence microscope. One of 10 representative micrographs is shown. Magnifications: × 400. **d** The production of IL-17a in the cell supernatant in the gastric cell lines (GES-1, MKN45 and MK74), the production of IL-17a in the cell supernatant in the neutrophils isolated from GC tissues and nontumoral tissues, neutrophils activated by TTCS and NTCS, the production of IL-17a in the co-culture system were quantified 24 h after change the culture medium by ELISA. \* *P* \< 0.05. **e** the expression level of IL-17a mRNA in the GC cells (MKN45 and MK74), GC cells co-cultured neutrophils, neutrophils, and neutrophils co-cultured GC cells were quantified 24 h after change the culture medium by qRT-PCR. \*\* *P* \< 0.001
The production of IL-17a in the cell supernatant of the gastric cell lines GES-1, MKN45, and MKN74 was 47.67 + 4.26 pg/ml, 141.7 ± 10.41 pg/ml, and 89.33 + 10.35 pg/ml. The production of IL-17a in the cell supernatant of neutrophils isolated from GC tissues and nontumoral tissues and neutrophils activated by TTCS and NTCS was 458.3 ± 31.14 pg/ml, 242.0 ± 14.05 pg/ml, 503.3 ± 35.63 pg/ml, and 254.07 ± 19.29 pg/ml, respectively. The production of IL-17a in the co-culture system was 740.3 ± 52.92 pg/ml and 663.3 ± 46.31 pg/ml, respectively (Fig. [3](#Fig3){ref-type="fig"}d).
The ELISA results showed that gastric cell lines (GES-1, MKN45 and MKN74) produced a low level of IL-17a and that neutrophils isolated from GC tissues produced a higher level of IL-17a than neutrophils isolated from nontumoral tissues (*P* \< 0.05). Moreover, neutrophils activated by TTCS produced a higher level of IL-17a than neutrophils activated by NTCS (*P* \< 0.05), and the co-culture system produced higher levels of IL-17a (*P* \< 0.05) (Fig. [3](#Fig3){ref-type="fig"}d).
In addition, qRT-PCR results also showed that the expression level of IL-17a mRNA in GC cells (MKN45 and MKN74) increased slightly after co-culture with neutrophils, while the expression level of IL-17a mRNA in neutrophils increased significantly after co-culture with GC cells (MKN45 and MKN74) (*P* \< 0.001 and *P* \< 0.001) (Fig. [3](#Fig3){ref-type="fig"}e). Neutrophils in the tumor microenvironment may be the main source of IL-17a.
Neutrophils promote migration and invasiveness and EMT of GC cells through IL-17a {#Sec19}
---------------------------------------------------------------------------------
To study the effect of neutrophils on GC cells, a Transwell migration/invasion assay was used to explore the effect of neutrophils on the migration and invasion ability of GC cells. The results showed that neutrophils promote GC cell (MKN45 and MKN74) migration (*P* \< 0.001 and *P* \< 0.001) and invasion (*P* \< 0.001 and *P* \< 0.001) (Fig. [4](#Fig4){ref-type="fig"}a and b), when an IL-17a neutralizing antibody was added to the Transwell co-culture chamber, GC cell (MKN45 and MKN74) migration (*P* \< 0.001 and *P* \< 0.001) and invasion were decreased (*P* \< 0.001 and *P* \< 0.001) (Fig. [4](#Fig4){ref-type="fig"}a and b). Therefore, neutrophil promotes the migration and invasiveness of GC cells through IL-17a.Fig. 4Neutrophils enhance the migration, invasiveness and EMT of GC cells through IL-17a. (**a**, **b**) The effect of neutrophils on the migration and invasion ability of GC cells (MKN45 and MKN74) was determined 24 h when IL-17a neutralizing antibody or IgG isotype control antibody was added to Transwell co-culture chamber. Magnifications: × 100. \*, *P* \< 0.05; \*\*, *P* \< 0.001. **c** Protein expression of E-cadherin, Vimentin, and ZEB1 in GC cells (MKN45 and MKN74) co-cultured with neutrophils were analyzed by western blot when IL-17a neutralizing antibody or IgG isotype control antibody was added to the Transwell co-culture system. Densitometric analysis of E-cadherin, Vimentin, and ZEB1 expression were shown. \*\*, *P* \< 0.001
EMT, a well-characterized embryological process, has been identified to play a critical role in tumor metastasis. This process is characterized by loss of epithelial markers (e.g. E-cadherin) and the acquisition of mesenchymal markers (e.g. Vimentin, ZEB1). To examine the role of TANs in mediating EMT in GC cells, we cultured GC cells (MKN45 and MKN74) with TANs in a previously described co-culture system.
Western blot results showed that neutrophils promote the expression of EMT related markers in GC cells (MKN45 and MKN74). E-cadherin expression was significantly decreased (*P* \< 0.05 and *P* \< 0.05 in MKN45 and MKN74 cells, respectively), and Vimentin (*P* \< 0.05 and *P* \< 0.05 in MKN45 and MKN74 cells, respectively) and ZEB1 (*P* \< 0.05 and *P* \< 0.05 in MKN45 and MKN74 cells, respectively) expression was significantly upregulated (Fig. [4](#Fig4){ref-type="fig"}c), when an IL-17a neutralizing antibody was added to the Transwell co-culture system, The expression of E-cadherin protein in the epithelium of GC cells was upregulated (*P* \< 0.05 and *P* \< 0.05 in MKN45 and MKN74 cells, respectively), while the expression of Vimentin protein in the interstitial space decreased significantly (*P* \< 0.05 and *P* \< 0.05 in MKN45 and MKN74 cells, respectively). The expression of the transcription factor ZEB1 was also significantly downregulated (*P* \< 0.05 and *P* \< 0.05 in MKN45 and MKN74 cells, respectively) (Fig. [4](#Fig4){ref-type="fig"}c). Therefore, neutrophils promote EMT in GC cells, and further promotes the migration and invasiveness of GC cells, which may be mediated by IL-17a.
IL-17a activates the STAT3 signalling pathway and promotes EMT and migration and invasiveness of GC cells {#Sec20}
---------------------------------------------------------------------------------------------------------
The canonical IL-17a signal transduction pathway is initiated by its binding to IL-17Ra and phosphorylation of STAT3 through JAK2 activation. To determine the role of the IL-17a/JAK2/STAT3 pathway in mediating TANs-induced migration, invasion and EMT of GC cells (MKN45 and MKN74), we first explored the activation of the IL-17a/JAK2/STAT3 pathway in GC cells after co-culture with TANs. Western blot results showed that neutrophils promoted the phosphorylation of JAK2 (*P* \< 0.05 and *P* \< 0.05 in MKN45 and MKN74 cells, respectively) and STAT3 (*P* \< 0.05 and *P* \< 0.05 in MKN45 and MKN74 cells, respectively) but had no significant effect on the expression of JAK2 and STAT3 in GC cells (MKN45 and MKN74) (Fig. [5](#Fig5){ref-type="fig"}a and b). In contrast, the addition of an IL-17a neutralizing antibody or the JAK2 protein tyrosine kinase inhibitor AG490 to the co-culture system significantly reversed the TANs-mediated phosphorylation of JAK2 (*P* \< 0.05 and *P* \< 0.05 in MKN45 and MKN74 cells, respectively) and STAT3 (*P* \< 0.05 and *P* \< 0.05 in MKN45 and MKN74 cells, respectively) and had no effect on the expression of JAK2 and STAT3 in GC cells (MKN45 and MKN74) (Fig. [5](#Fig5){ref-type="fig"}a and b). A Transwell migration/invasion assay showed that AG490 decreased GC cell migration (*P* \< 0.001 and *P* \< 0.05 in MKN45 and MKN74 cells, respectively) and invasiveness (*P* \< 0.001 and *P* \< 0.05 in MKN45 and MKN74 cells, respectively) (Fig. [5](#Fig5){ref-type="fig"}c and d). Western blot analysis showed that AG490 inhibited the expression of EMT-related markers in GC cell, as demonstrated by increased expression of E-cadherin (*P* \< 0.05 and *P* \< 0.05 in MKN45 and MKN74 cells, respectively) and significantly decreased expression of Vimentin (*P* \< 0.05 and *P* \< 0.05 in MKN45 and MKN74 cells, respectively) and ZEB1 (*P* \< 0.05 and *P* \< 0.05 in MKN45 and MKN74 cells, respectively) (Fig. [5](#Fig5){ref-type="fig"}e). The IL-17a/JAK2/STAT3 pathway plays an important role in TANs-induced migration, invasiveness and EMT of GC cells.Fig. 5IL-17a activates the STAT3 signalling pathway and promotes EMT, migration and invasiveness of GC cells. **a**, **b** Protein expression of p-JAK2, JAK2, p-STAT3, and STAT3 in GC cells (MKN45 and MKN74) co-cultured with neutrophils were analyzed by western blot when IL-17a neutralizing antibody or AG490 was added to the Transwell co-culture system. Densitometric analysis of p-JAK2, JAK2, p-STAT3, and STAT3 expression were shown. \*, *P* \< 0.05. **c**, **d** The effect of neutrophils on the migration and invasion ability of GC cells (MKN45 and MKN74) were determined when AG490 was added to the Transwell co-cultured chamber. Magnifications: × 100. \*, *P* \< 0.05; \*\*, *P* \< 0.01. **e** Protein expression of E-cadherin, Vimentin, and ZEB1 in GC cells (MKN45 and MKN74) co-cultured with neutrophils were analyzed when AG490 was added to Transwell co-culture system. Densitometric analysis of E-cadherin, Vimentin, and ZEB1 expression were shown. \*, *P* \< 0.05
Discussion {#Sec21}
==========
TANs, which are activated neutrophils in the tumor stroma, are an essential component of the tumor microenvironment and play a role in tumor progression, therefore, they should be appraised carefully \[[@CR12]\]. In this study, we showed that neutrophils were highly enriched within GC and that neutrophils in the invasion margin of GC tissues were negatively correlated with patient survival. In addition, we demonstrate that high levels of IL-17a were present in GC tissues and that neutrophils produced IL-17a. In an in vitro experiment, we observed that IL-17a secreted by TANs plays an important role in the progression of GC. TANs-derived-IL-17a promoted the migration, invasiveness and EMT of GC cells via the activation of the JAK2/STAT3 pathway. Inhibition of this pathway with IL-17a an neutralizing antibody or the JAK2-specific inhibitor AG490 reversed these TAN-induced phenotypes in GC cells. Therefore, the activation of the JAK2/STAT3 pathway by IL-17a may play a central role in the interplay between TANs and GC cells.
Importantly, our findings also revealed the clinical relevance of neutrophils in GC. Specifically, we found that an increased frequency of intratumoral neutrophils predicted a poor prognosis. Given that the clinical outcomes of patients with GC remains poor and that few prognostic factors currently exist for this disease following surgery \[[@CR38]\], intratumoral neutrophil cell frequency might prove to be a useful clinical marker in the future. Moreover, neutrophils may influence tumor progression through the paracrine release of cytokines and chemokines with protumor or antitumor functions, depending on the tumor microenvironment \[[@CR13]\]. Previous studies have shown that neutrophils can produce IL-17a in inflammatory and autoimmune diseases \[[@CR27], [@CR28]\]. In addition, neutrophils can promote angiogenesis through IL-17a in GC. In the present study, both clinical samples analysis and an experimental study suggested that IL-17a is predominantly expressed by neutrophils.
Epithelial to mesenchymal transition (EMT) is a process by which epithelial tumor cells lose their epithelial features and gain a mesenchymal phenotype \[[@CR39]\]. EMT is considered as the key step by which tumor cells gain the higher ability of invasive and metastatic abilities. Tumor cells take advantage of EMT as an intermediary phenotype to achieve self-renewal and to adapt to their microenvironments \[[@CR40], [@CR41]\]. Experimentally, EMT can be induced not only by loss of cellular contact (for example, due to degradation of basement membranes or other modifications of the microenvironment), but also by numerous cytokines, especially by TGF-β \[[@CR42]\]. TANs can induce EMT in intratumoral cancer cells, but the molecular mechanisms are poorly understood. Studies have shown that loss of surface-associated E-cadherin, at least in part due to cleavage by neutrophil-derived elastase and the subsequent weakening of the cell-to-cell contacts by loss of cell polarity, is a crucial step in the EMT transition process \[[@CR42]\]. In addition, a plethora of factors that are abundant in infiltrated cells that infiltrate the tumor microenvironment (TANs in particular) such as IL-6, IL-8, IL-1β, and TNFα \[[@CR43]\] are also able to induce EMT in GC. However, very little is currently known about the mechanisms underlying the polarization of IL-17a + neutrophils and their role in GC progression. In the present study, we have shown that IL-17a secreted by TANs induced EMT of GC cells; this process is characterized by loss of the epithelial markers E-cadherin and the acquisition of the mesenchymal markers Vimentin and ZEB1. Then these EMT changes then contribute to the enhanced capability for active locomotion of GC cells, which is demonstrated by increased migratory ability triggered by TANs.
IL-17 is positively correlated with the degree of activation of STAT3 signalling pathway activation \[[@CR44]\]. The activation of STAT3 with the phosphorylation of Tyr705 is facilitated by the JAK signalling pathway \[[@CR33], [@CR45]\]. Accumulating evidences shows that activation of the IL-17a/JAK2/STAT3 signalling pathway by growth factors or cytokines plays an active role in tumor growth and progression. However, the role of TANs and IL-17a in GC has not been well addressed. Our present study has shown that TANs induced the phosphorylation of JAK2 and STAT3 in GC cells via the secretion of IL-17a. Our study also showed that inhibiting JAK2/STAT3 pathway activation with AG490 significantly impaired TAN-induced migration and invasion, as well as EMT of GC cells induced by TANs in vitro. TANs are known to secrete multiple growth factors and chemokines such as TNFα, CCL2, IL-8, and IL-17a into the tumor microenvironment, where they promote the growth and invasion of the underlying tumor by triggering multiple pathways \[[@CR9], [@CR26]\]. In the present study, we found that an IL-17a neutralizating antibody partly suppressed the JAK2 or STAT3 phosphorylation, which suggested that IL-17a partially contributed to the tumor-promoting effects of TANs on GC cells. Although we can not preclude the likely involvement of other growth factors and/or cytokines, the studies of neutralizing IL-17a or the inhibition of JAK2/STAT3 pathway activation with AG490 reveal that IL-17a is an important mediator of the tumor-promoting effects of TANs, which promote EMT via the activation of the JAK2/STAT3 signalling pathway in GC.
Conclusions {#Sec22}
===========
In summary, we find that neutrophils are highly enriched within GC, and are negatively correlated with patient survival and are associated with disease progression. In vitro data established a link between TANs and EMT of GC cells through IL-17a/JAK2/STAT3 signalling. Therefore therapies that target IL-17a or STAT3 signalling may provide future treatment efficacy in GC and are thus important for clinical study.
Additional files
================
{#Sec23}
Additional file 1:**Table S1** Association of CD66b+cells with clinicopathological feathers in Non, IM and TC of gastric cancer (DOCX 19 kb) Additional file 2:**Table S2** Univariate and Multivariate analyses of factors Associated with Disease-free Survival (DFS) with gastric adenocarcinoma (DOCX 16 kb) Additional file 3:**Table S3** Univariate and Multivariate analyses of factors Associated with Disease Special Survival (DSS) with gastric adenocarcinoma (DOCX 16 kb) Additional file 4:**Figure S1**. Kaplan-Meier curves of DFS and DSS based on the number of IL-17a+cells in GC. (a, b) Higher number of 17a+cells in GC tissues were closely correlated with poor DFS and DSS (*P* \< 0.001 and *P* \< 0.001). (DOCX 144 kb)
DAPI
: 4′,6-diamidino-2-phenylindole
DFS
: Disease-free survival
DSS
: Disease-specific survival
ELISA
: Enzyme-linked immunosorbent assay
EMT
: Epithelial mesenchymal transition
GC
: Gastric cancer
HIF-1α
: Hypoxia-inducible factor-1α
IL-17a
: Interleukin-17a
IL-6
: Interleukin-6
JAK2/STAT3
: Janus kinase 2/signal transducers and activators of transcription
JAKs
: Janus kinases
NETs
: Neutrophil extracellar traps
NTCS
: Non-tumor tissue culture supernatants
PMN
: Polymorphonuclear
QRT-PCR
: Quantitative real-time PCR
STAT
: Signal transducers and activators of transcription
TANs
: Tumor-associated neutrophils
TGF-β
: Transforming growth factor
TTCS
: Preparation tumor tissue culture supernatants
We thank Xiliang Cong, Xiuwen, Lan Hongyu Gao, and Zhiguo Li for their excellent technical assistance. We thank Wenpeng Wang, Shubin Song, and Yimin Wang for data collection and analysis. We thank Chunfeng Li and Hongfeng Zhang for fruitfull help.
Funding {#FPar1}
=======
This study was supported by a grant from the Harbin Medical University Cancer Hospital. No: Nn10PY2017--03.
Availability of data and materials {#FPar2}
==================================
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
SL Conception, design, data analysis, and writing-original draft; XC, HG, and XL: Provision of study materials or patients, data analysis and interpretation; ZL, WW, and SS: Collection and assembly of data; YW, CL, HZ, YX and YZ: Financial support, technical help and fruitful discussion. All authors read and approved the final manuscript.
Ethics approval and consent to participate {#FPar3}
==========================================
The present study was authorized by the Ethics Committee of Harbin Medical University Cancer. All procedures performed in studies were in accordance with the ethical standards. Informed consent was obtained from all patients and volunteers before they were included in the study.
Consent for publication {#FPar4}
=======================
Not applicable.
Competing interests {#FPar5}
===================
The authors declare that they have no competing interests.
Publisher's Note {#FPar6}
================
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
| {
"pile_set_name": "PubMed Central"
} |
**Session:** 51. Antimicrobial Stewardship: Interventions to Improve Outcomes
*Thursday, October 4, 2018: 12:30 PM*
| {
"pile_set_name": "PubMed Central"
} |
Background
==========
MicroRNAs (miRNAs) are short oligonucleotides (approximately 22 bp) that regulate gene expression. Target genes are determined by sequence complementarity between the 3\' untranslated region (UTR) and the mature miRNA, particularly in a 6 bp \'seed\' region \[[@B1],[@B2]\]. A range of algorithms have been developed to predict the genes targeted by specific miRNAs \[[@B3]\]. For example, \'TargetScan\' \[[@B4],[@B5]\] searches for conserved 8-mer and 7-mer sites in 3\' UTRs that match the seed region of a known miRNA. It is possible, therefore, to obtain lists of the potential target mRNAs for each miRNA. Plant miRNAs, which are often perfectly matched to their target sequences, act primarily by directing mRNA cleavage and degradation \[[@B6],[@B7]\]. In contrast, animal miRNAs have been shown to exert their effect largely via post-transcriptional inhibition of protein synthesis \[[@B8]\]. However, it has been shown that miRNAs expressed in animal cells can affect mRNA levels, not only when they share almost complete complementarity with their target site \[[@B9]\], but more generally when base-pairing is partial \[[@B10]-[@B12]\]. When, for example, miR-124, which is known to be characteristic of neuronal tissue, was overexpressed, the genes that were down-regulated at the mRNA level included a preponderance of those expressed at lower levels in neuronal compared to other tissues \[[@B11]\]. Conversely, silencing of miR-122 with a complementary, single-stranded RNA analogue, or \'antagomir\', resulted in increased expression of mRNAs that were enriched in miR-122 recognition motifs \[[@B13]\] and miR-122 can direct cleavage of a reporter gene. Depletion of proteins required for miRNA processing has been shown to cause widespread alteration in mRNA levels \[[@B14],[@B15]\].
The suggestion that miRNAs can affect mRNA levels led to the prediction that a miRNA expressed at a high level in a specific tissue might leave a signature on the mRNA expression profile. Sood *et al*. \[[@B16]\] and Farh *et al*. \[[@B17]\] demonstrated that the predicted target genes of known tissue-specific miRNAs (for example, miR-122 in liver; miR-1 in heart/skeletal muscle and miR-7 in pituitary) were expressed at significantly lower levels, as determined by microarray analysis, in their cognate tissue relative to all other tissues.
The conclusive demonstrations that miRNAs can alter mRNA levels suggested to us that, within a specific tissue, the expression of genes predicted to be targeted by a specific mature miRNA might have a detectable inverse relationship with the expression level of that miRNA. This approach has been made feasible by advances in microarray technology and provision of comprehensive gene coverage, which have made global gene expression data increasingly reliable and reproducible \[[@B18],[@B19]\]. Concomitantly, public repositories such as Gene Expression Omnibus (GEO) \[[@B20],[@B21]\] and ArrayExpress \[[@B22]\] have made data from a huge range of tissues available to the scientific community. A method for extracting miRNA signatures from an mRNA expression dataset would be invaluable because it could immediately be applied to analyze miRNA activity in any situation for which microarray gene expression data are available.
Others have had limited success in detecting a significant effect of miRNAs within a single gene expression profile using a non-parametric approach based on gene expression ranking \[[@B16]\]. However, by employing different predicted target and control datasets we were able to observe significant miRNA effects using a similar approach and by direct analysis of absolute target gene expression values (by the term \'target gene expression\' we refer to the expression of predicted target genes). We were able to predict many of the previously characterized, highly expressed and/or tissue-specific miRNAs (for example, 14 of 25 in brain). This approach will facilitate investigation of the activity of miRNAs upon mRNA expression, without the need for ranking gene expression of each gene across a series of tissues \[[@B11],[@B23]\].
Results and discussion
======================
Detection of miRNA signatures within endogenous gene expression profiles
------------------------------------------------------------------------
miRNAs can down-regulate target mRNAs; therefore, one would expect that the target genes of a highly expressed miRNA might be expressed at a significantly lower level than those of a lowly expressed miRNA. In this case it might be possible to detect the presence of miRNAs from the relative expression of their predicted target genes. The profile of miRNAs expressed in one tissue differs from that in another and to test whether different \'signatures\' were detectable, we first downloaded mRNA expression profiles for a range of tissues from GEO \[[@B20],[@B21]\]. A range of algorithms have been developed to predict miRNA target genes \[[@B3]\]. However, the scarcity of experimentally confirmed interactions has made it difficult to develop reliable algorithms and validate existing methods. The relevance of existing rules is uncertain \[[@B24]\] and additional factors such as co-factor binding and relative positions of target sites \[[@B25],[@B26]\] undoubtedly play a role. Of the publicly available algorithms, we chose to initially use TargetScan \[[@B27]\] because its requirement for a perfect match to the seed region and cross-species conservation reduce the false-positive rate \[[@B3]-[@B5]\]. The resulting higher specificity of this algorithm maximizes the ability to detect effects on expression of real miRNA target genes. After detecting a signal we subsequently tested alternative miRNA target gene prediction algorithms (see below). For every mRNA expression dataset, the mRNA expression of predicted targets was mapped onto the respective miRNA families. The average number of predicted target genes for a single miRNA expressed in a given tissue was 134 (± 9 standard error; the number of predicted target genes for each miRNA expressed in all tissues is shown in Additional data file 1). We then tested the ability of three analytical approaches to detect the effects of variable endogenous miRNA expression on mRNA levels.
Wilcoxon rank sum test
----------------------
Our first analysis followed the \'tissue-centric\' approach described by Sood and colleagues \[[@B16]\]. A vector of expression values for each set of specific miRNA target genes was compared to a vector of expression values for all predicted target genes. For all tissues, miRNAs with significantly low target gene expression were detected (Wilcoxon rank sum test), with lowest *p*-values ranging from 1.29 × 10^-5^in brain to 7.23 × 10^-3^in skeletal muscle. The results for all tissues are shown in Figure [1](#F1){ref-type="fig"}. It is notable that well characterized tissue-specific miRNAs, such as miR-122 in liver and miR-124 in brain, are all detected in the expected tissue and not elsewhere. This suggests that the \'signature\' that a miRNA exerts upon mRNA expression can be detected within a single gene expression profile, without relation to levels in other tissues as previously reported \[[@B16]\].
{#F1}
Ranked ratio
------------
In an alternative approach to analyzing the relative expression levels of all the predicted target genes of each miRNA within a particular tissue, we adapted the \'ranked ratio\' (RR) described by Yu *et al*. \[[@B23]\]. They first ranked the expression levels of each gene across a series of tissues. For each tissue the ranked genes were divided into two halves, one with high and one with low ranks. The RR values were then calculated by dividing the number of targeted genes in the \'low\' ranked group by the \'high\' ranked group. Instead of considering a range of tissues we ranked the targeted genes within a single expression dataset and for each miRNA calculated an RR value by dividing the number of predicted target genes with expression levels below the median absolute expression value by the number of predicted target genes above this value (comparison with other methods suggested that this was more effective than dividing genes into upper and lower halves - see below). This RR value is, therefore, an indicator of the distribution of a miRNA\'s target genes within a single mRNA population. A high RR indicates low expression in a greater proportion of target genes and is, therefore, indicative of miRNA expression in that tissue. The RR values for all miRNAs were calculated for all eight tissues and the ranked RR values for brain and liver are shown in Figure [2](#F2){ref-type="fig"} (for all other tissues analyzed, see Additional data file 2). As expected, known tissue-specific miRNAs have high RR values in their cognate tissue.
{#F2}
Mean absolute expression
------------------------
We next investigated whether an approach involving absolute target gene expression could be used to detect miRNA signatures. This could potentially identify miRNAs missed above, but runs the risk of being unduly influenced by single genes with a large change in expression. The technique is outlined in Figure [3a](#F3){ref-type="fig"}. The miRNAs were ordered by the mean expression value of their predicted target mRNAs, as shown for liver in Figure [3b](#F3){ref-type="fig"}. Of all the miRNAs in the liver, the lowest mean target gene expression value was that of miR-122a, a well characterized liver-specific miRNA \[[@B28]\]. To determine the likelihood that this observed reduction in mRNA expression is due to the selection of mRNAs with specific miRNA targets, we calculated the probability (*t*-test) that these samples are drawn at random from amongst all those genes expressed in the tissue and that contain a predicted miRNA target sequence. The resulting probabilities for all tissues are plotted in Figure [3c](#F3){ref-type="fig"} and those miRNAs with low target gene expression include many known tissue-specific examples, such as miR-124a in brain (*p*= 6.2 × 10^-4^) and miR-1 in skeletal muscle (*p*= 1.9 × 10^-2^).
{#F3}
To test the reliability of this approach and the robustness of available microarray expression data, it was applied to an independent mouse expression dataset generated in several different laboratories (see Materials and methods). The sets of miRNAs predicted from the two datasets were very similar for all tissues and the extent of overlap is depicted in Figure [4](#F4){ref-type="fig"}. Mammalian miRNAs and their target sites are highly conserved; indeed, sequence conservation is a requirement of the TargetScan predictions \[[@B4]\]. Accordingly, miRNA expression is conserved between species \[[@B29]\], at least for organisms with similar physiology \[[@B30]\] and miRNAs may have a role in reducing cross-species variation in mRNA expression \[[@B31]\]. Analysis of mRNA expression profiles from human tissues (Additional data file 3) revealed that approximately one-third of the human miRNAs with low target gene expression corresponded to those predicted in equivalent murine tissues (30.6% and 35.5% for mouse datasets 1 and 2, respectively). For example, of 18 human miRNAs predicted in brain, 7 were common with mouse dataset 1 (Figure [4](#F4){ref-type="fig"}), rather than the lower number (approximately 2) expected if the groups of miRNAs were independent (the observed numbers were similarly high for all other tissues and the second mouse dataset). This provides further evidence for conserved miRNA expression and independent validation of the prediction method.
{#F4}
Comparison of miRNA signature detection methods
-----------------------------------------------
We next compared the results of the three methods, Wilcoxon rank sum test, RR and absolute expression *t*-test, using a 10% significance level and an equivalent number of miRNAs from the RR method. For all tissues there was significant overlap amongst predicted miRNAs (Figure [5](#F5){ref-type="fig"}), with the Wilcoxon rank sum test and absolute expression *t*-test in strongest agreement. To evaluate how well the miRNA signature detected in target gene expression predicts actual miRNA expression, we compared the tissue distribution of miRNAs predicted by at least two of the methods with that derived from experimental evidence (cloning and Northern blots). Table [1](#T1){ref-type="table"} illustrates the accordance between the tissues in which miRNA activity (upon target genes predicted by TargetScan) is computationally predicted and those for which there is experimental evidence of miRNA presence (particularly when more recently characterized miRNAs are excluded). This is supported by positive Matthews correlation coefficients (MCC) \[[@B32]\] for all tissues, ranging from 0.2-0.5 (average value 0.34; Additional data file 4).
{#F5}
######
Correlation between predicted and previously characterized tissue-specific miRNA expression
miRNAs Brain Heart Kidney Liver Lung Ovary SM Testes
----------- --------- ----------- ----------- --------- ------- ------- ------- --------
let-7 \*1,2,3 \*1,2,3,4 \*1,3,4,6 \*2 \*1,8 \* \*1 \*3,9
miR-1/206 2 \*1,2,4,5 1 \*2 1 1,5
miR-10 \*4,6,7 \*7
miR-101 \*2 4 \*1 \* 9
miR-122a 3 \*1,2,3 9
miR-124a \*1,2
miR-125 \*1,2 \*1,3,4 \*1,4 1 \*1,3 \*
miR-128 \*1,2 \* \*1 \* \*
miR-133 2,3 \*1,2 1 \*1,2
miR-136 2 \*
miR-137 \*1,2 \* \* \*
miR-140 \* 4
miR-144 2 \*
miR-146 3 2,4 4 \* \*
miR-15 \*2,3 1,2,3 \*4,6 \*1,3 \* \*3,9
miR-150 4 \*
miR-153 \*1,2 \* \* \*
miR-190 1 \*7
miR-193 1,7,4 1 \*
miR-196 \* \* \*1,4 3 \*7 \* \*
miR-199 4 4 1 \*
miR-204 \* \*
miR-24 2,3 \*2 1,4,6 \*1,8 \*
miR-26 1,2,3 1,4 1,4,6 1,2 1 \* 1
miR-27 \*2,3 \*2 \*4,6 \*1,8 \* \* \*9
miR-29 \*1,2 2,4 \*4,6 1
miR-30 \*1,2,3 \*2,4 \*1,4,6 \*1,2 \*1 \* \*1 \*9
miR-31 \*
miR-326 \* \*
miR-33 4 \* \*
miR-330 \*
miR-331 \* 3 \* \* \*3 \*
miR-335 \*
miR-34 3 \* \*9
miR-365 3 \*
miR-370 \* \* \* \* \*
miR-375 \*
miR-378 \* \* \*
miR-448 \* \* \* \* \* \* \*
miR-503 \*3 3 3 3 3
miR-9 \*1,2
All miRNAs for which expression of predicted target genes (according to TargetScan) was lower than expected in ≥ 1 tissue (*p*\< 0.1) are listed and the tissues marked with an asterisk. Tissues in which miRNA expression has been experimentally characterized are indicated by the number of the appropriate reference: 1, Sempere *et al*. \[29\]; 2, Lagos-Quintana *et al*. \[28\]; 3, Gu J *et al*. \[47\]; 4, Naraba and Iwai \[48\]; 5, Zhao Y *et al*. \[49\]; 6, Lagos-Quintana *et al*. \[50\]; 7, Lagos-Quintana *et al*. \[51\]; 8, Hayashita *et al*. \[52\]; 9, Yu *et al*. \[53\]). Cells with both a reference number and asterisk indicate the overlap between our predicted expression pattern and the experimental data. SM, skeletal muscle.
Correlation of miRNA signatures with miRNA expression levels
------------------------------------------------------------
miRNA microarrays are now available that provide a global indication of miRNA expression within a tissue. We therefore compared our predictions of miRNAs that alter mRNA expression with the actual expression of the miRNAs themselves, as determined by miRNA microarrays \[[@B33]\]. For all tissues the expression levels of miRNAs with low target gene expression, determined by the absolute expression method (10% significance level), were significantly lower (*t*-test, *p*\< 0.05) than those miRNAs having no detectable effect on their target genes (Figure [6](#F6){ref-type="fig"}). This provides further confirmation that the miRNA signatures we have detected are a consequence of miRNA expression in the cognate tissue. In addition to miRNAs with low target gene expression, we detected a set of miRNAs whose target genes were expressed at significantly higher levels than the background set (Figure [3b](#F3){ref-type="fig"}). The expression of these miRNAs, as determined by microarrays, was not significantly different from those with no effect on mRNA expression.
![Correlation between miRNAs with predicted effects on mRNA expression and miRNA expression levels detected by miRNA microarrays. miRNAs were divided into those with significantly lower than expected target mRNA expression (labeled \'low\'), those with no detectable effect on their target expression (labeled \'mid\') and those with significantly high target expression (labeled \'high\'). The boxplots show the expression values (y-axis), determined by Thomson *et al*. \[33\], of the miRNAs in each group (x-axis). The expression of miRNAs with low target gene expression is significantly higher (*t*-test, *p*\< 0.05) than that of those with mid or high target expression (in all tissues except heart). Thomson *et al*. \[33\] labeled the microRNAs from each tissue with Cy3 and used a reference oligonucleotide set corresponding to all mature microRNAs, labeled with Cy5 (red channel) in all hybridizations. This reference set provided an internal hybridization control for every probe on the array. The miRNA microarray expression values used in our analyses are median centered normalized log ratio Cy3/Cy5 values.](gb-2008-9-5-r82-6){#F6}
Recently, comprehensive miRNA expression data for human tissues determined by reverse transcription PCR (RT-PCR) have become available \[[@B34]\]. This revealed an even clearer relationship between human miRNA copy number and level of predicted target gene expression (Figure [7](#F7){ref-type="fig"}). In an attempt to demonstrate the similarity between human and mouse miRNAs, the human orthologs of miRNAs predicted from analysis of murine data to have \'low\' or \'mid\' predicted target gene expression were selected. Surprisingly, significant differences were detected between the copy numbers in human tissues of these two groups of miRNAs, which had been selected based upon murine target gene expression (Additional data file 5). This is testament to the degree of conservation of miRNAs and their target genes between mice and humans and the accuracy of the RT-PCR measurements.
![Correlation between miRNAs with predicted effects on mRNA expression and miRNA expression levels detected by RT-PCR. miRNAs were divided into groups according to predicted target gene expression in human tissues, as described previously. The expression, as determined by RT-PCR \[34\] (y-axis), of the miRNAs in these groups (x-axis) is depicted by boxplots that illustrate the significantly higher expression (*t*-test, *p*ranging from 0.0014 in kidney to 0.0385 in brain) of miRNAs with low target gene expression relative to those with mid or high target expression (in all tissues except heart). Where necessary to present the median and interquartile ranges effectively, up to two outliers were omitted.](gb-2008-9-5-r82-7){#F7}
Some of those miRNAs with a significant effect on mRNA expression were highly expressed (for example, miR-122a, miR-124a, miR-125) and their observed lowering of mRNA levels could reasonably be attributed to a weak mRNA degradative activity secondary to their principal action directed at translation. However, other miRNAs that significantly affected target gene expression were not highly expressed, perhaps indicating a greater efficiency in mRNA degradation for these particular miRNAs. Therefore, the extent to which specific miRNAs cause mRNA degradation might be influencing our ability to detect their presence. We reasoned that the difference between miRNAs would be most marked between those highly expressed but having no detectable affect on mRNA expression and those expressed at a low level but with a significant impact on target mRNA expression. Other than extensive complementarity \[[@B9]\], the features of the miRNA-target interaction required for miRNAs to direct mRNA cleavage are unclear, although a number of features of site context, including position, local AU content and pairing with miRNA 3\' residues have been shown to increase site efficacy \[[@B2]\]. In a preliminary attempt to characterize the distinguishing properties of the potential classes of miRNAs described above, we analyzed the lengths of contiguous complementarity between miRNAs and predicted sites, but there were no significant differences.
Perturbation of miRNA expression affects cognate mRNA expression
----------------------------------------------------------------
In order to validate the miRNA signatures observed in mRNA profiles of normal tissues, we applied our approach to several publicly available gene expression datasets measured following an experimental perturbation of miRNA expression that resulted in either a decrease \[[@B13]\] or increase \[[@B12]\] in the activity of a specific miRNA. In each case the expected response was observed in target gene expression. Krutzfeldt *et al*. \[[@B13]\]demonstrated that intravenous injection of chemically modified oligonucleotides, or \'antagomirs\', complementary to miRNAs could specifically reduce the endogenous levels of the corresponding miRNA in mice. When we analyzed gene expression data from liver in which miR-122a had been silenced by use of an antagomir \[[@B13]\] the list of miRNAs with an influence on mRNA levels was very similar but miR-122a no longer had a detectable effect (Figure [8](#F8){ref-type="fig"}). This conclusively demonstrates that the signature of miR-122a on target gene expression that we observe is due to the physical presence of miR-122a rather than any evolutionary pressure on the expression of target genes co-expressed with their cognate miRNA.
![Detection of altered miRNA signatures following manipulation of miRNA expression. In the top and bottom panels miRNAs (x-axis) are ranked in order of average target gene expression (y-axis) with, for clarity, only miR-122a and miR124 labeled, respectively. Following inhibition of miR-122 by an antagomir \[13\] the position of the miR-122 average target gene expression in liver moved from the left to right of those for all ranked miRNAs (upper panel). This was reflected in a reduced probability (y-axis) that miR-122a predicted target genes were under-expressed, whilst all other miRNA effects remained relatively unaltered by the miR-122a antagomir (red versus dashed grey line in center left panel). Conversely, overexpression of miR-124 for 24 hours in the HepG2 cell line \[12\] caused miR-124 target gene expression to change from significantly highly over-expressed to significantly under-expressed (bottom panel). The selective drop in expression of miR-124 target gene expression is shown in the center right panel (this is apparent for the miR-122a and miR-122u isoforms present in the version of TargetScan used for this analysis). Whilst the data in this Figure are from analysis of mean absolute expression, the alternative methods produced similar results (data not shown).](gb-2008-9-5-r82-8){#F8}
Wang and Wang \[[@B12]\] transfected the HepG2 cell line with RNA duplexes that mimicked the miR-124 precursor. They demonstrated the effect of miR-124 over-expression by measuring mRNA expression profiles and showing that predicted targets of miR-124 were over-represented amongst the mRNAs down-regulated following this treatment, compared to a negative control RNA duplex. As expected, analysis of miRNA signatures in these expression datasets revealed miR-124 activity in cells in which it was overexpressed but not in controls (Figure [8](#F8){ref-type="fig"}).
Characteristics of predicted target gene groups
-----------------------------------------------
We could not find any increase in target complementarity for miRNAs with a significant effect on mRNA levels. However, we noticed that those miRNAs with low overall target gene expression in a specific tissue generally had a greater number of target genes expressed in that tissue. This was true across tissues and supports the observations of Farh *et al*. \[[@B17]\], who demonstrated that tissue-specific miRNA target genes are generally expressed in the cognate tissue, but at lower levels than other tissues.
In certain tissues some miRNA predicted target gene sets were expressed at significantly higher levels (10% probability) than those of other miRNAs. We observed that these often involved a highly expressed, tissue-specific miRNA in tissues in which the miRNA was not normally expressed, for example, miR-1 in liver (Figure [2](#F2){ref-type="fig"}). However, this phenomenon does not appear to be indicative of reduced suppression of miRNA levels due to low miRNA expression in these tissues (Figures [6](#F6){ref-type="fig"} and [7](#F7){ref-type="fig"}).
The targeting of mRNAs by miRNAs has been associated with 3\' UTR GC content and length \[[@B16],[@B35]\]. However, we found no consistent differences between the 3\' UTR GC content of predicted target genes of miRNAs with significantly low target gene expression (\'low\' genes) and those of background genes. However, for all tissues, the predicted target genes of miRNAs with significantly high target gene expression (\'high\' genes) had significantly lower GC content (Additional data file 6). The 3\' UTR length was significantly longer than background for \'low\' genes, but \'high\' genes were even longer. The biological significance of these observations is unclear. One might have expected genes with lower expression to be more AU rich because miRNAs have been implicated in degradation of mRNAs containing AU-rich elements \[[@B35]\]. The longer 3\' UTRs of \'low\' genes would be predicted to have more miRNA target sites and, therefore, be subject to more degradation, but this is inconsistent with the even longer 3\' UTRs observed for \'high\' genes.
Use of alternative target prediction methods
--------------------------------------------
The correlation demonstrated between expression of certain miRNAs and that of their predicted target genes indicates that at least some of the interactions suggested by TargetScan are valid. Random assignment of miRNA target genes rather than use of TargetScan predictions resulted in fewer miRNAs with low target gene expression and significantly less overlap between those miRNAs with low target gene expression in replicate expression datasets (*p*\< 0.00001).
We next investigated whether we could detect variations in expression of the targets predicted by alternative algorithms. Many of the highly expressed, known tissue-specific miRNAs identified above (Table [1](#T1){ref-type="table"}) were not observed when the equivalent analyses were performed with target gene sets predicted by miRanda \[[@B36],[@B37]\] or RNAhybrid \[[@B38],[@B39]\]. For example, in brain the TargetScan-based analyses predicted 14 of 24 known miRNAs whilst miRanda and RNAhybrid predicted 4 and 5, respectively. In Liver, TargetScan, miRanda and RNAhybrid predicted five, one and one, respectively, out of eight, and in skeletal muscle five, three and one out of five. The predicted targets of miR-122a were not lowly expressed in liver and, therefore, no effect could be observed following treatment with antagomir. Likewise, the expression levels of the predicted targets of miR-124 were not significantly lower following its overexpression. However, the miRNAs with low predicted target gene expression in a particular tissue were expressed at higher levels than others in that tissue (Additional data files 7 and 8), albeit less markedly than those miRNAs derived from equivalent analyses with TargetScan predictions (Additional data file 5). This suggests that these prediction methods do detect real miRNA targets, but that there may be more false predictions and/or that the target genes detected by these criteria are less susceptible to miRNA-mediated reduction in mRNA levels. The miRNAs detected in the analyses based upon miRanda or RNAhybrid target predictions are listed in Additional data file 9.
It has been suggested that target mRNAs can be repressed by miRNA binding sites in the 5\' UTR \[[@B40]\]. Analysis of all mouse 5\' UTRs for target sites using RNAhybrid yielded fewer predictions than in 3\' UTRs and there was no obvious link between miRNAs with low target gene expression and known, highly expressed, tissue-specific miRNAs such as miR-1 in heart or miR-122a in liver. However, there is evidence from the miRNA expression data of Liang *et al*. \[[@B34]\] that those miRNAs with low predicted target gene expression are more highly expressed (Additional data file 10). This suggests that functional interactions are occurring between miRNAs and 5\' UTRs *in vivo*.
Conclusion
==========
It is perhaps surprising that given the wide range of regulatory mechanisms acting on gene expression, we are able to detect the effect of miRNAs on their diverse collection of target genes. The effectiveness of three analytical approaches, including the use of absolute expression values that might be unevenly skewed by single extreme values, illustrates the extent of the effect and the correlation between independent datasets is remarkable. These findings provide further evidence that miRNAs play an important role in regulating mRNA expression and can affect the mRNA levels of multiple genes. Although the effects on mRNA expression profiles are highlighted following artificial manipulation of miRNA levels \[[@B12],[@B13]\], these are real *in vivo*effects because they are observed in data derived from endogenous situations. One limitation of the TargetScan predictions that we used, is that they cannot discriminate between miRNA family members with the same seed sequence (this has been addressed in release 4.0).
The mechanism by which miRNAs are causing degradation of their cognate mRNAs is unclear \[[@B8],[@B41]\] and may be a side-effect of the translation inhibition process and be related to high miRNA expression levels. In rare cases miRNAs can direct slicer-mediated cleavage if there is extensive complementarity with the target site, as reported for miR-196 and HOXB8 mRNA \[[@B9]\]. This is not the case for the miRNA-target interactions reported here, but they may be inducing deadenylation and subsequent cleavage \[[@B42]\]. The ability of specific miRNAs to reduce the level of certain target mRNAs very efficiently might explain why they have a detectable effect upon gene expression in tissues in which their expression has been shown to be very low, for example, miR-196 in brain. This raises the possibility that predicted miR-196 target sites included in this analysis may have distinctive features. An updated release of TargetScan (4.0) includes scores for features that contribute to predicted target site efficacy, which are summarized in a \'context score\' \[[@B2]\]. The context scores for the predicted targets of miR196 expressed in brain are not significantly higher than those of other miRNAs. However, there are several predicted target genes, including *HOXB7*and *HOXA5*, with high context scores - efficient degradation of these targets could contribute to a detectable signal from low levels of miR-196. Analyses of miRNA signatures in gene expression profiles will help to pinpoint those miRNAs acting at the mRNA level and determine to what extent this results from high miRNA expression or reflects specific mechanisms related to certain miRNAs.
It is difficult to validate miRNA target predictions from effects on translation due to the technical difficulties of genome-wide quantitative protein profiling. Analysis of the effects of miRNAs on target mRNA expression provides a potential alternative. The target predictions we employed \[[@B27],[@B37],[@B39]\] were sufficiently accurate to enable detection of miRNA signatures within gene expression profiles. The ability to detect these miRNA signatures also attests to the sensitivity and reproducibility of current microarray technology. The strength of signal detected could be used to assess the effectiveness of different target prediction algorithms. The lack of significant effects in previous studies \[[@B16]\] might be because the target gene predictions employed were less effective at predicting miRNA-target interactions that result in mRNA degradation.
A unique advantage of this approach is that it is possible to infer miRNA activity from a single gene expression profile rather than having to first rank the expression of each gene across a series of experiments \[[@B11],[@B16],[@B17]\]. This will facilitate the analysis of individual datasets of interest from the vast repository of gene expression data that are steadily accumulating \[[@B20],[@B22]\]. It is applicable to data from any sufficiently sensitive and comprehensive platform and to gene expression profiles generated from biological sources too scarce to permit direct miRNA detection.
Our analysis of a limited number of gene expression profiles has demonstrated the major impact of miRNAs upon mRNA expression. The programs are amenable to provision of an online service that would enable individual researchers to gain an indication of miRNA activity in their tissue/cells of interest. This is a convenient approach to gain additional biological insight from gene expression profiles.
Materials and methods
=====================
Stand Alone Java and R programs \[[@B43]\], in conjugation with MS Access and MS Excel, were used for the following analyses.
Gene expression data
--------------------
Mouse and human mRNA expression datasets that covered a range of tissues were downloaded from GEO \[[@B20],[@B21],[@B44]\]. In addition, a second mouse dataset covering the same tissues was compiled from data submitted by different laboratories. The tissues and GEO sample and platform accession numbers are listed in Additional data file 3. Evidence for tissue-specific miRNA expression supported by cloning, Northern hybridization or expressed sequence tag mapping was determined by literature review.
Calculation of miRNA target gene expression
-------------------------------------------
Only single gene-specific Affymetrix probesets (suffix_at) were considered and only those expressed in each dataset analyzed (designated by a \'present\' call if available, or alternatively a signal strength greater than the median value). Probeset IDs were converted to their cognate gene symbols, using the Biomart online suite \[[@B45]\]. Lists of miRNA families and their predicted target genes published by Lewis *et al*. \[[@B4]\] were downloaded from the TargetScan website (version 3.1) \[[@B27]\]. For mouse (Mouse Genome 430 2.0 genechip), 15,682 probesets were mapped to gene symbols and of these, between 3,120 and 3,792 were both predicted miRNA targets and expressed in the tissues analyzed (for human, 17,325 probesets and 4,135-5,906 expressed targets were analyzed). Additional miRNA target genes predicted by miRanda \[[@B36],[@B37]\] were downloaded from the miRBase website \[[@B46]\]. Sites with scores and conservation values (P_org) greater than average were selected for analysis. The RNAhybrid algorithm \[[@B39]\] was run with the following parameters: *p*\< 0.05, energy \< -10 kcal/mol and with enforced binding to bases 2-7.
For each mRNA expression dataset, lists of the expressed genes predicted to be targeted by each miRNA and their respective expression levels were compiled (the average value was calculated for genes represented by \> 1 probeset). Only miRNAs with \> 50 predicted targets were considered further. These were sorted according to the average expression values (absolute or logarithmically transformed according to the primary microarray normalization algorithm used to analyze the data) of their predicted target genes and when necessary divided into upper and lower halves.
Statistical analysis
--------------------
Three different analytical methods were used to compare the expression of the predicted targets of a specific miRNA in a particular tissue, with the set of expression values of all the genes predicted to be targeted by miRNAs in the same tissue. To analyze whether there was significant difference between the medians of the ranked gene expression values of the two sets, the nonparametric one-sided Wilcoxon rank sum test was employed. To test the hypothesis that the genes in each set are from the same population, we used a one-tailed Student\'s *t*-test (considering unequal variance and using log-normalized data). For every miRNA with *p*\< 0.1, we considered that the expression of all the genes predicted to be targeted by that miRNA in that tissue was significantly more or less than the average expression of all the predicted target genes.
To calculate an RR value, predicted target genes were ordered by expression value and then divided into two groups by either the median gene rank or absolute expression value. For each miRNA, the number of genes targeted in the lower group was divided by the number in the higher to give an RR value.
MCCs \[[@B32]\] were calculated as a measure of performance.
Calculation of GC content and length of predicted target mRNA 3\' UTRs
----------------------------------------------------------------------
Mouse 3\' UTR data were downloaded from Biomart \[[@B45]\]. The GC content and the lengths of the UTRs were then calculated for the nonredundant sets of genes predicted to be targeted by groups of miRNAs of interest.
Analysis of miRNA-target site complementarity
---------------------------------------------
For sets of miRNAs of interest, the number of predicted targeted genes having greater than or equal to *x*contiguous complementary nucleotides after the 6 bp seed was noted (where *x*ranged from 0-8, 0 being the seed itself and 8 the maximum complementarity observed after the seed region). The number of predicted target genes was calculated with and without consideration of multiple miRNA family members.
Abbreviations
=============
GEO, Gene Expression Omnibus; MCC, Matthews correlation coefficients; miRNA, microRNA; RR, ranked ratio; RT-PCR, reverse transcription PCR; UTR, untranslated region.
Authors\' contributions
=======================
DACS conceived the study. AA collected the data and performed the data analyses. DACS and AA interpreted the results. DACS drafted the manuscript with assistance from AA. Both authors read and approved the final manuscript.
Additional data files
=====================
The following additional data are available with the online version of this paper. Additional data file [1](#S1){ref-type="supplementary-material"} is a figure showing the number of target genes predicted by TargetScan for each miRNA and expressed in each mouse tissue. Additional data file [2](#S2){ref-type="supplementary-material"} is a figure displaying the RR values for all miRNAs in heart, kidney, lung, ovary, skeletal muscle and testes. Additional data file [3](#S3){ref-type="supplementary-material"} is a table listing the sources of gene expression data from the NCBI GEO website. Additional data file [4](#S4){ref-type="supplementary-material"} is a table listing the MCCs calculated for each tissue. Additional data file [5](#S5){ref-type="supplementary-material"} is a series of box plots showing the correlation between mouse miRNAs with lowly expressed mouse target genes predicted by TargetScan and expression levels of orthologous human miRNAs detected in cognate tissues by RT-PCR. Additional data file [6](#S6){ref-type="supplementary-material"} is a figure showing the GC content and lengths of the 3\' UTRs of miRNA predicted target mRNAs in a range of tissues. Additional data file [7](#S7){ref-type="supplementary-material"} is a series of box plots showing the correlation between miRNAs with lowly expressed target genes predicted by miRanda and miRNA expression levels detected by RT-PCR. Additional data file [8](#S8){ref-type="supplementary-material"} is a series of box plots showing the correlation between miRNAs with lowly expressed target genes predicted by RNAhybrid and miRNA expression levels detected by RT-PCR. Additional data file [9](#S9){ref-type="supplementary-material"} is a table listing miRNAs with significantly low target gene expression determined by miRanda and RNAhybrid.
Additional data file [10](#S10){ref-type="supplementary-material"} is a series of box plots showing that miRNAs with lowly expressed predicted target genes, as defined by putative 5\' UTR sites, are expressed at higher levels than those with no effect on target gene expression.
Supplementary Material
======================
###### Additional data file 1
The number of predicted target genes (y-axis) for each miRNA (x-axis) is displayed with a different symbol for each tissue (grey circle, brain; green triangle, kidney; blue square, lung; green diamond, skeletal muscle; white circle, heart; red triangle, liver; purple square, ovary; yellow diamond, testes). miRNAs with less than 50 predicted target genes expressed in a specific tissue were not considered (see dashed line).
######
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###### Additional data file 2
Each panel displays the data from a single tissue. The miRNAs are arranged in ascending order according to RR value, shown on the left-hand y-axis and displayed as a red line. The higher values reflect lower target gene expression and are, therefore, indicative of miRNA activity. The number of predicted target genes is shown on the right-hand y-axis and the values for each miRNA indicated by a dashed line.
######
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###### Additional data file 3
Sources of gene expression data from the NCBI GEO website.
######
Click here for file
###### Additional data file 4
The parameters were obtained from the predictions and evidence presented in Table [1](#T1){ref-type="table"}.
######
Click here for file
###### Additional data file 5
Box plots depict the copy numbers (y-axis) of groups of microRNAs (x-axis) whose mouse orthologs have low, mid or high predicted target gene expression (as defined in the text). Where necessary to present the median and interquartile ranges effectively, up to two outliers were omitted.
######
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###### Additional data file 6
The graphs on each row show data from a single tissue, with the first column depicting 3\' UTR length and the second GC content. The predicted target genes are divided into three groups, low (red), medium (orange) and high (yellow). Those in the low group are targeted by miRNAs with overall significantly low target gene expression, those in the medium group are targeted by miRNAs with overall target gene expression within the expected range and those in the high group are targeted by miRNAs with overall high target gene expression. The average lengths in bases or %GC content (y-axis) of the 3\' UTRs of each group of genes (x-axis) are shown with standard error bars.
######
Click here for file
###### Additional data file 7
miRNAs were divided onto those with significantly lower than expected target mRNA expression (labeled \'low\') and those with no detectable effect on their target expression (labeled \'mid\'). The boxplots show the copy number (y-axis) of the miRNAs in each group (x-axis) and illustrate the significantly higher expression of miRNAs with low target gene expression. The scale of the y-axis (copy number) was chosen to facilitate visual comparison between groups and necessitated omission of up to three outliers from several graphs.
######
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###### Additional data file 8
miRNAs were divided into those with significantly lower than expected target mRNA expression (labeled \'low\') and those with no detectable effect on the expression of their predicted targets (labeled \'mid\'). The boxplots illustrate the significantly higher expression of miRNAs with low target gene expression. The scale of the y-axis (copy number) was chosen to facilitate visual comparison between groups and necessitated omission of up to three outliers from several graphs.
######
Click here for file
###### Additional data file 9
miRNAs with significantly low target gene expression determined by miRanda and RNAhybrid.
######
Click here for file
###### Additional data file 10
miRNAs were divided into groups according to gene expression of targets predicted from potential 5\' UTR miRNA binding sites. For each tissue (brain, heart, kidney, liver, lung, ovary, skeletal muscle and testes) a box plot shows the miRNA copy number, as determined by RT-PCR \[[@B34]\] (y-axis) of the miRNAs in each group (\'low\' and \'mid\', x-axis).
######
Click here for file
Acknowledgements
================
We would like to acknowledge the contribution of all the researchers who have made their gene expression data publicly available and without whom this study would not have been possible. We are grateful to Drs Tom Gardiner, Gareth McKay, Tim Curtis and Colin Willoughby for helpful discussion. AA was supported by a SPUR studentship.
| {
"pile_set_name": "PubMed Central"
} |
Background
==========
Neuroblastoma is a childhood tumor originating from the peripheral sympathetic nervous system. It is characterized of two different patterns of disease progress. One, frequently occurring in very young children and without amplification of the *MYCN*gene, is often associated with good prognosis and sometimes even with spontaneous regression. The other group of tumors, however, often involving slightly older children and with *MYCN*amplification is associated with poor prognosis \[[@B1]\]. A common feature of highly malignant neuroblastoma is the acquisition of multidrug resistance \[[@B2]\].
Protein kinase C (PKC) constitutes a family of closely related protein serine/threonine kinase which are sub-grouped into classical (PKCα, βI, βII, and γ), novel (PKCδ, ε, η, and θ), and atypical (PKCι and ζ) isoforms. The basis for this classification is different domain structure and activator requirements of the isoforms \[[@B3]\]. The members of the PKC family are involved in the regulation of numerous cell processes including proliferation, apoptosis, and differentiation. It is likely that each isoform has a specific role in a given cell.
We have shown that neuroblastoma cells express PKCα, βI, and βII of the classical isoforms and PKCδ and ε of the novel isoforms \[[@B4],[@B5]\]. Of these isoforms PKCε is a positive regulator of neurite outgrowth during differentiation of these cells \[[@B6],[@B7]\] whereas PKCβI seems to have a positive role for neuroblastoma cell proliferation \[[@B5]\]. The latter study also indicated that inhibition of PKCβ could potentiate the growth suppressive effect of microtubule-interacting anticancer drugs.
The aim of this study was to investigate whether inhibition of PKCβ isoforms could be utilized to potentiate the effects of chemotherapeutic drugs on neuroblastoma cells. For that purpose three cell lines, one without *MYCN*amplification (SH-SY5Y), and two *MYCN*-amplified (IMR-32 and SK-N-BE(2)), were screened for the combinatorial effects of the PKCβ inhibitor and several chemotherapeutic drugs. One of these cell lines, SK-N-BE(2), has been shown to exhibit resistance to a broad range of anti-cancer compounds.
We found that the specific PKCβ inhibitor LY379196 suppressed the growth of all three neuroblastoma cell lines studied and that it potentiated the growth-suppressive effect of all investigated chemotherapeutics, except carboplatin, on the drug-resistant SK-N-BE(2) cell line. Furthermore, LY379196 potentiated the accumulation of \[^3^H\]vincristine in the SK-N-BE(2) cells suggesting that an effect on the elimination of the chemotherapeutic drugs is the mechanism whereby LY379196 influences the effect on cell growth.
Methods
=======
Cell lines
----------
IMR-32, SH-SY5Y, and SK-N-BE(2) neuroblastoma cells were maintained in Eagle\'s minimal essential medium supplemented with 10% FCS, 100 IU/ml penicillin and 100 μg/ml streptomycin (all cell culture reagents were from Gibco).
Cell viability analysis
-----------------------
Cells were seeded at a density of 5000 cells per well in 96 well plates and cultured for three (SK-N-BE(2)) or four (IMR-32 and SH-SY5Y) days. Drugs had been added to the wells prior to addition of cells. LY379196 (kindly provided by Eli Lilly Research Laboratories), GF109203X and Gö6976 (Calbiochem), and etoposide and paclitaxel (Sigma) were solubilized in DMSO. Vincristine (Sigma) and carboplatin and doxorubicin (ICN) were solubilized in water. The amount of viable cells in the wells were analyzed with an MTT assay (Promega) according to the supplier\'s protocol. To calculate the drug concentration that gives 50% viable cells compared to control conditions a non-linear curve fit, *y*= *A*~2~- (*A*~1~- *A*~2~)/(1 + *B*/*x*), was performed on the experimental data. With the parameter values obtained from the curve fit, we calculated the anticancer drug concentration that reduced the amount of viable cells to 50%. This was done in two ways to both show the effect of LY379196 on the potency of the chemotherapeutic drug and to illustrate the total effect of the combination of LY379196 and the chemotherapeutic drug on neuroblastoma cell growth. In the first case, the amount of cells in the absence of chemotherapeutic drug but in the presence of the particular LY379196 concentration was set to 100%, and the concentration of anticancer drug that gave 50% was calculated. In the second case, the amount of cells in the absence of both PKC inhibitor and chemotherapeutic drug was set to 100%. For each LY379196 concentration, the concentration of chemotherapeutic drug that suppressed the amount of cells to 50% was thereafter calculated.
Accumulation of \[^3^H\]vincristine
-----------------------------------
SK-N-BE(2) cells were seeded at a density of 100,000 cells/well in a 24-well plate. The next day PKC inhibitors were added at indicated concentrations and 15 min later \[^3^H\]vincristine (final concentration 1 μM, 0.25 Ci/mmol \[Amersham Pharmacia Biotech\]) was added. After 2 h the plate was put on ice and cells were washed three times with PBS. Cells were lysed in 10 mM NaH~2~PO~4~, pH 7.4, 1% Triton X-100, and 0.2% SDS and thereafter transferred to vials for scintillation counting.
Results
=======
In a previous study we found that inhibition of PKCβ with LY379196 suppresses the proliferation and growth of SK-N-BE(2) cells \[[@B5]\]. In order to examine whether this is a cell line-specific effect, or if it is more general in terms of neuroblastoma cells, three neuroblastoma cell lines -- IMR-32, SH-SY5Y, and SK-N-BE(2) -- were cultured in the presence of increasing concentrations of LY379196 (Figure [1](#F1){ref-type="fig"}). This showed that all cell lines were sensitive to LY379196. At a concentration of 300 nM, LY379196 suppressed the number of viable cells with 16--24% for the three cell lines, which indicates that a PKCβ isoform has a positive effect on neuroblastoma cell growth.
{#F1}
To elucidate a putative synergistic effect of the PKCβ inhibitor and different anticancer drugs used for neuroblastoma therapy the three cell lines were cultured in the presence of increasing concentrations of doxorubicin, etoposide, paclitaxel, vincristine, or carboplatin together with different concentrations of LY379196 (Figure [2](#F2){ref-type="fig"}, Table [1](#T1){ref-type="table"}). The cell lines displayed different sensitivity to the anticancer drugs. IMR-32 was consistently the most sensitive to all drugs investigated. SH-SY5Y and IMR-32 were equally sensitive to the topoisomerase inhibitors doxorubicin and etoposide whereas SH-SY5Y cells were three- to four-fold less sensitive to the microtubule interacting agents paclitaxel and vincristine and to the DNA intercalator carboplatin. SK-N-BE(2) was the most resistant cell line being more than fifty-fold more resistant to doxorubicin, etoposide and carboplatin than the other cell lines. SK-N-BE(2) cells also displayed a substantially higher resistance to vincristine and paclitaxel.
![**The effect of the combination of LY379196 and anticancer drugs on neuroblastoma cell growth.**IMR-32, SH-SY5Y, and SK-N-BE(2) cells were grown in medium supplemented with increasing concentrations of doxorubicin, etoposide, paclitaxel, vincristine, and carboplatin. Different amounts of LY379196 were also included in the medium yielding final concentrations of 0 (■, black), 20 (●, red), 100 (▲, green) or 500 (▼, blue) nM. After three (SK-N-BE(2)) or four (IMR-32 and SH-SY5Y) days in culture the amount of viable cells was analyzed with an MTT assay. The concentrations of the anticancer drugs are indicated on the x-axes as lg(\[drug\]/1M). Data are expressed as percent of values obtained in the absence of drugs and are mean ± SEM (n = 8--9).](1471-2407-3-10-2){#F2}
######
Effects of LY379196 on ED~50~values of chemotherapeutic drugs.
**Doxorubicin** **Etoposide** **Paclitaxel** **Vincristine** **Carboplatin**
---------------- ----------------- --------------- ---------------- ----------------- ----------------- ------- ------- ------- ------- -------
**IMR-32** (nM) (nM) (nM) (nM) (nM)
LY379196 **A** **B** **A** **B** **A** **B** **A** **B** **A** **B**
0 nM 6.3 110 1.7 0.51 760
20 nM 5.5 5.3 101 118 1.6 1.7 0.47 0.57 780 740
100 nM 5.5 4.7 88 98 1.4 1.6 0.40 0.47 880 720
500 nM 6.1 2.8 103 78 1.6 1.0 0.37 0.30 880 380
**SH-SY5Y** (nM) (nM) (nM) (nM) (μM)
LY379196 **A** **B** **A** **B** **A** **B** **A** **B** **A** **B**
0 nM 9.3 109 8.3 1.41 1.9
20 nM 8.2 9.8 93 114 7.3 8.0 1.20 1.45 2.2 2.3
100 nM 7.2 6.7 74 81 4.8 4.3 0.82 0.89 2.1 1.8
500 nM 5.0 2.0 75 55 4.3 2.7 0.57 0.33 2.0 0.7
**SK-N-BE(2)** (nM) (μM) (nM) (nM) (μM)
LY379196 **A** **B** **A** **B** **A** **B** **A** **B** **A** **B**
0 nM 920 7.2 117 42 \>100
20 nM 650 690 6.4 6.6 108 109 32 33 \>100 \>100
100 nM 450 380 5.3 4.8 105 94 21 21 \>100 \>100
500 nM 270 160 3.9 2.8 40 27 5.2 3.9 \>100 \>100
This table shows the concentrations (ED~50~) of the chemotherapeutic drugs, in the presence of different LY379196 concentrations, needed to suppress the number of viable cells after three (SK-N-BE(2)) or four (IMR-32 and SH-SY5Y) days in culture to 50% of the amount obtained in the absence of the drug. The data used for calculations are obtained from Figure [2](#F2){ref-type="fig"}. Values are geometric mean of three separate experiments with triplicate measurements. The ED~50~values were calculated using the amount of viable cells in the absence of chemotherapeutic drug but in the presence of LY379196 as 100% (values in A columns) or the amount of viable cells in the absence of anticancer drug and LY379196 (values in B columns). Thus, values in column A reflect the potency of the chemotherapeutic drug since the inherent growth-suppressing effect of LY379196 has been eliminated in the calculations, whereas values in column B reflect the combined growth-suppressing effect of LY379196 and chemotherapeutic drug. For carboplatin treatment of SK-N-BE(2) cells a reduction in viable cell amount by 50% was not obtained within the concentration range used.
When LY379196 was included in the medium there was a clear difference in effect depending on the cell line studied. For IMR-32, the least drug-resistant cell line, no effect of LY379196 on the potency of the chemotherapeutic drugs was observed. With 500 nM there was a suppression of the number of viable cells but this reflects the inherent growth-suppressive effect of LY379196 (Figure [1](#F1){ref-type="fig"}) and was not due to an enhancement of the potency of anticancer agents on IMR-32 cells.
For SH-SY5Y cells, concomitant exposure to 500 nM LY379196 led to approximately a doubling of the potency of doxorubicin, paclitaxel, and vincristine. The ED~50~values were decreased from 9.3, 8.3 and 1.41 nM, respectively, in the absence of LY379196 to 5.0, 4.3 and 0.57 nM (column A in Table [1](#T1){ref-type="table"}), respectively, in the presence of 500 nM LY379196. These values reflect the potency of the chemotherapeutic drugs, since the growth-suppressing effect of LY379196 has been compensated for in the calculation. If the combined effect of 500 nM LY379196 and the chemotherapeutic drugs is studied, the drug concentration necessary to suppress the number of viable cells to 50% of the amount obtained in the absence of both chemotherapeutic agent and LY379196 is further reduced to 2.0 nM, 27 nM and 0.33 nM (column B in Table [1](#T1){ref-type="table"}) for doxorubicin, paclitaxel and vincristine, respectively. This correlates to some degrees to the spectrum of drug resistance of SH-SY5Y cells compared to IMR-32 cells since SH-SY5Y cells are more resistant to both paclitaxel and vincristine. However, no effect was observed on the potency of carboplatin, despite the fact that SH-SY5Y cells are less sensitive to this compound.
The effect of LY379196 was most pronounced on SK-N-BE(2) cells, the cell line with the lowest sensitivity to the anticancer drugs. When 500 nM LY379196 was included in the medium, there was an eight-fold decrease in the ED~50~value of vincristine (from 42 to 5.2 nM), approximately a three-fold decrease of the values for doxorubicin (from 920 to 270 nM) and paclitaxel (from 117 to 40 nM), and also an enhancement of the etoposide effect (column A in Table [1](#T1){ref-type="table"}). These changes reflect an effect of LY379196 on the potency of the chemotherapeutic compounds since the inherent growth-inhibitory effect of LY379196 was compensated for in the calculations. As for SH-SY5Y cells, there was no effect of LY379196 on the cytotoxic/growth suppressive effect of carboplatin (Figure [2](#F2){ref-type="fig"}, Table [1](#T1){ref-type="table"}).
If the potentiating effect on the chemotherapeutic drugs is mediated via an inhibition of PKCβ isoforms, other inhibitors of PKC would be expected to yield a similar effect. Therefore the effect of two PKC inhibitors, GF109203X which inhibits all PKC isoforms and Gö6976 which only inhibits classical isoforms, on the growth-inhibiting effects of vincristine on SK-N-BE(2) cells was investigated (Figure [3](#F3){ref-type="fig"}, Table [2](#T2){ref-type="table"}). This experiment demonstrated that both these inhibitors increase the sensitivity of SK-N-BE(2) cells to vincristine. When the growth-suppressing effects of the inhibitors were compensated for, we found that the ED~50~of vincristine was roughly halved in the presence of 500 nM of either inhibitor (column A in Table [2](#T2){ref-type="table"}). Gö6976 by itself also caused a substantial decrease of the amount viable cells.
![**The effect of the combination of PKC inhibitors and vincristine on SK-N-BE(2) cell growth.**SK-N-BE(2) cells were grown in medium supplemented with increasing concentrations of vincristine. Different amounts of GF109203X (A) or Gö6976 (B) were also included in the medium yielding final concentrations of 0 (■, black), 20 (●, red), 100 (▲, green) or 500 (▼, blue) nM. After three days in culture the amount of viable cells was analyzed with an MTT assay. The concentration of vincristine is indicated on the x-axes as lg(\[drug\]/1M). Data are expressed as percent of values obtained in the absence of drugs and are mean ± SEM (n = 8--9).](1471-2407-3-10-3){#F3}
######
Effects of GF109203X and Gö6976 on ED~50~values of vincristine.
**GF109203X** **Gö6976**
--------------- --------------- ------------ ------- -------
(nM) (nM)
PKC inhibitor **A** **B** **A** **B**
0 nM 41 49
20 nM 30 35 35 35
100 nM 26 26 39 31
500 nM 28 18 24 NA
This table shows the concentrations (ED~50~) of vincristine, in the presence of different concentrations of GF109203X or Gö6976, needed to suppress the number of viable SK-N-BE(2) cells after three days in culture to 50% of the amount obtained in the absence of the drug. The data used for calculations are obtained from Figure [3](#F3){ref-type="fig"}. Values are geometric mean of three separate experiment with triplicate measurements. The ED~50~values were calculated using the amount of viable cells in the absence of vincristine but in the presence of PKC inhibitor as 100% (values in A columns) or the amount of viable cells in the absence of vincristine and PKC inhibitor (values in B columns). Thus, values in column A reflect the potency of vincristine since the inherent growth-suppressing effect of the PKC inhibitors has been eliminated in the calculations, whereas values in column B reflect the combined growth-suppressing effect of PKC inhibitor and vincristine.*NA*-- not available because culture in the presence of 500 nM Gö6976 reduced the number of cells to less than 50% of the number obtained in the absence of the compound.
The largest effect of LY379196 was observed for chemotherapeutic agents of the natural compound group and no effect was seen together with carboplatin. Resistance towards natural products has mostly been associated with a multidrug resistance phenotype, which has been suggested to be due to an increased expression of proteins, which pump out the compounds from the cells. To investigate whether inhibition of PKCβ influences this mechanism of resistance, the accumulation of \[^3^H\]vincristine was analyzed in the presence of increasing concentrations of PKC inhibitors (Figure [4](#F4){ref-type="fig"}). All inhibitors used (LY379196, GF109203X, and Gö6976) caused an enhancement of the accumulation of \[^3^H\]vincristine. The specific PKCβ inhibitor, LY379196, was the most potent compound and the concentrations which augmented the vincristine accumulation reflected the concentrations that sensitized the SK-N-BE(2) cells to vincristine.
![**PKC inhibitors augment the accumulation of \[^3^H\]vincristine in SK-N-BE(2) cells.**Different concentrations of LY379196, GF109203X and Gö6976 were added to SK-N-BE(2) cells 15 min prior to a 2 h incubation with \[^3^H\]vincristine. The amount of the radioactivity that had accumulated in the cells was thereafter measured. Data are expressed as percent of \[^3^H\]vincristine that had accumulated in cells that had not been exposed to inhibitor and are mean ± SEM of 8 determinations.](1471-2407-3-10-4){#F4}
Discussion
==========
We have previously seen that inhibition of a PKCβ isoform, conceivably PKCβI, suppresses the proliferation of the SK-N-BE(2) neuroblastoma cell line \[[@B5]\]. The data in this study indicate that the proliferation supporting effect of a PKCβ isoform may be general for neuroblastoma cells since the growth of the two other cell lines tested also were sensitive to the PKCβ inhibitor. When 300 nM LY379196 was included in the medium there were fewer cells after four days in culture of all cell lines suggesting that the effect may be general for neuroblastoma cells.
The data in this study also demonstrate that the PKCβ inhibitor augments the effect of natural product chemotherapeutic agents on multidrug-resistant neuroblastoma cells. In contrast to the effect on proliferation, which influenced all cell lines, the augmentation of the anticancer drugs was related to the grade of drug resistance of the cell line. It is therefore conceivable that LY379196 interferes with mechanisms that confer drug resistance to neuroblastoma cells, although it can not be excluded that other factors than the degree of drug resistance explain the difference in sensitivity to LY379196 in the presence of chemotherapeutic drugs.
The acquisition of multidrug resistance upon treatment with anti-cancer drugs is a common phenomenon for neuroblastomas \[[@B2]\]. This is a major reason for the high frequency of fatal outcome of the disease. Drug resistance of cancer cells has been suggested to be caused by increased expression of proteins of the ATP-binding cassette transporter family such as the *MDR1*protein product P-glycoprotein and MRP. High expression of *MDR1*in neuroblastomas was shown to be related to previous chemotherapeutic treatment \[[@B8]\], to drug resistance \[[@B9]\] and to bad prognosis in a subset of cases \[[@B10]\]. However, it was also shown to correlate inversely to *MYCN*amplification \[[@B11]\] and to good prognosis \[[@B12]\]. High expression of *MRP*has been reported to be prognostic for pour outcome \[[@B13]\] although other studies have not found this connection \[[@B14],[@B15]\]. The expression of *MRP*is positively influenced by *MYCN*\[[@B16]\], which is frequently amplified in neuroblastomas with pour outcome. Thus, the specific role of the *MDR1*and *MRP*genes in the acquisition of drug-resistance of neuroblastomas has not yet been settled.
Another study has instead indicated a role for mutation and inactivation of the *TP53*tumor suppressor gene in drug resistant neuroblastomas \[[@B17],[@B18]\]. Furthermore a recent report has shown that chromosome reassortments can lead to drug resistance in cells that lack the *MDR1*and *MRP1*genes that are thought to confer multidrug resistance \[[@B19]\].
The data in this study demonstrate a correlation between the potentiation of cytotoxic/growth suppressing effects of natural compound anti-cancer drugs and an enhancement of \[^3^H\]vincristine accumulation by PKC inhibitors in neuroblastoma cells. This suggests that the drug resistance of the SK-N-BE(2) cells is at least partially mediated by increased elimination of natural products by efflux pumps such as products of the *MDR1*and *MRP*genes. This is supported by studies which have shown that agents that interfere with these pumps sensitizes drug resistant neuroblastoma cells to chemotherapeutic agents of the natural compound class \[[@B20],[@B21]\]. Furthermore, LY379196 did not augment the effect of carboplatin, which is not a substrate for pumps of the P-glycoprotein family.
PKC activity was initially thought to support the activity of P-glycoprotein since it was shown to be a PKC substrate \[[@B22]-[@B24]\] and PKC inhibitors were reported to suppress the activity of the protein \[[@B22],[@B24],[@B25]\]. However, later studies demonstrated that phosphorylation of P-glycoprotein did not influence its function \[[@B26],[@B27]\]. It has also been shown that GF109203X directly influences the activity of P-glycoprotein by competing with other substrates \[[@B28]\] and a similar mechanism of action has also been suggested for the effect of GF109203X on MRP \[[@B29]\] and for the reversal of drug resistance by the PKC inhibitor Ro 32-2241 \[[@B30]\]. The effect of LY379196 may therefore be due to a competition with the anticancer drugs for P-glycoprotein or other members of this family.
However, there are other reports demonstrating attenuating effects of PKC inhibitors on drug resistance that do not involve P-glycoprotein. Safingol and a myristoylated PKC pseudosubstrate peptide have been shown to inhibit PKC and potentiate drug accumulation independently of effects on P-glycoprotein \[[@B31]-[@B33]\]. The data in this study indicate that a PKCβ isoform could be a relevant isoform to inhibit in order to attenuate PKC-supported drug resistance. It is notable that the inhibitor with highest specificity for PKCβ also had the most profound effect on the cytotoxicity of vincristine. The smaller effect of the other inhibitors could for instance be due to inhibition of pro-apoptotic PKC isoforms such as PKCδ, which has been shown to be important for apoptosis in several cell types \[[@B34]-[@B38]\]. It should be noted that Gö6976 alone causes a substantial decrease of viable neuroblastoma cells, which may imply toxic effects attendant to the lack of isoform specificity. This could mask a potentiating effect on the cytotoxicity of vincristine and also preclude the use of Gö6976 at effective concentrations.
Conclusions
===========
this study raises the potential to use PKCβ isoforms as targets to both partially suppress proliferation and to attenuate the multidrug resistance of neuroblastoma cells.
Competing interests
===================
None declared.
Author\'s Contributions
=======================
KS carried out all experiments. CL was responsible for the progress of the work and preparation of the manuscript. Both authors read and approved the final manuscript.
Pre-publication history
=======================
The pre-publication history for this paper can be accessed here:
<http://www.biomedcentral.com/1471-2407/3/10/prepub>
Acknowledgements
================
Eli Lilly Research Laboratories are gratefully acknowledged for supplying LY379196. This work was supported by the Swedish Cancer Society, the Children\'s Cancer Foundation of Sweden, the Swedish Society for Medical Research, the Royal Physiographic Society of Lund, the Crafoord; Magnus Bergvall; Gunnar, Arvid and Elisabeth Nilsson; Ollie and Elof Ericsson; and Malmö University Hospital Research Funds.
| {
"pile_set_name": "PubMed Central"
} |
INTRODUCTION
============
Competitive sports for disabled individuals have entered into a fast development process during the recent years. These sports have gained a place in a wide spectrum reaching open athletes and elite athletes. Paralympic games have raised awareness for the participation of disabled individuals in these sports. These games form an exercise characteristic for preventive health and improve cardio-metabolic aptness. Furthermore, they develop social integration of the disabled individuals by improving their self-confidence, self-competency and life quality ([@b1-jer-13-1-62]). Wheelchair (WC) basketball is the most popular paralympic sport ([@b8-jer-13-1-62]). This sport consists of activities demanding explosive strength and speed and intermediate intensity ([@b18-jer-13-1-62]).
WC-use requires the active and coordinated usage of upper extremity muscles especially shoulder complex muscles actively and coordinately. The shoulder flexors make the major contribution during WC-pushing stage and the shoulder extensors are dominant during the return stage. There is increased shoulder muscle cocontraction seen during the passage stage between these two stages ([@b17-jer-13-1-62]; [@b20-jer-13-1-62]).
The upper extremity injuries are seen in WC-using individuals considerably ([@b2-jer-13-1-62]). Shoulder pain is the most frequently seen complaint ([@b7-jer-13-1-62]). [@b6-jer-13-1-62] reported in their study conducted on WC-using individuals that the persons suffered shoulder pain caused by many reasons including biceps tendinitis and shoulder instability, and 29% of these people had pain during relaxation as well. [@b4-jer-13-1-62] showed in their study that 90% of women WC basketball athletes had shoulder and upper extremity pain. Muscle strength unbalance in the shoulder region and relative weakness in the depressors of humeral head can be affective for the development and continuation of rotator cuff compression syndrome ([@b3-jer-13-1-62]). In addition to shoulder complexities, elbow and wrist injuries occur frequently in WC athletes. Wrist extension and wrist joint movement speed with deviations are the factors affecting dynamic performance and causing injuries ([@b15-jer-13-1-62]). Both wrist extension and flexion movement gap and muscle strength are the factors affecting performance of WC basketball players ([@b21-jer-13-1-62]).
High level of condition is required for winning a competition in WC basketball like in all sports. The major factors affecting WC basketball performance are muscle strength and sprint speed. Having high level of muscle strength in WC basketball will ensure both performance improvement in basic movements unique to basketball and independence in daily life activities. The objective of this study was to reveal the isokinetic muscle strength characteristics of upper extremity in WC basketball athletes. The descriptive information will lead to the description of injury-preventing programs and more specific training programs.
MATERIALS AND METHODS
=====================
This study was conducted to reveal upper extremity isokinetic muscle strength characteristics in WC basketball players at the Ministry of Youth and Sports, Sports General Directorship, Health Affairs Department Directorship (Athlete Training and Health Research Center). All the athletes who accepted to participate in the study were informed about the study purpose, the assessments contained in the study and the benefits of the study before starting the study and the study was based on volunteerism, and approval was obtained. The necessary permit and approval was obtained from the Ethics Committee of Ankara Yıldırım Beyazıt University to conduct the study (13/03 \[346\]). The participants in the present study consisted of 12 male WC basketball players, aged 23--40 years, who are members of the Turkish national WC basketball team and their scores ranged between 2.5--4.5. The descriptive characteristics, age, height, weight and disability types of the athletes were recorded.
Isokinetic muscle strength assessment
-------------------------------------
Isokinetic muscle strength was assessed by ISOMED 2000 (D. & R. Ferstl GmbH, Hemau, Germany) device. Prior to the test, the athletes did general and special warming workouts for 10 min. Following the warming, the athletes were taken to the isokinetic device one by one for measurement and the device was adjusted according to their individual anthropometric structures. During the test, their weights were entered in the computer and the program was set. The suitability of the movement range of the joint to be tested to the angles that are to be tested was determined by making the athletes do a sample movement at a very low speed. At the same time, the gravity effect was set to zero. The joint angles were adjusted in the assessment by considering the joint movement ranges of the persons and the measurement characteristics of the device. An assessment was made for the shoulder flexion/extension movement between the angles of 30° flexion and 120° flexion, and for the wrist flexion/extension movement between the angles of 50° flexion and 60° extension. The assessment protocol: the athletes warmed by doing the flexion/extension movement at 90°/sec with five repeats as submaximal and they were ensured to perceive the movement. Following the warming movement and a 30-sec rest, they did maximal flexion/extension movement at 60°/sec speed and with five repeats ([@b12-jer-13-1-62]) and again following a 30-sec rest, they did maximal flexion/extension movement at 240°/sec speed and with 15 repeats and the test was completed. The assessments were made bilaterally for each joint and first the dominant side was assessed and 3 min later the nondominant side was assessed. The same protocol was applied the following day for the wrist joint.
Statistical analysis
--------------------
The data collected regarding the isokinetic strength of the athletes were analyzed by IBM SPSS Statistics ver. 22.0 (IBM Co., Armonk, NY, USA). The descriptive statistics of all variables were determined. The results were indicated as mean±standard deviation.
RESULTS
=======
Demographic characteristics and disability types of the athletes are shown in [Table 1](#t1-jer-13-1-62){ref-type="table"}.
The peak torque values and peak torque/weight values of dominant - nondominant shoulder flexion and extension movements of the athletes and wrist flexion and extension movements at 60°/sec and 240°/sec velocity, and the angles of these values and repeat times are shown in [Table 2](#t2-jer-13-1-62){ref-type="table"}.
The extension peak torque was found to be higher than the flexion peak torque in the shoulder joint, and the flexion peak torque value was higher in the wrist. The peak torque values were generated with 2--3 repeats at 60°/sec velocity at both movements for the wrist and shoulder. It occurred at 240°/sec velocity with an average of 5--6 repeats for both movements, and it occurred at 3--4 repeats for the wrist. Considering the angle values where the peak torque value is generated, it was at 60°/sec velocity in the shoulder joint as similar in the dominant and nondominant sides approximately 81°--86° and 112°--109° for flexion and extension respectively. At 240°/sec velocity, it was 110°--97°; 108°--106°. For the wrist, at 60°/sec velocity, it was approximately 16°--24° at flexion and at 5°--6° extension as similar in the dominant and nondominant sides respectively for the flexion and extension; at 240°, for flexion and extension, it was respectively 22°--25° flexion and 13°--6° extension.
The percentages of the shoulder and wrist flexion/extension movements of the athletes at 60°/sec and 240°/sec velocity for the dominant and nondominant sides and between the right and left, and the standard deviation values are shown in [Table 3](#t3-jer-13-1-62){ref-type="table"}.
The extension isokinetic muscle strength in the shoulder joint was higher than the flexion isokinetic muscle strength, and the flexion isokinetic muscle strength in the wrist was higher than the extension isokinetic muscle strength ([Table 3](#t3-jer-13-1-62){ref-type="table"}).
DISCUSSION
==========
In this study, the peak torque, peak torque/kg, right/left ration percentage and flexion/extension ratio percentage of WC basketball athletes were determined.
There are two studies in the literature assessing isokinetic muscle strength in healthy individuals. [@b5-jer-13-1-62] categorized according to age and gender and measured the wrist flexion and extension and shoulder flexion and extension at 60°/sec velocity, and [@b9-jer-13-1-62] published two studies determining isokinetic muscle strength in 178 healthy individuals at angular velocities that were different than ours ([@b5-jer-13-1-62]; [@b9-jer-13-1-62]). Moreover, [@b16-jer-13-1-62] published a study evaluating isokinetic muscle strength in WC tennis athletes as similar to our study. Our values at 60°/sec, and the flexion and extension peak torque values for the dominant and nondominant sides of [@b16-jer-13-1-62], and the shoulder and wrist flexion and extension peak torque values for 20--29 age group and 30--39 age group of [@b5-jer-13-1-62] are shown in [Table 4](#t4-jer-13-1-62){ref-type="table"}.
The peak torque of the shoulder flexion and extension in WC basketball athletes included in our study was approximately 2 times of those of the healthy participants in [@b5-jer-13-1-62] study and also of WC tennis athletes in [@b16-jer-13-1-62] study. It is thought that this outcome is caused by the fact that WC basketball sports requires intense upper extremity muscle strength for coordinated movement of WC and ball, and these athletes are included in the Paralympic class. [@b5-jer-13-1-62] found in their study that the wrist flexion/extension ratio was close to 1 in healthy individuals, and in our study, it was determined to be almost 1/3 in the favor of the flexors. Despite a high wrist flexor strength seems to be in favor of the athletes; it must be remembered that the muscle strength imbalance between the wrist flexors and extensors will be a preparatory factor for sports injuries. It was seen that the athletes had tennis player elbow complaint in their backgrounds as parallel to our results. We think that providing information on this issue to athletes and health team, and establishment of training programs for strengthening wrist extensors will prevent overuse injuries. There are numerous studies in the literature showing that individuals become prone to injuries due to muscle imbalances ([@b1-jer-13-1-62]; [@b14-jer-13-1-62]). Since there were only the peak torque data in [@b5-jer-13-1-62] study, peak torque/kg and other characteristic data were not able to be compared. Isokinetic assessments vary according to angular velocity. Therefore, it is not possible to make a comparison with this study since [@b9-jer-13-1-62] assessed the wrist in angular velocities different than ours. The flexion/extension difference and right and left difference in our study are shown in percentages in [Table 3](#t3-jer-13-1-62){ref-type="table"}. These data are critical for the existence of a balance between agonist/antagonist muscle forces and the disruption of the balance make the person prone to injuries ([@b13-jer-13-1-62]). [@b19-jer-13-1-62] found this ratio as 75%--85% for shoulder flexion and extension in their study. In our study, this ratio was found as approximately 71.2% as parallel to this result.
[@b10-jer-13-1-62] assessed the antagonist/agonist ratio between the wrist flexion and extension in their study and concluded that there was not a significant difference between the dominant and nondominant sides. In our study, we found that the wrist flexion/extension percentage in both sides varied between 260%--331%. The difference was outside of the normal limits and we suggest that the wrist extensors are strengthened and muscle imbalance is overcome to prevent sports injuries due to muscle strength imbalance.
[@b11-jer-13-1-62] reported in their study on isokinetic assessment that more than 10% of difference between the right and left extremities was abnormal. In our study, it was determined that the difference between the right and left side at the shoulder flexion and wrist extension at 240°/sec velocity was more than 10%. Therefore, workout programs must be added to the training programs to improve the strength for right and left bilaterally in activities requiring speed for especially the shoulder flexion and wrist extension. This puts forward the significance of neuro-reactive workouts in the training programs.
The results of our study and the percentages between the right-left and extension/flexion in [@b16-jer-13-1-62] study conducted on WC tennis players are shown in [Table 5](#t5-jer-13-1-62){ref-type="table"}. It was seen that the percentage of the right-left flexion and the percentage of the dominant side flexion/extension in WC tennis players were higher than those of the basketball players included in our study. We think that this difference is caused by the inclusion of WC using athletes by both groups however tennis is more asymmetric in comparison to basketball.
In addition, in our study the repeat number and angles where the peak strength occurs are indicated. In our study, there were athletes reaching the peak torque in the first repeat, there were also athletes reaching the torque strength at the 14th repeat. The peak occurred at 60°/sec velocity for the shoulder and wrist flexion and extension at an average of 2--3 repeats; and the peak occurred at 240°/sec for the shoulder flexion and extension at the fifth--sixth repeat, and it occurred at the third--fourth repeat for the wrist flexion and extension. This outcome of our study shows that the repeat number must be increased as the assessment speed increases. Studies need to be conducted on this issue with high number of athletes.
It was determined in our study that the peak torque occurred at the angles when the shoulder was in an elevated position and at the angles when the wrist was at a functional holding position. We think that this angle was affected by the fact that the athletes were basketball players and basketball throwing position affected the angle for peak torque generation in the wrist. In this context, the angles where the peak torque is generated should be considered when training programs are established.
The limitations of our study were failure to compare the peak torque values according to the disability type and age due to the low number of athletes.
In conclusion, WC basketball sport is the most popular one among paralympic sports branches and in Turkey. It is a critical tool for the rehabilitation of disabled persons and their integration in society. WC basketball sport is exercised at a professional level in the world and Turkey. Prevention of sports injuries is important in WC basketball like in other sports branches. The workout and training programs is an issue that needs attention for both prevention of sports injuries and achieving high performance in this sports branch. As our study revealed, determination of characteristics of muscle strength of WC athletes and especially using objective isokinetic tools will be guiding for the training program planning and prevention of sports injuries in long term.
CONFLICT OF INTEREST
No potential conflict of interest relevant to this article was reported.
######
Demographic characteristics of the athletes who participated in the study
Characteristic Value
--------------------------- -------------
Age (yr) 28.91±5.00
Height (cm) 75.64±11.79
Weight (kg) 72.73±10.2
Body mass index (kg/m^2^) 30.44±8.08
Disability type
Spina Bifida 1
Polyomyelitis 3
Amputation 2
Paraparesis 2
Paraplegia 2
Meningomyelocele 1
Values are presented as mean±standard deviation or number.
######
Peak torque, peak torque/weight, repeat times, and angle values of wheelchair basketball athletes for shoulder and wrist flexion-extension movement at 60°/sec and 240°/sec velocity
Angular velocity Dominant Nondominant
--------------------------------------- --------------- -------------- -------------- --------------
Shoulder
60°/sec (peak torque) (N/m) 109.63±28.58 150.71±38.75 101.15±28.47 146.98±36.07
60°/sec (peak torque/weight) (N/kg) 13.87±42.77 17.52±53.43 13.75±42.81 22.23±70.02
Angle (°) 81.66±14.36 112.16±16.44 86.16±15.39 109.75±15.12
Repeat number 2.33±1.49 2.91±1.44 3.33±1.49 3.00±1.20
240°/sec (peak torque) (N/m) 93.83±29.64 130.22±36.93 84.31±26.37 119.61±4.34
240°/sec (peak torque/weight) (N/kg) 11.09±33.92 15.6±47.8 12.37±38.9 17.11±53.56
Angle (°) 110.75 ±64.88 108.16±10.02 97.58±16.37 106.58±6.81
Repeat number 6.75±4.88 6.66±3.62 6.41±3.70 4.75±3.74
Wrist
60°/sec (peak torque) (N/m) 32.45±7.96 12.74±3.19 35.75±7.96 13.54±3.23
60°/sec (peak torque/weight) (N/kg) 0.44±0.09 0.17±0.04 0.50±0.13 0.18±0.05
Angle (°) 16.91±20.41 −6.91±16.15 24.41±11.88 −5.25±19.7
Repeat number 2.75±1.54 2.41±1.31 3.00±1.41 3.25±1.28
240°/sec (peak torque) (N/m) 24.15 ±3.35 12.10±9.24 26.39 ±6.78 13.30±8.24
240°/sec (peak torque/weight) (N/kg) 0.33±0.04 0.12 ±0.02 0.36±0.09 0.11±0.03
Angle (°) 22.83±9.61 −13.66±18.69 25.50 ±8.09 −6.91±31.16
Repeat number 4.08±3.94 4.50±3.94 3.66±2.66 3.16±1.26
Values are presented as mean±standard deviation.
######
The percentage between the right and left for the shoulder and wrist flexion and extension movements and the percentage of flexion/extension for all movements
Joint Right/left ratio (%) Flexion/extension ratio (%)
---------------- ---------------------- ----------------------------- -------------- --------------
Shoulder joint
60°/sec 109.1±18.8 103.2±21.3 73.28±9.21 69.12±9.63
240°/sec 113.1±31.1 106.6±22.4 73.26±13.6 70.2±13.3
Wrist joint
60°/sec 93.86 ±26.57 97.65±27.99 260.28±52.78 267.55±54.21
240°/sec 96.17±23.71 119.32±29.71 267.11±50.08 331.48±72.99
Values are presented as mean±standard deviation.
######
Literature values of the shoulder and wrist flexion and extension peak torque at 60°/sec velocity
Joint angular velocity Dominant Nondominant
------------------------------- -------------- -------------- -------------- --------------
Shoulder
60°/sec (our study) 109.63±28.58 150.71±38.75 101.15±28.47 146.98±36.07
60°/sec ([@b16-jer-13-1-62]) 51.4 ±13.9 75.2±20.1 41.4±14.4 68.3±21.2
60°/sec ([@b5-jer-13-1-62])
20--29 yr 51.8±10.6 72.1±19.5
30--39 yr 54.7±7.3 65.6±15.1
Wrist
60°/sec (our study) 32.45±7.96 12.74±3.19 35.75±7.96 13.54±3.23
60°/sec ([@b5-jer-13-1-62])
20--29 yr 20.6±4.4 10.4±3.3
30--39 yr 19.3±4.7 11.0±1.2
######
The literature values of the comparisons of the right-left and flexion/extension for the shoulder joint at 60°/sec velocity
Shoulder joint Right/left ratio (%) Flexion/extension ratio (%)
------------------------------ ---------------------- ----------------------------- ------------ ------------
60°/sec (our study) 109.1±18.8 103.2±21.3 73.28±9.21 69.12±9.63
60°/sec ([@b16-jer-13-1-62]) 79.1±12.3 90.0±10.6 149.0±23.1 173.7±46.0
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#s1}
============
Bluetongue (BT) is an infectious noncontagious viral disease of ruminants caused by the bluetongue virus (BTV). The virus, consisting of 24 different serotypes, is transmitted to its vertebrate host by a few species of biting midges of the *Culicoides* genus (*Diptera*: *Ceratopogonidae*) [@pone.0005171-Mellor1]. BT is a reportable disease of considerable socioeconomic concern and of major importance in the international trade of animals and animal products. From 1998 through 2005, at least 6 BTV strains belonging to 5 serotypes (BTV-1, BTV-2, BTV-4, BTV-9, and BTV-16) were continously present in the Mediterranean Basin [@pone.0005171-Barros1], [@pone.0005171-Breard1], [@pone.0005171-Mellor2]. Since August 2006, BTV-8 has caused severe epizootic outbreakes in northern Europe [@pone.0005171-Saegerman1]. The emergence of BT in parts of Europe never before affected was attributed mainly to climate change and linked to the Northern expansion of the major Old World vector *Culicoides imicola*. Additionally, there is also evidence for the involvement of other novel indigenous European vector species of *Culicoides* (*C. obsoletus* and *C. pulicaris* ) [@pone.0005171-Purse1].
BTV has a genome composed of ten linear segments of double-stranded RNA (dsRNA) and is classified as the type species of the genus *Orbivirus* within the family *Reoviridae* [@pone.0005171-Mertens1]. The BTV genome encodes 7 structural (VP1 through VP7) and 4 nonstructural proteins (NS1 through NS3/NS3A). The outer capsid is composed of two major structural proteins, VP2 and VP5 (segments 2 and 6, respectively), involved in cell attachment and virus entry. VP2 is known to contain the major neutralization determinant of BTV while VP5 influences virus neutralization through its conformational interaction with VP2 [@pone.0005171-DeMaula1]. The outer capsid covers the inner capsid that is composed of two major structural proteins VP3 and VP7 (encoded by segments 3 and 7, respectively) and three distinct minor proteins (VP1, VP4, and VP6 corresponding to segments 1, 4, and 9 respectively) [@pone.0005171-Verwoerd1], in addition to the viral genome. Four other nonstructural proteins, produced during the viral cycle (NS1, NS2, and NS3/NS3A) (segments 6, 8, and 10 respectively), are more conserved among serotypes [@pone.0005171-Verwoerd2].
Studies involving the natural hosts of BTV are limited by the complexity of the system, the scarce knowledge of their immune system, and the need to have an animal facility with biosafety level 3. To circumvent some of these problems, an adequate system would be the use of adult mice because of the knowledge of its genetics and its manageability. BTV infects newborn mice [@pone.0005171-Brewer1], [@pone.0005171-Letchworth1], [@pone.0005171-Carr1], but an adult mouse model will be necessary to allow studies of acquired immune responses and vaccination against BTV.
Bluetongue virus is a potent interferon alpha (IFN-α) inducer [@pone.0005171-Jameson1], [@pone.0005171-Fulton1], [@pone.0005171-MacLachlan1]. In addition, a temporal relationship between viremia and IFN-α activity has been observed in sheep infected with BTV, where IFN peak concentrations induced approximately a 90% decrease in virus titer [@pone.0005171-Foster1]. IFN-α plays an essential role in the antiviral innate immune response. Virus-derived dsRNAs are detected by Toll-like receptors on type I IFN-producing cells (mostly notably on plasmacytoid dendritic cells). Secreted IFNs bind to the type I IFN receptor (IFNAR) on the surface of neighbouring cells, activating the Janus kinase (Jak)/signal transducer and activator of transcription (Stat) signalling pathway. This in turn induces the transcriptional activation of target genes, the products of which render the cells resistant to virus replication through various mechanisms, including degradation of viral mRNAs, inhibition of viral translation, and inhibition of cell growth [@pone.0005171-Schindler1], [@pone.0005171-Staeheli1], [@pone.0005171-Muller1], [@pone.0005171-Sen1]. Blocking IFN-α/β activity in mice leads to a dramatically increased sensitivity to many viruses. Genetically targeted (knockout) mice lacking the β subunit of the IFN-α/β receptor (IFNAR^(−/−)^ mice) are unable to establish an antiviral state and, as a consequence, are highly susceptible to many viral infections, despite the presence of an otherwise intact immune system [@pone.0005171-Muller1], [@pone.0005171-Fiette1]. Recently, Ida-Hosonuma et al [@pone.0005171-IdaHosonuma1] found that the deletion of the IFNAR gene in the IFNAR^(−/−)^ mice resulted in the disruption of IFN- α/β-induced signaling, which is an important determinant of the tissue tropism and pathogenicity of poliovirus. Similarly, it has been reported that IFN- α/β plays an important role in the pathogenicity and tissue tropism of some viruses. The lack of an IFN system allows the virus to replicate more efficiently and IFNAR^(−/−)^ mice have been used as a laboratory animal model to study the immune response and pathogenicity of coronaviruses, vaccinia virus Ankara strain (MVA), measles virus, Rift valley fever virus and West Nile virus [@pone.0005171-Ohka1], [@pone.0005171-Ohno1], [@pone.0005171-Bouloy1], [@pone.0005171-Waibler1]. All these data, and the presence of an otherwise intact immune system in these mice [@pone.0005171-Muller1], [@pone.0005171-Fiette1] suggest that IFNAR^(−/−)^ mice could be a good animal model to study BTV infections and to evaluate vaccine strategies against this virus.
Here, we report the establishment of a new laboratory animal model suitable for the evaluation of vaccination strategies against BTV. Adult IFNAR^(−/−)^ mice support the *in vivo* growth of BTV-4 after intravenous inoculation. BTV-4 replicated in spleen, lung, thymus, and lymph nodes (popliteal, inguinal, mediastinal, and mesenteric) of IFNAR^(−/−)^ mice reproducing the tropism observed during calf and sheep infections. In addition, IFNAR^(−/−)^ mice vaccinated with a BTV-4 inactivated vaccine show complete protection against a lethal dose of BTV-4
Results {#s2}
=======
BTV-4 causes a lethal infection in adult IFNAR^(−/−)^ mice {#s2a}
----------------------------------------------------------
In order to develop an adult murine model for BTV infection in which mice showed disease symptoms, we tested the susceptibility of C57BL/6 and IFNAR^(−/−)^ mice to BTV infection. Since blood is the natural route of BTV infection in ruminants, adult C57BL/6 and IFNAR^(−/−)^ mice (males, 8 weeks old) were infected intravenously (i.v.) with 10^6^ PFUs of BTV-4. Under these conditions, C57BL/6 mice did not show any disease symptom or death following viral infection. By contrast, IFNAR^(−/−)^ mice were susceptible to BTV-4 infection ([Fig. 1A](#pone-0005171-g001){ref-type="fig"}), showing disease symptoms characterized by ocular discharges and apathy starting at 48 h.p.i. Disease progression led to animal death within 60 h.p.i. The LD~50~ value was obtained by i.v. inoculation with 10-fold dilutions of BTV-4, resulting in a LD~50~ value of 10^2.6^ PFU ([Fig. 1B](#pone-0005171-g001){ref-type="fig"}). At low infectious doses (10^2^ PFU or less) mice survived up to 21 days, at which point the experiment was terminated (data not shown).
{#pone-0005171-g001}
To determine virus dissemination, viral titers in blood samples after i.v. inoculation with 10^4^ PFUs of BTV-4 were analyzed. According to the previous data, no viremia was detected in C57BL/6 mice (data not shown). In contrast, viremia was observed in IFNAR^(−/−)^ mice at day 2 post-infection ([Fig. 2A](#pone-0005171-g002){ref-type="fig"}), with peak titers of 5×10^4^ PFU/ml at day 4 post-infection (p.i.), before animal death. IFNAR^(−/−)^ mice inoculated with 10^2^ PFU did not show any viremia but titers up to 3×10^4^ PFU/ml were observed at 3 and 4 days p.i. in mice inoculated with 10^3^ PFUs. Viral spread was determined in tissue samples. The first tissue to be reached by the virus was the spleen ([Fig. 2B](#pone-0005171-g002){ref-type="fig"}) where infectious virus was detected as early as 24 h.p.i. (5×10^3^ PFU/gr), with viral titers increasing thereafter until death (reaching titers of 2×10^6^ PFU/gr). By 48 h.p.i. significant titers of BTV-4 were detected in spleen, lung, thymus, and popliteal and mesenteric lymph nodes. At 72 h.p.i., titers up to 10^6^ PFU/gr of BTV-4 were recovered from the spleen, lung, thymus, and lymph nodes (popliteal, inguinal, mediastinal and mesenteric). No infectious virus was detected in liver, brain, heart, tongue, skin, and testicles at any time points examined, even when the tissues were analyzed by RT-PCR (data not shown). Interestingly, the virus was not detected in the blood until 48 h.p.i. These results suggest that the virus leaks into the blood stream after replicating in the spleen.
{ref-type="sec"}. Each point represents the mean values of the viral titer of six animals, and standard deviations are shown as error bars. (B) Mice (8 weeks old, 6 mice per group) were inoculated intravenously with 10^4^ PFUs of BTV-4. Virus was extracted from the indicated tissues at 24, 48 and 72 hours after infection for virus titration. Standard deviations are given. Procedures are detailed in [Materials and methods](#s4){ref-type="sec"}.](pone.0005171.g002){#pone-0005171-g002}
Susceptibility of IFNAR^(−/−)^ mice to other BTV serotypes {#s2b}
----------------------------------------------------------
To determine whether IFNAR^(−/−)^ mice were susceptible to BTV of different serotypes, IFNAR^(−/−)^ mice were inoculated with ten-fold serial dilutions of BTV-8. Mice infected with serotype 8 showed similar symptoms to those shown after infection with serotype 4. However, BTV-8 showed higher virulence than BTV-4 with only 10 PFUs of BTV-8 being enough to kill 100% of the mice at day 7 p.i. ([Fig. 3A](#pone-0005171-g003){ref-type="fig"}), while BTV-4 was not lethal at the same infective dose ([Fig. 1B](#pone-0005171-g001){ref-type="fig"}). The viremia was determined after i.v. inoculation with 10-fold dilutions of BTV-8 ([Fig. 3B](#pone-0005171-g003){ref-type="fig"}). BTV-8 was first detected in blood of mice inoculated with 10 PFUs at day 4 post-infection, and the titer increased up to 2×10^4^ PFU/ml at day 5 p.i. Titers up to 8×10^3^ PFU/ml were observed at 3 and 4 days p.i. in mice infected with 10^3^ and 10^2^ PFUs, respectively. In the three dilutions of virus analyzed, viral titers increased thereafter until animal death. Although BTV-8 showed higher virulence than BTV-4 in IFNAR^(−/−)^ mice, BTV-8 and BTV-4 titers in blood were equivalent, indicating that the higher virulence of serotype 8 is not due to a higher level of viral replication. These data suggest that IFNAR^(−/−)^ mice could be an adequate small animal model to study differences in virulence among BTV serotypes.
{ref-type="sec"}. Each point represents the mean values of the viral titer of six animals, and standard deviations are shown as error bars.](pone.0005171.g003){#pone-0005171-g003}
BTV-4 infection of IFNAR^(−/−)^ adult mice causes microscopic lesions in several tissues {#s2c}
----------------------------------------------------------------------------------------
In order to study the pathological effects of the infection in the organs where BTV replicates, histological analysis were performed on material of several organs extracted from BTV infected and uninfected IFNAR^(−/−)^ mice at 48 h.p.i. Gross pathological alterations were characterized by widespread oedema, haemorrhages especially in spleen and lungs, and enlarged spleen and lymph nodes. Histological examination of lungs of BTV infected mice showed hyperemia and increased septum size with infiltration of lymphocytes, inactivated macrophages and a few neutrophils ([Fig. 4](#pone-0005171-g004){ref-type="fig"}). The peribronchial and perivascular connective tissue contained a few infiltrating round cells ([Fig. 4](#pone-0005171-g004){ref-type="fig"}). These histopathological findings are consistent with bronchointerstitial pneumonia. There was also a moderate oedema in the alveolar cavity with presence of abundant alveolar macrophages and a few detached epithelial cells (descamative alveolitis). The infected spleen showed a marked lymphoid depletion with infiltration of neutrophils in the white pulp ([Fig. 4](#pone-0005171-g004){ref-type="fig"}). This lymphoid depletion was also observed in the thymus as well as the loss of thymic architecture with the medulla and the cortex becoming not distinguishable (data not shown). The results suggest that BTV-4 produces similar tissue lesions in IFNAR^(−/−)^ mice than in the natural host.
{#pone-0005171-g004}
Immunized IFNAR^(−/−)^ mice are completely protected against a lethal BTV-4 challenge {#s2d}
-------------------------------------------------------------------------------------
All the previous data indicated that BTV-4 caused a lethal infection in adult IFNAR^(−/−)^ mice. To provide further proof that BTV-4 was the causative agent of the disease and death in these animals and that IFNAR^(−/−)^ mice are a good animal model for BTV vaccination studies, vaccination protection experiments were performed. Adult IFNAR^(−/−)^ mice were immunized with a ZULVAC-BTV-4 inactivated BTV-4 preparation. The vaccine was administered by two consecutive subcutaneous injections of the equivalent to 3×10^5^ TCID~50~ of BTV-4 at 3 weeks intervals. After the second immunization, VP2 antibody titers were determined by ELISA. All immunized animals, but not the control animals, developed an antibody response ([Fig. 5A](#pone-0005171-g005){ref-type="fig"}) indicating a successful immunization. In addition, the immunization induced neutralizing antibodies against BTV-4 in IFNAR^(−/−)^ mice (VNT 1.53±0.32) as detected by virus neutralization tests. Three weeks after the second immunization, immunized and control IFNAR^(−/−)^ mice were challenged intravenously with 10^3^ PFUs of BTV-4. While all nonimmunized animals died, 100% of the immunized animals were protected against a lethal challenge ([Fig. 5B](#pone-0005171-g005){ref-type="fig"}). Infectious viral titers were analyzed in the blood of immunized and nonimmunized IFNAR^(−/−)^ mice by plaque assay after intravenous infection with BTV-4 ([Fig. 5C](#pone-0005171-g005){ref-type="fig"}). No Infectious viruses were detected in immunized mice; however, we observed titers up to 3×10^4^ PFU/ml in nonimmunized animals at day 5 post-challenge. In addition, we analyzed by RT-qPCR the presence of BTV genomes in the blood of inmmunized and nonimmunized IFNAR^(−/−)^ mice challenged with BTV-4. The results are summarized in [Table 1](#pone-0005171-t001){ref-type="table"}. BTV genomes were readily detected in nonimmunized mice at days three and four after BTV infection (C*t*: 27--29) and increased (C*t*: 23--26) thereafter until the death of the animal. In contrast, the RT-qPCR reaction yield no positive results for the majority of the immunized mice (n = 5) at all days post-challenge analyzed (*C*t≥38). Viral genomes were detected in one of the vaccinated mice (C*t*: 30) at days four and five post-challenge and in two of them at day 5 (C*t*: 32). However, in these three immunized mice the C*t* was higher than in the nonimmunized mice and the presence of BTV-genomes reverted to negative at day 7 post-challenge. Overall, these data indicate that protective immunity was achieved after vaccination. These results confirm that BTV-4 was the causative agent of disease and death of adult IFNAR^(−/−)^ mice.
{ref-type="sec"}. (B) Survival rates of immunized and nonimmunized IFNAR^(−/−)^ mice after inoculation with BTV-4. The mice were observed every 24 h for 14 days. (C) Titers of BTV-4 recovered in blood of immunized and nonimmunized IFNAR^(−/−)^ mice after challenge with BTV-4. Virus was extracted from blood and determined as described in [Materials and Methods](#s4){ref-type="sec"}. Each point represents the mean values of the viral titer of eight animals, and standard deviations are shown as error bars.](pone.0005171.g005){#pone-0005171-g005}
10.1371/journal.pone.0005171.t001
###### Detection of BTV-4 in blood of immunized and nonimmunized IFNAR^(−/−)^ mice after challenge with BTV-4 by RT-qPCR_S5.
{#pone-0005171-t001-1}
Animals Days post-challenge
--------- --------------------- ------- ---------------------------------- ---------------------------------- ------
**C-1** 29.07 25.05 [†](#nt103){ref-type="table-fn"}
**C-2** neg. 27.38 23.78 [†](#nt103){ref-type="table-fn"}
**C-3** 29.75 26.80 [†](#nt103){ref-type="table-fn"}
**C-4** neg. 27.57 23.45 [†](#nt103){ref-type="table-fn"}
**I-1** neg. neg. neg. neg. neg.
**I-2** neg. 30.57 30.74 neg. neg.
**I-3** neg. neg. neg. neg. neg.
**I-4** neg. neg. neg. neg. neg.
**I-5** neg. neg. neg. neg. neg.
**I-6** neg. neg. neg. neg. neg.
**I-7** neg. neg. 31.94 neg. neg.
**I-8** neg. neg. 32.06 neg. neg.
Results expressed as *C*t and transferred to negative (neg.) according to the cut-off *C*t≥38 described by Toussaint et al. (2007).
I, immunized mice. C, nonimmunized mice.
, death mice.
Discussion {#s3}
==========
For years, different groups have tried to establish a laboratory animal model to facilitate the studies of pathogenesis, immune response and vaccination against BTV. Natural hosts, although excellent tools for studies of pathogenesis and vaccination, are expensive and required specialized laboratories. Here, we characterize a new animal model based on adult IFNAR^(−/−)^ mice that support the *in vivo* growth of BTV-4 and BTV-8 after intravenous inoculation. A tremendous advantage of this mouse model is the availability of a wide variety of reagents that can be used to study many aspects of the immune response to the virus. In addition, we propose this animal model as an adequate system for testing BTV vaccines, an important issue because the cost of testing new vaccines in target species is a major obstacle for laboratories and industries. The new mouse model for the study of BTV infection has unique features that open the possibility to study in the same host-virus system susceptibility, virulence, immunobiology of infection, and vaccine efficacy, in ways that are not approachable with the natural host.
BTV infects newborn mice [@pone.0005171-Brewer1], [@pone.0005171-Letchworth1], [@pone.0005171-Carr1], [@pone.0005171-Franchi1], but an adult animal model will be necessary to allow studies of acquired immune responses and vaccination against BTV. Different approaches have been followed to avoid this problem. The protective effects of baculovirus expressed BTV-10 minor and non-structural proteins were assessed by measuring virus titers in the ovaries of mice challenged with homologous recombinant vaccinia virus (rVV). Protection mediated by CTLs specific for the BTV minor or nonstructural proteins could not be evaluated by challenging mice with BTV-10, as the virus does not cause disease in adult mice [@pone.0005171-Jones1]. The infection of fetal mice with BTV leads to severe cerebral malformation. Infection of 2-week-old mice resulted in very limited multiplication without sequelae, and infection of 4-week-old mice did not show clinical disease and no viral multiplication was detected [@pone.0005171-Narayan1]. Thus, the use of highly immature mice results in pathologies that do not resemble those found in the natural host. Another aspect to consider is the route of inoculation that may determine the outcome of infection. In this work, we have used the intravenous inoculation to resemble the natural infection route by mosquito biting. Thus, mice infected with BTV i.v. develop clinical symptoms, as it happens in natural hosts. This makes the mouse model even more atractive to be used as a tool for studying immune response to BTV and vaccination strategies.
Up-regulation of type I IFN is one of the earliest cellular responses upon contact with infectious agents. The rapid induction of type I IFN reflects the crucial role that this cytokine plays in the inhibition of viral spread before the generation of a specific immune response. Given that viruses must at least partially circumvent the IFN response, it is not surprising that an inability to do this can restrict host range. There are many examples where this is known. For example, neither measles virus nor polio virus will replicate efficiently in mouse models unless the IFN system has been compromised [@pone.0005171-Ohka1], [@pone.0005171-Mrkic1]. BTV is a potent type I interferon inducer in mice and cattle [@pone.0005171-Jameson1], [@pone.0005171-MacLachlan1], [@pone.0005171-Jameson2]. Our results show that BTV is pathogenic for IFNAR^(−/−)^ mice mainly due to the lack of the IFN-I receptor, allowing the virus to circumvent the IFN-I response in the host. In addition, IFNAR^(−/−)^ mice serve as a good animal model for BTV, reproducing many aspects of its natural host infection. First, IFNAR^(−/−)^ mice infected with BTV showed viremia and disease symptoms, in contrast with C57BL/6 mice, which did not show them, even when these animals were infected at the highest viral dose tested (10^6^ PFUs/mouse). The appearance of viremia after inoculation in infected IFNAR^(−/−)^ mice was dependent on the viral dose, althought the highest titers observed in blood were not dose-dependent. Second, the differential virulence of serotypes 4 and 8 were maintained in this animal model. Some BTV serotypes such as serotype 8, which recently caused infection in northern Europe, exhibit enhanced virulence in cattle [@pone.0005171-Saegerman1], in contrast to BTV-4 that usually exhibits subclinical infections in this species. In infected IFNAR^(−/−)^ mice, BTV-8 killed 100% of the animals with a dose as low as 10 PFUs per mouse. In contrast 10^3^ PFUs of BTV-4 were needed to kill 100% of the mice. In addition, the virus titers found in the blood of infected mice were similar at the same virus doses for the animals infected with BTV-8 than with BTV-4, indicating that BTV-8 exhibited a higher virulence than BTV-4 in the IFNAR^(−/−)^ model, as is observed in cattle, one of the BTV natural hosts. Third, BTV dissemination in IFNAR^(−/−)^ mice reproduced BTV infection in cattle and sheep. High titers of BTV are present in the lungs, precapsular and mesenteric lymph nodes, thymus, and spleen of infected calves [@pone.0005171-MacLachlan2], [@pone.0005171-BarrattBoyes1]. In IFNAR^(−/−)^ mice, the virus was detected in spleen before was isolated from other organs, including blood. Release of BTV from spleen was followed by an increased in viremia and dissemination to other organs (lymph nodes, lung and thymus), followed by its dissemination to lymph nodes, lung, and thymus after intravenous infection. This has been suggested also for calves [@pone.0005171-BarrattBoyes1] indicating a similar BTV spreading pattern in IFNAR^(−/−)^ mice. Histological analyses of BTV infected mice at 48 h.p.i. showed many similarities with lessions described in natural hosts. The permeability disorders of the vascular system described for ruminants [@pone.0005171-SchwartzCornill1] is reflected in the petecheias observed in spleen in BTV infected mice. The enlargement of lymph nodes reflects an ongoing immune response. However, the observed lymphoid depletion suggests that other cells are migrating to these organs or that a general aedema results in organ enlargement. The lungs are especially susceptible to permeability disorders of the vasculature induced by BTV, and this is consistent in BTV infected mice as well as in ruminants. In general, pathology in natural hosts has not been profoundly studied. The described pathology similarities between IFNAR^(−/−)^ mice and ruminants after infection with BTV indicate that our mouse model may be a good tool to study new findings in BTV pathology.
The cost of testing new vaccines in target species is a major obstacle for laboratories and industries. For this reason, the intracerebral inoculation of newborn mice with BTV vaccines has been used as an animal model to evaluate the level of attenuation of live attenuated BTV vaccines [@pone.0005171-Franchi1]. Our results show that adult IFNAR^(−/−)^ mice serve as a good animal model to test BTV vaccines. Even though the lack of type I interferon signals may have an effect in the development of acquired immune responses, our results and previous studies [@pone.0005171-Muller1], [@pone.0005171-Fiette1], [@pone.0005171-Ohka1], [@pone.0005171-Ohno1], [@pone.0005171-Bouloy1], [@pone.0005171-Waibler1] show that the IFNAR^(−/−)^ infection model is useful for the definition of effective vaccine candidates against BTV. Indeed, in our study all immunized animals developed an antibody response specific of BTV-4 with the production of neutralizing antibodies against the same serotype, indicating a successful immunization. The protection mediated by inactivated whole BTV vaccine in IFNAR^(−/−)^ mice infected with a lethal dose of BTV-4 was complete, and 100% of the animals did not show any symptoms associated with infection or died. Viremia was not detected in the blood of immunized animals after challenge with BTV-4 when analyzed by plaque assay. The presence of BTV genomes in the blood of immunized mice after BTV-4 challenge analyzed by RT-qPCR, a more sensible method than the plaque assay, showed the presence of viral genomes in the blood of some of the immunized mice but in all the cases, the *C*ts were higher than in the nonimmunized mice and those that were RT-qPCR positive reverted to negative at day seven post-challenge. Similar RTqPCR results have been observed in cattle and sheep vaccinated with ZULVAC-BTV-4 inactivated BTV-4 preparation [@pone.0005171-Paradell1] These results strongly support the involvement of BTV in the pathological processes described in this work and, moreover, suggest that the mouse model is adequate to evaluate vaccine candidates. Future work will determine whether the ability to protect mice with other vaccine formulations mimics the capacity to protect the natural host.
In summary, we have characterized a novel small animal model for BTV infection based on IFNAR^(−/−)^ adult mice that reproduces many aspects of its natural host infection. This animal model may facilitate the understanding of the mechanisms of BTV pathogenicity in its natural host and a faster advance in the development of new BTV vaccines.
Materials and Methods {#s4}
=====================
Virus and cells {#s4a}
---------------
Baby hamster kidney cells (BHK-21), and Vero cells were grown in Dulbecco\'s modified Eagle\'s medium (DMEM) supplemented with 2mM glutamine and 10% fetal calf serum (FCS). Standard virus titrations were performed in Vero cells. BTV serotypes 4 (Spain/01) and 8 (Belgium/06), originally isolated from an infected sheep in Spain in 2001 and a calf in Belgium in 2006, respectively, were used in the experiments. Virus stocks were generated by infection of confluent BHK-21 cells using a multiplicity of infection (MOI) of 1. At 48 h.p.i. or when total cytopathic efect (CPE) was visible, the cells and supernatants were harvested and centrifuged. The virus was released from the cells by three freeze and thaw cycles.
Mice {#s4b}
----
C57BL/6 mice were purchased from Harlan Interfauna Ibérica S.L. IFN α/βR^o/o^ IFNAR^(−/−)^ mice [@pone.0005171-Muller1], on a C57BL/6 genetic background, were generously provided by Professor R. Zinkernagel (Institute of Experimental Medicine, Zurich). All mice used were matched for sex and age (males 8 weeks). Mice were maintained under pathogen-free conditions and allowed to acclimatize to the biosafety level 3 (BSL3) animal facility at the Centro de Investigación en Sanidad Animal, INIA, Madrid, for 1 week before use in our experiments. All experiments with live animals were performed under the guidelines of the European Community (86/609) and were approved by the site ethical review committee.
Animal inoculation and processing of samples {#s4c}
--------------------------------------------
Mice were infected intravenously with different doses of virus. Mice were examined for clinical symptoms daily. LD50 values were calculated by the method of Reed and Muench (1938) [@pone.0005171-Reed1], after inoculation of mice with 10-fold serial dilutions of virus (from 10^6^ to 10^1^ PFU/mouse). Whole blood was collected in EDTA from all animals at regular intervals after inoculation. At varying times post-infection, several mice were sacrificed by perfusion with phosphate-buffered saline (PBS), and several organs (spleen, lung, thymus, liver, brain, heart, tongue, skin, and testicles), and lymph nodes (popliteal, inguinal, mediastinal, and mesenteric) were harvested. Tissues were homogenized in PBS using a Tissue Lyser homogenizer (Quiagen). The viruses were released from whole blood and homogenized tissues by three freeze/thaw cycles. The amount of infectious virus was measured by plaque assay on Vero cells.
Murine immunizations {#s4d}
--------------------
Groups of 6 IFNAR^(−/−)^ mice were immunized by two consecutive subcutaneous injections of either ZULVAC-BTV-4 (1.5×10^6^ TCID~50~BTV-4) inactivated BTV-4 preparation (Fort Dodge Veterinaria, S.A.) or phosphate-buffered saline (PBS) (controls), administered 3 weeks apart. Mice were intravenously inoculated with 10^3^ PFUs of BTV-4 (lethal dose) 3 weeks after the last immunization. Mice were bled before each immunization and virus challenge. Sera were tested for BTV-4 neutralizing antibodies by Virus Neutralization Test (VNT).
Histopathology {#s4e}
--------------
Samples from different tissues and organs were taken and fixed in 10% buffered formalin (pH 7.2) for histopathological studies. After fixation, samples were dehydrated through a graded series of alcohol to xylol and embedded in paraffin wax. Sections of 4-im-thick were cut and stained with hematoxylin and eosin (H & E) for histopathological analyses.
BTV-VP2 antibody detection by indirect ELISA {#s4f}
--------------------------------------------
MaxiSorp plates (Nunc, USA) were coated with VP2 baculovirus expressed protein (164 ng per well) and incubated overnight at 4°C. Plates were saturated with blocking buffer (PBS-0.05% Tween 20 and 5% skimmed milk). The animal sera, diluted in blocking buffer were added and incubated for 1 hour at 37°C. After three washes in PBS-0.05% Tween 20, plates were incubated for 1 hour at 37°C with an anti-mouse-HRP secondary antibody (Biorad, USA) at a 1/2,000 dilution in blocking buffer. Finally, after three washes in PBS-0.05% Tween 20, the reaction was developed with 50 µl of substrate solution 3,3′, 5,5′--tetramethylbencidine liquidsupersensitive (TMB) (Sigma) and stopped by adding 50 µl of 3N H~2~SO~4~. Results were expressed as optical densities (ODs) measured at 450 nm.
BTV-4 neutralizing antibody detection in immunized mice by virus neutralizing test (VNT) {#s4g}
----------------------------------------------------------------------------------------
The VNT was used to determine neutralizing antibody titers against BTV-4. For plaque reduction assays, 2 fold dilutions of sera were mixed with 100 PFU of BTV-4, incubated for 1 hour at 37°C and then plated onto monolayers of Vero cells. After 1 hour, agar overlays were added and the plates were incubated for 5 days. The titer was determined as the highest dilution that reduced the number of plaques by 50%.
RT-qPCR specific for BTV segment 5 {#s4h}
----------------------------------
Whole blood was collected in EDTA from all animals at regular intervals after inoculation and BTV challenge. Total RNA was extracted from blood with TRI Reagent Solution (Ambion), according to the method recommended by the manufacturer. The real-time RT-qPCR specific for BTV segment 5 was performed as described by Toussaint et al. (2007) [@pone.0005171-Toussaint1].
We thank Fort Dodge Veterinaria SA for facilitating the BTV inactivated vaccine ZULVAC-BTV-4.
**Competing Interests:**The authors have declared that no competing interests exist.
**Funding:**This work was supported by grants from the National Institute of Agronomics Research (INIA) (RTA-2006-169), the Comision Interministerial de Ciencia y Tecnologia (CICYT) (AGL2008-00646/GAN), and the EU Network of Excellence EPIZONE (Contract No FOOD-CT-2006-016236). Eva Calvo-Pinilla and Teresa Rodriguez-Calvo received fellowships from INIA and Comunidad de Madrid, respectively. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
[^1]: Conceived and designed the experiments: ECP NS JO. Performed the experiments: ECP TRC NS JO. Analyzed the data: ECP TRC JA NS JO. Contributed reagents/materials/analysis tools: JO. Wrote the paper: ECP NS JO.
| {
"pile_set_name": "PubMed Central"
} |
INTRODUCTION {#s1}
============
Aneurysms of the internal mammary artery (IMAA) are uncommon clinical entities \[[@RJU125C1]\] and usually occur in patients after sternotomy, placement of a central venous catheter or pacemaker leads \[[@RJU125C2]\]. Less common, these aneurysms are associated with vasculitis (e.g. Kawasaki disease \[[@RJU125C3]\]), connective tissue disorders (e.g. Marfan syndrome \[[@RJU125C4]\]), chest wall infections \[[@RJU125C5]\] or atherosclerosis \[[@RJU125C6]\]. However, changes in the structure of the vascular wall at the cellular level such as cystic medial necrosis or hyperplasia lead to loss of elasticity and formation of aneurysms \[[@RJU125C6]\].
Since rupture of IMAA leads to haemothorax and life-threatening conditions, early diagnosis and treatment is indispensable. We here report the case of a 46-year-old man with an idiopathic IMAA.
CASE REPORT {#s2}
===========
A 46-year-old man was presented to the Department of Vascular Surgery with distinct varicosis. In the context of preparation for vein stripping, chest X-ray was performed and revealed a nodular shadow attached to the pleural cupula (Fig. [1](#RJU125F1){ref-type="fig"}). Exact census of the anamnesis revealed increasing surge and numbness of the fingers depending on the load of the arm (comparable to the thoracic outlet syndrome). To further evaluate the nodular shadow on X-ray, computed tomography (CT) scan of the thorax with contrast medium has been drafted. The CT scan revealed a 3.6 × 4.4 cm inhomogeneous tumorous process dorsal of the left clavicle with impression of the neighbouring lung. Furthermore, it was suspected that the tumour infiltrated the left internal jugular and subclavian vein. Therefore, an additional phlebography was initiated. Phlebography confirmed the perceptions of the CT scan, but failed to shed any further light on the nature of the tumour (Fig. [2](#RJU125F2){ref-type="fig"}). Finally, magnetic resonance imaging (MRI) scan was performed in order to demonstrate the exact origin of the tumour, but also failed to obtain new findings (Fig. [3](#RJU125F3){ref-type="fig"}). Figure 1:Chest X-ray. Figure 2:Phlebography reveals a compression of the left subclavian vein.
Due to the symptoms and the suspicion of malignant tumour, an open resection was intended. Therefore, the patient was placed in a lateral position with the right arm freely moveable. Transverse incision was made below the axillary hairline between the pectoralis major and latissimus dorsi muscle, followed by careful preparation of the first rib, layer by layer. Resection of the first rib was done among sternocostal and costovertebral joint after accurate separation from the underlying pleura. Medial of the pleural cupula a boorish tumour was palpable and the left IMA was walled by this tumour. Therefore, the tumour and the left mammary artery were removed after cautious preparation. After careful arrest of bleeding, the wound was stepwise closed with single button sutures.
Histological examination of the removed tumour revealed a thrombotic obliterated aneurysm of the IMA. After operation, the patient recovered well. The peripheral pulses were palpable postoperatively and neurological tests did not show any abnormalities. X-ray during follow-up showed normal findings. An arterial duplex scan of the left subclavian arteries showed a good vascular flow (Fig. [3](#RJU125F3){ref-type="fig"}). Six months after operation, the postoperative course was uneventful and our patient was in a good physical condition. Figure 3:MRI scan of the thorax shows a tumour at the left pleural cupula.
DISCUSSION {#s3}
==========
The IMA arises from the first portion of the subclavian artery and immediately passes downwards close to the pleura within the upper intercostal space. Further distal, it proceeds anteriorly to the transversus thoracic muscle to end in the sixth intercostal space by dividing into the superior epigastric and musculophrenic artery \[[@RJU125C7]\]. This anatomical course makes the IMA vulnerable to severe deceleration or penetrating injuries \[[@RJU125C7]\].
Infections or genetic disorders are also frequent causes of IMAAs \[[@RJU125C3]--[@RJU125C5], [@RJU125C8]\]. A spontaneous development of an IMAA without a rememberable trauma or injury as described in our case is a rarity. Due to the low prevalence of IMAAs, informations regarding diagnosis and secure treatment options are scarce \[[@RJU125C1]\]. IMAAs typically appear with haemoptysis, cough and dyspnoea. In some patients, IMAAs only emerge as a bulging chest mass \[[@RJU125C8]\].
However, chest X-ray should be performed as first step when IMAA is suspected. An additional CT scan conceivably in combination with an angiography is a valuable method to localize the IMAA and to plan the surgical procedure \[[@RJU125C1]\]. In our patient, classical symptoms of an IMAA were missing and the IMAA was an incidental finding within routine X-ray. The compression symptoms of the IMAA were only found after accurate request and examination. The additional imaging procedures failed to confirm the diagnosis.
Prior to therapy, treatment options should be carefully considered. Open surgical repair, stent implantation or coil embolization has been described in the literature \[[@RJU125C1], [@RJU125C9], [@RJU125C10]\]. Even though long-term results are missing, coil embolization or stent implantation has become the treatment option of choice in minor aneurysms due to the minor invasivity of these procedures \[[@RJU125C3], [@RJU125C10]\]. Despite this advantage, we were not convinced with coil embolization or stent implantation because of the following reasons: A malignant process could not be excluded by the imaging prior to surgery.Even if the diagnosis of an IMAA would have been made, the compression of the adjacent vessels and nerves (which caused symptoms) would not have been treated by an endovascular procedure.Histological examination of the tumour revealed a thrombotic obliterated IMAA. The aneurysm was finally classified as idiopathic due to the fact that tissue disorders and inflammatory processes were not detectable, trauma or medical interventions were not rememberable, vessel anomalies could be excluded and atherosclerosis ruled out because of inconspicuous medical history and clinical examination.
An idiopathic IMAA is an extremely rare entity. The diagnosis may be difficult, when clinical symptoms are missing. When the diagnosis of IMAA is made, prompt therapy is essential in avoidance of life-threatening events. Selection of the ideal approach depends on the underlying pathogenesis. Open surgical resection represents an invasive procedure with the option of decompressions of neighbouring structures, good postoperative outcome and without complications in our case.
CONFLICT OF INTEREST STATEMENT {#s4}
==============================
The authors declare no conflict of interest.
FUNDING {#s5}
=======
The author(s) received no financial support for the authorship, and/or publication of this article.
| {
"pile_set_name": "PubMed Central"
} |
1. Introduction {#sec1-pharmaceutics-11-00020}
===============
According to the Joint United Nations Programme on HIV and AIDS (UNAIDS) factsheet from July 2018, between 31.1 and 43.9 million people currently live with HIV, although fewer than 23 million of them have access to antiretroviral therapy. 1.8 million people contracted the infection in 2017 indicating that, although the transmission of the disease has fallen by 47% since 1996, the problem is still far from being solved. The situation of young women is particularly worrying, as 7000 women between the ages of 15 and 24 become infected with the virus each week. In Sub-Saharan Africa particularly, this group is twice as likely to live with HIV as men, and 75% of new infections occur in women aged 15--19 \[[@B1-pharmaceutics-11-00020]\].
This high incidence of HIV in young women can be attributed to age-disparate sexual relationships, poor negotiating power with respect to the use of preventive tools (such as condoms) and intimate partner violence; moreover, the lack of prevention systems for women does not allow them to protect themselves from HIV transmission. The development of effective systems to prevent HIV infection initiated by women themselves is a challenge that has yet to be solved, and pre-exposure prophylaxis (PrEP) is one of the most innovative tools for increasing the options for self-protection in the population at high risk of HIV infection \[[@B2-pharmaceutics-11-00020],[@B3-pharmaceutics-11-00020]\].
Among the drugs used in vaginal microbicide development, reverse transcriptase inhibitors act on viral enzyme reverse transcriptase, which is responsible for transforming the viral RNA into DNA, thus inhibiting viral replication. Of these, tenofovir (TFV) has been shown to be effective and safe, and has a long half-life \[[@B4-pharmaceutics-11-00020]\]. TFV gel for vaginal application proved effective in the CAPRISA 004 trial when used pericoitally, reducing women's risk of infection by 39% compared to a placebo \[[@B5-pharmaceutics-11-00020],[@B6-pharmaceutics-11-00020]\]. It has also been demonstrated that vaginal administration of TFV can achieve concentrations in vaginal tissue that are 130 times higher with an oral administration regimen \[[@B7-pharmaceutics-11-00020]\].
Nevertheless, currently available vaginal dosage forms have several limitations, such as leakage, messiness and low residence time. As these issues lead to low adherence rates---the key factor in achieving protection \[[@B8-pharmaceutics-11-00020]\]---novel dosage forms are required. For example, vaginal films for the release of TFV have been developed and have even reached clinical trials, the FAME-04. Films users reported less product leakage than gels users. However, moderate vaginal leakage was still observed and daily administration was required, pointing to the need for further work. More sophisticated polymer-based systems for the controlled release of TFV are currently in the early stages of development; these include freeze-dried bigels \[[@B9-pharmaceutics-11-00020]\], hydrogels containing polymeric nanocapsules \[[@B10-pharmaceutics-11-00020]\], spray-dried microspheres \[[@B11-pharmaceutics-11-00020]\] or complex polymeric tablets \[[@B12-pharmaceutics-11-00020]\]. All these systems propose the use of natural mucoadhesive polymers, which are a promising option for the development of biocompatible controlled-release drug-delivery systems \[[@B13-pharmaceutics-11-00020]\].
Chitosan is a natural copolymer obtained by partial deacetylation of chitin, which is found in the exoskeleton of crustaceans, and also produced extracellularly by fungi and brown algae \[[@B14-pharmaceutics-11-00020]\]. This polymer is composed of β1→4)-linked 2-acetamido-2-deoxy-β-[d]{.smallcaps}-glucopyranose (*N*-acetylglucosamine) and α(1→4)-linked 2-amino-2-deoxy-β-[d]{.smallcaps}-glucospyranose (glucosamine). The percentage of glucosamine determines its physicochemical properties \[[@B15-pharmaceutics-11-00020]\], of which the most notable is its cationic nature, and especially interesting if we consider that most natural polymers present neutral or anionic charges \[[@B16-pharmaceutics-11-00020]\]. The mechanical properties of chitosan-based systems can therefore be modified by forming a polyelectrolyte complex with anionic compounds \[[@B17-pharmaceutics-11-00020]\]. This polymer offers several advantages from a pharmaceutical point of view, as it is biocompatible and biodegradable \[[@B18-pharmaceutics-11-00020]\]. It has been used in the development of numerous systems for vaginal administration such as tablets, films and gels \[[@B19-pharmaceutics-11-00020],[@B20-pharmaceutics-11-00020],[@B21-pharmaceutics-11-00020]\]. Formulations containing this excipient show sustained drug release and mucoadhesive properties \[[@B15-pharmaceutics-11-00020],[@B19-pharmaceutics-11-00020],[@B22-pharmaceutics-11-00020],[@B23-pharmaceutics-11-00020],[@B24-pharmaceutics-11-00020]\], and it may also be useful in pharmaceutical systems for the prevention of STDs due to its intrinsic antimicrobial activity \[[@B25-pharmaceutics-11-00020],[@B26-pharmaceutics-11-00020],[@B27-pharmaceutics-11-00020],[@B28-pharmaceutics-11-00020]\] and immunostimulant capacity \[[@B14-pharmaceutics-11-00020]\]. As the loss of the formulation is the main limitation to its therapeutic efficacy \[[@B29-pharmaceutics-11-00020]\], the association with other substances may offer a good alternative. The polymers than have been studied for use in combination with chitosan include locust bean gum \[[@B30-pharmaceutics-11-00020]\], pectin \[[@B31-pharmaceutics-11-00020]\], hydroxypropylmethylcellulose \[[@B29-pharmaceutics-11-00020]\] and tragacanth gum \[[@B32-pharmaceutics-11-00020]\] among others.
The development and evaluation of a formulation based on mixtures of chitosan and locust bean gum \[[@B30-pharmaceutics-11-00020]\] has been previously reported for buccal administration; it was observed that the second is capable of buffering the effect of acid pH on the drug release from chitosan-based formulations \[[@B33-pharmaceutics-11-00020]\]. This polymer is a neutral galactomannan vegetable gum obtained from *Ceratonia Silqua* L. \[[@B34-pharmaceutics-11-00020],[@B35-pharmaceutics-11-00020]\]. It consists of a linear chain of (1→4)-linked-[d]{.smallcaps}-mannopyranosyl units with (1→6)-linked side chains of -[d]{.smallcaps}-galactose, in a mannose/galactose ratio of 4:1. In the pharmaceutical industry, it has shown potential in the inhibition of gastrointestinal diseases and has also been used as a carrier agent for the controlled release of drugs, either alone or in combination with other polymers \[[@B35-pharmaceutics-11-00020]\].
Chitosan and pectin are able to form complexes through electrostatic interactions between positively charged amino groups in chitosan and negatively charged carboxylate groups in pectin \[[@B36-pharmaceutics-11-00020]\]. This combination has been explored with promising results for the development of different dosage forms \[[@B37-pharmaceutics-11-00020]\]. Pectin is a natural heteropolysaccharide obtained from apple or citrus peel. This polymer contains at least 65% galacturonic acid units. It is formed of (1→4)-linked α-D-galactosyluronic acid residues and neutral sugars such as rhamnose, galactose, arabinose and others. The acid groups of the galacturonic units can be methoxlyated and amidated to different degrees \[[@B38-pharmaceutics-11-00020],[@B39-pharmaceutics-11-00020]\]. It has been assessed as an excipient in several vaginal formulations such as gels, tablets and moisturizers \[[@B13-pharmaceutics-11-00020],[@B40-pharmaceutics-11-00020],[@B41-pharmaceutics-11-00020]\].
With this background, the aim of the present work was to develop chitosan-based vaginal mucoadhesive tablets for the controlled release of tenofovir by combining chitosan with other complementary polymers to achieve four-day release and mucoadhesion periods with moderate swelling profiles. This would be comfortable for the patient and ensure therapeutic compliance.
2. Materials and Methods {#sec2-pharmaceutics-11-00020}
========================
2.1. Materials {#sec2dot1-pharmaceutics-11-00020}
--------------
Tenofovir (TFV, lot: FT104801401, MW: 287.21 g/mol) was provided by Carbosynth Limited (Berkshire, UK). Chitosan (lot: 0055790), was supplied by Guinama (Valencia, Spain). Locust bean gum (lot: 010 M0087) and pectin (lot: BCBK7271V) were supplied by Sigma Aldrich (Saint Louis, MO, USA). Magnesium stearate PRS-CODEX (MgS; lot: 85269 ALP) was acquired from Panreac (Barcelona, Spain). All other reagents used in this study were of analytical grade and used without further purification. Demineralized water was used in all cases.
2.2. Polymers Characterization {#sec2dot2-pharmaceutics-11-00020}
------------------------------
Intrinsic viscosity is one of the characteristic values of polymers, and at preset conditions, only depends on the molecular weight of molecules \[[@B42-pharmaceutics-11-00020]\]. Diluted gels of increasing concentrations of the three polymers were prepared (from 0.0125 g/dL to 0.1 g/dL). Locust bean gum and pectin gels were prepared in water \[[@B43-pharmaceutics-11-00020],[@B44-pharmaceutics-11-00020]\]. As it does not gel in water, an aqueous solution of acetic acid 0.3 mol/L and sodium acetate 0.2 mol/L was used to prepare chitosan gels \[[@B45-pharmaceutics-11-00020]\]. Their absolute viscosities (*η*) were measured with a Brookfield viscosimeter (Viscoelite, Fungilab^®^, Barcelona, Spain). Solvent viscosity (*η*~0~) was also measured. From η and *η*~0~, the reduced viscosities (*η~sp~*) were determined by the following equation (Equation (1)):$$\eta_{sp} = \eta - \eta_{0}/\eta_{0}$$
The intrinsic viscosity \[*η*\] was obtained from *η~sp~* of the different polymers, according to the Huggins equation (Equation (2)) \[[@B44-pharmaceutics-11-00020]\]:$$\eta_{sp}/c = \left\lbrack \eta \right\rbrack + Hc$$ where *c* is the concentration of the gel n g/dL and *H* is a constant which depends on the polymer under evaluation. The relationship between intrinsic viscosity and molecular mass has been previously described by the Mark--Houwink--Sakurada equation (Equation (3)) \[[@B44-pharmaceutics-11-00020]\]:$$\left\lbrack \eta \right\rbrack = K\left( M_{w} \right)^{\alpha}$$ where *K* and *α* are constants that depend on the polymer's nature, solvent, and measuring temperature; *M~w~* is the viscosity-average molecular mass. All samples were measured at room temperature (25 °C).
The molar fraction of *N*-acetylglucosamine units in the chain of chitosan--referred to as degree of deacetylation--was experimentally determined in triplicate through the titration method proposed by Czechowska-Biskup et al. \[[@B46-pharmaceutics-11-00020]\]. 0.100 g of chitosan was dissolved in 10 mL of hydrochloric acid 0.1 M and the solution was titrated with NaOH 0.1 M. (Equation (4)):$$NH_{2}\left( \% \right) = \left( {\left( {C_{HCl} \times V_{HCl} - C_{NaOH} \times V_{NaOH}} \right) \times M_{w}/g} \right) \times 100$$ where *C~HCl~* and *C~NaOH~* (mol/L) are the concentrations of HCl and NaOH, respectively; *V~HCl~* and *V~NaOH~ (L)* is the volume of HCL or NaOH employed, respectively; *M~w~* is the molecular weight (NH~2~ = 16 g/mol) and *g* is the weight of CH in g. Assuming that 9.94% is the theoretical NH~2~ percentage, the degree of deacetylation was calculated as follows (Equation (5)):$$DD\ \left( \% \right) = NH_{2}\left( \% \right)/9.94 \times 100$$
As regards pectin, the ratio of esterified galacturonic acid groups to galacturonic acids groups---defined as the degree of esterification (DE)---was experimentally determined in triplicate attending to the method described by Liew et al. \[[@B47-pharmaceutics-11-00020]\]. 0.200 g of pectin was dissolved in 20 mL hydrochloric acid 0.1 M and the solution was titrated with NaOH 0.1 M up to the complete neutralization of free acid groups, the result being the initial titration volume (ITV(mL)). Then, 10 mL of NaOH 0.1 M were added to neutralize polygalacturonic acid and allowed to stand at room temperature for 2 h to de-esterify pectin. 10 mL of HCl 0.1 M were added to neutralize NaOH and the sample was titrated with NaOH 0.1 M, the volume used being recorded as final titration volume (FTV (mL)). The DE was then calculated using the formula (Equation (6)):$$DE\ \left( \% \right) = \left\lbrack {FTV/\left( {ITV + FTV} \right)} \right\rbrack \times 100$$
2.3. Preparation of the Tablets {#sec2dot3-pharmaceutics-11-00020}
-------------------------------
Physical mixtures, whose composition is indicated in [Table 1](#pharmaceutics-11-00020-t001){ref-type="table"}, were compacted at a pressure of 3.68 × 10^8^ Pa during 240 s, with a press similar to the one used to prepare of samples for the IR technique, and 13 mm dies. Blank batches (without drug) and loaded batches (with 30 mg and 100 mg of TFV) were obtained.
2.4. Tablets Characterization {#sec2dot4-pharmaceutics-11-00020}
-----------------------------
Tablets were weighted and thickness, diameter and hardness of all the batches were measured in triplicate using a TA.XT*plus* Texture Analyser (Stable Micro Systems, Surrey, UK). Results were processed, and average values and standard deviations were calculated.
2.5. Swelling Test {#sec2dot5-pharmaceutics-11-00020}
------------------
The swelling behavior of the tablets was assessed using the method previously described by Ruiz-Caro et al. \[[@B48-pharmaceutics-11-00020]\]. Each sample was fixed to a stainless-steel disc using a cyanoacrylate adhesive. This preparation was then placed in a beaker containing 100 mL of simulated vaginal fluid (SVF, pH = 4.2) \[[@B49-pharmaceutics-11-00020]\]. The beakers were placed in a shaking water bath at 37 °C and 15 opm. To adequately characterize the swelling of the tablet, samples were weighed at preset times until the complete dissolution or erosion of the tablet. Each analysis was done in triplicate, and swelling ratio (SR) was determined according to the formula (Equation (7)):$$SR\left( \% \right) = \left\{ {\left\lbrack {\left( {W_{t} - W_{0}} \right)/W_{0}} \right\rbrack/P_{np}} \right\} \times 100$$ where *W~0~* and *W~t~* correspond to the weight of the dry and swollen tablet respectively; and *P~np~* is the proportion of natural polymer, which is the swellable component of the tablet.
Maximum swelling ratios (*SR*~max~) and time to maximum swelling (*t*~max~) for each batch were then statistically processed through a two-way ANOVA considering polymer nature and drug content as factors (α = 0.05).
2.6. Characterization of Swelling Witnesses. Hg Porosimetry and SEM {#sec2dot6-pharmaceutics-11-00020}
-------------------------------------------------------------------
Swelling witnesses were prepared for further structural characterization \[[@B19-pharmaceutics-11-00020]\]. Tablets from each batch were fixed to stainless-steel discs and placed in beakers containing SVF. The preparation was maintained in the shaking water bath (37 °C, 15 opm) until the maximum SR was achieved for each formulation (data previously determined in the swelling test). The swollen tablets were then extracted and lyophilized (Lio-Labor^®^; Telstar, Barcelona, Spain), obtaining porous structures called swelling witnesses. The lyophilization conditions were identical for all the formulations, in order to guarantee that the differences in the porosities of the formulations are a consequence of their composition and not of the lyophilization process. In this way, the comparison among the different systems is justified.
The pore size distributions (PSD) of the witnesses were determined by mercury porosimetry using an Autopore II 9215 (Micromeritics Corp., Norcross, GA, USA). The corresponding PSD~m~ were calculated from the intrusion curves assuming cylindrical pore shapes in all cases. The swelling witnesses were observed by electron microscopy using a field emission scanning electron microscope (JEOL JSM-6335F, Tokyo, Japan).
2.7. Drug Release {#sec2dot7-pharmaceutics-11-00020}
-----------------
The release study was performed according to the methodology proposed by Sánchez-Sánchez et al. \[[@B18-pharmaceutics-11-00020]\]. Each tablet was introduced in a borosilicate glass flask containing 80 mL of SVF in sink conditions \[[@B19-pharmaceutics-11-00020]\] and placed in a shaking water bath at 37 °C and 15 opm. At given times, 5 mL were removed from the flask and immediately replaced with 5 mL of tempered SVF. The aliquot was filtered and the concentration of TFV was determined by UV spectroscopy at a wavelength of 260 nm (Shimadzu^®^ UV-1700 spectrophotometer, Kyoto, Japan). The study was performed in triplicate and statistically processed by determining the similarity factor f2 \[[@B50-pharmaceutics-11-00020]\].
2.8. Mucoadhesion Assessment {#sec2dot8-pharmaceutics-11-00020}
----------------------------
In order to determine the tablets' capacity to adhere to the vaginal mucosa at the time of administration, the work and force necessary for detachment was assessed using the TA.XT*plus* Texture Analyser (Stable Micro Systems) through a new ex vivo method. The samples were fixed to a 20 mm stainless-steel probe. Square fragments of 2 × 2 cm of bovine vaginal mucosa (obtained from a local slaughterhouse) were fixed to a petri dish with a cyanoacrylate adhesive. The probe with the tablet was moved at a speed of 1 mm/s until it came into contact with the mucosa, applying a contact force of 500 g for 30 s. The probe was then separated from the mucosa at a speed of 0.1 mm/s until the complete detachment of the tablet. The force applied during the detachment of the formulation was measured at a rate of 500 pps. The force applied vs. the distance travelled by the probe was measured, and the maximum force required to separate the tablet from the mucosa was recorded. Also, the area under the curve between the force--distance profiles was determined \[[@B51-pharmaceutics-11-00020]\]. Each batch was evaluated in duplicate, and the results were statistically processed using a two-way ANOVA, with drug dose and polymer as the factors (α = 0.05).
However, the tablets must remain adhered to the mucosa while the drug is being released, in order to afford the woman protection against the sexual transmission of HIV for the longest possible time. The optimal formulation must not only release the drug in a sustained manner, but also remain adhered to the vaginal mucosa during a similar period, so the residence time of the tablet in the vaginal mucosa was assessed through an ex vivo mucoadhesion test \[[@B19-pharmaceutics-11-00020]\]. A sample of bovine vaginal mucosa was fixed with a cyanoacrylate adhesive to an 8.5 cm × 5 cm stainless steel plate. Each tablet was then adhered to the mucosa, applying a given pressure (500 g for 30 s). The preparation was placed at an angle of 60° inside a beaker containing SVF, and then in the shaking water bath (Selecta^®^ UNITRONIC320 OR, Barcelona, Spain) at 37 °C and 15 opm. The residence time was assessed by observation of the samples. All batches were evaluated in duplicate.
3. Results and Discussion {#sec3-pharmaceutics-11-00020}
=========================
3.1. Polymers Characterization {#sec3dot1-pharmaceutics-11-00020}
------------------------------
In [Figure 1](#pharmaceutics-11-00020-f001){ref-type="fig"}, *η~sp~*/*c* (dL/g) vs. concentration (g/dL) for the three polymers is displayed. All polymers showed a direct relation between *η~sp~*/*c* and the concentration of the gel, locust bean gum-based gels being the ones showing higher differences in viscosity depending on the concentration. Pectin- and chitosan-based gels show similar profiles, with a less steep slope compared to locust bean gum gels. However, it must be noted that chitosan does not gel in water, which makes these results hardly extrapolable to the performance of the tablet. The value of \[*η*\] was therefore obtained for all the polymers employing these plots. The *K* and α values for Mark--Houwink--Sakurada equation in each polymer had been previously described in literature, making it possible to determine *M*~w~, displayed in [Table 2](#pharmaceutics-11-00020-t002){ref-type="table"}. DD (%) for chitosan and DE (%) for pectin are also shown in [Table 2](#pharmaceutics-11-00020-t002){ref-type="table"}.
The molecular masses obtained for pectin and locust bean gum are within the normal range of molecular masses for both polymers \[[@B43-pharmaceutics-11-00020],[@B52-pharmaceutics-11-00020]\]. These results indicate that the chitosan employed for the study was a low-molecular-weight chitosan \[[@B53-pharmaceutics-11-00020]\] with a degree of deacetylation of (50.47--58.99)%. In the case of pectin, it was shown that it was a highly methoxylated pectin \[[@B54-pharmaceutics-11-00020]\].
3.2. Tablets Characterization {#sec3dot2-pharmaceutics-11-00020}
-----------------------------
Thickness, diameter and weight values for the batches are displayed in [Table 3](#pharmaceutics-11-00020-t003){ref-type="table"}.
The thickness of the tablets was directly related to their weight. In all the cases, the thickness of the tablets was higher than 1.5 mm and lower than 2.5 mm. An average diameter of 13 mm was obtained in all cases due to the fact that the same dye was used in all cases. The average weights obtained fit with theoretical values for all the batches.
Hardness data for the tablets evaluated are represented in [Figure 2](#pharmaceutics-11-00020-f002){ref-type="fig"}. Tablets constituted exclusively by polymers showed low hardness, lower than 10 N in all cases, due to the amorphous nature of these substances, which are not suitable for tableting without compaction excipients. However, the addition of a drug leads to a significant increase in hardness in all cases. The more TFV there is in the system, the higher the hardness of the tablet. This indicates that TFV, which is a crystalline substance, may act as structural agent, allowing the correct transmission of force from the punch to the mass being compacted. However, the degree of influence of the drug is significantly lower in the case of locust bean gum-based tablets, which may be due to a low interaction between the drug and the polymer. According to data, addition of TFV to the tablets significantly improves their handling, all loaded systems being suitable for handling and self-administration.
3.3. Swelling Test {#sec3dot3-pharmaceutics-11-00020}
------------------
[Figure 3](#pharmaceutics-11-00020-f003){ref-type="fig"} shows the swelling profiles, where weight increases were observed in all cases due to the uptake of SVF in the tablets. After reaching the maximum SVF uptake (maximum SR, *SR*~max~), the weight of the formulations decreases due to the erosion or dissolution of the system in the medium. Nevertheless, the swelling profiles are conditioned by the nature of the polymer.
[Figure 3](#pharmaceutics-11-00020-f003){ref-type="fig"}A contains the swelling profiles of the blank batches. C tablets show a sudden entry of SVF in the first hours. However, the gelation of the polymer is incomplete in this medium, as gelling in chitosan is pH-dependent and require diluted acids \[[@B55-pharmaceutics-11-00020],[@B56-pharmaceutics-11-00020]\]. The erosion of the tablet is observed after reaching a low maximum SR value after no more than 24 h. P tablets swell completely in this medium, adequately hydrating the tablet and forming a gel in 24 h. P tablets have the highest swelling rate, as pectin is an acidic polymer that is able to gel in this medium, whose pH is above the pKa of pectin (2.9--3.2) \[[@B31-pharmaceutics-11-00020]\], which enables it to capture a large amount of SVF. The behavior observed in L batch is similar to that of the P batch, although in this case a more consistent gel is obtained \[[@B57-pharmaceutics-11-00020]\], leading to more moderate swelling that is also more sustained over time. The swelling behavior in batches of tablets containing a combination of two polymers is conditioned by the nature of each polymer and the possible interaction between them. Thus, CL tablets show moderate swelling profiles with a lower dissolution time in the medium than observed when the polymers are evaluated separately. Locust bean gum is a neutral polymer that does not modify its charge in the medium, so the partial gelation of the chitosan induces the separation of the polymer chains in the locust bean gum, drastically reducing the high consistency of the gel. In contrast, CP tablets reveal a synergy between chitosan and pectin due to their ability to form polyelectrolyte complexes, as previously described \[[@B37-pharmaceutics-11-00020]\]. Although the penetration of SVF is slower than in the tablets containing only pectin---and therefore has a lower maximum SR---more time is required for the complete dissolution of the system. Pectin releases protons into the medium, which remains negatively charged. Chitosan captures the protons released by the pectin and the chains of both polymers become joined by means of electrostatic interactions, generating a very compact structure which slows the entry of SVF.
[Figure 3](#pharmaceutics-11-00020-f003){ref-type="fig"}B shows the swelling profiles of the batches containing 30 mg of TFV. It can be seen that the entire swelling and erosion process is delayed for batch C30, indicating that the tablets containing TFV maintained their structure in SVF for a longer period, as the TFV acted as a structural agent that prevents the destructuring of the system. L30 and P30 tablets show overlapping swelling profiles for L and P respectively, because the presence of TFV is unable to alter the water uptake capacity of both polymers. This indicates that locust bean gum and pectin generate systems that are robust enough not to modify the physicochemical properties of the tablets in the presence of the drug. However, after mixing locust bean gum or pectin with chitosan, CL30 and CP30 show a delay in the swelling process that can be attributed to the structural behaviour of TFV in the presence of chitosan.
This behaviour is even more evident in [Figure 3](#pharmaceutics-11-00020-f003){ref-type="fig"}C, which shows the swelling data obtained from systems containing 100 mg TFV. C100 requires more time to imbibe SVF and erode in the medium, so the more TFV the C tablets contain, the greater the structuring action of the drug. The swelling profiles of L100 and P100 overlap with the corresponding systems containing no drug or 30 mg of TFV, so the drug is unable to modify the characteristic swelling behaviour of either of the two polymers. CL100 and CP100 batches show the structuring capacity of TFV on chitosan and their swelling and complete erosion processes are even more delayed compared to the systems containing either no drug or 30 mg of TFV.
[Figure 4](#pharmaceutics-11-00020-f004){ref-type="fig"} contains the values of *SR*~max~ and *t*~max~ from the corresponding swelling profiles. No significant differences are obtained in the *SR*~max~ from the statistical analysis of C, C30 and C100 batches, but the presence of TFV delays the entry of SVF, so the greater the amount of drug in the tablet, the longer the *t*~max~, as the structuring power of the drug slows the entry of the medium in the chitosan tablet. No significant differences were detected in the statistical analysis between L, L30 and L100 in either the *SR*~max~ or in the *t*~max~, and the same true for the P, P30 and P100 batches. This suggests that the L and P formulations are robust and SVF can access the tablet as the characteristic three-dimensional arrangement of the polymer chains does not depend on the presence of TFV. When chitosan is combined with locust bean gum (CL, CL30 and CL100), the presence of the drug significantly reduces the *SR*~max~ and increases *t*~max~. This can be explained by the fact that the drug is interposed between the ungelled chitosan and the locust bean gum gel, so the entry of SVF is less abrupt and the separation of the locust bean gum chains is less marked; the amount of SVF that is able to enter the system is therefore lower and impeded, leading to a higher *t*~max~. In batches where chitosan is mixed with pectin (CP, CP30 and CP100), both polymers are joined by electrostatic interactions so *SR*~max~ is not modified by the presence of TFV, although *t*~max~ is delayed, as SVF entry is slowed by the structuring power of TFV.
3.4. Microstructure of Witnesses. Hg Porosimetry and SEM {#sec3dot4-pharmaceutics-11-00020}
--------------------------------------------------------
The PSD of the swelling witnesses samples are plotted in [Figure 5](#pharmaceutics-11-00020-f005){ref-type="fig"}, and the corresponding microstructures are shown in [Figure 6](#pharmaceutics-11-00020-f006){ref-type="fig"}.
All PSD are plotted in terms of their maximum pore volume (mL/g), and it can be seen that for the un-mixed polymers (chitosan, locust bean gum and pectin), the pore volume increases in the order C \< L \< P, agreeing with the data from the swelling test. However, when chitosan is mixed with locust bean gum or pectin the pore volume is in the order CL \> CP, also due to the aforementioned swelling behaviour, as the PSD reflects the water present in the hollows at *t*~max~, and which was removed during lyophilization during the preparation of the swelling witnesses.
The addition of TFV to these polymers leads to important changes in their PSD. The incorporation of TFV to chitosan produces an increase in porosity and the pore sizes become smaller. These changes continue if the concentration of TFV increases, due to the TFV's structuring power over chitosan, which improves the system's capacity to imbibe water and causes the appearance of pores when water is removed during the lyophilization process for obtaining the swelling witnesses. Data obtained from the swelling test indicate that C30 and C100 generated systems with higher swelling, and the number of pores is related to this value.
For the locust bean gum polymer, the addition of TFV causes little change in both pore volume and PSD, although a slight increase in pore size is observed at higher concentrations of TFV in the polymer. In this case it can be concluded that the presence of the drug does not modify the behaviour of this polymer in the medium. In other words, locust bean gum gel is so robust that its structure---and therefore its porosity---is not conditioned by the presence of TFV. This can be related with the results of hardness evaluation, as it was shown that locust bean gum-based tablets were the least affected by the presence of drug. Finally, the incorporation of TFV to the pectin polymer produces a significant decrease in the total pore volume and a smaller pore size; these changes are also independent of the amount of TFV. One possible explanation is that even though the presence of TFV does not modify either the rate or the amount of medium imbibed by pectin in its gelling process (the swelling profiles for P, P30 and P100 are practically overlapping), the visual observation of the systems during the swelling test revealed that swelled systems with the drug (P30 and P100) were more compact than P swelled tablets.
The addition of TFV to the polymer mixtures (i.e., CL and CP) caused major changes in the PSD data. In the case of CL tablets, the incorporation of TFV (CL30 and CL100) leads to less porous swelled systems; in other words, lower pore volume and pore diameter. These data agree with the data from the swelling test, as CL30 and CL100 had a lower SR~max~ and slower uptake rates of the medium (higher *t*~max~), indicating that the presence of TFV modifies the arrangement of the chains in both polymers in the water uptake process and reduces their swelling capability compared to the CL batch. The addition of TFV to CP batches forms systems (CP30 and CP100) with smaller pores (lower pore diameter) but their pore volume data are higher than the corresponding CP system, which can be attributed to TFV's structuring power over chitosan. The entry of the aqueous medium in the system is therefore hindered during the swelling process, and although the swelling capacity is not modified by the presence of TFV (similar *SR*~max~ data for CP, CP30 and CP100 batches), the time required to achieve *SR*~max~ is longer (higher *t*~max~ of CP30 and CP100 respect to CP data). This is the reason that smaller pores are observed in swelling witnesses of CP30 and CP100.
In summary, the addition of a gelling polymer to chitosan-based systems leads to a modification of the microstructure of the system due to the redistribution of the polymer chains during the swelling process caused by the interaction between the polymers.
[Figure 6](#pharmaceutics-11-00020-f006){ref-type="fig"} shows the microstructure of all the swelling witnesses that corroborate the porosity results described above.
3.5. Drug Release {#sec3dot5-pharmaceutics-11-00020}
-----------------
The TFV release profiles from the tablets are shown in [Figure 7](#pharmaceutics-11-00020-f007){ref-type="fig"}. Sustained release of over 24 h are observed in all cases. However, the TFV release profiles showed to be conditioned by the composition of the tablets.
According to [Figure 7](#pharmaceutics-11-00020-f007){ref-type="fig"}A, chitosan-based systems (C30 and C100) are able to release the drug over 120 h, because although the system starts eroding from 24 h (as seen in the swelling profiles in [Figure 3](#pharmaceutics-11-00020-f003){ref-type="fig"}), the drug continues to be released from the fragments of the eroded tablets. There are no significant differences between C30 and C100 batches; this may be related to the porosity results, which indicate that the volume of pores is proportional to the amount of drug, so the release of TFV through the pores is independent of the amount of drug
The TFV dissolution profiles from the locust bean gum batches (L30 and L100) in [Figure 7](#pharmaceutics-11-00020-f007){ref-type="fig"}B show the sustained releases of TFV for up to 120 h due to a progressive diffusion of the drug through the high-consistency gel generated in the medium according to Chakravorty et al. \[[@B45-pharmaceutics-11-00020]\]. Significant differences can be observed in the release profiles, which can be attributed to the drug being released by diffusion; there is more drug in the gel when the medium penetrates into the system, so more time is required to dissolve the drug. Since the volume of pores in the system is constant regardless of the amount of drug in the tablet, the greater the amount of TFV in the tablet, the slower the drug release through the gel.
Drug release from batches based on pectin (P30 and P100) lasts a maximum of 72 h, as seen in [Figure 7](#pharmaceutics-11-00020-f007){ref-type="fig"}C. The low consistency of the gel formed allows a greater entry of SVF, so the drug diffuses faster through it. In this case, there are also significant differences depending on the amount of drug. As the drug is also released through diffusion, higher concentrations of TFV within the tablet slow its dissolution in the small amount of fluid in the gel. According to the PSD results, there are no differences either in pore volume or pore size for P30 and P100, so the release of the drug through the pores is slower for larger amounts of drug.
[Figure 7](#pharmaceutics-11-00020-f007){ref-type="fig"}D shows a drug release of up to 72 h in the case of batches of chitosan combined with locust bean gum (CL30 and CL100). This is shorter than observed in the batches prepared with each of the polymers separately. The explanation is that CL30 and CL100 tablets are unable to gel and undergo the destructuring that is responsible for releasing the drug. This destructuring of CL30 and CL100, once they are introduced in SVF, can be seen in the micrographs of the corresponding swelling witnesses ([Figure 6](#pharmaceutics-11-00020-f006){ref-type="fig"}).
According to [Figure 7](#pharmaceutics-11-00020-f007){ref-type="fig"}E, batches prepared with the association of pectin and chitosan (CP30 and CP100) show overlapping TFV controlled-release profiles that are not dependent on the amount of drug they contain. This is because the combination of both polymers forms a polyelectrolyte complex, as previously described in the swelling test section, creating a robust gelled system able to control the release of TFV during 120 h. Of all the systems evaluated, those based on the combination of chitosan and pectin are the only ones with a sustained TFV release profile that does not depend on the amount of drug in the tablet. This mixture of polymers can therefore be said to form the most robust formulation in the evaluation.
3.6. Mucoadhesion Assessment {#sec3dot6-pharmaceutics-11-00020}
----------------------------
[Figure 8](#pharmaceutics-11-00020-f008){ref-type="fig"} contains the results of the mucoadhesion forces and mucoadhesion works for the batches. It can be seen that all the blank formulations (C, L, P, CL and CP) are able to attach to the vaginal mucosa, and force values of between 0.075 N and 0.16 N are required to detach the tablets. This confirms the mucoadhesiveness of all the polymers assayed, whose mechanisms of adhesion have been previously described (chitosan through electrostatic interactions \[[@B46-pharmaceutics-11-00020]\] and pectin and locust bean gum through hydrogen bonds \[[@B58-pharmaceutics-11-00020]\]). Pectin is the polymer with the highest mucoadhesion force and work values in quantitative terms compared to locust bean gum and chitosan. When chitosan and locust bean gum are mixed (CL tablets), the mucoadhesion force and work are similar to that obtained from tablets prepared with the unmixed polymers (C tablets and L tablets). However, the association of chitosan and pectin (CP tablets) has a mucoadhesion force and work close to those obtained from tablets prepared with pectin exclusively (P tablets), confirming the high mucoadhesiveness of pectin even when mixed. The addition of TFV to the systems leads in all cases to an increase in the mucoadhesion force and work. The mucoadhesion force cannot be related to the amount of drug in the systems. In fact, the results of the ANOVA analysis indicate that there are no significant differences in the mucoadhesion force and work among the blank batches (C, L, P, CL and CP), those loaded with 30 mg (C30, L30, P30, CL30 and CP30) and those containing 100 mg (C100, L100, P100, CL100 and CP100) of TFV, as shown in [Table 4](#pharmaceutics-11-00020-t004){ref-type="table"}.
Once it was verified that all the formulations had mucoadhesive properties, the next step was to determine how long they remained bonded to the vaginal mucosa.
[Figure 9](#pharmaceutics-11-00020-f009){ref-type="fig"} shows the residence times of the batches. All the blank systems remain mucoadhered for at least 24 h. Chitosan-based tablets (C tablets) are lost through erosion due to the destructuration of the system. Tablets containing locust bean gum (L tablets) get detached from the mucosa while they are still embibing water, so the whole swollen tablet is lost. Tablets with pectin (P tablets) showed the lowest residence times, as it formed a fluid gel unable to maintain its structure, and adherence to the mucosa, so the detachment occurs when the *SR*~max~ determined in the swelling tests ([Section 3.3](#sec3dot3-pharmaceutics-11-00020){ref-type="sec"}) is reached. The combination of chitosan and locust bean gum leads to a lower residence times due to the destructuration of the system described in the swelling studies ([Section 3.3](#sec3dot3-pharmaceutics-11-00020){ref-type="sec"}), while the combination of chitosan and pectin (CP tablets) had the longest mucoadhesion residence times, as the tablets do not detach from the mucosa, so the system is dissolved in the medium still being attached to the surface of the mucosa. This confirms the robustness of the chitosan-pectin combination due to the formation of the previously described polyelectrolyte complex \[[@B37-pharmaceutics-11-00020]\] and also to the swelling and porosity data. However, as in the case of the mucoadhesion force measurements, the ANOVA results indicate that there are no significant differences in mucoadhesion residence times among the blank batches (C, L, P, CL and CP), those loaded with 30 mg (C30, L30, P30, CL30 and CP30) and those containing 100 mg (C100, L100, P100, CL100 and CP100) of TFV, as shown in [Table 5](#pharmaceutics-11-00020-t005){ref-type="table"}.
Although the TFV controlled-release profiles, mucoadhesion force and mucoadhesion residence times are the parameters used to select the best formulation, the patient's comfort is a factor that cannot be overlooked when seeking to improve adherence to the treatment. The most suitable formulation is therefore the one with a moderate swelling behaviour that is able to remain in the vaginal mucosa while the drug is being released. Although chitosan-based batches swell very little, they disintegrate very quickly; this would hinder their presence at the site of action since the fragments would be expelled from the vagina, leading to reduced mucoadhesion times \[[@B29-pharmaceutics-11-00020]\]. In the case of locust bean gum and pectin batches, although the structure is maintained during the SVF uptake process, the swelling is so great that they cannot strictly be considered an option for vaginal administration, since the patient's discomfort would compromise their therapeutic compliance \[[@B19-pharmaceutics-11-00020]\]. Although the combination of chitosan and locust bean gum reduces the maximum swelling, the formulation dissolves quickly in the medium, so vaginal leakage could be expected to be high. However, the association of chitosan and pectin generates systems with a moderate swelling but which maintain their structure for prolonged periods of time, so they may be good candidates to fulfil the objective of this work. Vaginal turnover must also be taken into account. This is the physiological mechanism whereby any possible harmful elements such as pathogens or foreign particles are expelled from the vaginal environment \[[@B59-pharmaceutics-11-00020]\]. This mechanism represents a limitation for mucoadhesive vaginal systems, as this cyclical process occurs after 96 h and barely allows the permanence of formulations during prolonged periods in in vivo studies. Fortunately, CHP systems for the controlled release of TFV (C30 and C100) deliver almost the entire drug amount before vaginal turnover occurs, and the mucoadhesion time exceeds this period.
4. Conclusions {#sec4-pharmaceutics-11-00020}
==============
The association of pectin and chitosan generates an electrostatic interaction between both polymers in SVF, thus obtaining robust and highly-structured gelling systems in this medium, with a moderate swelling that ensures therapeutic compliance, and a mucoadhesion residence time and controlled release of tenofovir for 4 days, the time corresponding to vaginal turnover.
It has been demonstrated that the antiretroviral drug tenofovir can be integrated in the matrix of these tablets regardless of its quantity, so they can also be said to be robust systems that are not modified by the amount of drug they contain. The tablets based on chitosan and pectin are therefore interesting candidates for the prevention of sexual transmission of HIV through the controlled release of tenofovir for 4 days.
Cazorla-Luna R. and Martín-Illana A. are beneficiaries of University Professor Training fellowships granted by the Spanish Ministry of Education, Culture and Sport and Notario-Pérez F. is the beneficiary of a Research Training Fellowship granted by the Spanish Ministry of Science, Innovation and Universities. We are grateful to the Carnes Barbero slaughterhouse (El Barraco, Ávila, Spain) for supplying the biological samples. We would also like to thank María Hernando, veterinarian of the Junta de Castilla y León, for verifying the suitability of the biological samples. Electron microscopy using a field emission scanning electron microscope (JEOL JSM-6335F, Tokyo, Japan) was done in the Research Support Centres and Unique Science and Technology Facility at the Complutense University of Madrid.
Investigation, R.C.-L., F.N.-P. and A.M.-I.; Writing---original draft, R.R.-C., A.T. and J.R.; Writing---review & editing, M.D.V.
This work was supported by projects MAT2012-34552 from the Spanish Ministry of Economy, Industry and Competitiveness, and MAT2016-76416-R financed by the Spanish Research Agency and the European Regional Development Fund (AEI/FEDER, UE).
The authors declare no conflict of interest.
{#pharmaceutics-11-00020-f001}
{#pharmaceutics-11-00020-f002}
{#pharmaceutics-11-00020-f003}
{#pharmaceutics-11-00020-f004}
{#pharmaceutics-11-00020-f005}
{#pharmaceutics-11-00020-f006}
{#pharmaceutics-11-00020-f007}
{#pharmaceutics-11-00020-f008}
{#pharmaceutics-11-00020-f009}
pharmaceutics-11-00020-t001_Table 1
######
Composition of the batches prepared in mg/tablet.
Batch Chitosan Locust Bean Gum Pectin MgS TFV
------- ---------- ----------------- -------- ----- -----
C 290 3
C30 290 3 30
C100 290 3 100
L 290 3
L30 290 3 30
L100 290 3 100
P 290 3
P30 290 3 30
P100 290 3 100
CL 145 145 3
CL30 145 145 3 30
CL100 145 145 3 100
CP 145 145 3
CP30 145 145 3 30
CP100 145 145 3 100
pharmaceutics-11-00020-t002_Table 2
######
Results obtained from the characterization of the polymers employed.
\[*η*\] *K* (dL/g) α *M*~w~ (kDa) DD (%) DE (%)
----------------- --------- ------------------------------------------------- ---------------------------------------- -------------- -------------- --------------
Chitosan 24.74 9.30 × 10^−3^ \[[@B45-pharmaceutics-11-00020]\] 0.76 \[[@B45-pharmaceutics-11-00020]\] 3.21 × 10^1^ 54.73 ± 4.26
Locust bean gum 16.42 3.04 × 10^−4^ \[[@B43-pharmaceutics-11-00020]\] 0.80 \[[@B43-pharmaceutics-11-00020]\] 2.78 × 10^3^
Pectin 20.00 1.4 × 10^−6^ \[[@B44-pharmaceutics-11-00020]\] 1.34 \[[@B44-pharmaceutics-11-00020]\] 2.19 × 10^2^ 79.91 ± 1.66
pharmaceutics-11-00020-t003_Table 3
######
Data of thickness (mm), diameter (mm) and weight (mg) of the tablets prepared.
Batch Thickness (mm) Diameter (mm) Average Weight (mg) Theoretical Weight (mg)
------- ---------------- ---------------- --------------------- -------------------------
C 1.683±0.006 13.027 ± 0.021 292.00 ± 0.82 293
C30 1.857 ± 0.040 13.033 ± 0.015 323.23 ± 0.47 323
C100 2.340 ± 0.052 13.070 ± 0.104 394.45 ± 0.07 393
L 1.833 ± 0.029 13.020 ± 0.104 293.17 ± 0.32 293
L30 1.970 ± 0.020 13.120 ± 0.010 322.40 ± 1.27 323
L100 2.367 ± 0.006 13.097 ± 0.012 392.57 ± 0.68 393
P 1.747 ± 0.006 13.057 ± 0.006 292.63 ± 1.10 293
P30 1.953 ± 0.029 13.090 ± 0.010 323.67 ± 1.07 323
P100 2.347 ± 0.042 13.057 ± 0.006 392.40 ± 1.55 393
CL 1.977 ± 0.015 13.183 ± 0.015 292.90 ± 0.36 293
CL30 2.113 ± 0.015 13.150 ± 0.036 321.63 ± 0.32 323
CL100 2.410 ± 0.010 13.080 ± 0.017 391.70 ± 0.62 393
CP 1.700 ± 0.069 13.083 ± 0.015 292.10 ± 1.39 293
CP30 1.907 ± 0.029 13.053 ± 0.012 323.27 ± 0.55 323
CP100 2.277 ± 0.031 13.043 ± 0.006 393.77 ± 0.21 393
pharmaceutics-11-00020-t004_Table 4
######
Results of the ANOVA processing of the mucoadhesion forces of the batches; the factors are the nature of the polymer and the drug content. *p*-values below α = 0.05 indicate no significant differences.
Polymer Nature Drug Content Interaction
---------------------- ---------------- -------------- -------------
*Mucoadhesion force* 9.47×10^-5^ 9.61×10^-6^ 1.01×10^-3^
*Mucoadhesion work* 8.28×10^-4^ 3.12×10^-2^ 3.61×10^-1^
pharmaceutics-11-00020-t005_Table 5
######
Results of the ANOVA processing of the mucoadhesion times of the batches; the factors are the nature of the polymer and the drug content. *p*-values below α = 0.05 indicate no significant differences.
Polymer Nature Drug Content Interaction
---------------- -------------- -------------
4.1×10^-6^ 1.58×10^-3^ 1.75×10^-2^
| {
"pile_set_name": "PubMed Central"
} |
Introduction {#s1}
============
Within a species, organ size is remarkably reproducible. While extrinsic factors like hormones are required for growth, classic transplantation experiments indicate that intrinsic factors within organs determine size ([@bib7]). For example, embryonic limb buds transplanted from a large species of salamander onto a small species grow to the size characteristic of the donor ([@bib52]). Similar findings have been made in quail and chick limbs ([@bib26]; [@bib57]), rat hearts and kidneys ([@bib10]; [@bib43]), and mouse thymuses ([@bib32]). Consistently, developing *Drosophila* wings transplanted into adult abdomens grow to the proper size, indicating that the information determining size is located within the developing organ ([@bib13]). Indeed, the *Drosophila* wing is a classic model system for studying organ size, as its size is highly replicable ([@bib15]; [@bib13]), and all adult precursor cells are located within the pouch region of the developing larval imaginal disc ([@bib16]) ([Figure 1A](#fig1){ref-type="fig"}, grey). Despite extensive work, the molecular mechanisms underlying intrinsic organ size control remain unclear ([@bib54]). While morphogens direct both patterning and growth of developing organs ([@bib50]), a link between patterning molecules and growth control pathways has not been established ([@bib41]).10.7554/eLife.11491.003Figure 1.Localized JNK activity exists in the developing wing.(**A**) Schematic of wing precursor cells (grey) in the developing disc (A, anterior; P, posterior). (**B-F**) Antibody staining against active, phosphorylated JNK (pJNK, green; DAPI, blue) labels a stripe in wildtype (**B-C**) but not JNKK mutant (**D-E**, *hep^r75/Y^*) third instar discs. Boxed region in (**B**) and (**D**) is magnified in (**C**) and (**E**), respectively. Weak pJNK signal is also detected along the dorsal/ventral boundary. pJNK stripe staining is lost in JNKK mutant clones (**F**, *hep^r75^*, clone is negatively marked in **F'**). (**G-I**) pJNK localizes to the same cells in which *ptc* is expressed (**G**, *ptc\>RFP,* red) along the A/P boundary, and is lost following JNK phosphatase expression (**H**, *ptc\>puc, RFP*, red) or RNAi-mediated knockdown of *bsk* within the *ptc* domain (**I**, *ptc\>bsk^RNAi^, RFP,* red). Bar: 50 um (**B-F**, **H-I**) and 25 um (**G**). See also [Figure 1---figure supplement 1](#fig1s1){ref-type="fig"}.**DOI:** [http://dx.doi.org/10.7554/eLife.11491.003](10.7554/eLife.11491.003)10.7554/eLife.11491.004Figure 1---figure supplement 1.pJNK recognizes endogenous JNK activity in developing wing discs.Related to [Figure 1](#fig1){ref-type="fig"}. (**A-C**) Wildtype *Canton-S* wing discs stained for DAPI (blue) and pJNK (green) during (**A**) early third instar (L3), (**B**) mid-third instar, and (**C**) late third instar. (**D**) Wing disc stained for DAPI (blue), pJNK (green), and *puc-lacZ* (red). Boxes indicate areas enlarged in **E** and **F**. (**E**) Notum cells are positive for pJNK and *puc-lacZ*. (**F**) Blade cells show a stripe of pJNK staining but no detectable *puc-lacZ*. (**G-H**) A second, independently generated antibody against pJNK from Promega shows a similar pattern in third instar discs. (**G**) Whole wing disc and (**H**) wing blade. (**I**) Inhibition of JNK signaling in the dorsal compartment reduces pJNK staining (green) (*ap\>puc*). (**J**) *ptc-Gal4* expresses in a stripe in early L3 stage. (**K**) Inhibition of JNK in all wing blade cells (*rn\>bsk^RNAi\#1^, RFP*) or (**L**) in *ptc* cells (*ptc\>bsk^RNAi\#2^, RFP*) eliminates pJNK (green) signal. (**M**) Western blot analysis of larval extracts from *Canton-S* (Lane 1) and *hep^r75/Y^* (Lane 2) animals. pJNK is predicted to be \~43kD. Loading control (bottom) is alpha-tubulin. Bar: 50 um.**DOI:** [http://dx.doi.org/10.7554/eLife.11491.004](10.7554/eLife.11491.004)
The Jun N-terminal Kinase (JNK) pathway promotes proliferation during regeneration and tumor growth ([@bib6]; [@bib23]; [@bib40]; [@bib44]; [@bib58]). In fact, JNK-induced proliferation is often non-autonomous ([@bib12]; [@bib35]; [@bib40]; [@bib47]; [@bib58]). Basket (Bsk) is the singular *Drosophila* JNK and is activated by phosphorylation by the JNKK Hemipterous (Hep) ([@bib18]; [@bib45]). Canonical JNK signaling acts through the transcription co-factor Jun, which regulates migration and apoptosis ([@bib45]). Although the role of JNK in activating Yorkie signaling and growth during regeneration and tumorigenesis is clear ([@bib12]; [@bib47]; [@bib48]), it is not known to regulate proliferation and growth during developmental size control.
Here we show that localized JNK activity in the developing wing is established by Hedgehog (Hh) signaling and controls wing size through a non-canonical, Jun-independent signaling mechanism that inhibits the Hippo pathway.
Results and discussion {#s2}
======================
JNK is active in the developing *Drosophila* wing pouch {#s2-1}
-------------------------------------------------------
Two independently generated antibodies that recognize the phosphorylated, active form of JNK (pJNK) specifically label a stripe in the pouch of developing wildtype third instar wing discs ([Figure 1B--C](#fig1){ref-type="fig"} and [Figure 1---figure supplement 1G--H](#fig1s1){ref-type="fig"}). Importantly, localized pJNK staining is not detected in hemizygous *JNKK* mutant discs ([Figure 1D--E](#fig1){ref-type="fig"}; *hep^r75^/Y*), in clones of *JNKK* mutant cells within the stripe ([Figure 1F](#fig1){ref-type="fig"}; *hep^r75^, FRT10/Ubi-GFP, FRT10;; MKRS, hs-FLP/+*), following over-expression of the JNK phosphatase *puckered (puc*) ([Figure 1---figure supplement 1I](#fig1s1){ref-type="fig"}; *ap-Gal4, UAS-puc*), or following RNAi-mediated knockdown of *bsk* using two independent, functionally validated RNAi lines ([Figure 1---figure supplement 1K--L](#fig1s1){ref-type="fig"}; *rn-Gal4, UAS-bsk^RNAi\#1^*or *ptc-Gal4, UAS-bsk^RNAi\#2^*; see Experimental Genotypes for full genotypes and conditions) ([@bib18]; [@bib29]; [@bib30]; [@bib36]; [@bib55]; [@bib59]).
The stripe of localized pJNK staining appeared to be adjacent to the anterior-posterior (A/P) compartment boundary, a location known to play a key role in organizing wing growth, and a site of active Hedgehog (Hh) signaling ([@bib4]; [@bib49]; [@bib60]). Indeed, pJNK co-localizes with the Hh target gene *patched (ptc*) ([Figure 1G](#fig1){ref-type="fig"}; *ptc-Gal4, UAS-RFP*). Expression of the JNK phosphatase *puc* in these cells specifically abrogated pJNK staining ([Figure 1H](#fig1){ref-type="fig"}; *ptc-Gal4, UAS-puc*), as did RNAi-mediated knockdown of *bsk* ([Figure 1I](#fig1){ref-type="fig"} and [Figure 1---figure supplement 1L](#fig1s1){ref-type="fig"}; *ptc-Gal4, UAS-bsk^RNA\#i1or2^*). Together, these data indicate that the detected pJNK signal reflects endogenous JNK signaling activity in the *ptc* domain, a region of great importance to growth control. Indeed, while at earlier developmental stages pJNK staining is detected in all wing pouch cells ([Figure 1---figure supplement 1A](#fig1s1){ref-type="fig"}), the presence of a localized stripe of pJNK correlates with the time when the majority of wing disc growth occurs (1000 cells/disc at mid-L3 stage to 50,000 cells/disc at 20 hr after pupation, ([@bib14]), so we hypothesize that localized pJNK plays a role in regulating growth.
Localized JNK activity regulates global wing size {#s2-2}
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Inhibition of JNK signaling in the posterior compartment previously led to the conclusion that JNK does not play a role in wing development ([@bib31]). The discovery of an anterior stripe of JNK activity spurred us to re-examine the issue. Since *bsk* null mutant animals are embryonic lethal, we thus conditionally inhibited JNK signaling in three independent ways in the developing wing disc. JNK inhibition was achieved by RNAi-mediated knockdown of *bsk (bsk^RNAi\#1or2^*), by expression of JNK phosphatase (*puc*), or by expression of a dominant negative *bsk (bsk^DN^*). These lines have been independently validated as JNK inhibitors ([@bib29]; [@bib30]; [@bib36]; [@bib55]). Inhibition of JNK in all wing blade cells (*rotund-Gal4, rn-Gal4*) or specifically in *ptc-*expressing cells (*ptc-Gal4*) resulted in smaller adult wings in all cases, up to 40% reduced in the strongest cases ([Figures 2A--F, 2J--K](#fig2){ref-type="fig"}, and [Figure 2---figure supplement 1D](#fig2s1){ref-type="fig"}). Importantly, expression of a control transgene (*UAS-GFP*) did not affect wing size ([Figure 2---figure supplement 1B--C](#fig2s1){ref-type="fig"}; *ptc-Gal4, UAS-GFP*). This contribution of JNK signaling to size control is likely an underestimate, as the embryonic lethality of *bsk* mutations necessitates conditional, hypomorphic analysis. Nevertheless, hypomorphic *hep^r75^/Y* animals, while pupal lethal, also have smaller wing discs ([Figure 2---figure supplement 1G](#fig2s1){ref-type="fig"}), as do animals with reduced JNK signaling due to *bsk^DN^* expression ([Figure 2---figure supplement 1H--I](#fig2s1){ref-type="fig"}; *ap-Gal4, UAS-bsk^DN^*). Importantly, total body size is not affected by inhibiting JNK in the wing. Even for the smallest wings generated (*rn-Gal4, UAS-bsk^DN^*), total animal body size is not altered ([Figure 2---figure supplement 1A,E](#fig2s1){ref-type="fig"}).10.7554/eLife.11491.005Figure 2.Modulation of localized JNK signaling changes wing size.Inhibition of JNK in all wing blade cells (**B-E, J**) or within the *ptc* domain (**F, K**) decreases adult wing size compared to controls (**A, C-E, J**, *rn\>)* or (**F, K**, *ptc\>*). Note that autonomous reduction between longitudinal veins 3 and 4 accounts for a small portion of the global reduction. Apoptosis inhibition does not rescue the small wing phenotype (red, **G**, *rn\>p35, bsk^DN^*). (**H-I, L**) Increased JNK signaling within the *ptc* domain following *eiger* expression causes an increase in disc size (**I**, *ptc\>egr, RFP*, red; DAPI, blue) compared to controls (**H**, *ptc\>RFP*, red). (**L**) This is increase is dependent on *bsk (ptc\>egr, bsk^DN^*) but not affected by *diap1* or *p35* expression (*ptc\>egr, diap1* or *ptc\>egr, p35*). Due to high pupal lethality, disc size was analyzed when animals reached the wandering third instar stage. (**M-O**) JNK inhibition does not affect cell size (**N-O**, *rn\>bsk^DN^*). (**P-Q**) Increased JNK signaling within the *ptc* domain causes an increase in proliferation (**Q**, *ptc\>egr, RFP*, red; EdU, green) compared to controls (**P**, *ptc\>RFP*, red; EdU, green). EdU of boxed region in (**P**) and (**Q**) is shown in (**R**) and (**S**), respectively. (**T**) Quantification of mean EdU signal in wing pouch regions between *ptc\>RFP* and *ptc\>egr* animals. Whiskers are SD. For box plots of area quantifications, whiskers represent maximum and minimum values (**J-L**, **O**). \*-\*\*\*\*=p\<0.05--0.0001. n.s.= not significant. Bar: 50 um. See also [Figure 2---figure supplements 1](#fig2s1){ref-type="fig"}--[4](#fig2s4){ref-type="fig"}.**DOI:** [http://dx.doi.org/10.7554/eLife.11491.005](10.7554/eLife.11491.005)10.7554/eLife.11491.006Figure 2---figure supplement 1.JNK inhibition does not affect body size or cell death, but rather cell proliferation.Related to [Figure 2.](#fig2){ref-type="fig"} (**A**) Control *rotund-Gal4 (rn\>*) alone female fly (left). Inhibiting JNK in the entire wing (*rn\>bsk^DN^*) leads to a female fly with smaller, well-patterned wings (right). Black bars highlight difference in wing size. (**B-C**) Expression of a control transgene (*UAS-GFP*) does not affect wing size (*ptc\>GFP*). (**D**) Quantification of relative wing size for knockdown of *bsk* with a second RNAi line (*bsk^RNAi\#2^*). (**E**) Adult body length is not affected by inhibiting JNK by *rn-Gal4*. (**F**) Inhibition of JNK with *rn-GAL4* delays development. (**G**) *hep^r75^/Y* animals have smaller wing discs than controls (*Canton-S* or *hep^r75^/+*), even when adjusted for delayed developmental time (7d AEL). (**H-I**) JNK inhibition (red, dorsal half) causes a reduction in wing pouch size compared to its matched control (blue, ventral half) (*ap\>bsk^DN^*, red). (J) JNK inhibition (dorsal half) reduces cell proliferation by phosphorylated histone 3 (PH3) staining (green) compared to its matched control (ventral half) (*ap\>bsk^DN^*). (**K**) Control discs (*ap\>RFP*, blue) do not show a difference in PH3 staining between dorsal and ventral halves (ratio = 1.04), while JNK inhibited ones do (Ratio = 0.86, red). (**L**) Control wing pouch (*rn\>RFP*, red) stained for cleaved Caspase 3 (CCP3, green). (**M**) Inhibition of JNK in all pouch cells (*rn\>bsk^DN^, RFP*, red) does not induce apoptosis as assayed by CCP3 staining (green). (**N**) Positive control expression of wildtype JNK (*bsk^AY^*) causes apoptosis and CCP3 staining (green). Two-sided student's t-test: \*-\*\*\*p\<0.05--0.001. Bar: 50 um.**DOI:** [http://dx.doi.org/10.7554/eLife.11491.006](10.7554/eLife.11491.006)10.7554/eLife.11491.007Figure 2---figure supplement 2.Activating JNK signaling increases wing disc size independent of cell death or developmental timing.Related to [Figure 2](#fig2){ref-type="fig"}. (**A-C**) Age-matched wing discs expressing *RFP* by *ptc-GAL4* (control, A) or *RFP* and *egr* by *ptc-GAL4* (**B**). (**C**) Wing disc area quantification for A-B. (**D-F**) Induction of apoptosis in the *ptc* domain reduces wing disc size. (**D**) Control *ptc\>RFP* wing. (**E**) Expression of *UAS-hid (ptc\>hid*) decreases wing size. (**F**) Quantification of **D-E**. (**G**) Size increase due to *egr* expression depends on *bsk* activity (*ptc\>egr, bsk^DN^*), but is not affected by expression of *diap1* (**G**, *ptc\>egr, diap1*) or *p35* (**I**, *ptc\>egr, p35*). Quantification of **G-I** is presented in [Figure 2L](#fig2){ref-type="fig"}. Two-sided student's t-test: \*-\*\*p\<0.05--0.01. Bar: 100 um.**DOI:** [http://dx.doi.org/10.7554/eLife.11491.007](10.7554/eLife.11491.007)10.7554/eLife.11491.008Figure 2---figure supplement 3.JNK inhibition does not affect Dpp or EGFR signaling.Related to [Figure 2](#fig2){ref-type="fig"}. (**A-C**) Wing discs stained for the EGFR reporter pERK (green). (**A**) Control wing disc (*ap\>RFP,* red). (**B**) Inhibition of EGFR signaling in the dorsal half of the disc (*ap\>EGFR^RNAi^, RFP,* red) decreases dorsal pERK (green) staining, while (**C**) inhibition ofJNK signaling (*ap\>bsk^DN^, RFP,* red) does not. (D-F) Wing discs stained for the Dpp reporter pSMAD (green). (**D**) Control (*ap\>RFP,* red). (**E**) Inhibition of Dpp signaling in the dorsal half of the disc (*ap\>dpp^RNAi^, RFP,* red) abolishes dorsal pSMAD (green) staining, while (**F**) inhibition of JNK signaling (*ap\>bsk^DN^, RFP,* red) does not. (**G**) Quantification of pSMAD fluorescence, as a ratio of dorsal to ventral staining. *ap\>dpp^RNAi^* causes a dramatic decrease in the ratio, while JNK inhibition (*ap\>bsk^DN^*) does not produce a statistically significant change (p=0.17). (**H**) pSMAD gradient fluorescence plot by distance along the A-P axis. Ventral (blue) is control, while dorsal (red) is knockdown of *dpp*. (**I**) pSMAD gradient fluorescence plot by distance along the A-P axis. Inhibiting JNK signaling (dorsal, red) does not affect pSMAD gradient formation (compare blue to red). (**J**) Control *rn-Gal4* alone control. (**K**) RNAi-mediated knockdown of *dpp* causes a reduction in wing veins and a more pronounced effect on AP than PD length. (**L**) Inhibition of JNK does not cause wing vein loss, but does cause a global reduction in size. AFU.: arbitrary fluorescence units. Bar: 50 um.**DOI:** [http://dx.doi.org/10.7554/eLife.11491.008](10.7554/eLife.11491.008)10.7554/eLife.11491.009Figure 2---figure supplement 4.Inhibiting EGFR or Dpp signaling does not affect pJNK establishment.Related to [Figure 2](#fig2){ref-type="fig"}. Inhibition of EGFR (**A**) or Dpp (**B**) by RNAi does not have an effect on pJNK (green). Bar: 50 um.**DOI:** [http://dx.doi.org/10.7554/eLife.11491.009](10.7554/eLife.11491.009)
To test whether elevation of this signal can increase organ size, we expressed *eiger (egr*), a potent JNK activator ([@bib22]), within the *ptc* domain (*ptc-Gal4, UAS-egr*). Despite induction of cell death as previously reported ([@bib22]) and late larval lethality, we observed a dramatic increase in wing disc size without apparent duplications or changes in the shape of the disc ([Figures 2H--I and 2L](#fig2){ref-type="fig"}; *ptc-Gal4, UAS-egr*). While changes in organ size could be due to changing developmental time, wing discs with elevated JNK signaling were already larger than controls assayed at the same time point ([Figure 2---figure supplement 2A--C](#fig2s2){ref-type="fig"}; *ptc-Gal4* and *ptc-Gal4, UAS-egr*). Similarly, inhibition of JNK did not shorten developmental time ([Figure 2---figure supplement 1F](#fig2s1){ref-type="fig"}; *rn-Gal4, UAS-bsk^DN^*). Thus, changes in organ size by modulating JNK activity do not directly result from altering developmental time. Finally, the observed increase in organ size is not due to induction of apoptosis, as expression of the pro-apoptotic gene *hid* does not increase organ size ([Figure 2---figure supplement 2D--F](#fig2s2){ref-type="fig"}). In contrast, it causes a decrease in wing size ([Figure 2---figure supplement 2D--F](#fig2s2){ref-type="fig"}). Furthermore, co-expression of *diap1* or *p35* did not significantly affect the growth effect of *egr* expression (p\>0.05; [Figure 2L](#fig2){ref-type="fig"} and [Figure 2---figure supplement 2H--I](#fig2s2){ref-type="fig"}; *ptc-Gal4, UAS-egr, UAS-diap1* and *ptc-Gal4, UAS-egr, UAS-p35*), while the effect was dependent on Bsk activity (p\<0.05; [Figure 2L](#fig2){ref-type="fig"} and [Figure 2---figure supplement 2G](#fig2s2){ref-type="fig"}; *ptc-Gal4, UAS-egr, UAS-bsk^DN^*).
In stark contrast to known developmental morphogens, we did not observe any obvious defects in wing venation pattern following JNK inhibition ([Figure 2A--B](#fig2){ref-type="fig"}), suggesting that localized pJNK may control growth in a pattern formation-independent manner. Indeed, even a slight reduction in Dpp signaling results in dramatic wing vein patterning defects ([Figure 2---figure supplement 3K](#fig2s3){ref-type="fig"}). Second, inhibiting Dpp signaling causes a reduction in wing size along the A-P axis, while JNK inhibition causes a global reduction ([Figure 2---figure supplement 3J--L](#fig2s3){ref-type="fig"}). Furthermore, ectopic Dpp expression increases growth in the form of duplicated structures ([@bib60]), while increased JNK signaling results in a global increase in size ([Figure 2H--I](#fig2){ref-type="fig"}). Molecularly, we confirm that reducing Dpp signaling abolishes pSMAD staining, while quantitative data shows that inhibiting JNK signaling does not ([Figure 2---figure supplement 3D--I](#fig2s3){ref-type="fig"}). Furthermore, we also find that Dpp is not upstream of pJNK, as reduction in Dpp signaling does not affect pJNK ([Figure 2---figure supplement 4B](#fig2s4){ref-type="fig"}). Together, the molecular data are consistent with the phenotypic results indicating that pJNK and Dpp are separate programs in regulating growth. Consistent with our findings, during the revision of this manuscript, it has been suggested that Dpp does not play a primary role in later larval wing growth control ([@bib1]). Finally, we found that inhibition of JNK does not affect EGFR signaling (pERK) and that inhibition of EGFR does not affect the establishment of pJNK ([Figure 2---figure supplement 3A--C](#fig2s3){ref-type="fig"} and [4A](#fig2s4){ref-type="fig"}).
A difference in size could be due to changes in cell size and/or number. We examined wings with reduced size due to JNK inhibition and did not detect changes in cell size or apoptosis ([Figure 2M--O](#fig2){ref-type="fig"} and [Figure 2---figure supplement 1L--N](#fig2s1){ref-type="fig"}; *rn-Gal4, UAS-bsk^DN^*), suggesting that pJNK controls organ size by regulating cell number. Consistently, the cell death inhibitor *p35* was unable to rescue the decreased size following JNK inhibition ([Figure 2G](#fig2){ref-type="fig"}; *rn-Gal4, UAS-p35, UAS-bsk^DN^*). Indeed, inhibition of JNK signaling resulted in a decrease in proliferation ([Figure 2---figure supplement 1J--K](#fig2s1){ref-type="fig"}; *ap-Gal4, UAS-bsk^DN^*), while elevation of JNK signaling in the *ptc* domain resulted in an increase in cell proliferation in the enlarged wing disc ([Figure 2P--T](#fig2){ref-type="fig"}; *ptc-Gal4, UAS-egr*). Importantly, this increased proliferation is not restricted to the *ptc* domain, consistent with previous reports that JNK can promote proliferation non-autonomously ([@bib12]; [@bib35]; [@bib40]; [@bib47]; [@bib58]).
Non-canonical JNK signaling regulates size {#s2-3}
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To determine the mechanism by which pJNK controls organ size, we first considered canonical JNK signaling through its target Jun ([@bib24]). Interestingly, RNAi-mediated knockdown of *jun* in *ptc* cells does not change wing size ([Figure 3A--B](#fig3){ref-type="fig"} and [Figure 3---figure supplement 1C--F](#fig3s1){ref-type="fig"}; *ptc-Gal4, UAS-jun^RNAi\#1or2^*; Both RNAi lines can effectively inhibit *jun* activity, [Figure 3---figure supplement 1A--B](#fig3s1){ref-type="fig"}), which is consistent with previous analysis of *jun* mutant clones in the wing disc ([@bib28]). Furthermore, in agreement with this, a reporter of canonical JNK signaling downstream of *jun (puc-lacZ* \[[@bib39]\]) is not expressed in the pJNK stripe ([Figure 1---figure supplement 1F](#fig1s1){ref-type="fig"}). Finally, knockdown of *fos (kayak, kay*) alone or with *jun^RNAi^* did not affect wing size ([Figure 3---figure supplement 1G--H](#fig3s1){ref-type="fig"}; *rn-Gal4, UAS-kay^RNiA\#1or2^ and rn-Gal4, UAS-jun^RNAi\#1^, UAS-kay^RNiA\#1or2^*). Together, these data indicate that canonical JNK signaling through *jun* does not function in the pJNK stripe to regulate wing size.10.7554/eLife.11491.010Figure 3.Non-canonical JNK signaling regulates wing size.RNAi-mediated knockdown of *Jun* within the *ptc* stripe does not change adult wing size (**A-B**, red, *ptc\>jun^RNAi^* compared to blue, *ptc\>*). RNAi-mediated knockdown of *jub* does change global wing size (**C-D**, red, *ptc\>jub^RNAi^* compared to blue, *ptc\>*). Expression of *yki* in all wing cells (E-F, red, *rn\>yki, bsk^DN^* compared to blue, *rn\>*) or within the *ptc* stripe (**G-H**, red, *ptc\>bsk^DN^, yki* compared to blue, *ptc\>*) rescues wing size following JNK inhibition. RNAi-mediated knockdown or overexpression of *yki* in *ptc* cells decreases or enlarges wing size, respectively (**I-J**, red, *ptc\>yki^RNAi^*, blue, *ptc\>*, and K-L, red, *ptc\>yki*, blue, *ptc\>*). (**M-N**) Inhibition of JNK signaling does not enhance the phenotype of Yki inhibition alone (M, red, *ptc\>bsk^DN^, yki^RNAi^*; blue, *ptc\>yki^RNAi^*). (**O-P**) RNAi-mediated knockdown of *fj* modifies the Yki growth phenotype (**O**, red, *ptc\>yki, fj^RNAi^*; blue, *ptc\>yki*). For box plots, whiskers represent maximum and minimum values. \*\*\*\*=p\<0.0001. See also [Figure 3---figure supplements 1](#fig3s1){ref-type="fig"}--[2](#fig3s2){ref-type="fig"}.**DOI:** [http://dx.doi.org/10.7554/eLife.11491.010](10.7554/eLife.11491.010)10.7554/eLife.11491.011Figure 3---figure supplement 1.*Jun* RNAi line validation and loss of *kayak* phenotypes.Related to [Figure 3](#fig3){ref-type="fig"}. RNAi-mediated knockdown of *Jun* in *ap* domain cells decreases *puc* expression (*puc-lacZ*, green) (**B**) compared to controls (**A**). Dotted line indicates *puc+* cells that co-localize with *ap-Gal4*. Note decreased *puc-lacZ* staining in this domain following *Jun* inhibition. However, (**C-D**) inhibition of *Jun* in all wing cells by RNAi-mediated knockdown does not show a phenotype. (**E-F**) A second *Jun* RNAi line does not show a phenotype when expressed in *ptc*-expressing cells. (**G-H**) Inhibition of *kayak/fos* (red, *rn\>kay^RNAi^*) does not affect wing size, nor does inhibiting *jun* and *kay* together (green, *rn\>kay^RNAi^, jun^RNAi^*). Individually, *kay^RNAi^* lines induced a thorax closure defect when driven by *ap-Gal4*. For box plots, whiskers represent maximum and minimum values. Bar: 5**DOI:** [http://dx.doi.org/10.7554/eLife.11491.011](10.7554/eLife.11491.011)10.7554/eLife.11491.012Figure 3---figure supplement 2.JNK interacts with Yki to cause global changes in wing size.Related to [Figure 3](#fig3){ref-type="fig"}. (**A**) Schematic for measuring the ratio of anterior to posterior wing area. (**B**) Local (*ptc*-driven) inhibition of JNK or increased Yki expression affects the anterior and posterior compartments equally. (**C-D**) The effect of inhibiting JNK signaling can be partially suppressed in a *lats* heterozygous mutant background (**C**, red, *rn\>bsk^DN^; lats^e26-1^/+*). (**G-H**) Inhibition of *fj* alone does not change wing size (**G**, red, *ptc\>fj^RNAi^*, blue, *ptc\>*), albeit it slightly changes wing shape, likely due to its effect on polarity. (**I-J**) Over-expression of *fj* causes a decrease in wing size (**I**, red, *ptc\>fj*). For box plots, whiskers are maximum and minimum values. Two-sided student's t-test: \*-\*\*\*\*p\<0.05--0.0001.**DOI:** [http://dx.doi.org/10.7554/eLife.11491.012](10.7554/eLife.11491.012)
In search of such a non-canonical mechanism of JNK-mediated size control, we considered the Hippo pathway. JNK signaling regulates the Hippo pathway to induce autonomous and non-autonomous proliferation during tumorigenesis and regeneration via activation of the transcriptional regulator Yorkie (Yki) ([@bib3]; [@bib12]; [@bib47]). Recently it has been shown that JNK activates Yki via direct phosphorylation of Jub ([@bib48]). To test whether this link between JNK and Jub could account for the role of localized pJNK in organ size control during development, we performed RNAi-mediated knockdown of *jub* in the *ptc* stripe, and observed adults with smaller wings ([Figure 3C--D](#fig3){ref-type="fig"}; *ptc-Gal4, UAS-jub^RNAi\#1or2^*). Indeed, the effect of JNK loss on wing size can be partially suppressed in a heterozygous *lats* mutant background ([Figure 3---figure supplement 2C--D](#fig3s2){ref-type="fig"}; *rn-Gal4, UAS-bsk^DN^, lats^e26-1^/+*) and increasing downstream *yki* expression in all wing cells ([Figure 3E--F](#fig3){ref-type="fig"}; *rn-Gal4, UAS-yki, UAS-bsk^DN^*) or just within the *ptc* domain ([Figure 3G--H](#fig3){ref-type="fig"}; *ptc-Gal4, UAS-yki, UAS-bsk^DN^*) can rescue wing size following JNK inhibition. These results suggest that pJNK controls Yki activity autonomously within the *ptc* stripe, leading to a global change in cell proliferation. This hypothesis predicts that the Yki activity level within the *ptc* stripe influences overall wing size. Consistently, inhibition of JNK in the *ptc* stripe translates to homogeneous changes in anterior and posterior wing growth ([Figure 3---figure supplement 2A--B](#fig3s2){ref-type="fig"}). Similarly, overexpression or inhibition of Yki signaling in the *ptc* stripe also results in a global change in wing size ([Figure 3I--L](#fig3){ref-type="fig"} and [Figure 3---figure supplement 2A--B](#fig3s2){ref-type="fig"}; *ptc-Gal4, UAS-yki; ptc-Gal4, UAS-yki^RNAi^*).
It is important to note that the *yki* expression line used is wild-type Yki, which is still affected by JNK signaling. For this reason, the epistasis experiment was also performed with activated Yki, which is independent of JNK signaling (*UAS-yki^S111A,S168A,S250A.V5^;* [@bib34]). Expression of this activated Yki in the *ptc* stripe caused very large tumors and lethality (data not shown). Importantly, inhibiting JNK in this context did not affect the formation of these tumors or the lethality (data not shown; *ptc-Gal4, UAS-yki^S111A,S168A,S250A.V5^, UAS-bsk^DN^*). Furthermore, inhibiting both JNK and Yki together does not enhance the phenotype of Yki inhibition alone ([Figure 3M--N](#fig3){ref-type="fig"} and [Figure 3---figure supplement 2E--F](#fig3s2){ref-type="fig"}; *ptc-Gal4, UAS-yki^RNAi^, UAS-bsk^DN^ and ptc-Gal4, UAS-yki^RNAi^, UAS-puc*), further supporting the idea that Yki is epistatic to JNK, instead of acting in parallel processes.
Mutants of the Yki downstream target *four-jointed (fj*) have small wings with normal patterning, and *fj* is known to propagate Hippo signaling and affect proliferation non-autonomously ([@bib2]; [@bib20]; [@bib46]; [@bib53]; [@bib56]). Although RNAi-mediated knockdown of *fj* in *ptc* cells does not cause an obvious change in wing size, it is sufficient to block the Yki-induced effect on increasing wing size ([Figure 3O--P](#fig3){ref-type="fig"} and [Figure 3---figure supplement 2G--H](#fig3s2){ref-type="fig"}*; ptc-Gal4, UAS-yki, UAS-fj^RNAi^*and *ptc-Gal4, UAS-fj^RNAi^*). However, overexpression of *fj* also reduces wing size, which makes it not possible to test for a simple epistatic relationship (*ptc-Gal4, UAS-fj*; [Figure 3---figure supplement 2I--J](#fig3s2){ref-type="fig"}). Overall, these data are consistent with the notion that localized pJNK regulates wing size not by Jun-dependent canonical JNK signaling, but rather by Jun-independent non-canonical JNK signaling involving the Hippo pathway.
Hh sets up pJNK by elevating *dTRAF1* expression {#s2-4}
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While morphogens direct both patterning and growth of developing organs ([@bib50]), a link between patterning molecules and growth control pathways has not been established ([@bib41]). pJNK staining is coincident with *ptc* expression ([Figure 1G](#fig1){ref-type="fig"}), suggesting it could be established by Hh signaling. During development, posterior Hh protein travels across the A/P boundary, leading to activation of the transcription factor Cubitus interruptus (Ci) in the stripe of anterior cells ([@bib11]; [@bib42]). To test whether localized activation of JNK is a consequence of Hh signaling through Ci, we performed RNAi-mediated knockdown of *ci* and found that the pJNK stripe is eliminated ([Figure 4A--B](#fig4){ref-type="fig"}; *ptc-Gal4, UAS-ci^RNAi\#1or2^*). Consistently, adult wing size is globally reduced ([Figures 4D and 4G](#fig4){ref-type="fig"}). In contrast, we do not observe a change in pJNK stripe staining following RNAi-mediated knockdown of *dpp* or *EGFR* ([Figure 2---figure supplement 4A--B](#fig2s4){ref-type="fig"}). Expression of non-processable Ci leads to increased Hh signaling ([@bib38]). Expression of this active Ci in *ptc* cells leads to an increase in pJNK signal and larger, well-patterned adult wings ([Figures 4C,E](#fig4){ref-type="fig"}, and 4G; *ptc-Gal4, UAS-Ci^ACT^*). The modest size increase shown for *ptc\>Ci^ACT^* is likely due to the fact that higher expression of this transgene (at 25°C) leads to such large wings that pupae cannot emerge from their cases. For measuring wing size, this experiment was performed at a lower temperature (20°C, see Experimental Genotypes) so that the animals were still viable. Furthermore, inhibition of JNK in wings expressing active Ci blocks Ci's effects, and resulting wings are similar in size to JNK inhibition alone ([Figure 4F--G](#fig4){ref-type="fig"}*; ptc-Gal4, UAS-Ci^ACT^, UAS-bsk^DN^*). Together, these data indicate that Hh signaling through Ci is responsible for establishing the pJNK stripe.10.7554/eLife.11491.013Figure 4.Hh signaling through Ci establishes localized pJNK.RNAi-mediated knockdown of *Ci* in *ptc* cells abrogates pJNK (green) staining (**A-B**, *ptc\>Ci^RNAi^, RFP* compared to *ptc\>RFP*) and results in smaller adult wings (**D**, red, *ptc\>Ci^RNAi^* compared to blue, *ptc\>*). Expression of activated *Ci* in the *ptc* domain leads to increased pJNK staining (green) (**C**, *ptc\>Ci^ACT^, RFP*) and a larger wing (**E**, red, *ptc\>Ci^ACT^* compared to blue, *ptc\>*). Inhibition of JNK signaling in these cells blocks the effect of activated Ci (red, F, *ptc\>Ci^ACT^, bsk^DN^).* For the box plot (**G**), whiskers represent maximum and minimum values. \*\*\*-\*\*\*\*=p\<0.001--0.0001. Bar: 50 um.**DOI:** [http://dx.doi.org/10.7554/eLife.11491.013](10.7554/eLife.11491.013)
To determine the mechanism by which Ci activates the JNK pathway, we compared transcriptional profiles of posterior (red, *hh+*) and *ptc* domain cells (green, *ptc+*) isolated by FACS from third instar wing discs ([Figure 5A](#fig5){ref-type="fig"}; Materials and methods). Of the total 12,676 unique genes represented on the microarray, 50.4% (6,397) are expressed in *ptc* domain cells, posterior cells, or both (log~2~ normalized expression ≥6.5; [Figure 5---figure supplement 1A--D](#fig5s1){ref-type="fig"}; [Supplementary file 1](#SD1-data){ref-type="supplementary-material"}; Materials and methods). We thresholded on a false discovery rate \<0.01 and fold change ≥1.5 and found that 5.7% (363) of expressed genes were upregulated in *ptc* cells and 3.8% (242) were downregulated ([Figure 5---figure supplement 1D](#fig5s1){ref-type="fig"}; [Supplementary file 2](#SD2-data){ref-type="supplementary-material"}; Materials and methods). Hh pathway genes known to be differentially expressed are identified ([Figure 5B](#fig5){ref-type="fig"}). We next asked whether any JNK pathway genes are differentially expressed and found that *dTRAF1* expression is more than five-fold increased in *ptc* cells ([Figure 5C](#fig5){ref-type="fig"}), while other JNK pathway members are not differentially expressed ([Figure 5C](#fig5){ref-type="fig"}; [Supplementary file 1](#SD1-data){ref-type="supplementary-material"}; [Supplementary file 2](#SD2-data){ref-type="supplementary-material"}).10.7554/eLife.11491.014Figure 5.Hedgehog signaling establishes pJNK by elevating *dTRAF1* expression.(**A**) *ptc* cells (green, *ptc+*) and posterior cells (red, *hh+*) from third instar wing discs were dissociated and sorted by FACS. RNA was isolated and hybridized to microarrays. Differentially expressed genes were identified. (**B**) Hedgehog pathway genes known to be differentially expressed are identified. Genes upregulated in *ptc* cells (*ptc+*) compared to posterior (*hh+*) cells are highlighted in green and downregulated in red. Genes with log~2~ normalized expression ≥6.5 are considered expressed. (**C**) JNK pathway gene *dTRAF1* is \>5-fold upregulated in *ptc* cells. (**D-I**) RNAi-mediated knockdown of *dTRAF1* eliminates pJNK (green) staining (**E**, *ptc\>dTRAF^RNAi\#1^, RFP*, red) and leads to smaller adult wings (**F-I**, *rn\>dTRAF^RNAi\#1^* or *ptc\>dTRAF^RNAi\#1^*). (**J**) Ci inhibition causes a \~30% decrease in *dTRAF1* expression in 3^rd^ instar wing discs, relative to endogenous control *Rp49*. Whiskers are SD. For box plots, whiskers are maximum and minimum values (**H-I**). \*-\*\*\*\*=p\<0.05--0.0001. Bar: 50 um. See also [Figure 5---figure supplement 1](#fig5s1){ref-type="fig"}--[2](#fig5s2){ref-type="fig"}.**DOI:** [http://dx.doi.org/10.7554/eLife.11491.014](10.7554/eLife.11491.014)10.7554/eLife.11491.015Figure 5---figure supplement 1.Transcriptional profiling quality control and additional dTRAF1 validation.Related to [Figure 5](#fig5){ref-type="fig"}. Quality assessment analyses were conducted with post-normalized data. (**A**) Microarrays cluster by condition, indicating that biological effects are driving variability. (**B**) Principle components analysis similarly groups biological replicates. Outliers were not detected in (**A**) or (**B**). (**C**) Density plots of the log~2~ normalized expression in *ptc* domain (*ptc+*) or posterior (*hh+*) samples are very similar in shape and range, further suggesting comparable signal quality between the two arrays. Probe sets with a median log~2~ normalized expression ≥6.5 in *ptc*+ samples, *hh*+ cells, or both, were considered expressed ([Supplementary file 1](#SD1-data){ref-type="supplementary-material"}; Materials and methods). This corresponds to 6854 genic probe sets (47.3%). (**D**) Quantile-quantile plot for the differential expression analysis. Based on a criteria of minimum fold change ≥1.5 and false discovery rate (FDR) ≤0.01, 624 of 6,854 genic probe sets (9.1%) are differentially expressed, with 376 (5.5%) upregulated in *ptc+* samples (green) and 248 (3.6%) downregulated in *ptc+* samples (red, [Supplementary file 2](#SD2-data){ref-type="supplementary-material"}; Materials and methods). The dashed line indicates the tuning parameter, *delta*, which is chosen according to the specified FDR (≤0.01). Inhibition of *dTRAF1* expression by a second RNAi line also abolishes pJNK staining (**E**, *ptc\>dTRAF^RNAi\#2^*, and (**F**) leads to a smaller adult wing (red) compared to control (blue). (**G**) Quantification of adult wing size. (**H**) Multiple Ci binding sites (red lines) are identified within the *dTRAF1* gene region (green). Height of red line indicates significance of the binding site. Boxes indicate exons, and arrowed lines indicate introns in the direction of transcription. For box plot, whiskers represent maximum and minimum values. \*\*\*\*=p\<0.0001. Bar: 50 um.**DOI:** [http://dx.doi.org/10.7554/eLife.11491.015](10.7554/eLife.11491.015)10.7554/eLife.11491.016Figure 5---figure supplement 2.Inhibiting *dTRAF1* can modify an activated Ci phenotype.Related to [Figure 5.](#fig5){ref-type="fig"} (**A**) Compared to control wings (blue, *ptc\>*), inhibiting *dTRAF1* while activating Ci still leads to a *dTRAF1* phenotype of a smaller wing (red, *ptc\>Ci^ACT^, dTRAF1^RNAi^*). Compare to [Figure 4E,G](#fig4){ref-type="fig"}. For box plot, whiskers represent maximum and minimum values. \*\*\*=p\<0.001.**DOI:** [http://dx.doi.org/10.7554/eLife.11491.016](10.7554/eLife.11491.016)
*dTRAF1* is expressed along the A/P boundary ([@bib37]) and ectopic expression of *dTRAF1* activates JNK signaling ([@bib9]). Thus, positive regulation of *dTRAF1* expression by Ci could establish a stripe of pJNK that regulates wing size. Indeed, we identified Ci binding motifs in the *dTRAF1* gene ([Figure 5---figure supplement 1H](#fig5s1){ref-type="fig"}), and a previous large-scale ChIP study confirms a Ci binding site within the *dTRAF1* gene (Chr2L: 4367100- 4371393; \[[@bib5]\]). Consistently, a reduction in *Ci* led to a 29% reduction in *dTRAF1* expression in wing discs ([Figure 5J](#fig5){ref-type="fig"}; *ptc-Gal4, UAS-Ci^RNAi^*). Given that the reduction of *dTRAF1* expression in the *ptc* stripe is buffered by Hh-independent *dTRAF1* expression elsewhere in the disc ([@bib37]), this 29% reduction is significant. Furthermore, inhibition of *dTRAF1* by RNAi knockdown abolished pJNK staining ([Figure 5D--E](#fig5){ref-type="fig"} and [Figure 5---figure supplement 1E](#fig5s1){ref-type="fig"}; *ptc-Gal4, UAS-dTRAF1^RNAi\#1or2^*). Finally, these animals have smaller wings without obvious pattern defects ([Figure 5F--I](#fig5){ref-type="fig"} and [Figure 5---figure supplement 1F--G](#fig5s1){ref-type="fig"}). Conversely, overexpression of *dTRAF1* causes embryonic lethality (*ptc-Gal4, UAS-dTRAF1*), making it not possible to attempt to rescue a *dTRAF1* overexpression wing phenotype by knockdown of *bsk*. Nevertheless, it has been shown that dTRAF1 function in the eye is Bsk-dependent ([@bib9]). Finally, inhibition of *dTRAF1* modulates the phenotype of activated Ci signaling (*ptc-Gal4, UAS-dTRAF1^RNAi^, UAS-Ci^ACT^*; [Figure 5---figure supplement 2](#fig5s2){ref-type="fig"}). Together, these data reveal that the pJNK stripe in the developing wing is established by Hh signaling through Ci-mediated induction of *dTRAF1* expression.
Localized pJNK controls antenna and leg size {#s2-5}
--------------------------------------------
Finally, we detected localized centers of pJNK activity during the development of other imaginal discs including the eye/antenna and leg ([Figures 6A and 6G](#fig6){ref-type="fig"}). Inhibition of localized JNK signaling during development caused a decrease in adult antenna size ([Figures 6B--C and 6F](#fig6){ref-type="fig"}; *dll-Gal4, UAS-bsk^DN^*) and leg size ([Figures 6H--I and 6L](#fig6){ref-type="fig"}; *dll-Gal4, UAS-bsk^DN^*). Conversely, increasing JNK signaling during development resulted in pupal lethality; nevertheless, overall sizes of antenna and leg discs were increased ([Figures 6D--E and 6J--K](#fig6){ref-type="fig"}; *dll-Gal4, UAS-egr*). Together, these data indicate that localized JNK signaling regulates size in other organs in addition to the wing, suggesting a more universal effect of JNK on size control.10.7554/eLife.11491.017Figure 6.Modulation of localized JNK signaling within the developing antenna or leg changes organ size.pJNK (green) staining of wildtype antenna/eye (**A**) and leg (**G**) third instar discs. Inhibition of JNK in the developing antenna (**B-C, F**, *dll\>bsk^DN^*) or leg (**H-I, L**, *dll\>bsk^DN^)* leads to a smaller adult organ. Increased JNK activation within the antenna (**D-E**, *dll\>egr, RFP*, red) or leg disc (**J-K**, *dll\>eg*r*, RFP*, red) causes an increase in disc size. (**M**) Model of how localized JNK signaling regulates wing size during development. Engrailed (En) controls Hh signaling, leading to a stripe of active Ci along the A/P boundary. Ci increases transcription of *dTRAF1*, activating JNK (pJNK, green). JNK acts in a non-canonical, Jun-independent manner to regulate Yki or Yki-dependent signaling. As the human *dTRAF1* homolog, *TRAF4*, and Hippo components are amplified in numerous cancers, these findings provide a new mechanism for how the Hh pathway could contribute to tumorigenesis ([@bib8]; [@bib21]). For box plots, whiskers represent maximum and minimum values (**F, L**). \*\*\*\*=p\<0.0001. Bar: 100 um**DOI:** [http://dx.doi.org/10.7554/eLife.11491.017](10.7554/eLife.11491.017)
Intrinsic mechanisms of organ size control have long been proposed and sought after ([@bib7]; [@bib54]). Our study reveals that in developing *Drosophila* tissues, localized, organ-specific centers of JNK signaling contribute to organ size in an activity level-dependent manner. Such a size control mechanism is qualitatively distinct from developmental morphogen mechanisms, which affect both patterning and growth ([@bib60]). Aptly, this mechanism is still integrated in the overall framework of developmental regulation, as it is established in the wing by the Hh pathway ([Figure 6M](#fig6){ref-type="fig"}). Our data indicate that localized JNK signaling is activated by Ci-mediated induction of *dTRAF1* expression. Furthermore, we discovered that it is not canonical Jun-dependent JNK signaling, but rather non-canonical JNK signaling that regulates size, possibly through Jub-dependent regulation of Yki signaling, as described for regeneration ([@bib48]) ([Figure 6M](#fig6){ref-type="fig"}). As the human *dTRAF1* homolog, *TRAF4*, and Hippo components are amplified in numerous cancers ([@bib8]; [@bib21]), these findings provide a new mechanism for how the Hh pathway could contribute to tumorigenesis. More importantly, these findings offer a new strategy for potential cancer therapies, as reactivating Jun in Hh-driven tumors could lead tumor cells towards an apoptotic fate.
Materials and methods {#s3}
=====================
*Drosophila* stocks and husbandry {#s3-1}
---------------------------------
Fly crosses were maintained at 25°C on standard cornmeal-molasses media unless otherwise indicated (see Experimental Genotypes). When possible, crosses were established so that every experimental animal had an in-vial *Gal4* alone control. For experiments that necessitated precise developmental staging, 2 hr egg lays were conducted on apple juice agar plates with yeast paste. For all other experiments, females were allowed to lay eggs on standard media for 24 hr, after which they were removed and progeny were considered as 12 +/- 12 hr after egg lay. The following stocks were utilized: (1) *Canton-S* (02) *y, hep^r75^, FRT10.1/FM7iGFP* ([@bib18]) (2) *Ubi-GFP, FRT10.1;; hs-FLP, MKRS/TM6B* (3) *UAS-puc* (III) ([@bib30]) (4) *w; ap-GAL4, UAS-src-RFP; Sb/TM6B* (5) *w; ptc-GAL4, UAS-src-RFP; Sb/TM6B* (6) *UAS-bsk^RNAi^*(II and III) VDRC 34138 ([@bib36]) and BDSC 32977 (7) *w, UAS-bsk^DN^ (X)* (8) *w;; UAS-bsk^DN^/TM6B* (9) *w;; rn-GAL4/TM6B* (10) *y, UAS-p35; Adv/CyO; Sb/TM6B* (11) *w; Sp/CyO; UAS-egr/MKRS* (12) *UAS-diap1* (III) BDSC 6657 (13) *UAS-bsk^AY^* (II) BDSC: 6407 (14) *UAS-Ci^RNAi^*(II and III) BDSC 31236 and 31236 (15) *UAS-Ci5m/TK-GFP* ("*UAS-Ci^ACT^*") ([@bib38]) (16) *puc^E69^/TM6B* ("*puc-lacZ*") ([@bib39]) (17) *UAS-dTRAF1^RNAi^*(X and III) VDRC 21213 and 21214 (18) *UAS-jun^RNAi^* (III) BDSC 31595 and VDRC 10835 (19) *UAS-kay^RNAi\#1^* (III) BDSC 33379 and 31322 (20) *UAS-jub^RNAi^*(III and II) BDSC 32923 and 41938 (21) *y,w;; lats^e26-1^/TM6B* (22) *yw; UAS-yki.GFP; Sb/TM6B* BDSC 28815 ([@bib33]) (23) *UAS-yki^RNAi^/TM3* BDSC 31965 (24) *UAS-fj^RNAi^/TM3* BDSC 28009 (25) *UAS-fj.V5* (III) BDSC 44252 (26) *w; dll-Gal4, UAS-src.RFP/CyO* (27) *UAS-dpp^RNAi^*(III) BDSC 25782 (28) *UAS-EGFR^RNAi^*(III) BDSC 25781 (29) *UAS-yki^S111A.S168A.S250A.V5^*(III) BDSC 28817
Imaginal disc staining {#s3-2}
----------------------
Antibody staining was performed according to standard procedures for imaginal discs. The following antibodies were used: rabbit PhosphoDetect^TM^ anti-SAPK/JNK (pThr^183^, pTyr^185^) (1:100, Calbiochem, immunogenic sequence is 100% identical to *D. melanogaster bsk/JNK*), rabbit anti-ACTIVE® JNK (1:100, Promega, immunogenic sequence is 100% identical to *D. melanogaster bsk/JNK*), rabbit anti-cleaved-caspase 3 (1:250, Cell Signaling), mouse anti-betagalactosidase (1:500, Sigma), rabbit anti-pERK (1:75, Cell Signaling), rabbit anti-pSMAD (1:75, Cell Signaling), rabbit anti-phosphorylated histone 3 (1:250, Cell Signaling), goat Alexa-488-conjugated anti-rabbit IgG (1:250, Invitrogen), goat Alexa-488-conjugated anti-mouse IgG (1:250, Invitrogen), goat Alexa-555-conjugated anti-rabbit IgG (1:250, Invitrogen). EdU staining was performed according to established protocol ([@bib19]) using the Click-iT EdU cell proliferation assay kit (Invitrogen), Grace's Media (Invitrogen) and a 10 min EdU incubation.
Imaginal disc imaging {#s3-3}
---------------------
Imaginal discs to be imaged by confocal microscopy were mounted in Vectashield mounting media with DAPI (Vector Labs). Confocal images were taken with a Zeiss LSM510 Meta confocal microscope or a Leica TCS SP8 STEAD 3X confocal microscope with 405nm, 488nm, 561nm, and 633nm lasers. Both microscopes gave similar results. Measurements of disc size were performed from images of at least fifteen discs using NIH Image-J software.
Western blot analysis {#s3-4}
---------------------
Whole *Canton-S* and *hep^r75/Y^* larvae were lysed in standard RIPA buffer with protease and phosphatase inhibitors. Proteins were separated by SDS-PAGE using a 4--15% acrylamide gel (BioRad), transferred for 1 hr at 4°C, and probed with primary antibodies: rabbit anti-pJNK (Calbiochem, 1:1000) and mouse anti-alpha tubulin (Sigma, 1:4000). HRP-conjugated secondary antibodies (anti-rabbit and anti-mouse) were used at 1:5000. ECL (Pierce) was used for detection with film.
Adult organ imaging {#s3-5}
-------------------
Adult wings, legs, or antenna were dissected in 70% ethanol, mounted in Permount mounting media (Fisher Scientific), and imaged with a Leica DFC300FX camera on a Leica MZ FLIII stereomicroscope. Measurements of wing size were performed from images of twenty to sixty female flies using NIH Image-J software. Wing images were false-colored and overlayed to scale using Adobe Photoshop CS3 software. Cell size was measured by dividing the number of hairs (1 hair/cell) by a set area using Adobe Photoshop CS3 software. Mean EdU signal was measured in Adobe Photoshop CS3. Measurements of antenna or leg size were performed from images of at least twenty male flies for each genotype using NIH Image-J software.
Statistical analysis {#s3-6}
--------------------
To determine whether differences in area were statistically significant, two-sided student's t-tests were performed using raw data values, matched for temperature and sex. Box plots were generated where whiskers represent maximum and minimum, a plus sign indicates the mean, a horizontal line within the box indicates the median, and the box represents the 25--75% quartile range. Both parametric and non-parametric analyses were performed, and p-values less than 0.05 were considered significant. Data are presented as relative to the mean of the matched *Gal4*-alone control.
Gene expression profiling {#s3-7}
-------------------------
For each of three biological replicates, 200 pairs of wing imaginal discs were dissected from third instar larvae of the genotypes *hh-Gal4; UAS-mCD8GFP* or *ptc-Gal4; UAS-mCD8GFP*. Discs were stored in Schneider\'s *Drosophila* Media (21720, Invitrogen) plus 10% FBS (10438, Invitrogen) on ice for less than two hours prior to cell dissociation. Discs were washed twice with 1 ml cell dissociation buffer (Sigma, C-1544). Elastase (Sigma, E-0258) was diluted to 0.4 mg/ml in fresh cell dissociation buffer once discs were ready. Discs were incubated for 20 min at room temperature in 0.4 mg/ml elastase with stirring by a magnetic micro stirring bar. Undissociated tissue was spun out, cell viability was measured using the Beckman Vi-CELL Cell Viability Analyzer (\>80%), and cells were immediately isolated using the BD FACSAria II system within the Stanford FACS facility. Dead cells labeled with propidium iodide (P3566, Invitrogen) were excluded during FACS, and purity of sorted cells was greater than 99% by post-sorting FACS analysis. Total RNA was extracted from sorted cells (RNeasy, Qiagen), quality was assessed with the Agilent Bioanalyzer 2100 (RIN \> 7.0), and microarray analysis was performed in the Stanford Protein and Nucleic Acid Facility (Affymetrix *D. mel* GeneChip Genome 2.0 microarrays).
Identification of differentially expressed genes {#s3-8}
------------------------------------------------
All analyses were conducted in R version 3.1.1 (2014-07-10). Expression values were determined using the *affy* package ([@bib17]), available from BioConductor (<http://bioconductor.org>). The automatically downloaded *Drosophila* 2.0 CDF environment was utilized. Probe level data from the *CEL* files were imported using the function *ReadAffy* and converted to expression values using the function *rma* with default settings. This method implements robust multi-array average (RMA) for background correction followed by quantile normalization. PM correction was not performed. Probe level expression values were combined into probe set expression measures using *medianpolish,* the standard summary method employed in RMA ([@bib25]). Expression values are log~2~ transformed.
Post-normalization microarray quality assessment was conducted using the *arrayQualityMetrics* package ([@bib27]), available from BioConductor. Default settings were used, with *ptc* domain (*ptc+*) versus posterior (*hh+*) as the covariate in *intgroup*. Biological replicates cluster together in a dendrogram of inter-array difference, estimated as the mean absolute difference between the data of the arrays ([Figure 5---figure supplement 1A](#fig5s1){ref-type="fig"}), indicating that biological effects are stronger than any batch effects. Similarly, principle components analysis also separates biological replicates into two clusters ([Figure 5---figure supplement 1B](#fig5s1){ref-type="fig"}). Outliers were not detected by either of these methods.
Probe sets were mapped to genes using the *drosophila2.db* annotation package (version 3.0.0), available from BioConductor. 14,481 of 18,952 (76.4%) probe sets map to gene isoforms---12,676 (87.5%) of which correspond to unique genes (some genes are mapped by ≥1 probe set). In order to minimize technical artifacts, probe sets mapping to the same gene were not combined.
Based on the distribution observed in the density plot of normalized probe set expression values, probe sets (genes) with median log~2~ expression value ≥6.5 in at least one condition (*ptc+* and/or *hh+*) were considered to be expressed ([Figure 5---figure supplement 1C](#fig5s1){ref-type="fig"}). According to these criteria, 7,228 of 18,952 probe sets (38.1%) are expressed. This corresponds to 6,854 of 14,481 gene isoforms (47.3%), which corresponds to 6,397 of 12,676 unique genes (50.4%, [Figure 5---figure supplement 1D](#fig5s1){ref-type="fig"}, [Supplementary file 1](#SD1-data){ref-type="supplementary-material"}).
To identify probe sets (genes) differentially expressed between *ptc+* and posterior (*hh+*) samples, we used the *samr* package, an R implementation of significance analysis of microarrays ([@bib51]). This package is available from CRAN (<http://cran.r-project.org/>). Only expressed probe sets mapping to genes (6,854) were considered in this analysis. Differentially expressed probe sets were identified with the function *SAM*, using a two class unpaired response type, the t-statistic as the test statistic, and a false discovery rate (FDR) threshold of 0.01. The maximum number of possible permutations (720) was used. To ensure these results are biologically meaningful, we further trimmed this list to probe sets with a minimum 1.5 fold change between *ptc+* and *hh+* cells. Based on these criteria, 624 of 6,854 probe sets (9.1%) are differentially expressed, with 376 (5.5%) upregulated in *ptc+* samples and 248 (3.6%) downregulated in *ptc+* samples ([Figure 5---figure supplement 1D](#fig5s1){ref-type="fig"}, [Supplementary file 2](#SD2-data){ref-type="supplementary-material"}). A gene was considered differentially expressed if any mapped probe set was differentially expressed. Therefore, of the 6,397 unique expressed genes, 604 (9.4%) are differentially expressed, 363 (5.7%) upregulated and 242 (3.8%) downregulated. One gene, *Tie*, was mapped by probe sets both up- and down-regulated. The quantile-quantile plot in [Figure 5---figure supplement 1D](#fig5s1){ref-type="fig"} was prepared using the *samr.plot* function.
Real-time polymerase chain reaction {#s3-9}
-----------------------------------
Total RNA was extracted from third instar wing discs from *ptc-Gal4* or *ptc-Gal4, UAS-Ci^RNAi ^*animals using a standard TriZol extraction. RNA was reverse transcribed using the iScript cDNA Synthesis Kit (Bio-Rad) according to manufacturer's instructions. *dTRAF1* expression was quantified relative to *Rp49 (RpL32*- FlyBase, endogenous control) by real-time PCR performed in triplicate using the SYBR Green fast kit (Applied Biosystems) and an Applied Biosystems machine according to the manufacturer's instructions. The following primers were used: *dTRAF1*, 5'-GCACTCCATCACCTTCACAC-3' and 5'-TAGCTGATCTGGTTCGTTGG-3'; *Rp49*, 5′-GGCCCAAGATCGTGAAGAAG-3′ and 5′-ATTTGTGCGACAGCTTAGCATATC-3′.
Transcription factor binding site analysis {#s3-10}
------------------------------------------
The *Drosophila* Ci positional weight matrix from the BioBase TRANSFAC database was queried against the *Drosophila melanogaster* genome with a p-value \<0.0001 (chosen based on known Ci binding sites within *ptc*) using FIMO (MEME) and aligned back to the UCSC genome browser.
Experimental genotypes {#s3-11}
----------------------
### Crosses were maintained at 25°C unless otherwise indicated {#s3-11-1}
[Figure 1](#fig1){ref-type="fig"}: (B-C) *Canton-S* (D-E) *y, hep*^*r75*^*, FRT10.1 /Y* (F) *y, hep*^*r75*^*, FRT10.1/Ubi-GFP, FRT10.1;; hs-FLP, MKRS/+* (G) *w/+; ptc-GAL4, UAS-src.RFP/+* (H) *w; ptc-GAL4, UAS-src.RFP; UAS-puc* 29°C (I) *w; ptc-GAL4, UAS-src.RFP/UAS-bsk*^*RNAi*^ 29°C
[Figure 1---figure supplement 1](#fig1s1){ref-type="fig"}: (A-C, G-H) *Canton-S*, (D-F) *puc*^*E69*^*/+ *(I) *w; ap-Gal4/+; UAS-puc/+* (J) *w; ptc-Gal4, UAS-src.RFP/+* (K) *w/yv, UAS-bsk*^*RNAi\#1*^*/UAS-src.RFP; rn-Gal4/+* 29°C (L) *w/yv; ptc-Gal4, UAS-src.RFP/+; UAS-bsk*^*RNAi\#2*^*/+*
[Figure 2](#fig2){ref-type="fig"}: (A) *w/+;; rn-Gal4/+* (B) *w/w, UAS-bsk*^*DN*^*;; rn-Gal4/UAS-bsk*^*DN*^ (C) Blue: *w/+;; rn-Gal4/+* Red: *w/w, UAS-bsk*^*DN*^*;; rn-Gal4/UAS-bsk*^*DN*^(D) Blue: *w/+;; rn-Gal4/+* 29°C Red: *w; UAS-bsk*^*RNAi\#1*^*/+; rn-Gal4/+* 29°C (E) Blue: *w/+;; rn-Gal4/+* 29°C Red: *w;; rn-Gal4, UAS-puc/UAS-puc* 29°C (F) Blue: *w/+; ptc-Gal4, UAS-src.RFP/+; Sb/+* Red: *w, UAS-bsk*^*DN*^*/w; ptc-GAL4, UAS-src.RFP/Sp; UAS-bsk*^*DN*^*/Sb* (G) *w, UAS-bsk*^*DN*^*/w, UAS-p35;; rn-GAL4/UAS-bsk*^*DN*^ 29°C (H, P, R) *w/+; ptc-GAL4, UAS-src.RFP/+* (I, Q, S) *w; ptc-GAL4, UAS-src.RFP/+*, *UAS-egr/+* (M) *w/+;; rn-Gal4/+* (N) *w/w, UAS-bsk*^*DN*^*; Sp/+; rn-Gal4/UAS-bsk*^*DN*^
[Figure 2---figure supplement 1](#fig2s1){ref-type="fig"}: (A) Left: *w/+;; rn-Gal4/+* 25°C Right: *w/w, UAS-bsk*^*DN*^*;; rn-Gal4/UAS-bsk*^*DN*^25°C (B) Blue: *w/+; ptc-Gal4, UAS-src.RFP/+; Sb/+* Red: *w/+; ptc-Gal4, UAS-src.RFP/+; Sb/UAS-GFP* (H, J) *w, UAS-bsk*^*DN*^*/w; ap-Gal4, UAS-src.RFP/+; UAS-bsk*^*DN*^*/+* 29°C (L) *w/+;; rn-Gal4/+* 29°C (M) *w/w, UAS-bsk*^*DN*^*;; rn-Gal4/UAS-bsk*^*DN *^29°C (N) *w/+; UAS-bsk*^*AY*^*/+; rn-Gal4/+*
[Figure 2- figure supplement 2](#fig2s2){ref-type="fig"}: (A) *w/+; ptc-Gal4, UAS-src.RFP/+* 6 days AEL (B) *w/+; ptc-Gal4, UAS-src.RFP/+; UAS-egr/Sb* 6 days AEL (D) *w/+; ptc-Gal4, UAS-src.RFP/+* (E) *w/UAS-hid; ptc-Gal4, UAS-src.RFP/+* (G) *w, UAS-bsk*^*DN*^*/w; ptc-Gal4, UAS-src.RFP/+; UAS-egr/UAS-bsk*^*DN*^ (H) *w/+; ptc-Gal4, UAS-src.RFP/UAS-diap1; UAS-egr/Sb* (I) *w/w, UAS-p35; ptc-Gal4, UAS-src.RFP/+; UAS-egr/Sb*
[Figure 2---figure supplement 3](#fig2s3){ref-type="fig"}: (A, D) *w; ap-GAL4/UAS-src.RFP* (B) *w; ap-GAL4/UAS-src.RFP; UAS-EGFR*^*RNAi*^*/+* (C, F) *w/w, UAS-bsk*^*DN*^*; ap-GAL4/UAS-src.RFP; UAS-bsk*^*DN*^*/+* (E) *w; ap-GAL4/UAS-src.RFP; UAS-dpp*^*RNAi*^*/+* (J) *w/+;; rn-Gal4/+* (K) *w;; UAS-dpp*^*RNAi*^*/rn-Gal4* (L) *w/w, UAS-bsk*^*DN*^*;; rn-Gal4/UAS-bsk*^*DN*^
[Figure 2---figure supplement 4](#fig2s4){ref-type="fig"}: (A) *w/yv; ptc-Gal4, UAS-src.RFP/+; UAS-EGFR*^*RNAi*^*/+* (B) *w/yv; ptc-Gal4, UAS-src.RFP/+; UAS-dpp*^*RNAi*^*/+*
[Figure 3](#fig3){ref-type="fig"}: (A) Blue: *w/+; ptc-Gal4, UAS-src.RFP/+* Red: *w/+; ptc-Gal4, UAS-src.RFP/+ UAS-jun*^*RNAi\#1*^*/+* (C) Blue: *w/+; ptc-Gal4, UAS-src.RFP/+* 29°C Red: *w/+; ptc-Gal4, UAS-src.RFP/+; UAS-jub*^*RNAi\#1*^*/+* 29°C (E) Blue: *w/+;; rn-GAL4/+* 29°C Red: *w/w, UAS-bsk*^*DN*^*; UAS-yki.GFP/+; rn-GAL4/UAS-bsk*^*DN *^29°C (G) Blue: *w/+; ptc-Gal4, UAS-src.RFP/+* 29°C Red: *w/w, UAS-bsk*^*DN*^*; ptc-Gal4, UAS-src.RFP/UAS-yki.GFP; UAS-bsk*^*DN*^*/+* 29°C (I) Blue: *w/+; ptc-Gal4, UAS-src.RFP/+* Red: *w/+; ptc-Gal4, UAS-src.RFP/+; UAS-yki*^*RNAi\#1*^*/+* (K) Blue: *w/+; ptc-Gal4, UAS-src.RFP/+* 29°C Red: *w/+; ptc-Gal4, UAS-src.RFP/UAS-yki.GFP* 29°C (M) Blue: *w/+; ptc-Gal4, UAS-src.RFP/+; UAS-yki*^*RNAi\#1*^*/+* Red: *w/ UAS-bsk*^*DN*^*; ptc-Gal4, UAS-src.RFP/+; UAS-yki*^*RNAi\#1*^*/ UAS-bsk*^*DN*^ (O) Blue: *w/+; ptc-Gal4, UAS-src.RFP/UAS-yki.GFP* 29°C Red: *w/+; ptc-Gal4, UAS-src.RFP/UAS-yki.GFP; UAS-fj*^*RNAi*^*/+* 29°C
[Figure 3---figure supplement 1](#fig3s1){ref-type="fig"}: (A) *w/+; ap-Gal4, UAS-src.RFP/+; puc*^*E69*^*/+* (B) *w/+; ap-Gal4, UAS-src.RFP/UAS-jun*^*RNA\#1i*^*; puc*^*E69*^*/+* (C) Blue: *w/+;; rn-Gal4/+* Red: *w/+; UAS-jun*^*RNAi\#1*^*/+; rn-Gal4/+* (E) Blue: *w/+; ptc-Gal4, UAS-src.RFP/+* Red: *w/+; ptc-Gal4, UAS-src.RFP/UAS-jun*^*RNAi\#2 *^(G) Blue: *w/+;; rn-Gal4/+* Red: *w/+;; rn-Gal4/UAS-kay*^*RNAi *^Green: *w/+; UAS-jun*^*RNAi*^*/+; rn-Gal4/UAS-kay*^*RNAi*^
[Figure 3---figure supplement 2](#fig3s2){ref-type="fig"}: (C) Blue: *w/+;; rn-Gal4/+* Red: *w/w, UAS-bsk*^*DN*^*; UAS-bsk*^*DN*^*/+; rn-Gal4/lats*^*e26-1 *^(E) *w; ptc-Gal4, UAS-src.RFP/+; UAS-yki*^*RNAi\#1*^*/UAS-puc* (G) Blue: *w/+; ptc-Gal4, UAS-src.RFP/+* 29°C Red: *w/+; ptc-Gal4, UAS-src.RFP/+; UAS-fj*^*RNAi*^*/+* 29°C (I) Blue: *w/+; ptc-Gal4, UAS-src.RFP/+* Red: *w/+; ptc-Gal4, UAS-src.RFP/UAS-fj; Sb/+*
[Figure 4](#fig4){ref-type="fig"}:(A) *w/+; ptc-Gal4, UAS-src.RFP/+* (B) *w/yv; ptc-Gal4, UAS-src.RFP/+; UAS-Ci*^*RNAi*^*/+* (C) *w/+; ptc-Gal4, UAS-src.RFP/+; UAS-Ci*^*ACT*^*/+* (D) Blue: *w/+; ptc-Gal4, UAS-src.RFP/+* 20°C Red: *w/yv; ptc-Gal4, UAS-src.RFP/+; UAS-Ci*^*RNAi*^*/+* 20°C (E) Blue: *w/+; ptc-Gal4, UAS-src.RFP/+* 20°C Red: *w/+; ptc-Gal4, UAS-src.RFP/+; UAS-Ci*^*ACT*^*/+* 20°C (F) Blue: *w/+; ptc-Gal4, UAS-src.RFP/+; UAS-Ci*^*ACT*^*/+* 20°C Red: *w/UAS-bsk*^*DN*^*; ptc-Gal4, UAS-src.RFP/+; UAS-Ci*^*ACT*^*/UAS-bsk*^*DN*^ 20°C
[Figure 5](#fig5){ref-type="fig"}: (D) *w/+; ptc-Gal4, UAS-src.RFP/+* 29°C (E) *w/+; ptc-Gal4, UAS-src.RFP/+; UAS-dTRAF1*^*RNAi\#1*^*/+* 29°C (F) Blue: *w/+;; rn-Gal4/+* 29°CRed: *w/+;; UAS-dTRAF1*^*RNAi\#1*^*/rn-Gal4* 29°C (G) Blue: *w/+; ptc-Gal4, UAS-src.RFP/+* 29°C Red: *w/+; ptc-Gal4, UAS-src.RFP/+; UAS-dTRAF1*^*RNAi\#1*^*/+* 29°C
[Figure 5---figure supplement 1](#fig5s1){ref-type="fig"}: (E) *UAS-dTRAF1*^*RNAi\#2*^*/Y; ptc-Gal4, UAS-src.RFP/+; Sb/+* 29°C (F) Blue: *w/+; ptc-Gal4, UAS-src.RFP/+* 29°C Red: *w/UAS-dTRAF1*^*RNAi\#2*^*; ptc-Gal4, UAS-src.RFP/+* 29°C
[Figure 5---figure supplement 2](#fig5s2){ref-type="fig"}: (A) Blue: *w/+; ptc-Gal4, UAS-src.RFP/+* 20°C Red: *w/+; ptc-Gal4, UAS-src.RFP/+; UAS-Ci*^*ACT*^*/UAS-dTRAF1*^*RNAi\#1*^ 20°C
[Figure 6](#fig6){ref-type="fig"}: (A, G) *Canton-S* (B, D, H, J) *w; dll-Gal4, UAS-src.RFP/+* (C, I) *UAS-bsk*^*DN*^*/Y; dll-Gal4, UAS-src.RFP/+; UAS-bsk*^*DN*^/+ (E, K) *w; dll-Gal4, UAS-src.RFP/+; UAS-egr/+*
Funding Information
===================
This paper was supported by the following grants:
- http://dx.doi.org/10.13039/100000002National Institutes of Health R01CA069408-20 to Tian Xu.
- http://dx.doi.org/10.13039/100000002National Institutes of Health Graduate Student Training Fellowship to Helen Rankin Willsey.
- http://dx.doi.org/10.13039/100000011Howard Hughes Medical Institute HHMI Investigators to Philip A Beachy, Tian Xu.
- http://dx.doi.org/10.13039/100000002National Institutes of Health K99 to Xiaoyan Zheng, Philip A Beachy.
We thank the Vienna Drosophila RNAi Collection, the Transgenic RNAi Project, the Bloomington Drosophila Stock Center, and T Kornberg for fly stocks; R Nandez for technical assistance; R Harland for lab space, A Giraldez, V Greco, and L Cooley for helpful discussions. This work was supported in part by training grant T32 GM007499 to HRW and by award R01 CA069408-20 from the NIH/NCI to TXTX and PAB are Howard Hughes Medical Institute Investigators.
Additional information {#s4}
======================
The authors declare that no competing interests exist.
HRW, Performed all experiments except the microarray, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article.
XZ, Performed the gene expression profiling and suggested its use in the analysis, Acquisition of data, Contributed unpublished essential data or reagents.
JCP-P, Conception and design, Analysis and interpretation of data, Drafting or revising the article.
AJW, Identified differentially expressed genes, Analysis and interpretation of data, Drafting or revising the article.
PAB, Performed the gene expression profiling and suggested its use in the analysis, Conception and design, Drafting or revising the article, Contributed unpublished essential data or reagents.
TX, Conception and design, Analysis and interpretation of data, Drafting or revising the article.
Additional files {#s5}
================
10.7554/eLife.11491.018
###### Genes expressed in posterior (*hh+*) and/or *ptc* domain wing disc cells.
**DOI:** [http://dx.doi.org/10.7554/eLife.11491.018](10.7554/eLife.11491.018)
10.7554/eLife.11491.019
###### Differentially expressed genes between posterior (*hh+*) and *ptc* domain wing disc cells.
**DOI:** [http://dx.doi.org/10.7554/eLife.11491.019](10.7554/eLife.11491.019)
10.7554/eLife.11491.020
Decision letter
McNeill
Helen
Reviewing editor
The Samuel Lunenfeld Research Institute
,
Canada
In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.
Thank you for submitting your work entitled \"Localized JNK Signaling Regulates Organ Size During Development\" for consideration by *eLife*. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors and the evaluation has been overseen by the Reviewing Editor and Fiona Watt as the Senior Editor.
The reviewers have discussed the reviews with one another and the Reviewing editor has drafted this decision to help you prepare a revised submission.
Summary:
In this manuscript, Willsey et al. report a role for localized JNK signaling in regulating *Drosophila* organ size, particularly the developing wing. Mechanistically, Hh signaling from the posterior compartment activates JNK signaling in the A/P boundary by Ci-mediated transcriptional up-regulation of *dTRAF1*. The activated JNK signaling promotes cell proliferation and wing growth through Jun-independent, Jub-dependent Yki activation. While the connection between JNK and Yki has been previously reported, the findings of localized JNK signaling in organ size control and activation of JNK by Hh-Ci pathway are novel and important. However, there are concerns that should be addressed before this manuscript is considered for publication.
Essential revisions:
1\) To verify the specificity of antibody, the authors used *rn-Gal4* and *ptc-Gal4* to knock down *bsk* ([Figure 1I](#fig1){ref-type="fig"}, [Figure 1---figure supplement 1K--L](#fig1s1){ref-type="fig"}). Actually *ap-Gal4* should be used in this situation, as it is expressed only in the dorsal compartment, and the ventral part could serve as an internal control. Similarly, *ap-Gal4* should be used to knock down *hep*, since some p-JNK staining still presents in the *hep* mutant clone ([Figure 1F](#fig1){ref-type="fig"}\').
2\) A control UAS line (e.g. UAS-LacZ or *UAS -GFP*) should be included to exclude the possibility that expression of any protein by *ptc*- or *rn-Gal4* may disturb wing development and affect wing size.
3\) Is there any effect on wing disc size by blocking JNK activity?
4\) Does *ptc*\>*egr*-induced wing disc enlargement depends on Bsk?
5\) Does *ptc*\>*egr* increases the adult wing size?
6\) To show the increased *ptc*\>*egr* wing disc size is not a consequence of apoptosis, authors should block apoptosis by expressing p35.
7\) A positive control should be included to validate CCP3 staining ([Figure 2---figure supplement 1H, I](#fig2s1){ref-type="fig"}).
8\) There is no evidence that the endogenous JNK signaling regulates cell proliferation. The authors should check cell proliferation (Edu staining) in *rn*- or *ptc*\>*bsk^DN^* discs. The non-cell autonomous increase of cell proliferation in *ptc*\>*egr* discs could be triggered by caspase activation, rather than a direct outcome of JNK activation. To discriminate the two possibilities, *diap1* should be added to block caspase activation.
9\) Though Jun is not required by JNK to regulate wing size, what about Fos?
10\) The effect of *UAS-Yki* on *UAS-bsk^DN^* could be additive effect, but not rescue. The authors should check whether *ptc*\> or *rn*\>*bsk^DN^*-induced small wing phenotype could be suppressed in heterozygous *lats* mutants.
11\) *UAS-bsk^DN^* is probably not strong enough to enhance the *ptc*\>*yki^RNAi^*phenotype, what about *UAS-puc*?
12\) Though *fj* appears to be involved in ectopic Yki-triggered wing growth, is it required by endogenous *bsk* and *yki* to regulate wing growth? How do the authors explain this? Is *fj* acting via control of Ft or Ds in this process? To prove this point more clearly, the authors should overexpress *fj* with PtcGal4. Also, does the *fj-lacZ* reporter show a stripe pattern similar to pJNK in the 3rd instar larval wing disc?
13\) Does *dTRAF1^RNAi^* block CiACT-triggered wing growth?
14\) Does expression of *dTRAF1* increase wing size in a Bsk-dependent manner?
15\) Surprisingly, knockdown of Jun did not affect wing size-the authors invoke a non-canonical pathway, but could the knockdown have been incomplete? Also, is there redundancy with Kayak? Similarly, the argument that this signaling is non-canonical is based on the *puc-lacZ* reporter- could this instead be due to delayed reporter activity, or reduced sensitivity?
\[Editors\' note: further revisions were requested prior to acceptance, as described below.\]
Thank you for resubmitting your work entitled \"Localized JNK Signaling Regulates Organ Size During Development\" for further consideration at *eLife*. Your revised article has been favorably evaluated by Fiona Watt (Senior editor), a Reviewing editor, and two reviewers, one of whom is a member of our Board of Reviewing Editors. The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:
The authors have addressed all the concerns well, except two below.
1\) Does expression of *dTRAF1* increase wing size in a Bsk-dependent manner?
The authors responded that ptc\>*dTRAF1* is lethal. What about other *Gal4* drivers, e.g. *rn-Gal4, ap-Gal4*? What Cha et al. showed is that *dTRAF1*-induced cell death depends on JNK. It remains unknown whether *dTRAF1* regulates JNK-dependent cell proliferation and growth. The question is quite crucial for this manuscript.
2\) Redundancy of Kayak. Can the authors provide evidence of the efficacy of their Kayak knockdown. The provided experiment showing that double knockdown of Kayak and Jun has no effect on growth is only worthwhile if the knockdowns are effective.
10.7554/eLife.11491.021
Author response
1\) To verify the specificity of antibody, the authors used rn-Gal4 and ptc-Gal4 to knock down bsk ([Figure 1I](#fig1){ref-type="fig"}, [Figure 1---figure supplement 1K-L](#fig1s1){ref-type="fig"}). Actually ap-Gal4 should be used in this situation, as it is expressed only in the dorsal compartment, and the ventral part could serve as an internal control. Similarly, ap-Gal4 should be used to knock down hep, since some p-JNK staining still presents in the hep mutant clone ([Figure 1F](#fig1){ref-type="fig"}\').
We thank the reviewers for this suggestion. We now use *ap-Gal4* to inhibit JNK signaling in the dorsal compartment and see a specific reduction in pJNK in those cells (*ap*\>*puc*). This data is now included in the text and figures (paragraph one, subheading "JNK is active in the developing Drosophila wing pouch"; [Figure 1---figure supplement 1I](#fig1s1){ref-type="fig"}). *hep^r75^* (used in the clonal analysis) is likely not a null allele since hemizygous mutant embryos complete dorsal closure, which likely explains the minor residual pJNK staining in the mutant clones. Unfortunately none of the available *UAS-hep^RNAi^* lines are strong enough to induce the known *hep* mutant phenotype of a split thorax, nor are they strong enough to abolish pJNK staining (data not shown). So unfortunately we cannot perform this experiment precisely as requested. However, we note that we have done 10 independent experiments to validate the specificity of the antibody and are confident in its fidelity.
2\) A control UAS line (e.g. UAS-LacZ or UAS -GFP) should be included to exclude the possibility that expression of any protein by ptc- or rn-Gal4 may disturb wing development and affect wing size.
We now show that expression of *UAS-GFP* by *ptc-GAL4* does not affect wing size (*ptc*\>*GFP*). We have now included the new data in the text and in the figures (paragraph one, subsection "Localized JNK activity regulates global wing size"; [Figure 2---figure supplement 1B--C](#fig2s1){ref-type="fig"}).
3\) Is there any effect on wing disc size by blocking JNK activity?
We now show that blocking JNK causes a reduction in wing disc size (*ap*\>*bsk^DN^*). We have included the new data in the text and in the figures (paragraph one, aforementioned subsection; [Figure 2---figure supplement 1H--I](#fig2s1){ref-type="fig"}).
4\) Does ptc\>egr-induced wing disc enlargement depends on Bsk?
We now show that *ptc*\>*egr*-induced disc enlargement depends on *bsk (ptc*\>*egr, bsk^DN^*). In fact, these discs are significantly smaller than even control discs (p= 0.0078). We have now included the new data in the text and in the figures (paragraph two, aforementioned subsection; [Figure 2L](#fig2){ref-type="fig"}, disc image in [Figure 2---figure supplement 2G](#fig2s2){ref-type="fig"}).
5\) Does ptc\>egr increases the adult wing size?
We tried to assess whether *ptc*\>*egr* causes an increase in adult wing size, but these animals were larval lethal. The overgrowth of the disc likely precludes proper pupation. We have now added this point to the text (paragraph two, aforementioned subsection).
6\) To show the increased ptc\>egr wing disc size is not a consequence of apoptosis, authors should block apoptosis by expressing p35.
We now show that expression of *UAS-p35* with *UAS-egr* does not abolish the size effect of *UAS-egr (ptc*\>*egr, p35*). We have now included the new data in the text and in the figures (paragraph two, aforementioned subsection; [Figure 2L](#fig2){ref-type="fig"}, disc image in [Figure 2---figure supplement 2I](#fig2s2){ref-type="fig"}).
7\) A positive control should be included to validate CCP3 staining ([Figure 2---figure supplement 1H, I](#fig2s1){ref-type="fig"}).
We now show that expression of *UAS-bsk^AY^*, a constitutively active JNK allele, induces CCP3 staining in the wing. We have now included the new data in the figures ([Figure 2---figure supplement 1N](#fig2s1){ref-type="fig"}).
8\) There is no evidence that the endogenous JNK signaling regulates cell proliferation. The authors should check cell proliferation (Edu staining) in rn- or ptc\>bsk^DN^ discs. The non-cell autonomous increase of cell proliferation in ptc\>egr discs could be triggered by caspase activation, rather than a direct outcome of JNK activation. To discriminate the two possibilities, diap1 should be added to block caspase activation.
We now show that inhibiting JNK signaling causes a reduction in proliferation by phosphorylated histone 3 staining (*ap*\>*bsk^DN^*). We have included this new data in the text and in the figures (paragraph four, aforementioned subsection; [Figure 2---figure supplement 1J-K](#fig2s1){ref-type="fig"}). We now also show that expression of *UAS-diap1* does not block the growth effect of *UAS-egr (ptc*\>*egr, diap1*). We have included this new data in the text and in the figures (paragraph two, same subsection; [Figure 2L](#fig2){ref-type="fig"}, disc image in [Figure 2---figure supplement 2H](#fig2s2){ref-type="fig"}).
9\) Though Jun is not required by JNK to regulate wing size, what about Fos?
We now show that inhibiting Fos does not alter wing size (*rn*\>*kay^RNAi\#1,2^*). We have now included the new data in the text and in the figures (paragraph one, subsection "Non-canonical JNK signaling regulates size"; [Figure 3---figure supplement 1G-H](#fig3s1){ref-type="fig"}).
10\) The effect of UAS-Yki on UAS-bsk^DN^ could be additive effect, but not rescue. The authors should check whether ptc\> or rn\>bsk^DN^-induced small wing phenotype could be suppressed in heterozygous lats mutants.
We now show that the *rn*\>*bsk^DN^* wing phenotype can be partially suppressed in a heterozygous *lats* mutant background (*rn*\>*bsk^DN^; lats^e2b-1/+^*). We have now included the new data in the text and in the figures (paragraph two, same subsection; [Figure 3---figure supplement 2C--D](#fig3s2){ref-type="fig"}).
11\) UAS-bsk^DN^ is probably not strong enough to enhance the ptc\>yki^RNAi^ phenotype, what about UAS-puc?
We now show that *UAS-puc* does not enhance the *ptc*\>*yki^RNAi^*phenotype (*ptc*\>*yki^RNAi^, puc*). We have now included the new data in the text and in the figures (paragraph three, same subsection; [Figure 3---figure supplement 2E--F](#fig3s2){ref-type="fig"}).
12\) Though fj appears to be involved in ectopic Yki-triggered wing growth, is it required by endogenous bsk and yki to regulate wing growth? How do the authors explain this? Is fj acting via control of Ft or Ds in this process? To prove this point more clearly, the authors should overexpress fj with PtcGal4. Also, does the fj-lacZ reporter show a stripe pattern similar to pJNK in the 3rd instar larval wing disc?
We have now overexpressed *fj* and found it also reduces wing size. Therefore, we cannot simply conclude that *fj* is required by endogenous Bsk and/or Yki to regulate growth. We have now made this clear in the text and figures (paragraph four, same subsection; [Figure 3---figure supplement 2I--J](#fig3s2){ref-type="fig"}).
*fj-lacZ* is known to be present in a gradient in the wing disc, highest at the A/P and D/V boundaries, emanating distally (Villano and Katz, 1995). Overall, signaling downstream of Yki is intricate and has not been worked out.
13\) Does dTRAF1^RNAi^ block CiACT-triggered wing growth?
We now show that *UAS-dTRAF^RNAi^* can modulate Ci^ACT^-triggered wing growth (*ptc*\>Ci^ACT^, *dTRAF^RNAi^*), further strengthening our finding. We have now included the new data in the text and in the figures (paragraph three, subsection "Hh sets up pJNK by elevating *dTRAF1* expression"; [Figure 5---figure supplement 2](#fig5s2){ref-type="fig"}).
14\) Does expression of dTRAF1 increase wing size in a Bsk-dependent manner?
We tried to determine whether expression of *UAS-dTRAF1* in the *ptc* domain increases wing size in a Bsk-dependent manner, but unfortunately expressing *UAS-dTRAF1* is lethal. Nevertheless, it has been shown that *dTRAF1* function in the eye is Bsk-dependent (Cha et al., 2003). We have now included the new data and discussed this in the text (p. 11-2, para. 2, line 257-259).
15\) Surprisingly, knockdown of Jun did not affect wing size-the authors invoke a non-canonical pathway, but could the knockdown have been incomplete? Also, is there redundancy with Kayak? Similarly, the argument that this signaling is non-canonical is based on the puc-lacZ reporter- could this instead be due to delayed reporter activity, or reduced sensitivity?
Null mutant clones of *jun* do not show a phenotype in the wing (Kockel et al., 1997). Furthermore, *puc-lacZ* is both a sensitive and quick JNK signaling reporter, as indicated by its fast and robust response to JNK activation (McEwen and Peifer, 2005). We note that *UAS-jun^RNAi^* is strong enough to show an effect on *puc-lacZ* expression in the stalk region of the wing ([Figure 3---figure supplement 1A--B](#fig3s1){ref-type="fig"}). We now show that inhibition of *kayak* does not have an effect on wing size (*rn*\>*kay^RNAi^*), and is not redundant with Jun (*rn*\>*jun^RNAi^, kay^RNAi^*). These data are consistent with previous reports that *jun/fos* do not control wing growth (Kockel et al., 1997). We have now included the new data and discussed this in the text (Paragraph one, subsection "Non-canonical JNK signaling regulates size"; [Figure 3---figure supplement 1G--H](#fig3s1){ref-type="fig"}).
*\[Editors\' note: further revisions were requested prior to acceptance, as described below.\] 1) Does expression of dTRAF1 increase wing size in a Bsk-dependent manner?*
*The authors responded that ptc\>dTRAF1 is lethal. What about other Gal4 drivers, e.g. rn-Gal4, ap-Gal4? What Cha et al. showed is that dTRAF1-induced cell death depends on JNK. It remains unknown whether dTRAF1 regulates JNK-dependent cell proliferation and growth. The question is quite crucial for this manuscript.*
The other *Gal4* drivers mentioned (*rn-Gal4* and *ap-Gal4*) express *Gal4* in many more cells than *ptc-Gal4*, so over-expression of *dTRAF1* will certainly be lethal. We show that *dTRAF1* is required for growth and cell proliferation, as inhibition of *dTRAF1* in the wing leads to a small wing phenotype and a loss of pJNK staining ([Figure 5D--I](#fig5){ref-type="fig"}). Indeed, *dTRAF1* null mutants fail to grow during the larval stages and have very small imaginal discs (Cha et al., [Figure 5](#fig5){ref-type="fig"}). This loss of function experiments show that in addition to cell death, *dTRAF1* is involved in regulation of growth.
*2) Redundancy of Kayak. Can the authors provide evidence of the efficacy of their Kayak knockdown. The provided experiment showing that double knockdown of Kayak and Jun has no effect on growth is only worthwhile if the knockdowns are effective.*
These kayak RNAi lines induced the typical JNK-phenotype, thorax closure defect, when driven by *ap-Gal4*, confirming their efficacy. We have added this in the legend of [Figure 3---figure supplement 1](#fig3s1){ref-type="fig"}.
[^1]: Department of Anatomy and Regenerative Biology, School of Medicine and Health Sciences, The George Washington University, Washington, United States.
[^2]: School of Life Sciences, Tsinghua University, Beijing, China.
| {
"pile_set_name": "PubMed Central"
} |
Published: October 25, 2019
Introduction {#sec1}
============
In animal cells, cytokinesis leads to the separation of the cytoplasm into two daughter cells. Defects during this process can lead to polyploidy and aneuploidy, situations that are frequently observed in cancers ([@bib15]). Cytokinesis begins in anaphase with the formation of an actomyosin ring, which constricts the plasma membrane of the cells, finally leading to the formation of an intercellular bridge ([@bib28]). Future daughter cells are connected by this intercellular bridge for several hours before individual assortment by a process termed abscission, which generally occurs in the G1 phase of the cell cycle ([@bib16]). At the center of the intercellular bridge is the midbody, a crucial component of the abscission process. It plays a role as the recruitment platform for abscission and acts as a stabilizer of the intercellular bridge ([@bib28]). After abscission, the midbody remnant (MR) is asymmetrically inherited by one of the two daughters cells and then degraded by autophagic or non-autophagic lysosomal pathways ([@bib7], [@bib9]).
Centrosomal protein 55 kDa (CEP55) is a key regulator of cytokinetic abscission. This protein is predominantly an α-helical coiled-coil protein, which is able to oligomerize ([@bib43], [@bib5], [@bib29], [@bib13]). CEP55 is essential for the recruitment of the [E]{.ul}ndosomal [s]{.ul}orting [c]{.ul}omplex [r]{.ul}equired for the [t]{.ul}ransport (ESCRT) machinery to the intercellular bridge ([@bib29], [@bib5], [@bib6], [@bib25]). CEP55 directly interacts with two components of the ESCRT machinery, [AL]{.ul}G-2 [i]{.ul}nteracting protein [X]{.ul} (ALIX) and the ESCRT-I [T]{.ul}umor [s]{.ul}usceptibility [g]{.ul}ene 101 (TSG101), via its atypical α-helical coiled-coil dimeric region called EABR ([E]{.ul}SCRT-and [A]{.ul}LIX-[b]{.ul}inding [r]{.ul}egion). Once recruited at the midbody through CEP55, the ESCRT complex orchestrates membrane remodeling events leading to the physical separation of the two daughter cells. In addition to its function in cytokinesis, CEP55 has also a central role in the midbody integrity maintenance ([@bib43]) and in the MR degradation ([@bib12], [@bib23]).
CEP55 is recruited to the midbody in late mitosis ([@bib2]). This recruitment depends on the Mitotic kinesin-like protein 1 (MKLP-1), a component of the Centralspindlin complex, which has been shown to co-immuno-precipitate with CEP55 under endogenous conditions as well as in reticulocyte lysates after *in vitro* transcription/translation of both proteins ([@bib43], [@bib2]). It has been reported that CEP55 become phosphorylated during mitosis on its S425 and S428 residues by Cyclin-dependent kinase 1 (Cdk1) and Extracellular signal-regulated kinase 2 (Erk2), whereas its S436 residue is phosphorylated by Polo-like kinase 1 (PLK1) ([@bib13], [@bib2]). The PLK1 phosphorylation prevents CEP55 early recruitment to the mitotic spindle and its accumulation to the midbody, and its impairment leads to severe abscission defects and inhibition of the ESCRT machinery recruitment to the midbody ([@bib2], [@bib20]). It has been proposed that PLK1 could indirectly interact with CEP55 via the formation of a quaternary complex, which involves Myotubularin-related proteins 3 and 4 (MTMR3 and MTMR4) ([@bib37]). Both these myotubularins were shown to be important in the abscission process regulating CEP55 recruitment to the midbody ([@bib37]). Importantly, a severe human embryonic pathology called MARCH (multinucleated neurons, anhydramnios, renal dysplasia, cerebellar hypoplasia, and hydranencephaly) was recently associated with a deletion of the last 40 amino acids in CEP55, leading to a severe defect of CEP55 recruitment to the midbody ([@bib14]).
The post-translational modification of proteins by mono-ubiquitin or poly-ubiquitin chains of variable length and/or different linkage types is involved in a vast range of cellular processes, including autophagy, DNA repair, protein turnover, and many signal transduction events such as the nuclear factor (NF)-κB signaling pathway ([@bib21]). These chains of ubiquitin are specifically recognized by proteins that contain ubiquitin-binding domains (UBDs), allowing a large number of effector proteins to translate the modifications into specific outcomes. In the NF-κB cascade, NEMO ([N]{.ul}F-κB [E]{.ul}ssential [Mo]{.ul}dulator) contains two UBDs, which non-covalently bind to hybrid M1/K63 poly-ubiquitin chains to trigger the NF-κB activation ([@bib11]). The NEMO UBD referred to as NOA/UBAN domain (also called NUB or CC2-LZ) forms a long parallel α-helical coiled-coil dimer, which selectively binds to a Methionine 1 (M1) di-ubiquitin chain ([@bib34]). The second UBD is a classical CCHC-type zinc finger (ZF), which adopts a ββα fold and interacts with a weak preference to lysine 63 (K63) and M1 over lysine 48 (K48) poly-ubiquitin chains ([@bib8], [@bib32]).
During cytokinesis, the E2/E3 ubiquitin ligase BRUCE (BIR repeat containing ubiquitin-conjugating enzyme), the deubiquitinases USP8 (Ubiquitin specific peptidase 8), and AMSH (Associated molecule with the SH3-domain of STAM) were reported to be located at the midbody and play key roles in the cytokinetic abscission process ([@bib33]) ([@bib30]). However, despite an accumulation of mono-ubiquitin and different types of poly-ubiquitin chains at the midbody and intercellular bridge, little is known about the role of ubiquitin signaling during the abscission process ([@bib33], [@bib30], [@bib27]).
Here, we identified CEP55 as a member of the NEMO-related protein family. Indeed, we show that CEP55, like NEMO, contains two UBDs, namely, NOA and ZF, which differently regulate CEP55 functions. Both domains bind to poly-ubiquitin chains *in vitro*, but NOA is specifically involved in the abscission process, whereas we showed that ZF is critical for CEP55 recruitment to the midbody and is therefore crucial in CEP55 functions at the midbody. Altogether our data reveal a critical role for non-degradative ubiquitination linked to CEP55 function in the last steps of cell division.
Results {#sec2}
=======
CEP55 Contains Two Ubiquitin-Binding Domains, which Are Structurally Similar to NOA and ZF UBDs of NEMO {#sec2.1}
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The NEMO function depends on two UBDs in its C-terminal part, referred to as the NOA/UBAN domain ([@bib24], [@bib41]) and the ZF domain ([@bib8], [@bib32]). The NOA domain forms a long parallel α-helical coiled-coil dimer ([@bib34]), whereas the ZF domain exhibits an UBZ (Ubiquitin-binding zinc finger) architecture based on a classical ββα fold ([@bib8]). Multiple sequence alignments including analysis of protein sequence databases, combined with the Paircoil2 algorithm, predict that the CEP55 protein contains in its C-terminal part two UBDs. We refer to these as NOA (residues 304--396, 38% similarity) and ZF (residues 435--464, 47% similarity), respectively, owing to their significant homologies with the same domains in NEMO, Optineurin and ABIN2 ([Figures 1](#fig1){ref-type="fig"}A, [S1](#mmc1){ref-type="supplementary-material"}A, and S1B). To test this hypothesis, we first expressed in *E*.*coli* and purified to homogeneity a His-tag version of the human CEP55 NOA fragment (residues 304--396) ([Figure 1](#fig1){ref-type="fig"}B inset). Circular dichroism spectrum analysis in the far-UV showed that CEP55 His-NOA exhibits a high α-helical content of 80%, compatible with an α-helical coiled-coil structure ([Figure 1](#fig1){ref-type="fig"}B). To non-ambiguously determine the oligomeric state of CEP55 His-NOA in solution, we performed size exclusion chromatography experiments coupled in line with multi-angle light scattering (SEC-MALS), and showed that CEP55 His-NOA behaves mainly as monomers and dimers, with a very small amount of higher-order oligomers (3% of hexamers, [Figure 1](#fig1){ref-type="fig"}C). Based on these biochemical experiments, we generated a structural model of the NOA^CEP55^ using NOA^NEMO^ as the structural template ([Figure S1](#mmc1){ref-type="supplementary-material"}C) and observed that the C359 residue of one monomer is in close proximity with the C359 residue of the second monomer when NOA^CEP55^ forms an α-helical coiled-coil dimer ([Figure S1](#mmc1){ref-type="supplementary-material"}E). We utilized this ability of the CEP55 His-NOA construct to form an intermolecular disulfide bond at C359 to further confirm its dimeric state. As shown in [Figure S1](#mmc1){ref-type="supplementary-material"}F, CEP55 His-NOA migrated at the dimeric molecular mass in the absence of the reducing agent dithioerythritol (DTE), whereas it migrated at the monomeric molecular mass upon treatment with DTE, further confirming that CEP55 His-NOA likely forms a long parallel α-helical coiled-coil dimer like that of NEMO. However, this isolated NOA^CEP55^ domain appears to exhibit a much weaker dimerization affinity than that of NEMO NOA (6-fold less), which has been previously shown to have a dimerization constant around 30 μM ([@bib19]).Figure 1CEP55 Contains NOA and ZF Domains(A) Schematic representation of structural and functional domains of CEP55 and NEMO showing a similar C-terminal architecture encompassing two ubiquitin-binding NOA and ZF domains. Sequence numbering is given for the human proteins ([NP_001093327.1](ncbi-p:NP_001093327.1){#intref0040} and [NP_001120654.1](ncbi-p:NP_001120654.1){#intref0045}). KBD, Kinase binding domain; CC1-2, Coiled-coil 1 and 2; HLX2, Helical domain 2; EABR, ESCRT and ALIX binding region; NOA, NEMO, Optineurin and Abin domain (also called UBAN, CoZi, CC2-LZ and NUB); ZF, Zinc Finger; UBD, Ubiquitin-binding domain. Both EABR and NOA domains form parallel coiled-coil dimers.(B) Normalized far-UV circular dichroism spectrum of purified CEP55 His-NOA (0.03 mg/mL at 1°C), showing a high α-helix content compatible with a coiled-coil structure. In inset is depicted the SDS-PAGE analysis of CEP55 His-NOA after staining with Coomassie blue.(C) Analysis of CEP55 His-NOA by size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS). M, Monomer; D, Dimer; H, Hexamer.(D) Fluorescence emission spectra of CEP55 ZF V438W peptide (2 μM, hereafter denoted as ZF (W)) in the presence (black) or in absence of zinc ion (gray) after excitation of both tyrosine (Y447 and Y461) and tryptophan (W438) residues at 280 nm. Note that the emission shoulder peak observed in the 300--315 nm region, which is only observed in absence of zinc ion, reflects tyrosine fluorescence emission (Y447, Y461). This shoulder peak disappears and vanishes to the profit of tryptophan emission after ion zinc addition, owing to an increase of Tyr to Trp internal FRET. Inset: model of CEP55 ZF (W) pointing out that the zinc-induced folding brings close together the two Y447, Y461 and W438 residues, leading to internal FRET.(E) Fluorescence titration of CEP55 ZF (W) peptide (0.8 μM) using increasing zinc concentrations (0.1--1.4 μM) at excitation and emission wavelengths of 280 and 342 nm, respectively. Error bars represent the standard deviation of the fluorescence measurements of each titration point for 5 min.
To investigate whether the human CEP55 fragment containing the residues 435--464 adopts a zinc-finger fold, we generated a synthetic peptide mimicking this region and containing the V438W substitution (CEP55 ZF (W)). This substitution was previously described to act as a sensitive probe to monitor zinc-induced folding of the NEMO ZF without interfering with its overall structure ([@bib8]). We recorded the fluorescence emission spectrum of the CEP55 ZF (W) peptide at 300--450 nm using an excitation wavelength of 280 nm, allowing us to specifically irradiate the two tyrosines (Y447 and Y461) and tryptophan (W438) residues ([Figure 1](#fig1){ref-type="fig"}D). Upon zinc addition, CEP55 ZF (W) exhibits an increase of the fluorescence signal with a 7-nm blue shift of the tryptophan emission maximum (353--342 nm). Concomitantly, this leads to a decrease of the minor shoulder at 300--310 nm, which usually corresponds to fluorescence emission spectra of tyrosine residues. This clearly indicated that an internal FRET from tyrosine to tryptophan occurs only after the addition of zinc. In the presence and absence of zinc, the internal FRET efficiencies from Y to W were then determined and a 1.9 ± 0.06-fold increase was observed upon zinc addition ([Table S1](#mmc1){ref-type="supplementary-material"}). Because internal FRET efficiency is highly sensitive to the distance between the acceptor and the donor, these data clearly indicated that zinc induces a spatial confinement of the two Y447 and Y461 to W438, reflecting a proper folding of the domain as a zinc finger. We took advantage of this zinc-dependent fluorescence signal at 342 nm to titrate CEP55 ZF (W) by zinc ion ([Figure 1](#fig1){ref-type="fig"}E). A strong binding affinity less than 20 nM was observed with a binding stoichiometry of 1:1. This binding stoichiometry was further confirmed using a colorimetric assay as described in [Transparent Methods](#mmc1){ref-type="supplementary-material"}. Altogether, these data combined with multiple sequence alignment showed that the CEP55 fragment containing the residues 435--464 forms a CCHC-type ZF structure, which is likely built on a ββα fold. In a similar way to NOA^CEP55^, a structural model of ZF^CEP55^ was generated using NEMO ZF as template, emphasizing the four residues (C440, C443, H458, and C462) that tetra-coordinate the zinc atom ([Figure S1](#mmc1){ref-type="supplementary-material"}D) and that are strictly conserved among all CEP55 orthologues ([Figure S1](#mmc1){ref-type="supplementary-material"}A).
NOA^CEP55^ Preferentially Binds to Linear Di-ubiquitin Chain, whereas ZF^CEP55^ Binds to Non-degradative Linear and K63 Chains {#sec2.2}
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To assess the ability of CEP55 His-NOA to bind to poly-ubiquitin chains, we next performed qualitative Ni-NTA pull-down experiments in the presence of increasing concentrations of purified K63- and M1-linked tetra-ubiquitin (Ub~4~) chains ([Figures 2](#fig2){ref-type="fig"}A and 2B). As negative control, we used a His-tagged recombinant protein (His-DARPin labeled Ctrl), which does not bind to any mono or poly-ubiquitin chains even at high concentration (up to 10 mM ubiquitin site concentration). As shown in [Figures 2](#fig2){ref-type="fig"}A and 2B, a specific binding of CEP55 His-NOA to K63- and M1-Ub~4~ was observed in a dose-dependent manner. Interestingly, similar pull-down experiments using different ubiquitin linkage types revealed that CEP55 NOA preferentially recognizes non-degradative M1 and K63-linked Ub~4~ chains over degradative K48- and K11-linked Ub~4~ chains ([Figure 2](#fig2){ref-type="fig"}C), indicating that CEP55 His-NOA prefers to interact with open and even extended conformations of Ub~4~. These results were surprising since the isolated NOA/UBAN domain of NEMO as well as its NEMO-related proteins OPTN and ABIN2 were reported to exhibit a strong linkage specificity for short M1 over K63 di-ubiquitin chains ([@bib34], [@bib31], [@bib26]). A possible explanation could be that pull-down experiments are only qualitative and performed under non-equilibrium conditions. For this reason, we next characterized the interaction between CEP55 His-NOA and K63- or M1-Ub~4~ chains by determining association (kon) and dissociation (koff) rate constants using biolayer interferometry (BLI) technology ([Figures 2](#fig2){ref-type="fig"}D and 2E). In this experiment, CEP55 His-NOA was first immobilized on anti-penta-His biosensors and incubated with different concentrations of K63- or M1-Ub~4~. A K~D~ value of 27 μM for M1-linked Ub~4~ chains was observed ([Figure 2](#fig2){ref-type="fig"}E), which is 30-fold lower than that of K63-linked Ub~4~ ([Figure 2](#fig2){ref-type="fig"}D), indicating that CEP55 NOA preferentially binds to M1 poly-ubiquitin chains *in vitro*.Figure 2CEP55 NOA and ZF Form Specific Ubiquitin-Binding Domains, which Preferentially Bind to Non-degradative K63 and M1 Polyubiquitin Chains(A and B) His pull-down experiments of CEP55 His-NOA (8 μM) with increasing concentrations of purified (A) K63 tetra-ubiquitin and (B) linear tetra-ubiquitin (1, 2, 4, 8, and 16 μM). Ctrl, negative control (His-DARPin).(C) His pull-down experiment of CEP55 His-NOA (8 μM) with different linkages of tetra-ubiquitin chains (16 μM), including M1 (linear), K11, K48, or K63 tetra-ubiquitin as indicated. Ctrl, negative control (His-DARPin).(D and E) Quantitative ubiquitin binding of CEP55 His-NOA by BioLayer Interferometry (BLI) using various concentrations of K63 (D) or linear (E) tetra-ubiquitin chains (2.5, 5, 10, and 20 μM). Anti-penta-His biosensors were used in each binding experiment to immobilize 8 μM of His-NOA (see [Transparent Methods](#mmc1){ref-type="supplementary-material"}).(F) Quantitative ubiquitin binding of Strep-tag II CEP55 ZF by BLI with a mixture of K63 Ub~2-7~ polyubiquitin chains. Biotinylated K63 Ub~2-7~ (0.2 mg/mL) was immobilized on a streptavidin biosensor before adding various concentrations of Strep-tag II CEP55 ZF (2.5, 5, 10, or 20 μM).(G) Quantitative ubiquitin binding of Strep-tag II CEP55 ZF by BLI with an M1 (linear) di ubiquitin chain. M1 GST-Ub~2~ (20 μM) was immobilized on an anti-GST biosensor before the addition of Strep-tag II CEP55 ZF at different concentrations (1, 6.25, 12.5, or 25 μM).(H) Binding comparison of the Strep-tag II CEP55 ZF (10 μM) by BLI between a mixture of K63-Ub~2-7~ or K48 Ub~2-7~ polyubiquitin chains (0.2 mg/mL). For the experiments shown in (F)--(H), the Strep-tag II CEP55 ZF was each time freshly prefolded with 1 mM ZnCl~2~, which was maintained at the same concentration in the binding buffer.
In a similar way, we next characterized the ability of CEP55 ZF to bind to K63 and M1 poly-ubiquitin chains using BLI assays. A mixture of biotinylated K63 poly-ubiquitin chains (Ub~2-7~) and GST-Ub~2~ M1 were immobilized on streptavidin ([Figure 2](#fig2){ref-type="fig"}F) and anti-GST antibody ([Figure 2](#fig2){ref-type="fig"}G) biosensors, respectively, before being incubated with increasing concentrations of a StrepII-tag version of CEP55 ZF peptides (STII-CEP55 ZF). CEP55 ZF similarly binds K63 Ub~2-7~ poly-ubiquitin chains and GST-Ub~2~ M1, since similar K~D~ values of 2.1 and 1.6 μM were obtained, respectively. This ubiquitin-binding activity was specific because no binding was observed with a mixture of biotinylated K48 Ub~2-7~ chains ([Figure 2](#fig2){ref-type="fig"}H), indicating that CEP55 ZF preferentially recognizes K63 and M1 chains over K48 poly-ubiquitin chains. We also confirmed the specific binding of CEP55 ZF to mono- and K63 di-ubiquitin in solution by fluorescence polarization (FP) experiments using N-terminally fluorescein labeled CEP55 ZF and under more stringent buffer conditions ([Figures S1](#mmc1){ref-type="supplementary-material"}G and S1H). Of note, similar binding affinities were observed with mono-ubiquitin and K63 di-ubiquitin in solution, but they were much lower than those obtained with BLI experiments. This is presumably because poly-ubiquitin chains were immobilized in BLI experiments, whereas they were in solution in FP experiments (see [Discussion](#sec3){ref-type="sec"}). Taken together, these data clearly demonstrated that CEP55 contains two UBDs in its C-terminal part: a UBZ-type (UBZ^CEP55^) located at the C-terminal extremity that binds to non-degradative linear and K63 chains and an NOA domain (NOA^CEP55^) that preferentially interacts with linear Ub chains.
Structure-Based Identification of UBZ^CEP55^ Mutants Defective in Ubiquitin Binding {#sec2.3}
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Generation of ubiquitin binding defective single-point mutants of UBZ^CEP55^, without altering the overall ZF structure, could be a challenging task since recent crystal complexes of UBZ domains with ubiquitin revealed different modes of ubiquitin recognition ([Figure S2](#mmc1){ref-type="supplementary-material"}). Nevertheless, we could classify at least three groups of UBZ domains, including FAAP20/Polη ([@bib40]; PDB:[3WWQ](pdb:3WWQ){#intref0010} \[[@bib3]\]), RAD18/WRNIP1 ([@bib18]; PDB:[5VF0](pdb:5VF0){#intref0015} \[[@bib38]\], PDB:[3VHT](pdb:3VHT){#intref0020}), and NDP52 ([@bib42]; PDB:[4XKL](pdb:4XKL){#intref0025}), based on their crystal structures in complex with Ub ([Figures S2](#mmc1){ref-type="supplementary-material"}A and S2B). Structure-based sequence alignment analysis of UBZ^CEP55^ from different organisms with different UBZ groups showed that UBZ^CEP55^ displays the highest homology with the RAD18/WRINP1 UBZ group, suggesting a similar ubiquitin-binding mode ([Figure S2](#mmc1){ref-type="supplementary-material"}C). In particular, Ub-interacting residues of WRINP1-UBZ in the β1-strand, β-loop (the loop between both antiparallel β-strands), and the α-helix of the UBZ ββα fold are conserved or replaced by functionally equivalent residues in CEP55-UBZ ([Figure S2](#mmc1){ref-type="supplementary-material"}C). These residues are also strictly conserved in CEP55 from several organisms. We therefore generated a structural model of CEP55 ZF in complex with ubiquitin, using the crystal structure of the UBZ-GFPWRNIP1:ubiquitin complex as template ([Figure 3](#fig3){ref-type="fig"}). According to this model, the CEP55 residue E460, which is strictly conserved among all CEP55 orthologues ([Figure S1](#mmc1){ref-type="supplementary-material"}A), forms a strong binding determinant of the complex by making a dual salt bridge with R42 and R72 of ubiquitin ([Figure 3](#fig3){ref-type="fig"}A). To validate our hypothesis, we generated an N-terminally fluorescein-labeled CEP55 ZF mutant (F-ZF E460A) as well as a StrepII-tag version of CEP55 ZF mutant (STII-CEP55 ZF E460A) containing the E460A single point mutation. F-ZF E460A displayed an almost 20-fold weaker affinity compared with WT for mono-ubiquitin when determined in solution by FP ([Figure 3](#fig3){ref-type="fig"}B). We confirmed this drastic loss of affinity of the E460A mutant by BLI using immobilized GST-Ub2 M1, since binding affinity of the E460A UBZ mutant was 26-fold reduced compared with the WT ([Figure 3](#fig3){ref-type="fig"}C).Figure 3Structure-Based Identification of ZF Single-Point Mutant Defective in Ubiquitin Binding(A) Structural model of CEP55 ZF (green) in complex with mono-ubiquitin (orange) pointing out E460 as a critical residue that makes a specific salt bridge with R42 and R72 residues of ubiquitin.(B) Mono-ubiquitin titration in solution monitored by fluorescence polarization of fluorescein-labeled WT and E460A mutant CEP55 ZF (10 μM) in the presence of 1 mM ZnCl~2~.(C) Quantitative ubiquitin binding determined by BLI experiment of WT and E460A mutant CEP55 ZF with M1 (linear) di-ubiquitin chain. M1 GST-Ub2 was first immobilized on an anti-GST biosensor before adding various concentrations of WT and E460A mutant STII-CEP55 ZF (6.25, 12.5, and 25 μM). For the experiments shown in (B) and (C), all ZF peptides were freshly prefolded in the presence of 1 mM ZnCl~2~, which was maintained at the same concentration in the binding buffer.
We then verified whether the E460A mutation impedes the ability of UBZ^CEP55^ to interact with ubiquitin and not the overall structure of UBZ domain. For this purpose, we investigated the zinc-induced folding of the E460A mutant (ZF^CEP55^ (W) E460A) by measuring its internal FRET efficiencies from tyrosine to tryptophan ([Table S1](#mmc1){ref-type="supplementary-material"}). Similar fluorescence emission spectra of the WT and E460A mutant with or without the presence of Zn^2+^ indicated a similar folding ([Figure S2](#mmc1){ref-type="supplementary-material"}D). Consistently, similar gains of internal FRET efficiencies from tyrosine to tryptophan were observed for the WT and E460A after the zinc addition ([Table S1](#mmc1){ref-type="supplementary-material"}). Furthermore, Zn^2+^ titration experiments by fluorescence ([Figure S2](#mmc1){ref-type="supplementary-material"}E) combined with a PAR colorimetric assay (data not shown) showed similar binding affinities and binding stoichiometries of the WT and E460A UBZ mutant. Thus, these data in agreement with our structural model indicated that the E460A mutation in UBZ^CEP55^ is a crucial binding determinant for ubiquitin recognition but does not alter the proper folding of this ZF structure.
CEP55 ZF and Its Ubiquitin-Binding Ability Are Necessary for CEP55 Recruitment to the Midbody {#sec2.4}
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To study the functional role of UBZ^CEP55^ in the context of the full-length protein, we next carried out functional depletion/rescue experiments in HeLa cells. For this purpose, endogenous CEP55 was knocked down by small interfering RNA (siRNA) and transiently re-expressed with siRNA-resistant plasmids expressing either CEP55 WT (HA-CEP55 WT), a debilitating double mutant (HA-C440A/C443A), which is unable to tetracoordinate the zinc atom, or the E460A single-point mutant defective in the ubiquitin binding (HA-CEP55 E460A). As shown in [Figure 4](#fig4){ref-type="fig"}A, knockdown of the endogenous CEP55 by siRNA was very efficient, and ectopic expressions of the WT and its single and double mutants gave similar expression levels of CEP55 as judged by western blot. As previously reported in the literature, efficient knockdown of CEP55 leads to an increase of cells connected by an intercellular bridge ([Figures 4](#fig4){ref-type="fig"}A and [S3](#mmc1){ref-type="supplementary-material"}A) as well as multinucleated cells ([Figures 4](#fig4){ref-type="fig"}B and [S3](#mmc1){ref-type="supplementary-material"}A). These phenotypes were partially restored by the expression of the HA-CEP55 WT protein ([Figures 4](#fig4){ref-type="fig"}A, 4B, and [S3](#mmc1){ref-type="supplementary-material"}A). We do believe that the incomplete rescue was due to technical issues of simultaneously reaching very high transfections efficiencies with both DNA and siRNA and to intrinsic properties of the CEP55 protein, which lead to mainly insoluble protein under transient expression conditions, as previously described ([@bib23]) ([Figure S3](#mmc1){ref-type="supplementary-material"}C). In addition, it is likely that the insoluble form of CEP55 could trap the soluble and functional form of CEP55 and so contributes to an incomplete rescue. Nevertheless, this rescue was statistically significant and reproducible (n = 3 with more than 300 nuclei/cells analyzed by experiment) ([Figures 4](#fig4){ref-type="fig"}A and 4B). Strikingly, neither the CEP55 C440A/C443A double mutant nor the CEP55 E460A single-point mutant were able to functionally rescue normal cytokinesis as does the WT, as judged by the persistent increase of cells connected by intracellular bridges ([Figure 4](#fig4){ref-type="fig"}A) and the increase in the proportion of multinucleated cells ([Figure 4](#fig4){ref-type="fig"}B) induced by CEP55 knockdown. These data clearly indicated that CEP55 ZF and its ubiquitin-binding property are crucial for CEP55 function during abscission. Moreover, the C440A/C443A double mutant and the E460A single-point mutant, unlike the WT, were unable to functionally restore the accumulation of MRs induced by CEP55 depletion ([Figure S3](#mmc1){ref-type="supplementary-material"}B), highlighting a critical role of UBZ^CEP55^ in remnant midbody clearance.Figure 4CEP55 ZF Is Essential for CEP55 Recruitment to the Midbody and Ensuring Proper Cytokinetic AbscissionHeLa cells were concomitantly transfected with a non-targeting (NT) or CEP55 siRNA and with an empty vector or a CEP55 siRNA-resistant vector encoding HA-CEP55 WT, HA-CEP55 C440A/C443A, or HA-CEP55 E460A.(A and B) Analyses of the ZF mutant\'s ability to rescue cytokinesis defect induced by CEP55 depletion. The percentage of cells connected by midbodies (A) and the percentage of multinucleated cells (B) were quantified. Images are represented in [Figure S3](#mmc1){ref-type="supplementary-material"}A. Data represent the mean ± SD of three independent experiments, and the total number of nuclei (n) counted in the three experiments is indicated. The level of expression of CEP55 in the soluble fraction for the different conditions was controlled by WB.(C) Recruitment to the midbody of HA CEP55 WT, HA CEP55 C440A/C443A, and HA CEP55 E460A. Cells were stained with DAPI (blue) and immunolabeled with HA (yellow), MKLP-1 (red), and α-tubulin (cyan) antibodies. Insets show an expanded view of the midbody.(D) Relative quantification of CEP55 intensity at the midbody reporter in the different conditions. Cells were stained with DAPI, CEP55, MKLP-1, and β-tubulin antibodies. CEP55 intensity was normalized by MKLP-1 intensity at each detected midbody (MKLP-1 spot).Analyses were conducted on three independent experiments, n represents the total number of midbodies. Scale bar, 10 μM. \*\*\*p \< 0.001; \*p \< 0.05; ns, not significant (p ≥ 0.05).
Next, we assessed whether UBZ^CEP55^ could be involved in the recruitment of CEP55 to the midbody. Because transient expression of the ectopic CEP55 could interfere with the endogenous CEP55 via the formation of hetero-oligomers, similar functional knockdown and rescue experiments of endogenous CEP55 were performed. Interestingly, although HA-CEP55 WT was recruited to the midbody, neither the C440/C443A double mutant nor the E460A single-point mutant were found to co-localize with the midbody ([Figure 4](#fig4){ref-type="fig"}C). Image quantification using MKLP-1 as midbody reporter confirmed the defect of both CEP55 mutants in their recruitment to the midbody compared with the WT ([Figure 4](#fig4){ref-type="fig"}D). In line with this, the ALIX endogenous protein, which is recruited to the midbody via CEP55 binding, also exhibited a clear defect in midbody localization with both CEP55 ZF mutants, but not the WT, using again MKLP-1 as midbody reporter ([Figure S3](#mmc1){ref-type="supplementary-material"}D).
Taken together, these results reveal that the UBZ^CEP55^ UBD and its ubiquitin-binding properties are crucial for CEP55 recruitment to the midbody, allowing CEP55 to ensure proper abscission and MR clearance.
Structure-Based Identification of CEP55 NOA Mutants Unable to Bind Ubiquitin {#sec2.5}
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The precise mode of linear di-ubiquitin recognition by the NOA/UBAN domain originally found in the NEMO protein ([@bib34]) was more recently confirmed by two other NEMO-like proteins called Optineurin ([@bib31]) and ABIN2 ([@bib26]). Indeed, structural comparison of NEMO NOA in complex with an M1-linked di-Ub with the recent crystal structures of Optineurin with a linear di-Ub chain ([@bib31]) and ABIN2 with a linear tri-Ub chain ([@bib26]) revealed a highly conserved binding mode of NOA with two M1-linked units of ubiquitin ([Figure S4](#mmc1){ref-type="supplementary-material"}). Contrary to the UBZ family, the NOA UBD forms a long parallel α-helical coiled-coil dimer, in which one monomer mainly interacts with the distal Ub (second Ub), whereas the other monomer interacts with the proximal ubiquitin (first Ub). This explains why the NOA dimeric state is required for ubiquitin binding. Structure-based sequence alignment of NOA UBD family with CEP55 from different organisms reveal the presence of conserved or functionally equivalent ubiquitin-interacting residues with both proximal and distal ubiquitin. This sequence conservation suggests that CEP55 binds to a di-Ub chain in a similar manner to NOA family and the degree of conservation of each amino acid position was higher between CEP55 and ABIN2 NOA, especially when compared with residues interacting with proximal ubiquitin ([Figure S4](#mmc1){ref-type="supplementary-material"}). We then generated a structural model of CEP55 NOA in complex with a linear di-Ub chain using the linear diubiquitin:NOA complex of NEMO as structural template (PDB: [2ZVO](pdb:2ZVO){#intref0030}) ([Figure 5](#fig5){ref-type="fig"}A). According to the model, L351, Q354, and Q355 interact with the I44 centered hydrophobic patch of the distal ubiquitin ([Figure 5](#fig5){ref-type="fig"}A, left panel). L351 and Q355 residues are perfectly conserved in all the NOA family and CEP55 orthologs, whereas Q354 is conserved only in ABIN2. Moreover, D362 and F363 interact with the C-terminal tail of distal ubiquitin and are strictly conserved in all the NOA family ([Figures 5](#fig5){ref-type="fig"}A right panel and [S4](#mmc1){ref-type="supplementary-material"}).Figure 5Structure-Based Identification of CEP55 NOA Mutants Defective in Ubiquitin Binding(A) Modelization of CEP55 NOA (green) in interaction with linear di-ubiquitin pointing out critical residues in the recognition of the distal ubiquitin (orange) and proximal ubiquitin (cyan). Zoom on the interface between distal ubiquitin and CEP55 NOA. Left: residues involved in the recognition of the hydrophobic patch of the distal ubiquitin. Right: residues involved in the recognition of the C-terminal tail of the distal ubiquitin.(B) Comparison of the association constants (K~A~) of the different His-NOA mutants for linear tetra-ubiquitin measured by BLI. Curves are represented in [Figures S5](#mmc1){ref-type="supplementary-material"}A--S5C.
To validate the importance of these ubiquitin-interacting residues, we purified to homogeneity two His-NOA mutants containing either the L351A/Q354A/Q355A triple mutation or D362R/F363P double mutation and assessed their affinities with M1-linked tetra-ubiquitin by BLI ([Figures 5](#fig5){ref-type="fig"}B and [S5](#mmc1){ref-type="supplementary-material"}A--S5C). A deletion mutant of the residues 343--364 corresponding to the minimal NOA site was also generated taking into account the nature of the heptad repeats in the coiled-coil dimer. For all of the three mutants, we observed drastic losses of binding affinity for M1-linked Ub~4~ compared with the WT (more than 500-fold), indicating that the mutated residues within the double and triple mutants correspond to critical ubiquitin-binding determinants in agreement with our structural model. To determine whether these mutations also caused global disruption of the dimeric coiled-coil structure, we next analyzed the mutants by SDS-PAGE in the absence of the reducing agent DTE ([Figure S5](#mmc1){ref-type="supplementary-material"}D) and by SEC-MALS ([Figure S5](#mmc1){ref-type="supplementary-material"}E). The L351A/Q354A/Q355A triple mutant and the deletion mutant form considerably less stable dimers than the WT, as no dimers with both mutants were detected by SDS-PAGE in the absence of DTE and SEC-MALS. For the D362R/F363P double mutant, a significant amount of dimers could be detected in SDS-PAGE without DTE, indicating that this mutant forms more stable dimers than the triple and deletion mutants. Nevertheless, the D362R/F363P mutant dimer was less stable than the WT, as judged by the dimer/monomer ratio of the mutant in SDS-PAGE without DTE, which is significantly lower than the WT. Furthermore, no dimer with this mutant could be observed with SEC-MALS. Rather, we could detect a second form of monomer more compact, which likely results from the replacement of Phe363 by Pro363 in this mutant. Therefore, all structure-guided mutants generated from the structural model displayed a total loss of ubiquitin binding activity, but they also displayed a defect in dimerization properties to varying extents.
CEP55 NOA Is Absolutely Required for Normal Cytokinetic Abscission but Not for CEP55 and ALIX Recruitment to the Midbody {#sec2.6}
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To investigate the functional importance of CEP55 NOA UBD, we carried out functional depletion/rescue experiments in HeLa cells. To this end, we generated the D362R/F363P double mutant in the context of the full-length CEP55 protein. As shown in [Figure 6](#fig6){ref-type="fig"}A, the protein expression level of the D362R/F363P double mutant was similar to that of the WT. Nevertheless, it is worth noting that a significant portion of the WT and mutant proteins were found in the insoluble fraction of cell lysates, as we found for the CEP55 UBZ mutants ([Figures S3](#mmc1){ref-type="supplementary-material"}C and [S6](#mmc1){ref-type="supplementary-material"}C). Importantly, ectopic expression of the D362R/F363P double mutant was not able to rescue the cytokinetic defect induced by CEP55 knockdown, contrary to the WT ([Figures 6](#fig6){ref-type="fig"}A and [S6](#mmc1){ref-type="supplementary-material"}A). On the other hand, the amount of multinucleated cells was almost similar in cells expressing either the double mutant or the WT ([Figures 6](#fig6){ref-type="fig"}B and [S6](#mmc1){ref-type="supplementary-material"}A). Although an increase of cells connected by an intercellular bridge may reflect a delay in the abscission process, the appearance of multinucleated cells often results from more drastic abscission defects and/or an earlier event involved in the stabilization of the intercellular bridge and midbody integrity. In the case of CEP55, previous studies showed CEP55 plays crucial roles in midbody integrity ([@bib43], [@bib6]) as well as in the abscission process ([@bib29], [@bib5], [@bib25]). Consistent with previous studies, we conclude that the CEP55 NOA UBD plays an important role in CEP55 during abscission, albeit to a lesser extent than UBZ^CEP55^, and does not appear to be involved in the stabilization of the intercellular bridge or seems less important than CEP55 ZF in the abscission process.Figure 6CEP55 NOA Is Not Involved in CEP55 Recruitment to the Midbody but Is Absolutely Required to Ensure Proper Abscission(A and B) Effect of the double NOA mutant (D362R/F363P) on the ability to rescue cytokinesis defect induced by CEP55 depletion. HeLa cells were concomitantly transfected with a non-targeting (NT) or CEP55 siRNA and with an empty vector or a CEP55 siRNA-resistant vector encoding either HA-CEP55 WT or HA-CEP55 D362R/F363P. The percentage of cells connected by midbodies (A) and the percentage of multinucleated cells (B) were quantified. Representative images are shown in [Figure S6](#mmc1){ref-type="supplementary-material"}A. Data represent the mean ± SD of three independent experiments, and the total number of nuclei (n) counted in the three experiments is indicated. The level of CEP55 expression in the soluble fraction were controlled by western blot as indicated.(C) Recruitment to the midbody of HA-CEP55 WT and HA-CEP55 D362R/F363P. Same immunofluorescence experiments as (A). Cells were then stained with DAPI (blue) and immunolabeled with HA (yellow), MKLP-1 (red), and α-tubulin (cyan) antibodies. Insets show an expanded view of the midbody. Scale bar, 10 μM.(D) Relative quantification of CEP55 intensity at the midbody as indicated. Immunofluorescence experiments were the same as (A) except for CEP55 labeling. CEP55 intensity was normalized by MKLP-1 intensity at the midbody upon MKLP-1 dots. The total numbers of midbodies (n) that result from three independent experiments are indicated.\*\*\*p \< 0.001; \*p \< 0.05; ns, not significant (p ≥ 0.05).
Moreover, although the D362R/F363P double mutant exhibits a defect in the abscission process, it is normally recruited to the midbody, indicating that the detrimental effect of the mutant in cytokinesis occurs after its recruitment to the midbody ([Figures 6](#fig6){ref-type="fig"}C and 6D). Consistent with these data, we also observed that the D362R/F363P double mutant was able to recruit ALIX to the midbody as efficiently as the WT ([Figure S6](#mmc1){ref-type="supplementary-material"}D), suggesting that the functionality of the EABR dimeric domain was not affected by the ubiquitin-binding deficient mutation in the NOA domain in the context of the full-length protein.
Finally, we quantified the percentage of the remnants by cell using knocked down HeLa cells of CEP55 expressing either the WT or the double mutant ([Figure S6](#mmc1){ref-type="supplementary-material"}B). No difference was observed between the WT and the double mutant, indicating that CEP55 NOA is not involved in CEP55 MR clearance.
Altogether, our results showed that NOA UBD plays an important role in cytokinetic abscission, albeit to a lesser extent than UBZ^Cep55^ UBD, and is not involved in CEP55 nor in ALIX recruitment to the midbody neither in MR clearance.
UBZ^CEP55^ Functions as a Specific Targeting Domain to the Midbody in a Ubiquitin-Binding-Dependent Manner {#sec2.7}
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We next focused our studies on the UBZ-dependent recruitment of CEP55 to the midbody by examining the possibility that this domain alone could serve as a specific midbody localization signal. If UBZ^CEP55^ forms an autonomous UBD domain for this targeting activity to the midbody, thus, any fusion proteins that only contain the UBZ^CEP55^ domain should be specifically recruited to the midbody. For this purpose, we generated a GFP-UBZ^CEP55^ construct and investigated by fluorescence microscopy its cellular localization. As shown in [Figures 7](#fig7){ref-type="fig"}A and 7B, GFP-UBZ^CEP55^, but not GFP alone, co-localized with the midbody marker, MKLP-1, indicating that UBZ^CEP55^ exhibits a specific targeting activity to the midbody. This was not due to the differences of protein expression level because GFP and GFP-UBZ^CEP55^were expressed as similar levels in cells ([Figure 7](#fig7){ref-type="fig"}C). Because endogenous CEP55 could interfere with the GFP-UBZ^CEP55^ recruitment to the midbody, we then performed experiments in CEP55 knock-downed HeLa cells, concomitantly expressing GFP alone or GFP-UBZ^CEP55^ ([Figures S7](#mmc1){ref-type="supplementary-material"}A and S7B). No significant difference in the targeting activity was observed in cells transfected with an siRNA control and CEP55-specific siRNA, demonstrating that the endogenous CEP55 was not necessary for the recruitment of GFP-UBZ^CEP55^ to the midbody.Figure 7Ubiquitin-Binding Activity of CEP55 UBZ Is Required to Function as a Specific Cargo Receptor to the Midbody(A) HeLa cells were transiently transfected with plasmids encoding for GFP alone or GFP-UBZ^CEP55^ and then analyzed by immunofluorescence and fluorescence microscopy.(B) Quantification of the percentage of co-localization between GFP or GFP-UBZ^CEP55^ and MKLP-1 (midbody reporter) in transfected cells. Data represent the mean ± SD of six independent experiments. \*\*p \< 0.01.(C) Expression levels were controlled by western blot.(D) CRISPR-Cas9-mediated CEP55 knockout U2OS cell lines were transiently reconstituted following transfection with different plasmids encoding the WT, a debilitating double mutant unable to form a ZF structure (C440A/C443A), or the chimeric CEP55 proteins containing different ZF groups interacting with ubiquitin as indicated (left panel and [Figure S2](#mmc1){ref-type="supplementary-material"}). Reconstituted cells were then analyzed by immunofluorescence and the levels of expression controlled by western blot before quantifying the percentages of co-localization between CEP55 and MKLP-1 (midbody) for the indicated chimeric CEP55 proteins.Data represent the mean ± SE of two independent experiments; n represents the total number of midbodies. \*\*\*p \< 0.001; \*\*p \< 0.01; \*p \< 0.05; ns, not significant (p ≥ 0.05).
We next addressed the question whether this CEP55 UBZ-dependent targeting activity depends on its ability to bind to ubiquitin and whether this ubiquitin binding depends on one type of M1 or K63 linkage of polyubiquitin chains. For these experiments, we generated CRISPR-Cas9-mediated CEP55 knockout U2OS cell lines to increase the sensitivity of our functional complementation assay ([Figure S7](#mmc1){ref-type="supplementary-material"}C). We also generated different CEP55 chimeric proteins in which the CEP55 UBZ was replaced with other classes of UBD based on small ZF modules known to interact with ubiquitin such as UBZ^WRNP1^, UBZ^RAD18^, UBZ^NDP52^, nZF2^TAB2^, and A20^ZF7^ ([Figure 7](#fig7){ref-type="fig"}D). UBZ^WRNP1^ and UBZ^RAD18^ were chosen because their crystal structures showed a binding mode similar to that of ubiquitin, which is likely close to that of UBZ^CEP55^. In addition, UBZ^WRNP1^ exhibits no ubiquitin linkage specificity, whereas UBZ^RAD18^ exhibits a little preference for K63 over K48 polyubiquitin (4-fold) and an *in vitro* stronger polyubiquitin binding, owing to the extended LR motif (224--240) located at its C terminus ([@bib39]). We also used the UBZ^NDP52^, which is also built on a ββα fold structurally related to UBZ^WRNP1^ and UBZ^RAD18^. However, it uses a different mode of ubiquitin recognition ([Figures S2](#mmc1){ref-type="supplementary-material"}B and S2C) and was reported to have no specificity for one type of ubiquitin linkage ([@bib40]). By contrast, nZF2^TAB2^ and A20^ZF7^ display strong linkage specificity for K63 and M1 polyubiquitin chains, respectively, but they are likely structurally different from UBZ^CEP55^. CEP55 knockout U2OS cells generated by the CRISPR-Cas9 technology were transiently reconstituted following transfection with different plasmids encoding for either the WT or the chimeric CEP55 proteins bearing the different UBZs mentioned before or a debilitating double mutant (C440A/C443A, unable to form a ZF structure). Reconstituted cells were then analyzed by immunofluorescence to quantify the percentages of co-localization between CEP55 and the midbody marker, MKLP-1. The level of expression of the different constructs was controlled by western blot in parallel experiments. As shown in [Figure 7](#fig7){ref-type="fig"}D, CEP55 ΔZF-UBZ^RAD18^ totally restored the CEP55 recruitment to the midbody, similar to the WT, whereas the debilitating double mutant did not. All chimeric CEP55 proteins were expressed at a similar level in U2OS cells. The four other chimeric CEP55 proteins, CEP55 ΔZF-UBZ^NDP52^, ΔZF-UBZ^WRNP1^, ΔZF-nZF2^TAB2^, and ΔZF-A20^ZF7^, also complement the targeting activity of CEP55 but to in a lesser extent than the WT. Nevertheless, the CEP55 ΔZF-UBZ^NDP52^ significantly displayed a better restoration than those of CEP55 ΔZF-UBZ^WRNP1^, CEP55 ΔZF-nZF2^TAB2^, and CEP55 ΔZF-A20^ZF7^, which partially complement the CEP55 recruitment to the midbody at a similar level. Importantly, no significant difference was observed between CEP55 ΔZF-nZF2^TAB2^ and CEP55 ΔZF-A20^ZF7^, indicating that no predominant M1 or K63 linkage was required for the UBZ-dependent targeting activity of CEP55.
Next, we addressed the question of how the CEP55 UBZ contributes to cellular poly-Ub binding in the context of the full-length protein. To this end, His-tagged WT or mutant (C440/C443A) human full-length CEP55 purified from 293-F cells were incubated with whole-cell extracts containing free and anchored polyubiquitin chains. His-tagged WT and UBZ double mutant immobilized on Ni-NTA beads were then pulled down, and bound polyubiquitin chains were revealed by western blot using anti-Ub antibodies ([Figure S7](#mmc1){ref-type="supplementary-material"}D). As negative control, we used a mutant of the NOA/UBAN ubiquitin binding domain of NEMO (D311G), which is defective in ubiquitin binding. We observed that the CEP55 UBZ significantly contributes to polyubiquitin binding since the C440A/C443A debilitating double mutant exhibits a slight, albeit significant decrease in polyubiquitin binding activity compared with the CEP55 WT. Note that this polyubiquitin binding activity of the UBZ double mutant was not totally abolished when compared with the NOA/UBAN mutant of NEMO. This suggests that CEP55 NOA, as well as the EABR domain, which interacts with UBD-containing ALIX and TSG101 proteins, could contribute to the polyubiquitin binding activity of CEP55. Moreover, and unsurprisingly, we observed that CEP55 and polyubiquitin chains specifically colocalize with each other and are localized to the midbody at the late stage of cytokinesis ([Figure S7](#mmc1){ref-type="supplementary-material"}E).
Altogether, we conclude that the ubiquitin binding ability of CEP55 UBZ is required to specifically target the CEP55 protein to the midbody. In addition, no significant linkage preference for M1- or K63-linked chains was observed for this targeting activity, consistent with the ubiquitin-binding properties of CEP55 UBZ.
Discussion {#sec3}
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NEMO is the prototype of a family of proteins including Optineurin and ABIN2, which contain the same type of UBDs. Although the UBD of these proteins are conserved, they exert different functions such as NF-κB regulation in the case of NEMO, membrane trafficking and autophagy in the case of Optineurin, and NF-κB and ERK MAP kinase regulation in the case of ABIN2. Here we identified the CEP55 protein, a regulator of cytokinesis, as a member of this family protein, which contains both NOA/UBAN and UBZ domains in the C-terminal part of the protein. Although we did not determine the tridimensional structures of these UBDs, we showed strong evidences by structure-guided sequence alignment analysis and biochemical and biophysical experiments that the predicted CEP55 NOA fragment (residues 304--396) adopts an α-helical coiled-coil dimer and in the C-terminal region (residues 435--464) a ZF/UBZ architecture, which are structurally similar to those of NEMO, Optineurin, and ABIN2 proteins. Based on these experiments, we generated structural models of both CEP55 NOA and UBZ domains, which are fully consistent with mutagenesis data and allowed us to identify several mutants defective in ubiquitin binding. These generated mutants in each domain and in the context of the full-length protein also permitted the demonstration of the functional importance of these NOA and UBZ domains for the CEP55 function in cell division and specifically in cytokinetic abscission.
Despite an extensive search of crystallization conditions and generation of different constructs, no high-quality diffraction crystals of CEP55 NOA or CEP55 UBZ with or without ubiquitin could be obtained. For NOA, this was likely due to its high tendency to form higher-order oligomers (mainly hexamers), which probably result from the assembly of three dimeric units. In the case of CEP55 UBZ, this was due to its poor solubility at high concentration as judged by dynamic light scattering experiments (data not shown). Moreover, this tendency of recombinant or synthetic CEP55 UBZ to aggregate and collapse when used at a concentration above 50--70 μM prevented us to determine its structure by NMR, contrary to NEMO UBZ for which we successfully solved the NMR structure ([@bib8]).
Although CEP55 NOA is structurally similar to NEMO and its related proteins, Optineurin and ABIN2, some characteristic features of CEP55 were observed. First, the nature of the typical seven-residue periodicity (abcdefg)n, known as the heptad repeat, significantly differs from that of NEMO in the N-terminal part of the NOA domain, also called the CC2 region in NEMO. Indeed, four hydrophobic residues at a and d heptad positions in NEMO (a: L260, L274 M295; d: A270), which are crucial for the dimer stability, are replaced by polar and charged residues in CEP55 (a: N311, S325, Q346; d: E321). The presence of these polar and charged residues could destabilize the hydrophobic packing of the α-helical coiled-coil dimer in the N-terminal region of CEP55 NOA, providing a possible explanation why the CEP55 NOA forms a dimer less stable than that of NEMO, with a dimerization constant that is about 6-fold lower than that of NEMO ([@bib17]). In line with this, caution also should be taken on the apparent linkage selectivity for linear chains observed with the isolated NOA of CEP55. Indeed, previous crystal structures of NOA complexes with K63- and M1-linked di-ubiquitin chain revealed the interactions with the two ubiquitin moieties of M1-linked di-ubiquitin chain (distal and proximal), whereas only the distal ubiquitin moiety interacts with the K63-linked di-ubiquitin chain. These differences in ubiquitin-binding mode can lead to a more stabilizing effect on the NOA dimerization with the linear chain compared with K63 chains. Thus, the differences of affinity between linear and K63 chains for NOA CEP55 could be artificially enhanced by the dimerization effect induced by linear chain binding, possibly leading to an overestimation of linkage selectivity of CEP55 NOA for linear chains over K63 chains.
Even though size exclusion chromatography showed that the D362R/F363P eluates into two well-separated elution peaks, multi-angle light scattering analysis pointed out that the two peaks correspond to monomeric species with the same molecular mass, indicating that this NOA double mutant adopts two different monomeric forms in solution ([Figure S5](#mmc1){ref-type="supplementary-material"}E, low panel). One monomeric species appears to be more elongated with a higher frictional coefficient compared with the second monomeric species, which displays the same elution profile as that observed with the WT and the L351/Q354A/Q355A triple mutant. Because the proline can adopt two *cis* and *trans* conformations in protein, the presence of this less compact second monomeric species is compatible with the F363P substitution in the NOA double mutant.
Moreover, the SDS-PAGE analysis under non-reducing conditions points out that the dimerization properties of the D363R/F363P double mutant are partially altered as compared with the WT, whereas those of the L351/Q354A/Q355A triple mutant are totally abolished ([Figure S5](#mmc1){ref-type="supplementary-material"}D). Thus, the triple mutation induces a more destabilizing effect on CEP55 NOA dimerization than the double mutant. In the light of the structural model shown in [Figure 5](#fig5){ref-type="fig"}, it is tempting to speculate that each Q354 of one monomer makes a specific hydrogen bound with the sulfur atom of each M356 belonging to the second monomer, giving a possible explanation why a drastic destabilization of the CEP55 NOA dimeric state was observed with the L351A/Q354A/Q355A triple mutant. Notably, it is worth pointing out that a tight coupling between dimerization and ubiquitin binding was previously observed upon studies with the NOA/CC2-LZ domain of NEMO ([@bib17]). Therefore, finding mutations in CEP55 NOA, which only alter ubiquitin binding without interfering with the dimerization process, could be a challenging task, which could be insurmountable at the current stage without prior knowledge of the crystal structures of the apoform and complexed form of CEP55 NOA with a ubiquitin chain.
When analyzing ubiquitin-binding properties of CEP55 UBZ, different binding affinities were observed when we determined dissociation constants in solution by fluorescence polarization and BLI experiments. Even if we did not strictly compare the same type of K63-linked chain in FP (K63-UB~2~) and BLI experiments (K63-Ub~2-7~), these differences in binding affinity cannot probably be due to the differences of the chain length. First, the mixture of K63-linked UB~2-7~ chains used in BLI experiments mainly contains di-ubiquitin and tri-ubiquitin chains and only a minor amount of Ub~4~-~7~ chains. Second, the experimental conditions used in solution and BLI experiments were different. Indeed, the DDM detergent was added at 0.1% in FP experiments because it was required to prevent the non-specific binding between ubiquitin and the fluorescein conjugated to CEP55 UBZ. This may considerably reduce the ubiquitin-binding constant by disrupting the hydrophobic interactions on the I44-centered hydrophobic interface of ubiquitin. Moreover, the ubiquitin chain used was in solution in FP experiments, whereas it was immobilized in BLI experiments. This could affect the binding entropy and may contribute to the difference of binding affinity observed between solution and BLI binding studies. Similar conflicting results between solution and pull-down binding studies were also reported with TAB2 NZF to assess the linkage selectivity between K48 and K63 Ub~2~ and Ub~4~ chains ([@bib22]).
Strikingly, we observed that the CEP55 ΔZF-UBZ^RAD18^ chimeric protein completely restored CEP55 midbody recruitment like the WT, whereas all the other chimeric proteins tested only partially compensate for the phenotype ([Figure 7](#fig7){ref-type="fig"}D). A possible explanation could be the binding mode of UBZ^RAD18^ and its ubiquitin-linkage preference for K63 and linear chains over K48 chains, which appeared to be more similar to those of UBZ^CEP55^. However, one cannot rule out the possibility that this best compensation effect was also due to the additional ELRM motif located at the C terminus of UBZ^RAD18^ ([@bib39]), which can confer a better binding affinity to polyubiquitin chains compared with all the other UBZs and nZFs tested.
Importantly, recent studies reported that CEP55 mutations are responsible for two human pathologies named MARCH ([@bib14]) and Meckel-like syndrome ([@bib4], [@bib35]), which exhibit some similar clinical signs. Hypomorphic CEP55 mutations were found to be associated with MARCH, which is a severe and autosomal-recessive syndrome that affects neuronal mitosis. The developmental features of MARCH include hydranencephaly, anhydramnios, renal dysplasia, and cerebellar hypoplasia. Amorphic CEP55 mutations were reported to be associated with the Meckel-like syndrome and with *in utero* lethality. This syndrome is linked to cilia-related disorders, called ciliopathies, and some clinical signs detected in the affected fetus are similar with MARCH, including aberrant developments of brain and cerebellum, renal cysts, and oligohydramnios. Importantly, the MARCH-related p.S425X mutation (c.1274C \> A), which has a similar CEP55 expression level as that of a healthy patient control ([@bib14]), corresponds to a deletion of the UBZ. Strikingly, this p.S425X mutation exhibits a defect in CEP55 recruitment to the midbody like the C440A/C443A debilitating double mutant investigated in the present study. Therefore, it is tempting to speculate that all mutations of CEP55 that alter the UBZ structure by modifying the zinc-ion-coordinating residues (C440, C443, H458, and C462) or that impair its ubiquitin-binding properties like the E460A single mutant may also lead to the MARCH syndrome. It is also tempting to predict that the extent and severity of the clinical signs found in MARCH and Meckel-like syndromes could be correlated with mutations found in the CEP55 NOA and UBZ domains, taking into account our findings that mutations located in CEP55 UBZ lead to a more severe defect in cytokinetic abscission than those contained in NOA.
Live-cell imaging with super resolution microscopy using live PALM experiments supports the view that NEMO induces dynamic condensates with liquid-like properties ([@bib36]), which are reminiscent of the formation of membrane-less compartments induced by liquid phase separation ([@bib1]). We previously showed that these NEMO condensates are mediated by the C-terminal bipartite UBD of NEMO and the multivalent interactions with M1- and K63-linked polyubiquitin chains. Like NEMO, and its Optineurin and ABIN2 related proteins, CEP55 likely forms an elongated coiled-coil structure with two similar folded UBDs in its C-terminal part that are interspersed by a disordered linker of 30--50 amino acids rich in hydrophobic Pro residues. Such a bipartite domain and CEP55 oligomerization could favor multivalent-multivalent interactions with extended conformations of M1- and K63-linked polyubiquitin chains, facilitating formation of membrane-less biomolecular condensate through liquid phase separation. These multivalent interactions could guide the assembly with midbody-associated proteins required for cytokinesis such as MKLP-1 and ALIX proteins. Consistent with this hypothesis, we observed impaired interactions of the C440A/C443A debilitating UBZ mutant of CEP55 with MKLP-1 and ALIX in preliminary experiments. Further characterization of CEP55 and its ubiquitin-mediated protein assembly should provide deeper insights into the mechanism by which CEP55 is tightly regulated in cytokinesis and may contribute to open therapeutic strategies for cancers.
Limitations of the Study {#sec3.1}
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In this study, we have clearly shown that CEP55 contains two NEMO-like UBDs in its C-terminal part, which are both crucial for cytokinetic abscission in a ubiquitin-binding-dependent manner. However, neither the ubiquitinylated protein, which can be CEP55 itself, nor the ubiquitin ligase(s) that contribute to CEP55 recruitment to the midbody were identified in this study but are worth investigating in the future. Moreover, although structural models of CEP55 NOA and UBZ domains were validated by biophysical and biochemical data, determination of their true structures by NMR and/or crystallization, which proved more difficult than expected, is lacking and requires further investigation in the future.
Methods {#sec4}
=======
All methods can be found in the accompanying [Transparent Methods supplemental file](#mmc1){ref-type="supplementary-material"}.
Supplemental Information {#appsec2}
========================
Document S1. Transparent Methods, Figures S1--S7, and Table S1
We thank N. Tarantino for technical assistance, A. Haouz (Crystallography Core Facility), P. England (Molecular Biophysics Core Facility), S. Petres (Production and Purification of Recombinant Proteins Core Facility), as well as M. Matondo, T. Chaze, and J. Chamot-Rooke (UtechS MSBio). We acknowledge A. Echard and M. Mhlanga for their advice and support. Funding was supplied by Ligue Nationale Contre le Cancer, Global Care Initiative, and Institut Carnot Pasteur MS (to F.A.); Ligue Nationale Contre le Cancer comité du Val-d\'Oise and Fondation ARC pour la recherche sur le cancer (to E.L.).
Author Contributions {#sec6}
====================
Conceptualization, K.N.S.H., E.L., and F.A.; Methodology, K.N.S.H., E.F., A.B., M.C., L.D., M.B., E.L., and F.A.; Investigation, K.N.S.H., E.F., A.B., L.D., M.C., M.B., E.L., and F.A.; Formal Analysis, K.N.S.H., L.D., M.B., E.L., and F.A.; Resources, K.N.S.H., E.F., and M.C.; Project Administration, F.A.; Writing - Original Draft, K.N.S.H. and F.A.; Visualization, K.N.S.H., E.L., and F.A.; Writing - Review & Editing, K.N.S.H., R.W., A.I., E.L., and F.A.; Funding Acquisition and Supervision, E.L. and F.A.
Declaration of Interests {#sec7}
========================
The authors declare no competing interests.
Supplemental Information can be found online at <https://doi.org/10.1016/j.isci.2019.08.042>.
[^1]: Present address: Sorbonne Université, INSERM U1135, CNRS ERL 8255, Center d\'Immunologie et des Maladies Infectieuses (CIMI-Paris), 75013 Paris, France
[^2]: These authors contributed equally
[^3]: Lead Contact
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