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the methods of data collection and analysis utilized in this study are similar to those used in our previously published analysis investigating adverse cardiovascular outcomes and overall mortality risk with oral antidiabetes anti - hyperglycemic monotherapy (14). the source population was obtained from an ehr - derived clinical data repository at the cleveland clinic. this study was approved by the institutional review board. for the period 24 october 1998 to 12 october 2006, we identified all newly and previously diagnosed patients with type 2 diabetes using documented icd-9 and by identifying patients with at least two encounters for diabetes after visiting the cleveland clinic main campus or family health centers and who had a prescription for glyburide, glipizide, or glimepiride entered into the ehr. patients were stratified into three medication cohorts according to the initial prescription entered in the ehr at baseline. all patients were 18 years of age and had no history of dialysis at baseline. patients prescribed insulin or other injectable diabetes medications (as monotherapy or in conjunction with oral agents) and those on multiple oral agents at baseline were excluded. follow - up began on the day after the first prescription of the qualifying study drug was entered in the ehr. patients entered the cohort in a staggered fashion at any time point between 24 october 1998 and 12 october 2006 and from that time were followed until the date of mortality or censoring. patients with no observed mortality were censored on the last clinic encounter or the date of extraction of vital status from the social security death index (ssdi) minus a 6-month lag, whichever came last. a multivariable analysis was utilized to compare patients in each cohort, which allowed us to adjust for differences in baseline characteristics. variables were chosen and derived based on prior considerations of their clinical relevance with respect to the risk of mortality. the baseline medical history variables chosen for the overall mortality model were as follows : age, sex, race (caucasian versus non - caucasian), modification of diet in renal disease estimated glomerular filtration rate, hemoglobin a1c (a1c), bmi, systolic blood pressure, diastolic blood pressure, hdl cholesterol, ldl cholesterol, triglycerides, smoking status, ace therapy, or angiotensin receptor blocker therapy, aspirin therapy, clopidogrel therapy, cholesterol - lowering medication, new diabetes, cad, congestive heart failure, and median household income. we were unable to use family history or alcohol use as predictor variables due to inconsistent documentation in the ehr. the baseline variables were derived from the ehr on the date closest to the date of the first sulfonylurea prescription up to 21 days after baseline. missing baseline values were imputed by chained equations (mice) package, version 1.16 for r, without regard to the outcomes, using regression techniques that included all patients and all baseline values to predict the missing value. mortality was defined by documentation of death in the ehr or by being listed as deceased in the ssdi, which allowed us to identify those deceased individuals who were lost to follow - up in the ehr. analyses were performed using the statistical package r for windows, version 2.8.1 (r development core team 2008). multivariable cox proportional hazards models were used to derive hazard ratios for the three baseline medication group comparisons. after adjustments were made for the baseline covariates, the following comparisons were made in all patients and restricted to patients with a history of cad : glipizide versus glyburideglipizide versus glimepirideglyburide versus glimepiride glipizide versus glyburide glipizide versus glimepiride glyburide versus glimepiride follow - up began on the day after the first prescription of the qualifying study drug was entered in the ehr. patients entered the cohort in a staggered fashion at any time point between 24 october 1998 and 12 october 2006 and from that time were followed until the date of mortality or censoring. patients with no observed mortality were censored on the last clinic encounter or the date of extraction of vital status from the social security death index (ssdi) minus a 6-month lag, whichever came last. a multivariable analysis was utilized to compare patients in each cohort, which allowed us to adjust for differences in baseline characteristics. variables were chosen and derived based on prior considerations of their clinical relevance with respect to the risk of mortality. the baseline medical history variables chosen for the overall mortality model were as follows : age, sex, race (caucasian versus non - caucasian), modification of diet in renal disease estimated glomerular filtration rate, hemoglobin a1c (a1c), bmi, systolic blood pressure, diastolic blood pressure, hdl cholesterol, ldl cholesterol, triglycerides, smoking status, ace therapy, or angiotensin receptor blocker therapy, aspirin therapy, clopidogrel therapy, cholesterol - lowering medication, new diabetes, cad, congestive heart failure, and median household income. we were unable to use family history or alcohol use as predictor variables due to inconsistent documentation in the ehr. the baseline variables were derived from the ehr on the date closest to the date of the first sulfonylurea prescription up to 21 days after baseline. missing baseline values were imputed by chained equations (mice) package, version 1.16 for r, without regard to the outcomes, using regression techniques that included all patients and all baseline values to predict the missing value. mortality was defined by documentation of death in the ehr or by being listed as deceased in the ssdi, which allowed us to identify those deceased individuals who were lost to follow - up in the ehr. analyses were performed using the statistical package r for windows, version 2.8.1 (r development core team 2008). multivariable cox proportional hazards models were used to derive hazard ratios for the three baseline medication group comparisons. after adjustments were made for the baseline covariates, the following comparisons were made in all patients and restricted to patients with a history of cad : glipizide versus glyburideglipizide versus glimepirideglyburide versus glimepiride glipizide versus glyburide glipizide versus glimepiride glyburide versus glimepiride using the ehr, we were able to identify 4,279 initiators of monotherapy with glyburide, 4,325 initiators of monotherapy with glipizide, and 2,537 initiators of monotherapy with glimepiride, with and without a history of cad, 18 years of age and not on insulin or a noninsulin injectable at baseline. table 1 shows the distribution of the baseline categorical variables for the entire cohort as well as the subgroup of patients with a history of cad. baseline characteristics of the entire cohort and the subgroup of patients with cad : categorical variables baseline characteristics of the entire cohort and the subgroup of patients with cad : continuous variables data are means sd or percent. egfr, estimated glomerular filtration rate ; mdrd, modification of diet in renal disease. the cohorts contained a total of 1,921 mortality events in the entire cohort (n = 11,141) and 322 in the subgroup with a history of documented cad (n = 1,505). the survival curves for mortality, for both the entire cohort and for the subgroup with a documented history of cad, can be seen in fig. there were 1,753 patients lost to follow - up in the ehr but with vital status from the ssdi. the hazard ratios with 95% cis for the sulfonylurea monotherapy comparisons for mortality in the entire cohort, and the subgroup with documented cad, can be seen in table 3, after adjusting for baseline variables. overall mortality in the entire cohort (a) and subgroup with a documented history of cad (b), treated with sulfonylurea monotherapy. the decreasing numbers of patients at risk for mortality are secondary to the staggered entry of the study subjects, not loss to follow - up. hazard ratio (95% ci) for the sulfonylurea monotherapy comparisons for mortality in the entire cohort and the subgroup with documented cad mortality model adjusted for baseline covariates : age, sex, race (caucasian vs. non - caucasian), modification of diet in renal disease estimated glomerular filtration rate, a1c, bmi, systolic blood pressure, diastolic blood pressure, hdl cholesterol, ldl cholesterol, triglycerides, smoking status, ace or angiotensin receptor blocker therapy, aspirin therapy, clopidogrel therapy, cholesterol - lowering medication, new diabetes, cad, congestive heart failure, and median household income. for the period 24 october 1998 to 12 october 2006, no difference in overall mortality risk was found with glipizide versus glyburide (hazard ratio 1.04 [95% ci 0.941.15 ]), glipizide versus glimepiride (1.05 [0.921.19 ]), or with glyburide versus glimepiride (1.00 [0.891.14 ]). the subanalysis on patients with documented cad revealed a trend toward an increased overall mortality risk with glyburide versus glimepiride (1.36 [0.961.91 ]) and glipizide versus glimepiride (1.39 [0.991.96 ]). no difference (or trend) in overall mortality within the subgroup was appreciated with glipizide versus glyburide (1.03 [0.801.31 ]). the present study did not find a statistically significant difference in the risk of overall mortality among the various treatment options, suggesting that overall mortality is not substantially influenced by the choice of sulfonylurea. however, in the subanalysis of patients with documented cad, a trend toward an increased overall mortality risk with glyburide versus glimepiride (hazard ratio 1.36 [95% ci 0.961.91 ]), and surprisingly a trend toward an increased risk of mortality with the sur1-specific sulfonylurea glipizide versus glimepiride (1.39 [0.991.96 ]), were observed, suggesting that glimepiride may be the preferred sulfonylurea in those with underlying cad. although the study did not find any obvious difference in mortality risk between patients treated with specific sulfonylureas, it is still possible that some differences in mortality may truly exist. there were significantly fewer patients in the cad subanalysis, and the results showed a strong trend toward a reduced risk with glimepiride. it is quite possible that a larger sample size would have detected a significant difference. however, it would not be appropriate to perform a post hoc power calculation since nonsignificant p values will tend to be associated with low power even if the sample size was adequate (15). a clinically meaningful difference in mortality would seem unlikely in the main analysis of all patients given the large sample size. substantial multicollinearity in a regression model can cause erroneous conclusions about the association between individual variables (e.g., sulfonylurea type) and the outcome of interest. the variance inflation factors ranged from 1.93 to 1.95 in the entire cohort and 2.35 to 2.40 in the subset of patients with documented cad, which, according to snee (16), suggests substantial multicollinearity is unlikely to be present. there is a discrepancy within the literature regarding the risk of mortality (overall or cardiovascular mortality) with specific sulfonylureas. a recent report found no substantial (statistically significant) differences in either 30-day or 1-year mortality in users of various sulfonylureas after myocardial infarction (although use of gliclazide monotherapy showed a trend toward lower mortality [hazard ratio 0.70 { 95% ci 0.481.0 } ]), suggesting that mortality is not substantially influenced by the choice of sulfonylurea (17). (18) found that total mortality was lower for gliclazide and glimepiride versus glibenclamide (glyburide) treatment (0.33 [0.260.41 ], p < 0.001, and 0.605 [0.410.89 ], p < 0.01, respectively) as well as a reduced cardiovascular mortality with gliclazide versus glibenclamide (glyburide) (0.29 [0.210.38 ], p < 0.001). the point estimates (hazard ratios) differ greatly between the analyses conducted by horsdal. there are a variety of proposed mechanisms for an increased mortality risk with specific sulfonylureas. despite the differing effects of individual sulfonylureas on the sur receptors and myocardial ischemic preconditioning, there are also differing effects regarding the risk of hypoglycemia, independent of their sur - binding characteristics, which may be influencing mortality (13). among the sulfonylureas studied in our analysis, glyburide is the most common agent associated with documented hypoglycemia (19). glyburide has been shown to continue to stimulate insulin secretion in the setting of profound hypoglycemia to a greater extent when compared with glimepiride (20), in part because glyburide accumulates within the -cell (21), unlike other sulfonylureas, prolonging insulin secretion. thus, hypoglycemia could be playing a dominant role in increasing the risk of mortality (more so than differing selectivity and effects on the sur receptors and ischemic preconditioning, respectively), which has previously been reported with sulfonylureas, specifically when compared with metformin (14,2224). other than the increased risk of hypoglycemia documented with glyburide (and the differences in other pharmacologic properties inherent to the individual sulfonylureas : sur specificity and effects on ischemic myocardium), glipizide, glimepiride, and glyburide generally have very similar side - effect profiles. the analysis was based on exposure to a medication based on the initial prescription entered in the ehr ; however, there is no documentation of compliance with the prescribed medication. the prescribed medication at baseline defined which medication group the patient belonged ; however, the medication exposure times after baseline are unknown. current clinical practice procedures suggest that it is more likely for additional agents to be added to a baseline medication than to switch from one class of medication to another or from one sulfonylurea to another. approximately 70% of the cohort remained on a single drug (baseline medication) throughout their time in the cohort. the medication groups in our study were not balanced with respect to baseline variables and risk factors ; however, the multivariable analysis adjusted for the differences in baseline variables and risk factors that had the most relevance with respect to the risk of mortality. although some covariates may have changed over time, we would not anticipate these changes to favor one specific sulfonylurea versus another (besides the inherent characteristics of the individual agents). sulfonylurea monotherapy was not randomized in the present study, so selection bias may be present. it is possible that one sulfonylurea may have been chosen over another because of cost (the food and drug administration did not approve first - time generic formulations of glimepiride until november 2005), patient age, reduced glomerular filtration rate, risk of hypoglycemia, or perceived differing effects on myocardial ischemic preconditioning. however, although age and renal insufficiency are associated with an increased risk of death, the multivariable analysis adjusted for differences in baseline age and renal function, so this should not explain the results. to take into account the fact that generic glimepiride was not available throughout the entire duration of our study, we adjusted for socioeconomic status by including the median household income estimated from zip code data from the 2000 census in the multivariable analysis. the strengths of the study include a large cohort of patients followed up to 8 years and real - world effect of the medications in a diverse patient population. in addition, we adjusted for many baseline variables (accurately captured by the ehr) that have substantial effects on mortality. furthermore, linking our outcome to the ssdi allowed us to capture mortality in those patients lost to follow - up in the ehr. our results did not identify an increased mortality risk among the individual sulfonylureas (glyburide, glipizide, or glimepiride) in the entire cohort but did find evidence of a trend toward an overall mortality reduction with glimepiride in those with documented cad, suggesting that glimepiride may be the preferred sulfonylurea in those with underlying cad. the literature contains conflicting results regarding whether an increased overall mortality (or cardiovascular mortality) risk accompanies the various sulfonylureas (12,17,18). this discrepancy would support prospective studies to determine whether the difference in pharmacologic properties inherent to individual sulfonylureas translates into differences in the risk of adverse cardiovascular outcomes and overall mortality, especially in patients with preexisting cad. | objectivesulfonylureas have historically been analyzed as a medication class, which may be inappropriate given the differences in properties inherent to the individual sulfonylureas (hypoglycemic risk, sulfonylurea receptor selectivity, and effects on myocardial ischemic preconditioning). the purpose of this study was to assess the relationship of individual sulfonylureas and the risk of overall mortality in a large cohort of patients with type 2 diabetes.research design and methodsa retrospective cohort study was conducted using an academic health center enterprise - wide electronic health record (ehr) system to identify 11,141 patients with type 2 diabetes (4,279 initiators of monotherapy with glyburide, 4,325 initiators of monotherapy with glipizide, and 2,537 initiators of monotherapy with glimepiride), 18 years of age with and without a history of coronary artery disease (cad) and not on insulin or a noninsulin injectable at baseline. the patients were followed for mortality by documentation in the ehr and social security death index. multivariable cox models were used to compare cohorts.resultsno statistically significant difference in the risk of overall mortality was observed among these agents in the entire cohort, but we did find evidence of a trend toward an increased overall mortality risk with glyburide versus glimepiride (hazard ratio 1.36 [95% ci 0.961.91 ]) and glipizide versus glimepiride (1.39 [0.991.96 ]) in those with documented cad.conclusionsour results did not identify an increased mortality risk among the individual sulfonylureas but did suggest that glimepiride may be the preferred sulfonylurea in those with underlying cad. |
in the 21st century, cereals continue to constitute the most important crops with an annual output of 2 billion tons (according to fao in 2006 ; http://www.fao.org). in today 's worldwide production, barley ranks fourth among cereals and is preferentially used as feed grain, as a raw material for beer production and, to a smaller extent, as food. initially, barley was domesticated in the fertile crescent of the neolithic near east over 10 000 years ago. in the subsequent millennia, farmers continuously adapted local populations to their needs, leading to a great variety of landraces. about 100 years ago, these formed the basis for the development of modern cultivars by cross breeding. during this time, grain yield was more than doubled with an estimated genetic contribution to this increase of about 3050%. however, to meet the future challenges imposed by a changing environment, to feed a growing world population, and to provide renewable resources to satisfy the soaring demand for energy, genomics - based technologies have to be efficiently implemented to study the genetic basis of plant performance and to isolate agronomically important genes from the genetic diversity present in the gene pool of barley. a broad spectrum of resources has been developed during the last two decades to facilitate the systematic analysis of the barley genome. these include a large number of mapped molecular markers, comprehensive est collections, bac libraries, mutant collections, dna arrays, and enabling technologies such as the large scale production of doubled haploids and efficient transformation protocols. advances made in barley genomics and recent efforts made towards physical map construction and sequencing of the barley gene space (http://barleygenome.org) will largely contribute to a comprehensive understanding of gene functions in the context of agronomical important phenotypes (refer to figure 1 and table 1). recently, the techniques and methods employed in cereal genomics have been reviewed [38 ]. in this overview, we have tried to summarize progress in structural and functional genomics of barley and put emphasis on important agronomical aspects such as grain yield, seed quality traits, and implications for malting quality improvement. the seven barley chromosomes represent the basic genome of all triticeae species. still, the large genome (5500 mb), of which 80% is composed of repetitive dna is presently not amenable to whole genome sequencing. therefore, large scale sequencing programs for the development of expressed sequence tags (ests) from various cdna libraries have been initiated. the progress made in the last 5 years resulted in the generation of 437,713 ests covering different cdna libraries from various stages of plant development and tissues challenged with abiotic and biotic stresses (http://www.ncbi.nlm.nih.gov/dbest/dbest_summary.html, september 14th 2007 release). alignment of these ests led to the identification of a representative set of 50,453 unigenes with 23,176 tentative consensi and 27,094 singletons (http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=barley), representing possibly about 75% of all genes in the barley genome. an earlier estimate of the barley gene content based on 110,000 ests led to the prediction of around 30 000 unique genes. the same est data set, which was generated from different tissues covering the plant 's life cycle, was analyzed to gain insight into differential gene expression programs in diverse plant tissues by in silico expression studies. in this way, comprehensive analysis of extensive est resources generated from large genomes provides snap shots of the transcriptome aiding in gene discovery. this also allows identifying coregulated metabolic and regulatory networks [10, 11 ] and helps to establish high - density molecular maps [1214 ] which form the basis for comparative genomic studies, trait mapping, and map - based gene isolation. thus, in large genome cereal species like barley, est sequences facilitate a comprehensive overview of gene content and represent a resource to study the evolution and organization of a genome. regarding the latter, est - derived information remains limited as it fails to provide, for instance, regulatory information, since promoters and full length sequences are not available. physical maps represent an important link to connect the genetic level to the sequence level. similar to genetic maps, physical maps are available at different levels of resolution. wheat - barley addition lines are a useful resource to rapidly assign ests to an entire chromosome or to a chromosome arm. using this resource, 1787 genes present on the barley 1 genechip could be assigned to the six different chromosomes of barley (365 genes to 2h, 271 to 3h, 265 to 4h, 323 to5h, 194 to 6h, and 369 to 7h). at a higher resolution, a physical map of all the seven barley chromosomes has been prepared by mapping dna markers derived from both genomic as well as gene - based sequences relative to the translocation breakpoints of individual chromosomes that had been isolated using microdissection techniques. the resulting map is of particular value, as it can be directly aligned to the genetic map of barley by common markers and thus allows for the estimation of the ratio between genetic and physical distances. here the presence of a wheat gametocidal chromosome in a wheat barley addition line was exploited to select 90 progeny lines that carried differently sized fragments of barley chromosome 7h. these were subsequently used to determine the physical order and distance of markers located on barley chromosome 7h. during the past several years, core public resources have been established by generating bacterial artificial chromosome (bac) libraries from different barley cultivars : morex (; 313,344 clones), cebada capa (; 177,000 clones) and haruno nijo (http://www.intl-pag.org/10/abstracts/pagx_p393.html). based on fluorescence in situ hybridization (fish) techniques karyotype landmarks were derived for barley, which could be used in future to place the bac clones onto the physical map. this map shows that the genetic linkage maps are well covered with markers among all chromosomes. at the same time, the physical maps reveal large areas of the barley genome that have yet to be mapped. these unmapped areas mainly consist of heterochromatin and show very low recombination rates. in accordance with these findings, there is increasing evidence that genes are not randomly distributed across the barley genome but confined to a gene space, which mainly covers the distal parts of the chromosomes. experimental evidence for the existence of a gene space has been gained from screening a barley bac library with est - derived probes, which showed a significant nonrandom distribution across the bac clones. more direct evidence has been reached on the sequence level for barley and other triticeae species. although up to now only a limited amount of sequence data is available, the average density of annotated genes is much higher than that expected for a random distribution across the genome. the disproportionate gene number found is probably due to the preferential selection of gene containing bacs for sequence analysis. within single bacs, there is considerable variation ranging, in case of barley, from 1 gene in 12 kb up to 1 gene in 220 kb (for review see). thus even the gene space itself seems to be characterized by a highly variable distribution of genes against the backdrop of noncoding, mainly repetitive dna. the existence of a gene space also opens up new opportunities to focus analyses on gene - rich regions only. recently, international efforts have been gearing up to utilize the extensive barley est resources for bac anchoring and genetic mapping. an elegant approach of screening of the morex bac library using probes resulted in the identification of gene containing bacs. upon fingerprinting of a subset of 21 161 clones, 2262 contigs could be assembled covering approximately 9.4% of the barley genome. furthermore, a database has been set up to search screening results of bac libraries as well as to provide an integrative view of data from the existing barley genetic and physical maps (http://www.genome.clemson.edu). the identified bac - based gene - rich regions of the genome have been selected as a genomic reference from cultivar morex to initiate sequencing of all gene - containing regions of the barley genome by an international effort coordinated through the international barley sequencing consortium (ibsc, http://barleygenome.org). despite the lack of a barley genome sequence, functional genomics efforts have been initiated by taking advantage of the available est sequence information generated by multinational coordinated efforts (see above). as a first step, efforts were made to derive functional assignments of the available barley unigene set by annotation transfer from homologous sequences relying on the available plant whole genome sequences and by identifying common motifs from interpro. as a result, several ontology structures such as mips and mapman functional categories (n. sreenivasulu, unpublished data ; http://mapman.mpimp-golm.mpg.de/index.shtml) were developed. such computational methods also yielded putative regulatory networks as well as metabolic pathway interaction networks, but still about half of the genes have to be classified as unknown. the available barley est unigene resources played a profound role in developing several platforms for transcriptome analysis including cdna - based macroarrays [11, 25 ], microarrays, and oligonucleotide - based affymetrix arrays [10, 27 ]. other profiling techniques used in barley include cdna - aflp, sage (serial analysis of gene expression) [29, 30 ], and igentifier. the latter method combines elements of tag sequencing such as sage and fragment display. by successfully applying these techniques, barley transcriptome data have been collected from grain development [11, 25, 32 ], grain germination [33, 34 ], at least 15 different tissues / organs covering different growth stages, and abiotic [26, 3537 ] as well as biotic stress responses [38, 39 ]. the new insights gained from transcriptome analysis of host - pathogen studies have lately been reviewed by wise.. these large scale gene expression data sets serve as baseline experiments to generate a barley transcriptome atlas. also, an online plant expression database (plexdb), previously known as barleybase (http://www.plexdb.org/plex.php?database=barley) has been created to store, visualize, and statistically analyze barley 1 genechip data. while transcriptomics have brought about substantial progress in elucidating biochemical pathways of barley seed metabolism (see reviews [5, 42 ]), very recent findings shed light on the interplay of many cellular and metabolic events that are coordinated by a complex regulatory network during barley seed development [10, 11, 25 ]. studying expression data of nearly 12 000 seed - expressed genes revealed, for instance, the participation of tissue - specific signaling networks controlling aba - mediated starch accumulation (via snf1 kinase and a set of transcription factors) in the endosperm and participation of aba - responsive genes in establishing embryo desiccation tolerance. cpg methylation found in the promoters of prolamin box - binding factor and b - hordein genes suppresses transcript levels during the prestorage until the intermediate phase of grain development. this process coincides with the coexpression of methyltransferases, core histones and dna - unwinding atpases. thus storage protein gene expression may be regulated by cpg methylation. using a lys 3a mutant, it has been shown that demethylation of the b - hordein promoter does not occur in the mutant, hence transcripts encoding storage proteins such as b - hordeins and c - hordeins are almost absent in the developing endosperm of this mutant. transcriptome profiling of barley embryos using the 22k affymetrix barley 1 genechip revealed activation of developmentally distinct defense related gene sets including coregulated phenylpropanoid and phytoalexin related genes around 20 days after flowering (daf), followed by upregulation of antioxidant and pathogen related gene sets around 37 daf. the knowledge obtained on metabolic processes of seed quality traits could eventually be used to develop superior varieties by genetic engineering or by marker - assisted selection in conventional breeding programs. transcriptome analysis has also been carried out during barley grain germination at tissue - specific levels [10, 46 ]. using cdna array technology gene expression was analyzed in germinating seed samples, collected from ten different barley genotypes showing differential malting response. based on six different malting quality parameters related to hydrolytic events connected to protein, starch, and cell wall degradation 19 candidate genes were identified, whose transcript abundance showed a significant correlation with some of the malting quality parameters. analyzed seven different sage libraries derived from malted grains and identified 100 most abundant transcripts showing differential responses during eight different time points during malting. these transcripts are related to stress and defense response, hydrolytic processes and translational events. the list of candidate genes identified in the two studies [30, 46 ] was further validated by a genetical genomics approach in which gene expression studies were conducted with populations segregating for malting traits [34, 47 ]. a major aim of functional genomic studies is to understand the metabolic and regulatory networks within the structural and functional context of cells, tissues, and organs often changing with time. hence in this review, we update the functional genomic resources available (table 1) to study gene functions in barley using reverse genetics approaches and highlight the initial success achieved through genetic engineering based on the manipulation of individual genes. to determine gene - function relationships, large scale genome - wide reverse genetics approaches have been developed in barley (see for review) which includes both nontransgenic technology platforms such as tilling (targeting induced local lesions in genomes) and insertional mutagenesis systems based on transgenic technology [5054 ]. thus, the scottish crop research institute generated a large m2 tilling population in the barley cultivar optic with leaf material and seeds from 20 000 plants freeze dried and archived. ems induced mutations were scored at various growth stages under different conditions and documented [49, 55 ]. mutant phenotypes, candidate genes, and observed dna sequence variations can be queried in an scri mutant database (http://germinate.scri.ac.uk/barley/mutants/index.php?option=com_wrapper&itemid=35). in a more recent attempt, ipk developed a tilling population of 10 000 m2 plants in the cultivar similarly, a collection of 5000 m3 mutants of the cultivar morex is provided by the university of bologna (http://www.intl-pag.org/13/abstracts/pag13_p081.html). to aid functional gene analysis, insertional mutagenesis approaches were followed in barley during the last decade (i) to create loss - of - function mutations by the insertion of transposable elements into a gene of interest [5053 ] and (ii) use activation tagging (the random genomic insertion of either promoter or enhancer sequences) to generate dominant gain - of - function mutations [54, 56 ]. insertion lines have been generated by creating transgenic plants carrying ac and ds elements, and crossed them to induce ds transposition [5052 ]. ds elements were preferentially found in genic regions and exhibited a high - remobilization frequency [52, 53 ]. such ds launch pads, represented by barley lines with each harboring a single copy ds insertion at a well - defined position in the genome, will be valuable for future targeted gene tagging. similarly, dominant overexpression phenotypes [54, 56 ] will help to study gene functions in the large barley genome where loss - of - function mutations often may not cause phenotypes because of gene redundancy. in order to functionally characterize candidate genes identified in functional genomic studies, it was mandatory to establish a stable and efficient genetic transformation technique in barley. in contrast to the biolistic gene transfer technique, a more efficient agrobacterium mediated barley genetic transformation method based on immature embryos was developed in spring barley. in a recent attempt to further improve this technology, kumlehn. developed a transformation method for winter barley based upon the infection with agrobacterium of androgenic pollen cultures. by this approach, homozygous double haploid plants could be immediately obtained at high frequency through chromosome doubling. during the last decade, systematic efforts were made for genetic engineering of barley to improve seed quality traits including those related to malting (reviewed in). malting improvement has been addressed by altering the expression of hydrolytic enzymes related to the degradation of storage products such as starch (and - amylases, [61, 62 ]) and cell wall components. in another approach, several enzymes such as xylanase, glucanase, endo-, and exoprotease were over expressed in transgenic barley grains and preferably the enzyme mix necessary for malting process are provided by transgenic seeds. protein engineering has been used to produce thermostable 1, 3 ; 1, 4 - glucanases in transgenic barley grains [6466 ]. such grains can be used to enhance the feed quality of barley for poultry [67, 68 ]. in a similar approach, a hybrid cellulase gene driven by the endosperm specific rice glub-1 promoter was expressed and produced the enzyme up to 1.5% of total grain protein. in addition, functions of key genes involved in determining seed quality traits related to storage product accumulation were tested. for instance, antisense downregulation of limit dextrinase inhibitor showed reduced amylose over amylopectin levels and eventually reduced total starch. also overexpression of wheat thioredoxin h in the endosperm of transgenic barley grain leads to increased activity of the starch debranching enzyme limit dextrinase [71, 72 ]. further, a powerful approach of antisense oligodeoxynucleotide inhibition has been used to reveal sugar signaling networks. short stretches of 1225 nucleotide long single - strand sequences have been delivered to barley leaf cells to block the effect of susiba2, a key transcriptional activator involved in plant sugar signaling. recently, this approach has been successfully implemented to deliver antisense oligodeoxynucleotides to barley seed endosperm to suppress sugar related signaling genes. hvgamyb, a transcription factor initially identified in aleurone and shown to be upregulated by gibberellin, has been shown to be expressed also in barley anthers. the overexpressing hvgamyb transgenic lines show reduced anther size with a male sterility phenotype. our laboratory has recently characterized a new protein called jekyll, which is preferentially expressed in barley grain nucellar projection tissue. its downregulation decelerates autolysis of nurse tissue. as a result, proliferation of endosperm nuclei is impaired and less starch is finally accumulated in the endosperm. with respect to applied aspects in crop plants, a comprehensive knowledge of cellular and functional complexity as related to key agronomic traits a number of tools and databases were developed at our institute (leibniz institute of plant genetics and crop plant research / ipk) to store, analyze, and display the data derived from multiparallel - omics profiling studies at transcript, metabolite, and protein / enzyme level with the aim to eventually gain insight into the organization of function - related networks in barley [78, 79 ]. these include cr - est (it provides access to clustering and annotation data of ipk est projects), meta - all, and metacrop (they allow to access curated metabolic pathway information and kinetic reactions of crop plants), vanted (for visualization and analysis of metabolic and regulatory networks), hit - mds (for screening of coexpressed genes and validation of cluster centroids) as well as barley mapman and pageman [http://mapman.mpimp-golm.mpg.de ; to index and visualize overrepresented functional categories and detailed metabolic pathway charts from throughput transcriptome data ]. with the focus of using the developing seed as model for systems biology studies, we investigated transcriptional and metabolic networks during grain development [11, 25, 82 ], developed 3d models of the developing barley grain, implemented magnetic resonance - based techniques to establish 4d models as a framework to store different sets of data in their spatiotemporal context, visualized the spatial distribution of specific biochemical compounds by noninvasive nmr - based imaging methods and established kinetic models of primary metabolism (and e. grafahrend - belau and b. junker, unpublished data) as already worked out for potato. in addition, a proteomic platform has been successfully established to study barley grain development [88, 89 ]. the emerging model (largely qualitative) explaining how the barley grain develops and functions has to be further validated especially by the creation and analysis of different lines of transgenic plants with perturbations at putative key metabolic and/or regulatory sites (see figure 1). almost two decades ago, rflp markers were employed to develop the first comprehensive molecular marker maps in barley [9092 ]. using those rflp maps, a series of agronomic traits and characters including many quality traits and resistance against several diseases have been mapped (for review see [93, 94 ]). later, the availability of large numbers of ests facilitated the systematic development of functional markers, for example, by extracting ests containing simple sequence repeat (ssr) motifs using appropriate software tools. although est - based ssr markers have been shown to be less polymorphic than their genomic counterparts, this drawback is more than compensated for by the ease of their development. also, the availability of ests from multiple - genotypes / cultivars of barley provides the possibility to identify sequence polymorphisms (mainly single - nucleotide polymorphisms and small indels) in the corresponding est alignments. these in turn can be exploited for the development of markers [96, 97 ]. kota. developed the computer algorithm snipping for discovery of functional markers through browsing est assemblies in barley. also an snp2caps program has been published to facilitate the computational conversion of snp markers into caps markers. information generated from the diverse mapping projects was further enhanced by the development of consensus maps [14, 100102 ]. these provide integrative genetic information by featuring high marker densities. although the gel - based genotyping platforms offer the best quality marker systems, their low throughput encouraged researchers to explore high - throughput technologies that can simultaneously assay thousands of markers based on single nucleotide polymorphisms (snp). most recently, genome - wide scans using snp - based genotyping platforms such as illumina goldengate beadarrays and the diversity arrays technology (dart), which do not require any sequence information have been successfully established in barley. although darts are not systematically interrogating expressed sequences, the choice of appropriate enzymes facilitates their enriched representation. based on dart technology, a high - density consensus map has recently been established. a number of recent studies also reported the use of the affymetrix barley 1 genechip for identifying single - feature polymorphisms (sfps), which cover not only snps but also indels and polymorphisms generated due to alternative splicing and polyadenylation [34, 106 ]. an important application of the above discussed functional markers is marker - assisted selection (mas). mas is based on linking the dna polymorphisms revealed by marker analysis with agronomical traits allowing for their rapid selection in routine breeding programs. mas can be performed already at juvenile growth stages and before flowering, and thus provides breeders with the opportunity to implement faster back - crossing strategies and allele enrichment in complex crosses, which eventually reduces the time and costs required for the development of improved varieties. despite its inherent advantages, the application of mas in barley up to now has mainly been restricted to monogenic traits such as disease resistances. here, one of the most widespread examples is the marker assisted selection of the rym4 gene giving resistance to the barley yellow mosaic virus complex. for this gene, several closely linked and easily scorable markers have been developed [107, 108 ]. more recently, cloning of the gene facilitated the exploitation of functional polymorphisms within the coding region of the resistance gene to differentiate between alleles. using mas, several genes providing full resistance could be readily combined in complex crosses without time consuming progeny tests in the greenhouse or in the field (e.g., [110, 111 ]). (i) compared to monogenic traits, quantitative traits are characterized by lower heritabilities impairing their accurate scoring and entailing a less accurately defined genetic position of the corresponding quantitative trait locus (qtl). as a result, large chromosomal fragment needs to be selected for, resulting in the meiotic transfer of many potentially undesired genes. the feasibility of downtracking a qtl to a single gene has been initially demonstrated in tomato and requires the stepwise size reduction of a qtl fragment and its conversion into a near isogenic line by repeated backcrossing (for review see). in barley, this approach has been successfully employed to isolate the bot1 gene underlying a major qtl conferring boron tolerance. (ii) many of qtl alleles escape detection, when transferred into a different genetic background. the reasons for the disappearance of qtls include epistatic interactions, qtl x environment effects, the allelic states of the parental lines or the small contribution of a single qtl to the overall variance. as a result, only few common qtls were detected, when the results of mapping studies that were performed in different crosses were compared. although the number of successful examples for applying mas in barley breeding is still rather limited (see reviews by [114, 115 ], the recent implementation of high - throughput genotyping platforms (illumina, dart, and sfp identification by using barley 1 genechip affymetrix array) in barley will significantly increase the identification of marker trait associations, and the subsequent identification of potential candidate genes. finally, this will allow to treat qtls as monogenic traits and thus spur their marker assisted manipulation in breeding programs. in combination with a wide range of mapping populations developed for specific agronomic traits, this comprehensive resource of markers now allows the identification of polymorphisms in functionally defined sequences [12, 34, 105, 106 ]. functional markers will also be useful for (i) association studies based on linkage disequilibrium, (ii) detection of cis and trans - acting regulators either based on genetical genomics studies using well - defined mapping populations or by investigating allelic imbalance, (iii) identification of alleles influencing agronomically important traits using tilling / ecotilling approaches (ecotilling is a means to determine the extent of natural variation in selected genes), and (iv) genomics - assisted breeding (see figure 1). linkage disequilibrium is the nonrandom distribution of alleles in a sample population and forms the basis for the construction of genetic maps and the localization of genetic loci for a variety of traits. the principles leading to ld apply to both biparental mapping populations (f2, rils, etc.) and natural populations. therefore, ld mapping is the method of choice for genetic analysis in organisms like humans and animals, where experimental populations are either not available or difficult to establish. because of its inherent advantages, ld mapping approaches are increasingly being applied for plant species, in particular maize. due to the outbreeding character of this species, ld extends only over a few kb and thus leads to a high - genetic resolution, up to the level of individual candidate genes that can be associated with a given trait (see recent reviews [118, 119 ]). the use of association genetic analyses in inbreeding species such as barley has been limited so far however, recent studies have shown that ld extends over much longer genetic distances in barley than in maize. a european germplasm collection of 146 two - rowed spring barley cultivars was used to carry out ld mapping of yield traits using 236 aflp markers. associated markers were identified that are located in similar regions where qtls for yield had been found in barley [93, 121, 122 ]. a systematic survey of 953 gene bank accessions representing a broad spectrum of the genetic diversity in barley genetic resources revealed that ld extends up to 50 cm but is highly dependent on population structure [120, 123 ]. on the one hand, the high level of ld in barley is due to the inbreeding mating type of this species ; on the other hand, the selection of germplasm plays an important role. analysis of a germplasm collection of european cultivars, land races, and wild barley accession from the fertile crescent region provided hints that the level of ld decreases from cultivars to landraces to wild barley. similarly, morrell. reported low levels of ld in wild barley by examining ld within and between 18 genes from 25 accessions. local differences in ld have been observed at the grain hardness locus comprising four closely linked genes (hinb, hina, gsp, pg2). here, a high level of ld was observed in the intergenic region between hinb-1 and hina probably due to transposable elements present in this region, which influence the local recombination rate. by assaying 1524 genome - wide snps in elite northwest. concluded that whole - genome association scans can be exploited for trait mapping in barley. this was further exemplified by the identification of a marker that showed an association with the winter habit and which could be tracked to a cluster of cbf (c - repeat / dre - binding factor) gene homologs. in a recent whole genome ld - mapping approach, steffenson. used 318 wild barley accessions to perform association mapping studies using dart markers to identify rust resistance genes. in addition, ld analysis has been performed based on haplotypes derived from 131 accessions by covering 83 snps within 132 kb around the gene hveif4e, which confers resistance to barley yellow mosaic virus. the authors identified three haplogroups discriminating between the alleles rym4 and rym5. taken together, the above mentioned association studies provide starting points for a more systematic analysis of agronomic traits. these may be selected from the vast ex situ gene bank collections available for this crop. alone at the ipk gene bank some 20 000 different barley accessions represent an ample cross section of the genetic diversity present in this species. however, in order to fully exploit the potential of ld - based association analysis in this species, populations have to be carefully selected to minimize the confounding effects of population structure. this is particularly evident in modern barley germplasm, which is frequently structured into spring and winter as well as 2-rowed and 6-rowed types, forming distinct subpopulations (e.g.,). if these effects are not adequately accounted for during association analysis, the risk of detecting spurious associations increases. differentially expressed genes (but also proteins and metabolites) involved in metabolic and regulatory pathways and identified by high - throughput technologies are treated as phenotypes, and genetic variants that influence gene expression are identified in genetically related lines. this strategy has been successfully applied also in plant systems, and relevant data were reviewed elsewhere [128130 ]. here, we will focus on the latest development in expression qtl (eqtl) mapping in barley. using the barley 1 genechip affymetrix array sfp genotyping has been performed in 35 recombinant lines of a steptoe morex doubled - haploid population, enabling eqtl studies. using a high - throughput sfp genotyping platform, genome - wide linkage analysis has been performed based on 22 000 transcript data collected from 139 dh lines (steptoe morex). the most significant eqtls derived from germinating barley grain are linked to cis regulation. using the same mapping population, a serine carboxypeptidase 1 eqtl has been mapped on chromosome 3h to the same region where a qtl for the malting quality trait diastatic power has been mapped. in another study, instead of a segregating population a set of 47 bc3 dh introgression lines was employed (wild barley [h. spontaneum ] is introgressed in the genetic background of the elite line [h. vulgare ]) in order to understand gene expression networks controlling seed traits. initially, this bc3 dh population was used to identify qtls for yield and yield components. in further experiments, expression data from nearly 12 000 genes interrogated by using a barley seed specific array were used to calculate eqtls (c. pietsch., unpublished). although such initial studies provide evidence that genetical genomics is a promising concept which assists to expose gene - trait relationships, an extensive exploration of the technology needs the full barley genome sequence and improved high - throughput genotyping information. in recent years, we experienced a dramatic development of new tools and technologies for genome research and a concomitantly dramatic increase in data leading to a much improved and advanced knowledge base. barley research gained a lot of momentum from this development but the nonavailability of a whole genome sequence is still a serious limitation. however, due to consortial efforts (see above) and the rapidly developing sequencing technologies that are relevant for even complex genomes like that of barley this limitation will be largely overcome, hopefully within the next five years. high - throughput transcriptome analysis techniques have already provided numerous new insights in transcriptional networks. they will, together with rapidly improving protein and metabolite profiling techniques and in combination with new genetic analysis concepts such as genetical genomics and association genetics, improve our knowledge on the relationship between the genetic and the phenotypic architecture of agronomic traits and thus create a basis for knowledge - based molecular breeding. as a next step systems biology approaches are emerging, which attempt to model complex cellular or organismic functions in response to changing internal and external factors. until now molecular markers have had limited success in barley breeding programs, but due to recent advancement of barley genomics a stronger impact on breeding strategies is expected. for instance, marker technologies together with double haploid production have almost halved the time of variety development in australian wheat and barley breeding programs. however, new whole - genome breeding strategies have to be developed to make full use of the ever increasing knowledge about crop plant genomes and their behavior. | barley (hordeum vulgare), first domesticated in the near east, is a well - studied crop in terms of genetics, genomics, and breeding and qualifies as a model plant for triticeae research. recent advances made in barley genomics mainly include the following : (i) rapid accumulation of est sequence data, (ii) growing number of studies on transcriptome, proteome, and metabolome, (iii) new modeling techniques, (iv) availability of genome - wide knockout collections as well as efficient transformation techniques, and (v) the recently started genome sequencing effort. these developments pave the way for a comprehensive functional analysis and understanding of gene expression networks linked to agronomically important traits. here, we selectively review important technological developments in barley genomics and related fields and discuss the relevance for understanding genotype - phenotype relationships by using approaches such as genetical genomics and association studies. high - throughput genotyping platforms that have recently become available will allow the construction of high - density genetic maps that will further promote marker - assisted selection as well as physical map construction. systems biology approaches will further enhance our knowledge and largely increase our abilities to design refined breeding strategies on the basis of detailed molecular physiological knowledge. |
epigenetics mechanisms rely extensively on histone - mediated signaling, in which chemical modifications can make or break complex biological circuits [1, 2 ]. among the different histone marks, methylation of specific lysine and arginine side - chains can regulate chromatin compaction, repress or activate transcription, and control cellular differentiation [3, 4 ]. the transfer of a methyl group from the cofactor s - adenosyl - l - methione (sam) to substrate peptides can be catalyzed by two classes of enzymes [5, 6 ]. nine arginine protein methyltransferases (pmts) are known in human, whose function, structure, chemistry, and chemical inhibition have recently been reviewed [7 - 9 ] (yost. lysine methylation is catalyzed by set domain pmts, a family of about fifty proteins in human, and dot1l, an enzyme that lacks the canonical set domain, but shares the same fold as arginine pmts. the set domain is a sequence of 130 amino - acids, originally named after the drosophila genes su(var), e(z) and trithorax in which it was originally identified. it is defined by a specific fold organized around a pseudo - knot, and by the presence of two signature motifs, elxf / ydy and nhs / cxxpn, x being any amino - acid [12 - 14 ]. while the set domain is responsible for catalysis, the methyltransferase activity of pmts also depends on the presence of adjacent domains that recruit the substrate, or other structural modules, sometimes remote, that act as binding platforms for interaction partners within large multi - subunit complexes. for instance, the pmt ezh2 is only active within the prc2 complex when associated with eed and suz12 ; recruitment of eed is mediated by a region located 500 residues upstream of ezh2 s set domain. remote structural modules may not be necessary for pmt activity, but sometimes recognize the methylation substrate or reaction product. for instance, it was shown that an ankyrin repeat distinct from the catalytic domain of glp could recognize mono- or di - methylated lysine 9 of histone 3, the very reaction product of glp s set domain. as previously observed for histone deacetylases and histone acetyltransferases, it is becoming clear that histones are not the only subtrates of some pmts. these emerging signaling mechanisms, unrelated to the histone code, add to the already large body of evidence associating set domain pmts to multiple disease areas, and further drive the research community towards the development of chemical tools to better interrogate their function. the catalytic domain is composed of a core set domain that is structurally conserved and includes residues critical for catalysis, surrounded with a limited set of regions that vary in nature, sequence and shape (fig. these adjacent domains act like a shell around the set fold, and can be divided into two categories. first, the i - set and post - set domains (respectively inserted within, and immediately c - terminal to the set fold) form the binding groove for the substrate peptide, and, to a lesser extent, contribute to the cofactor binding pocket. a landmark feature of set domain pmts is that the substrate peptide and cofactor bind distinct sites, on different sides of the protein, and meet at the core of the structure where catalysis takes place. available ternary complexes of setd7 and glp reveal how the side - chain of the substrate lysine inserts into a narrow channel at the junction of the set, post - set, and i - set domains [14, 18 ]. in this configuration, the lysine is shielded from the solvent, which is believed to be required for catalysis. in the setd8 ternary structure, a wide pocket, rather than a channel, is occupied by the histone lysine and a flanking histidine [19, 20 ]. a catalytically inactive structure of mll1 features a more open peptide binding groove, which leaves the substrate lysine exposed to solvent. other protein binding partners, part of the mll complex, are expected to stabilize the active conformation, probably closer to that captured by the glp, setd7 and setd8 structures. since both i- and post - set domains are involved in substrate recognition, it comes as no surprise that both are present in all set domain pmts. the i - set domain has a fixed topology, and is structurally static, while the post - set domain adopts variable folds and is structurally dynamic, which has implications for the mechanism of substrate recognition (vide infra). available prdm structures reveal an extremely short and unfolded post - set, which may explain the absence of observed biochemical activity for these protein constructs. other domains surrounding the set domain include pre - set (n - terminal to set), n - set (n - terminal to set or pre - set), mynd (between set and i - set), and ctd (c - terminal to post - set) (fig. it is believed that some, if not all of these variable domains are acting as binding interfaces to other proteins or dna. a general concept would therefore be that different combinations of domains with diverse sequence, structure, and electrostatics, would dress the core set fold in very distinct ways, and allow selective recruitment of interaction partners, or facilitate specific positioning relative to the nucleosome, with functional implications. recent structures of smyd proteins illustrate how the ctd domain can adopt an open, catalytically competent conformation, as observed in smyd1, or an inactive conformation that partially occupies the substrate peptide binding site (fig. it was proposed that binding of hsp90, which activates smyd3, stabilizes the open conformation of the protein. domains adjacent to set may therefore not only act as protein interaction interfaces, but also as auto - inhibitory components. display of the molecular surface of pmts according to their electrostatic potential reveals that the substrate - binding groove is consistently electronegative, as illustrated fig. (4) for an h3k9, h3k4, and h4k20 pmt (glp, setd7 and setd8 respectively). this is in contrast with histone tails, which are enriched in lysine and arginine residues, and highly electropositive. this observation suggests a general mechanism whereby long - range electrostatic attractions can bring the pmts and their peptide substrates together in a loose complex, prior to sequence - specific recognition. a close inspection of pmt structures co - crystallized with substrate peptides reveals that the substrate lysine is anchored in a deep channel, and is the major contributor to binding enthalpy. surprisingly, in all available structures, an arginine side - chain located one to four residues upstream or downstream the substrate lysine is the next most important contributor to interaction, and makes extensive contacts with a well - defined cleft of the i - set domain (fig. interestingly, the shape, structural environment, and position of this cleft relative to the lysine binding channel varies from one enzyme to the other, suggesting that it could be exploited to design selective inhibitors. indeed, co - crystallized selective inhibitors were shown to occupy the arginine binding site, as discussed below [25, 26 ]. another observation with possible mechanistic consequences is the fact that histone residues projecting towards the groove are enriched in serine and threonine, two other sites of post - translational modification. it is tempting to speculate that this trend reflects a general structural mechanism where distinct combinations of histone marks would antagonize or possibly enhance substrate recognition by specific pmts. this hypothesis is supported by some experimental observations, but is beyond the scope of this study (see for instance [27 - 29 ]). as mentioned above, the i - set domain varies in sequence, but is structurally conserved across pmts. on the other hand, the post - set domain has variable topologies, sometimes organized around a coordinating zn atom, as is observed for instance in the h3k9 pmts g9a, or the h3k4 pmt mll1. setd7 was crystallized in its apo state, in a binary complex with cofactor, and ternary complex with cofactor and substrate peptide [14, 30, 31 ]. the i - set structure remains unchanged between the three states (with the exception of a tryptophan side - chain), while the conformation of the post - set domain varies considerably (fig. interestingly, a sequential mechanism seems to take place : the apo - conformation is completely unfolded. binding of the cofactor induces partial folding, where an helix contributing to the cofactor binding site adopts its final conformation. finally, proper positioning of the substrate peptide relative to the static i - set induces a final conformational adjustment of the post - set domain. based on similar observations, a model was proposed for the processivity of substrate methylation where an opening and closing motion of the post - set domain would allow release into the solvent of the cofactor and of a proton from the substrate lysine after a first methylation event. cofactor exchange and deprotonation of the substrate are both necessary before further methylation can take place. we propose a general structural mechanism integrating electrostatic phenomena, post - set dynamics, and histone mark cross - talk (fig. long range electrostatic attractions bring together the electropositive histone tail and a loose electronegative binding groove, composed of a pre - formed i - set and open post - set. the pmt may slide along the histone tail, held in place by non - specific electrostatics. once a specific combination of histone side - chains comes into register with i - set, the substrate lysine loses a proton to the solvent, and the complex clicks into a catalytically competent conformation where (1) a catalytic tyrsosine located at the c - terminus of the set domain completes formation of the lysine channel and projects towards the active site, (2) a conserved double - hydrogen bond flanking the substrate lysine is engaged with the i - set domain, (3) the post - set domain closes onto the bound peptide, shielding the catalytic center from solvent. histone marks deposited by other enzymes on flanking serine, threonine or arginine side - chains can affect the formation of this catalytically competent state. structures of the three h3k36 pmts setd2, setmar, and nsd1 (pdb codes 3h6l, 3bo5, 3ooi resp.), and the h3k9 tri - methylase suv39h2 (pdb code 2r3a) are lacking a peptide binding groove, which seems to contradict this model (fig. the i - set domain superimposes well with the i - set of active structures, such as histone - bound glp, but a side - chain of the post - set domain projects into what would be the substrate lysine channel, and flanking post - set residues occupy the peptide binding groove (fig. the functional relevance of this auto - inhibitory mechanism, originally reported for suv39h2, remains unknown at this time. catalysis takes place at the set domain, where the departing methyl of the cofactor lies in close proximity with the de - protonated -nitrogen of the substrate lysine, at the bottom of the lysine channel (fig. the nucleophilicity of the departing methyl group is enhanced by neighboring main chain carbonyl oxygens, and the hydroxyl end of a catalytic tyrosine. another surrounding tyrosine forms a hydrogen bond with the substrate lysine, thereby aligning the lone pair of the deprotonated -nitrogen with the scissile methyl - sulfur bond. a nucleophilic attack follows, which results in methylation of the lysine, and release of sah. a correlation has been observed between the number of residues susceptible of forming a hydrogen bond with the substrate lysine - generally a tyrosine - and the methylation state. indeed, adding hydrogen bonds restrains the rotational freedom of the nitrogen atom, which is necessary to align its lone pair with the scissile bond of the sulfonium group. mutational analyses have confirmed experimentally that a tyr - phe switch in the active site can effectively control the methylation product (fig. 7) [18, 32 - 34 ]. additionally, the extra bulk created by the tyrosine s hydroxy group, or, as shown in setd8, by a bound water molecule, can sterically prohibit higher methylation states. interestingly, this switch was recently reported as a frequent somatic mutation in lymphoma, changing the ezh2 from a multifunctional mono- di- and trimethylase to an enzyne with increased trimethylase activity, but little or no mono- and dimethylase activity [35, 36 ]. the cofactor and substrate peptide bind at two distinct pockets and meet at the catalytic site (fig. 2, top). this suggests two avenues for drug design : competitive inhibition of cofactor or peptide binding. currently the very homologous enzymes glp and g9a are the only two lysine pmts that were crystallized in complex with substrate peptide competitors : bix-01294 (ic50 ~1 m), e67 (ic50 ~ 270 nm), e72 (ic50 ~ 100 nm), unc0224 (ic50 ~ 15 nm) and unc0638 (ic50 ~ 10 nm) (fig. 1) [25, 26, 38 - 40 ]. we used the program sitemap (schrodinger, ny) to evaluate the druggability of the pockets exploited by these inhibitors (fig. 8). a druggability score (dscore), validated against a large training set, is calculated as a function of volume, hydrophobicity, and enclosure of the site. a score larger than 0.95 indicates that the site is druggable ; a value below 0.8 reflects poor druggability ; a dscore between 0.8 and 0.95 is in the gray zone, where no reliable conclusion can be drawn. bix-01294 occupies the open section of the peptide binding groove, but does not exploit the lysine channel (fig. 8). the druggability of the corresponding pseudo - site, which artificially excludes the lysine channel, is unclear. unc0638, another peptide competitor, recapitulates the binding pose of bix-01294, but has an additional aliphatic chain ending with a pyrrolidine that extends into the lysine channel (fig. 8). with a dscore of 1.05, the corresponding site is clearly druggable, as confirmed by the high potency of the ligand. we also calculated the druggability of the cofactor binding site, defined as the pocket occupied by sam, sah, or the close analogue synefungin. the dscore varied from 0.92 to 1.1 across all co - crystallized structures of human lysine pmts, with a mean value of 1.0 (fig. while this site appears more druggable in some lysine pmts than others, it is predicted to be druggable in all cases. the setmar structure is an exception, with a dscore of 0.92, due to its particularly high hydrophilicity. the cofactor site includes numerous polar groups that can not be buried by hydrophobic ligands without significant desolvation penalty. these must be matched by a complex and specific network of hydrogen bond donors and acceptors decorating the inhibitor. we have seen that in all available ternary structures, an arginine side - chain flanking the substrate lysine is an important contributor to binding enthalpy. it is interesting to note that the co - crystallized inhibitors all occupy the arginine binding site (fig. 8), a feature that could inspire by analogy the design of setd7 or setd8 inhibitors (fig. 4 bottom). interaction hot spots that should be exploited by potent chemical inhibitors can be predicted based on receptor - ligand contacts conserved across all available structures. at the peptide binding site, a conserved double hydrogen - bond between the backbone of the substrate lysine and a beta - strand of the i - set domain seems to be important for the interaction (fig. interestingly, this interaction is partially recapitulated by the pyrrolidine group of the potent inhibitor unc0638 (fig., a series of 6 hydrogen bonds engaged with five backbone atoms and one conserved asparagine side - chain of the set domain is observed in all available structures (fig. these hydrogen - bonds are clustered at two specific locations, acting as anchoring point for the cofactor, one at the adenine ring, the other at the methionine end. selective inhibition can only be achieved if the structural chemistry of the pocket is sufficiently specific to a given enzyme. it is therefore reasonable to infer that structural features used to read specific sequences can be exploited to design selective inhibitors. this is in part confirmed by the selectivity profile of unc0638 an inhibitor that specifically inhibit the h3k9 pmts g9a and glp, but not the h3k4 pmt setd7, the h4k20 pmt setd8, or even the h3k9 pmt suv39h2). the question of selectivity is not as clear for the cofactor site as it recognizes the same cofactor across all enzymes. the chemogenomic profiling of human kinases has demonstrated that selectivity can be engineered into atp competitors. a recent study shows that the structural diversity of the sam site in pmts is comparable to that of the atp site in kinases, suggesting that selective inhibition could be achieved at the pmt cofactor site. the selectivity profile of chaetocin, a fungal metabolite that competes with sam with some specificity for h3k9 pmts, reinforces the hypothesis that selective inhibition at the cofactor site is chemically tractable. finally, the peptide and cofactor pockets could be simultaneously targeted by bi - substrate competitors, a mode ofaction that was proposed for existing non - set domain pmt inhibitors [44, 45 ]. a variety of domains can dress the core set structure, and act as docking platforms for specific binding partners associated with diverse cellular events. sequence and post - translational modification status of substrate peptides are mainly recognized by the i - set domain, while a limited set of polar groups surrounding the substrate lysine control methylation specificity. the peptide and cofactor binding sites are chemically tractable, and can be targeted by selective small molecule inhibitors, independently or simultaneously. conserved interaction patterns observed in co - crystal structures strongly suggest the presence of a discrete number of interaction hotspots that can be exploited to achieve potent inhibition. the sgc is a registered charity (number 1097737) that receives funds from the canadian institutes for health research, the canadian foundation for innovation, genome canada through the ontario genomics institute, glaxosmithkline, karolinska institutet, the knut and alice wallenberg foundation, the ontario innovation trust, the ontario ministry for research and innovation, merck & co., inc., the novartis research foundation, the swedish agency for innovation systems, the swedish foundation for strategic research and the wellcome trust. | there are about fifty set domain protein methyltransferases (pmts) in the human genome, that transfer a methyl group from s - adenosyl - l - methionine (sam) to substrate lysines on histone tails or other peptides. a number of structures in complex with cofactor, substrate, or inhibitors revealed the mechanisms of substrate recognition, methylation state specificity, and chemical inhibition. based on these structures, we review the structural chemistry of set domain pmts, and propose general concepts towards the development of selective inhibitors. |
supplementary figure 1. procedures for long - term in vitro operant conditioning supplementary figure 2. protocol for camp injection and measurement of b51 membrane properties and plateau potentials | learning can lead to changes in the intrinsic excitability of neurons. however, it is unclear to what extent these changes persist and what role they play in the expression of memory. here, we report that in vitro analogues of operant conditioning produce a long - term (24 h) increase in the excitability of an identified neuron (b51) critical for the expression of feeding in aplysia. this increase in excitability, which is camp dependent, contributes to the associative modification of the feeding circuitry, providing a mechanism for long - term memory storage. |
labial adhesions are common in young girls and occasionally occur in elderly women. it is hypothesized that the relative hypoestrogenic states of these age groups predispose them to labial adhesions. although surgical dissection is sometimes required, topical oestrogen creams and gentle massage usually lead to successful breakdown of adhesions in these groups within a few weeks. labial adhesions have also been described in reproductive age women secondary to female circumcision, lichen sclerosis, herpes simplex, diabetes, pemphigoid, and caustic vaginitis [36 ]. to our knowledge, a 25-year - old woman, nulligravida, was divorced 5 years ago and was nondiabetic patient. because of some upper egypt traditions she refused to go to doctors (as approximately all surgeons in our localities are male). she had a severe itching with severe monilia infection, and she consulted only dermatologic doctors, the conditions worsened till complete adhesion occured leading to urine retention with difficulty in micturition, then urine retention with overflow. on taking the history, she was circumcised as a routine procedure done for young female in villages and rural area in upper egypt. on examination she had a complete vulval adhesion with only pinhole opening for micturation with complete adhesion of the labia and overflow of the urine with some leucodermic area from chronic irritations (figures 1(a) and 1(b)). the area was infiltrated with injection of adrenalin in concentration of 1 : 10000. separation of the adhesion and fusions was done, and bilateral vulvar flaps to reconstruct the defect of raw surface resulted. randomized mucocutaneous flaps were dissected posteriorly to be advanced to reconstruct the raw surface after separation of adhesion. urethral catheter was applied and lastly suturing of flaps using polyglactin (4/0) (ethicon). she was instructed for standard perineal care, which included spreading the labia periodically and washing with water and antiseptic. the patient was discharged 48 hours after surgery with no complications and instructed to continue routine perineal hygiene at home. examination revealed that her labia were completely healed (see figures 1(a), 1(b) and 2(a), 2(b)). labial fusion is most commonly noted between 3 months and 4 years of age and has a peak incidence of 3.3% between 13 months and 23 months of age. labial fusion is considered to be an acquired condition secondary to hypoestrogenism and vulvovaginitis [8, 9 ]. several previous authors hypothesized that the relative hypoestrogenic state of the immediate postpartum period contributes to the formation of labial adhesions [1, 10 ]. this middle age lady has no history of recent delivery and did not have any pattern of hypoestrogenic state. severe and recurrent infection with bad hygiene can also predispose to labial adhesion, especially monilia infection. the local cultural problems led to the late presentation of the case till complete urine retention occured. the fusion of distal based randomized mucocutaneous flap to reconstruct the defect has not been described in the literature before and the healing of the flap was excellent. | case. a 25-year - old woman presented with acute urine retention with overflow 6 months after an inadequate treatment of severe monilia infections. examination revealed complete adhesion between both labia majora. division of adhesion was done with reconstruction by labial mucocutaneous flap. complete recovery was achieved with good cosmetic outcome. conclusion. labial adhesions whatever their severity is can be surgically divided with complete correction by locally designed flap to reconstruct the introuitus with rapid recovery, good healing, and good cosmetic outcome. |
in order to strengthen public health systems decision makers need the right type of research evidence at the right time. strategies and resources that help decision makers access and utilize research evidence are crucial (frank. 2007 ; kiefer. many countries promote public health core competencies which require proficiency in using research evidence to inform decision making [e.g., canada (public health agency of canada 2010) ; united kingdom (public health resource unit 2008) ; united states (public health foundation 2010) ; australia (australian network of public health institutions 2009) ], however, the public health workforce generally lacks the skills and resources to accomplish these standards (bowen. inconsistent skill capacity for evidence - informed decision making combined with limited resources make effective implementation of evidence - informed public health a formidable task. even so, some recent initiatives have tackled these challenges in attempts to build individual and organizational capacity to better align public health programs and policies with the best available scientific evidence (e.g., dobbins. however, more can be done to support such efforts and to facilitate and improve evidence - informed decision making throughout public health systems. in 2007, the national collaborating centre for methods and tools (nccmt) was officially launched as a part of the government of canada s commitment to renew and strengthen public health across the country (medlar. the mandate of the nccmt is to improve public health stakeholders access to and use of knowledge translation (kt) methods and tools to support evidence - informed public health (frank. 2007). as defined by the canadian institutes of health research (cihr), kt involves dynamic and iterative processes of synthesizing, disseminating, exchanging and applying knowledge for the purposes of ensuring effective health services and products, enhancing the health care system and promoting health (canadian institutes of health research 2012). an environmental scan, key informant interviews and surveys conducted to inform the nccmt s work plans discerned the need to identify kt methods and tools relevant for public health in canada and to facilitate quick and easy access to these resources by decision makers (ciliska. one of the first projects undertaken by the nccmt was to create a searchable database of these resources and freely disseminate this information through its publicly accessible website. the nccmt s registry of methods and tools (http://www.nccmt.ca/registry/index-eng.html) was conceived and constructed as an interactive and expanding online database of resources to support public health decision makers to incorporate kt in public health practice. these resources include both methods (standardized processes, regular and systematic approaches, or sets of organized steps) and tools (standardized products such as instruments, surveys and checklists) that facilitate access to and use of research evidence in decision making and reflect the four types of kt activities as defined by cihr (synthesis, dissemination, exchange and application). in this paper, we describe : how the registry was developed and initially populated as a public health resource ; the methods and results of a recent systematic literature search for kt resources ; the current contents, features and users of the registry website ; and ongoing efforts to evaluate and enhance the registry. we conclude by discussing the relevance of the nccmt s registry of methods and tools as a unique, sustainable and internationally relevant resource for promoting evidence - informed public health. the development of the registry began in fall 2007 by establishing an advisory group comprised of 18 national and international public health policy and program decision makers, and kt researchers. work plans, processes and tools for building the database and website were developed with input from this group. in order to locate appropriate kt methods and tools, a comprehensive search strategy was developed that included both published and unpublished literature. to determine the relevance of identified resources an inclusion - screening tool was developed that contains three key assessment criteria : (1) describes a kt activity, (2) contains a method or a tool, and (3) can be used and/or adapted for use in canadian public health contexts. a data extraction tool was created to help distil relevant descriptive, implementation, measurement and development information for each resource including : the nature of the kt method or tool, relevance for public health, methodological strength, development history, and access issues (e.g., cost, format, language). tables 1 and 2 provide an overview of the inclusion and data extraction tools, which are available in full on the registry website. to support preparation of consistent syntheses of the extracted information finally, an interactive website with a searchable database was designed and built to house the registry. over the course of the first 2 years of the project these processes, tools and technology were piloted and refined.table 1questions from registry inclusion - screening tool (hamilton, on, canada, 2011)1. the resource contains a method or tool no (stop ; exclude) yes (continue)2a. the method / tool is used for knowledge translation (kt) no (stop ; exclude) yes (choose those that apply and continue) synthesis dissemination exchange application2b. the method / tool incorporates the following stage of activity for kt (choose those that apply and continue) planning doing evaluating3. the method / tool is relevant and/or can be adapted to the canadian public health context. no (stop ; exclude) yes (continue)table 2questions from registry data extraction tool (hamilton, on, canada, 2011)description 1. briefly describe any theories, models, frameworks, principles and/or philosophies used to develop the method / tool 3. briefly describe the method / tool (list questions, sections, elements, activities) 4 the method / tool was developed for use in public health contexts (yes or no) 5. the method / tool is transferable to public health contexts (yes or no) 6. provide a specific example for how the method / tool can be used in public healthimplementation 7. briefly describe the steps / process for using / implementing the method / tool 8. who is involved in the delivery and/or administration of the method / tool ? 9. who is involved as participants / respondents of the method / tool?evaluation and measurement characteristics 10. indicate whether the method / tool : has been evaluated, has not been evaluated, evaluation in progress, or information not available 11. indicate whether the validity properties of the method / tool : meet accepted standards, do not meet accepted standards, have not been tested, testing in progress, information not available, or validity properties not applicable 12. reliability properties of the method / tool : meet accepted standards, do not meet accepted standards, have not been tested, testing in progress, information not available, or reliability properties not applicable 13. choose an appropriate methodological rating for the method / tool : strong, moderate, weak, unknown / no evidence, or not applicablemethod / tool development 14. list the processes / steps used / taken to develop the method / tool 16. specify the year the method / tool was first released / made available for use, or when it was first put in practice 17. provide the contact information available for user support (name, position, organization, address, e - mail, phone) questions from registry inclusion - screening tool (hamilton, on, canada, 2011) questions from registry data extraction tool (hamilton, on, canada, 2011) the initial methods and tools considered for inclusion in the registry were drawn from kt resources identified in the nccmt s environmental scan which examined relevant published and grey literature between 1985 and 2006 (ciliska. 2006). in 20082009, project staff conducted a targeted search of over 100 websites and a keyword internet search using google scholar, hand searched reference lists and relevant journals, and contacted kt experts for recommendations. on an ongoing basis, potential published and grey literature resources are also identified by key informants, through list - serves, and by registry users requesting resources to address specific needs. the latter includes questions to address practice - based issues brought forward by public health professionals through an online discussion forum hosted by the nccmt (dialogueph http://www.nccmt.ca/forum/en/index.html). in 2011, a more comprehensive search of the published literature was undertaken with a systematic search strategy developed by a health sciences librarian that included six bibliographic databases, used 87 key terms and covered the period from january 2006 to january 2011 (see table 3). the titles and abstracts of all english language citations were reviewed for relevance by two independent screeners using the inclusion tool. all citations deemed potentially relevant by one or both screeners were retrieved for full - text review. agreement was reached through discussion and a third person was consulted to resolve persistent conflicts. using the extracted data and writing template, one project staff prepares the summary statements. summaries are then translated into french and files in both languages are posted on the registry website.table 3search strategy for methods and tools for knowledge translation in public health (hamilton, on, canada, 2011)dates : january 2006 to january 2011databases : cinahl, cochrane library, embase, medline, psych info, sociological abstractssearch terms (applied to title, abstract and subject headings):methodologies / toolsto facilitate useof public healthinterventionsfor decision makingby practitionersmethodfacilitatpublic healthinterventiondecisionpractitionermodelsynthesicommunity healthevidencepoliciesprofessionaltoolpromotpopulation healthpracticepolicyprovidertool kitaccesspreventiinformationplanstakeholderportalutilizhealth promotionstrategplansadministratorguideutilisrecommendationplanningpolicy makerbest practicetransferassessmentprioritpolicy makerclearinghouseimplementprogramanalyshealth personnelframeworkadoptresearchanalyzsettinginstrumentaggregatesystematic reviewevaluatphysicianknowledge transferanalyzliterature reviewdecision makerknowledge exchangeinformcritical appraisalorganizationknowledgeinfluencorganizationmanagementtranslatcommunitknowledge disseminationassistgovernmentknowledge translationcommunicatsocietdiffusion of innovationguideagencpathwayinstitutionaliworkforcerecommendationevaluatnurseknowledge brokerdisseminatopinion leaderchange agentthe asterisk is used as a truncation symbol to search for variations of the term (e.g., disseminat would retrieve dissemination, disseminate, disseminating, disseminated) search strategy for methods and tools for knowledge translation in public health (hamilton, on, canada, 2011) the asterisk is used as a truncation symbol to search for variations of the term (e.g., disseminat would retrieve dissemination, disseminate, disseminating, disseminated) as part of a broader evaluation of the nccmt s website services, in winter 2012, the registry underwent a formative review. research ethics approval was granted by hamilton health sciences / faculty of health sciences research ethics board (mcmaster university). the evaluation used a mixed - methods design with an online, cross - sectional survey and semi - structured telephone interviews with nccmt users. all registered nccmt users (n = 1,983) were invited to complete an online survey via e - mail. a purposeful sample of 50 nccmt users was invited to participate in individual telephone interviews. survey and interview respondents were asked to explore several topics including : their awareness of the registry, accessing registry resources, using these kt methods and tools in practice, and needs for specific types of methods and tools not currently featured in the registry. evaluation findings have been used to inform refinements to the user interface (e.g., website search and navigation features) and the registry products (e.g., summary statements), and to expand the pool of potential resources for populating the registry. the development of the registry began in fall 2007 by establishing an advisory group comprised of 18 national and international public health policy and program decision makers, and kt researchers. work plans, processes and tools for building the database and website were developed with input from this group. in order to locate appropriate kt methods and tools, a comprehensive search strategy was developed that included both published and unpublished literature. to determine the relevance of identified resources an inclusion - screening tool was developed that contains three key assessment criteria : (1) describes a kt activity, (2) contains a method or a tool, and (3) can be used and/or adapted for use in canadian public health contexts. a data extraction tool was created to help distil relevant descriptive, implementation, measurement and development information for each resource including : the nature of the kt method or tool, relevance for public health, methodological strength, development history, and access issues (e.g., cost, format, language). tables 1 and 2 provide an overview of the inclusion and data extraction tools, which are available in full on the registry website. to support preparation of consistent syntheses of the extracted information finally, an interactive website with a searchable database was designed and built to house the registry. over the course of the first 2 years of the project these processes, tools and technology were piloted and refined.table 1questions from registry inclusion - screening tool (hamilton, on, canada, 2011)1. the resource contains a method or tool no (stop ; exclude) yes (continue)2a. the method / tool is used for knowledge translation (kt) no (stop ; exclude) yes (choose those that apply and continue) synthesis dissemination exchange application2b. the method / tool incorporates the following stage of activity for kt (choose those that apply and continue) planning doing evaluating3. the method / tool is relevant and/or can be adapted to the canadian public health context. no (stop ; exclude) yes (continue)table 2questions from registry data extraction tool (hamilton, on, canada, 2011)description 1. briefly describe any theories, models, frameworks, principles and/or philosophies used to develop the method / tool 3. briefly describe the method / tool (list questions, sections, elements, activities) 4 the method / tool was developed for use in public health contexts (yes or no) 5. the method / tool is transferable to public health contexts (yes or no) 6. provide a specific example for how the method / tool can be used in public healthimplementation 7. briefly describe the steps / process for using / implementing the method / tool 8. who is involved in the delivery and/or administration of the method / tool ? 9. who is involved as participants / respondents of the method / tool?evaluation and measurement characteristics 10. indicate whether the method / tool : has been evaluated, has not been evaluated, evaluation in progress, or information not available 11. indicate whether the validity properties of the method / tool : meet accepted standards, do not meet accepted standards, have not been tested, testing in progress, information not available, or validity properties not applicable 12. reliability properties of the method / tool : meet accepted standards, do not meet accepted standards, have not been tested, testing in progress, information not available, or reliability properties not applicable 13. choose an appropriate methodological rating for the method / tool : strong, moderate, weak, unknown / no evidence, or not applicablemethod / tool development 14. list the processes / steps used / taken to develop the method / tool 16. specify the year the method / tool was first released / made available for use, or when it was first put in practice 17. provide the contact information available for user support (name, position, organization, address, e - mail, phone) questions from registry inclusion - screening tool (hamilton, on, canada, 2011) questions from registry data extraction tool (hamilton, on, canada, 2011) the initial methods and tools considered for inclusion in the registry were drawn from kt resources identified in the nccmt s environmental scan which examined relevant published and grey literature between 1985 and 2006 (ciliska. 2006). in 20082009, project staff conducted a targeted search of over 100 websites and a keyword internet search using google scholar, hand searched reference lists and relevant journals, and contacted kt experts for recommendations. on an ongoing basis, potential published and grey literature resources are also identified by key informants, through list - serves, and by registry users requesting resources to address specific needs. the latter includes questions to address practice - based issues brought forward by public health professionals through an online discussion forum hosted by the nccmt (dialogueph http://www.nccmt.ca/forum/en/index.html). in 2011, a more comprehensive search of the published literature was undertaken with a systematic search strategy developed by a health sciences librarian that included six bibliographic databases, used 87 key terms and covered the period from january 2006 to january 2011 (see table 3). the titles and abstracts of all english language citations were reviewed for relevance by two independent screeners using the inclusion tool. all citations deemed potentially relevant by one or both screeners were retrieved for full - text review. agreement was reached through discussion and a third person was consulted to resolve persistent conflicts. using the extracted data and writing template, one project staff prepares the summary statements. summaries are then translated into french and files in both languages are posted on the registry website.table 3search strategy for methods and tools for knowledge translation in public health (hamilton, on, canada, 2011)dates : january 2006 to january 2011databases : cinahl, cochrane library, embase, medline, psych info, sociological abstractssearch terms (applied to title, abstract and subject headings):methodologies / toolsto facilitate useof public healthinterventionsfor decision makingby practitionersmethodfacilitatpublic healthinterventiondecisionpractitionermodelsynthesicommunity healthevidencepoliciesprofessionaltoolpromotpopulation healthpracticepolicyprovidertool kitaccesspreventiinformationplanstakeholderportalutilizhealth promotionstrategplansadministratorguideutilisrecommendationplanningpolicy makerbest practicetransferassessmentprioritpolicy makerclearinghouseimplementprogramanalyshealth personnelframeworkadoptresearchanalyzsettinginstrumentaggregatesystematic reviewevaluatphysicianknowledge transferanalyzliterature reviewdecision makerknowledge exchangeinformcritical appraisalorganizationknowledgeinfluencorganizationmanagementtranslatcommunitknowledge disseminationassistgovernmentknowledge translationcommunicatsocietdiffusion of innovationguideagencpathwayinstitutionaliworkforcerecommendationevaluatnurseknowledge brokerdisseminatopinion leaderchange agentthe asterisk is used as a truncation symbol to search for variations of the term (e.g., disseminat would retrieve dissemination, disseminate, disseminating, disseminated) search strategy for methods and tools for knowledge translation in public health (hamilton, on, canada, 2011) the asterisk is used as a truncation symbol to search for variations of the term (e.g., disseminat would retrieve dissemination, disseminate, disseminating, disseminated) as part of a broader evaluation of the nccmt s website services, in winter 2012, the registry underwent a formative review. research ethics approval was granted by hamilton health sciences / faculty of health sciences research ethics board (mcmaster university). the evaluation used a mixed - methods design with an online, cross - sectional survey and semi - structured telephone interviews with nccmt users. all registered nccmt users (n = 1,983) were invited to complete an online survey via e - mail. a purposeful sample of 50 nccmt users was invited to participate in individual telephone interviews. survey and interview respondents were asked to explore several topics including : their awareness of the registry, accessing registry resources, using these kt methods and tools in practice, and needs for specific types of methods and tools not currently featured in the registry. evaluation findings have been used to inform refinements to the user interface (e.g., website search and navigation features) and the registry products (e.g., summary statements), and to expand the pool of potential resources for populating the registry. the initial search activities located 518 possible resources in the published and grey literature, 119 of which were full - text screened for inclusion. of these, 35 were deemed appropriate for the registry. with 8 summary statements completed, the registry was launched as a publicly accessible feature on the nccmt s website in fall 2009. data extraction continued for the remaining methods and tools located in the initial search and summary statements were posted to the registry as they became available. from the 2011 comprehensive search of the published literature a total of 562 citations were retrieved for full - text review, 105 of which were deemed appropriate for the registry and did not overlap with the previously included citations. a priority setting exercise was used to establish the order in which these methods and tools are being processed for inclusion in the database. precedence is being given to : (1) resources requested by registry users, (2) resources that fill gaps in registry categories (e.g., adapting research evidence, policy development) and (3) kt tools (i.e., practical instruments, surveys, checklists). as of november 2012, the registry included 133 summary statements : 65 on kt methods and 68 on kt tools. given efforts to advance the science and practice of kt, most methods and tools in the registry are recent with 62 % of the resources published and/or updated in 2006 or later. a total of 286 registered nccmt users (269 english, 17 french) responded to the web - based survey. eighty - five percent (184/217) of survey respondents were aware of the registry and 92 % (162/177) indicated that they would visit the registry again in the future. many participants (68 %, 122/179) indicated they have shared methods and tools found on the registry with colleagues and 42 % (73/175) have used a registry resource in their work. the main reasons given for accessing registry resources were to : assist with program planning (62 %, 109/176), share evidence with others (57 %, 100/176), critically appraise research evidence (50 %, 88/176), and support policy development (39 %, 69/176). interviews were transcribed, imported into a qualitative data management software program (nvivo) and content analysis was conducted to identify major themes in users experiences. participants thought the registry includes a substantial pool of kt methods and tools ; they appreciate that more than one resource is often available for a specific task and that website links are provided to facilitate access to the resources. some participants considered the summary statement feature a key strength of the registry ; they valued having access to comprehensive, useful and up - to - date information on each resource. however, both survey and interview respondents recommended streamlining the format and content of the summary statements and providing real life, practice - based examples to demonstrate how specific methods and tools have been used to support kt efforts in public health contexts. evaluation results highlighted the need to improve users experiences related to searching for appropriate kt resources. although most survey respondents (67 %, 120/179) indicated that they were able to access relevant methods and tools in the registry, one - third (33 %, 59/179) were neutral or negative with respect to being able to find resources. many people (43 %, 24/56) indicated that lack of time prevented effective searching, while others thought their key barriers were related to not understanding how to search (34 %, 19/56) and not knowing what resources were available to be found (27 %, 15/56). mostly, they reported a lack of results with the search terms they were using ; instead of entering the kt keywords (e.g., stakeholder analysis) they were using public health content keywords (e.g., immunization). acting on evaluation results, the registry indexing system was reorganized to make the types of resources available more explicit ; categories and labels were added that resonate with users and are specific to functions and tasks for applying research evidence in public health practice. in addition to tagging methods and tools according to the type of kt activity (synthesis, dissemination, application, exchange) and stage of activity (planning, doing, evaluating), registry entries are now categorized according to : (1) their status as a method or a tool, (2) which step(s) in the evidence - informed public health process the resource supports (i.e., define, search, appraise, synthesize, adapt, implement, evaluate) (national collaborating centre for methods and tools 2011), and/or (3) their association with particular kt tasks or issues (i.e., program planning, policy development, communication, capacity development, partnerships, networks, equity, theories). usability testing indicates this new categorization strategy better assists users in finding the kt resources that will meet their needs. incorporating user feedback, the summary statements have also been remodeled to enhance understanding and utilization of kt resources. website links to the specific methods and tools now appear at the front end of the summaries along with links to supplemental materials available to support practical application of the resources. when appropriate, statements describe how the method or tool can be used as a part of the evidence - informed public health process and identify complimentary nccmt resources that can be accessed to support such activities. in addition, summaries now contain links to other resources in the registry and articulate how the identified methods and tools can be used together to accomplish an overarching goal or task (e.g., combining resources for stakeholder mapping, consensus building and evidence synthesis to support groups interested in developing evidence - based guidance in collaboration with communities, practitioners and decision makers). introduced in fall 2011, another feature of the registry website is a user stories section that draws on real life experiences of implementing the kt methods and tools in public health settings. these short narratives are profiled on the registry s main page and the respective online summary statements. as of november 2012, there were four tools in the registry with a user story and more stories are currently in development. public health professionals are encouraged to submit their experiences with these and other kt methods and tools to expand this important strategy for dissemination. website statistics monitored by google analytics from january 1 to november 30, 2012 indicate almost half of users originate from canada (43 % of 45,081 visits). many other visitors are based in the united states (19 %) and the united kingdom (9 %) with the remaining users located in 188 other countries worldwide. for the first 11 months in 2012, google analytics counted 32,945 unique visitors accessing the registry with 84,490 page views. the surge in the number of visits may be explained, in part, by concurrent intensive efforts to populate and promote the registry. the registry was officially launched in june 2011 with webinars and oral presentations attended by target users, via an e - mail blast sent to distributed list - serves and through features in other organizations newsletters. between june 2011 and november 2012, 62 more summary statements were posted and a critical mass of methods and tools have been actively promoted by nccmt during national conference presentations and internationally accessible webinars. the initial search activities located 518 possible resources in the published and grey literature, 119 of which were full - text screened for inclusion. of these, 35 were deemed appropriate for the registry. with 8 summary statements completed, the registry was launched as a publicly accessible feature on the nccmt s website in fall 2009. data extraction continued for the remaining methods and tools located in the initial search and summary statements were posted to the registry as they became available. from the 2011 comprehensive search of the published literature a total of 562 citations were retrieved for full - text review, 105 of which were deemed appropriate for the registry and did not overlap with the previously included citations. a priority setting exercise was used to establish the order in which these methods and tools are being processed for inclusion in the database. precedence is being given to : (1) resources requested by registry users, (2) resources that fill gaps in registry categories (e.g., adapting research evidence, policy development) and (3) kt tools (i.e., practical instruments, surveys, checklists). as of november 2012, the registry included 133 summary statements : 65 on kt methods and 68 on kt tools. given efforts to advance the science and practice of kt, most methods and tools in the registry are recent with 62 % of the resources published and/or updated in 2006 or later. a total of 286 registered nccmt users (269 english, 17 french) responded to the web - based survey. eighty - five percent (184/217) of survey respondents were aware of the registry and 92 % (162/177) indicated that they would visit the registry again in the future. many participants (68 %, 122/179) indicated they have shared methods and tools found on the registry with colleagues and 42 % (73/175) have used a registry resource in their work. the main reasons given for accessing registry resources were to : assist with program planning (62 %, 109/176), share evidence with others (57 %, 100/176), critically appraise research evidence (50 %, 88/176), and support policy development (39 %, 69/176). interviews were transcribed, imported into a qualitative data management software program (nvivo) and content analysis was conducted to identify major themes in users experiences. participants thought the registry includes a substantial pool of kt methods and tools ; they appreciate that more than one resource is often available for a specific task and that website links are provided to facilitate access to the resources. some participants considered the summary statement feature a key strength of the registry ; they valued having access to comprehensive, useful and up - to - date information on each resource. however, both survey and interview respondents recommended streamlining the format and content of the summary statements and providing real life, practice - based examples to demonstrate how specific methods and tools have been used to support kt efforts in public health contexts. evaluation results highlighted the need to improve users experiences related to searching for appropriate kt resources. although most survey respondents (67 %, 120/179) indicated that they were able to access relevant methods and tools in the registry, one - third (33 %, 59/179) were neutral or negative with respect to being able to find resources. many people (43 %, 24/56) indicated that lack of time prevented effective searching, while others thought their key barriers were related to not understanding how to search (34 %, 19/56) and not knowing what resources were available to be found (27 %, 15/56). mostly, they reported a lack of results with the search terms they were using ; instead of entering the kt keywords (e.g., stakeholder analysis) they were using public health content keywords (e.g., immunization). acting on evaluation results, the registry indexing system was reorganized to make the types of resources available more explicit ; categories and labels were added that resonate with users and are specific to functions and tasks for applying research evidence in public health practice. in addition to tagging methods and tools according to the type of kt activity (synthesis, dissemination, application, exchange) and stage of activity (planning, doing, evaluating), registry entries are now categorized according to : (1) their status as a method or a tool, (2) which step(s) in the evidence - informed public health process the resource supports (i.e., define, search, appraise, synthesize, adapt, implement, evaluate) (national collaborating centre for methods and tools 2011), and/or (3) their association with particular kt tasks or issues (i.e., program planning, policy development, communication, capacity development, partnerships, networks, equity, theories). usability testing indicates this new categorization strategy better assists users in finding the kt resources that will meet their needs. incorporating user feedback, the summary statements have also been remodeled to enhance understanding and utilization of kt resources. website links to the specific methods and tools now appear at the front end of the summaries along with links to supplemental materials available to support practical application of the resources. when appropriate, statements describe how the method or tool can be used as a part of the evidence - informed public health process and identify complimentary nccmt resources that can be accessed to support such activities. in addition, summaries now contain links to other resources in the registry and articulate how the identified methods and tools can be used together to accomplish an overarching goal or task (e.g., combining resources for stakeholder mapping, consensus building and evidence synthesis to support groups interested in developing evidence - based guidance in collaboration with communities, practitioners and decision makers). introduced in fall 2011, another feature of the registry website is a user stories section that draws on real life experiences of implementing the kt methods and tools in public health settings. these short narratives are profiled on the registry s main page and the respective online summary statements. as of november 2012, there were four tools in the registry with a user story and more stories are currently in development. public health professionals are encouraged to submit their experiences with these and other kt methods and tools to expand this important strategy for dissemination. website statistics monitored by google analytics from january 1 to november 30, 2012 indicate almost half of users originate from canada (43 % of 45,081 visits). many other visitors are based in the united states (19 %) and the united kingdom (9 %) with the remaining users located in 188 other countries worldwide. for the first 11 months in 2012, google analytics counted 32,945 unique visitors accessing the registry with 84,490 page views. the surge in the number of visits may be explained, in part, by concurrent intensive efforts to populate and promote the registry. the registry was officially launched in june 2011 with webinars and oral presentations attended by target users, via an e - mail blast sent to distributed list - serves and through features in other organizations newsletters. between june 2011 and november 2012, 62 more summary statements were posted and a critical mass of methods and tools have been actively promoted by nccmt during national conference presentations and internationally accessible webinars. across public health systems there are increasing efforts to support and encourage knowledge of, access to and use of research evidence to inform program and policy decisions and a number of research programs have been initiated to study and evaluate what works to promote and achieve evidence - informed public health (e.g., armstrong. 2009a, b ; peirson. 2012 ; waters. 2011). in addition to workforce skills development and organizational change initiatives that focus on building capacity and infrastructure for evidence - informed public health, a variety of web - based resources have been created to provide decision makers with access to information, evidence, guidelines and tools. for example, there are several online, searchable and continually updated collections that house evidence syntheses on public health research. within the cochrane library there is a dedicated section for several dozen reviews that examine the effects of population - level public health interventions (http://ph.cochrane.org). the evidence for policy and practice information centre maintains a database that contains a few thousand systematic and non - systematic reviews of effectiveness in health promotion and public health (http://eppi.ioe.ac.uk/webdatabases/intro.aspx?id=2). the centre for reviews and dissemination provides access to an extensive library of systematic reviews on the effects of health care interventions and the delivery and organization of health services, economic evaluations of health care interventions, and health technology assessments ; more than 1,300 entries are tagged as public health related (http://www.crd.york.ac.uk/crdweb). created by the mcmaster health knowledge refinery, public health + is a database of over 1,200 methodologically sound primary studies and systematic reviews on public health topics, distilled from over 120 health journals, that have been rated as both relevant and newsworthy by volunteer experts (http://www.nccmt.ca/public_health_plus/all/1/list-eng.html). health - evidence is another web - based resource that provides access to more than 2,700 pre - processed (filtered, quality rated and summarized) reviews of health promotion and public health interventions (http://www.health-evidence.ca). a number of other online resources have been developed to provide the public health community with information, guides and tools to engage in evidence - informed decision making and program planning. for example, the uk national health service (nhs) website contains a section for public health evidence that includes 11 topic pages that provide guidance, implementation tools, case studies and information for the public (http://www.evidence.nhs.uk/nhs-evidence-content/public-health). also available on the nhs website is the national institutes of clinical excellence (nice) pathways information network that brings together all the nice guidance and related nice products concerning specific health topics, currently including 38 issues relevant to seven public health categories, and organizes them in a series of interactive flowcharts (http://pathways.nice.org.uk). supported by the centers for disease control and prevention in the united states, the guide to community preventive services provides access to systematic and economic reviews and evidence - based recommendations for effective public health interventions spanning 22 topic areas (http://www.thecommunityguide.org/index.html). the public health agency of canada s canadian best practices portal offers an inventory of policies, programs and interventions for chronic disease prevention and health promotion, and connects users to a broad range of products and other websites that : provide information and tools for evidence - informed public health and program planning ; contain public health related policy documents, resources and instruments ; and provide access to surveillance data, intervention strategies, systematic reviews and practice guidelines for ten public health topic areas (http://cbpp-pcpe.phac-aspc.gc.ca). notwithstanding the important contributions of these and other portals and databases for enhancing access to public health research and topic - specific information and resources, a critical gap in the evidence - informed public health landscape was recognized by the architects of the national collaborating centres and the public health community (ciliska. there was no existing resource, in canada or elsewhere, that was dedicated to identifying kt methods and tools relevant for public health contexts and that facilitated quick and easy access to these resources by decision makers. to our knowledge, the nccmt s registry of kt methods and tools remains unique in its goals, service and products. at present, the registry only includes methods and tools that are available in english and that are applicable to canadian public health contexts, although most resources are likely transferable to public health settings in other developed countries. these limitations mean the contents of the registry may not be sufficient to meet the needs of public health decision makers in non - english speaking parts of the world and in developing nations where public health systems may be encountering unique kt challenges related to their own development or to differences in population - based health priorities, technology, and so on. however, the registry s summary statements and companion materials provide all users with detailed information on which to base decisions about the applicability and transferability of the kt methods and tools for their specific needs and contexts. after 5 years of planning, development and implementation, the nccmt s registry of methods and tools is an operational, interactive database populated with over 130 resources (with many more in the queue) to support kt activities in public health. evaluation results suggest that the registry is a valued addition to the complement of resources currently available to support evidence - informed public health. recent interest in advancing the science and practice of developing and implementing methods and tools for kt suggests there will be a substantial pool of resources for continuing to populate the nccmt s registry and sustaining this database as a key resource for supporting the complex, dynamic and critical work of public health. as part of the ongoing efforts to build the capacity and infrastructure needed to support evidence - informed public health, the registry facilitates access to kt resources that can help decision makers integrate research knowledge into practice. the increasing number of visitors to the registry and the expanding interest in and use of kt methods and tools reinforces the relevance of this unique and practical resource for public health decision makers worldwide. | objectivesthis paper examines the development of a globally accessible online registry of knowledge translation methods and tools to support evidence - informed public health.methodsa search strategy, screening and data extraction tools, and writing template were developed to find, assess, and summarize relevant methods and tools. an interactive website and searchable database were designed to house the registry. formative evaluation was undertaken to inform refinements.resultsover 43,000 citations were screened ; almost 700 were full - text reviewed, 140 of which were included. by november 2012, 133 summaries were available. between january 1 and november 30, 2012 over 32,945 visitors from more than 190 countries accessed the registry. results from 286 surveys and 19 interviews indicated the registry is valued and useful, but would benefit from a more intuitive indexing system and refinements to the summaries. user stories and promotional activities help expand the reach and uptake of knowledge translation methods and tools in public health contexts.conclusionsthe national collaborating centre for methods and tools registry of methods and tools is a unique and practical resource for public health decision makers worldwide. |
since the art of medicine is based on diagnostic abilities to provide good treatments, strategies to treat dissatisfaction among physicians should use the same approach. treatments for doctors unhappiness must be followed by adequate diagnoses and recognition of its causes. dissatisfaction of physicians has been recognized as a worldwide phenomenon, and it has been linked with low levels of patient satisfaction, patient compliance with treatments, and quality of health services. furthermore, dissatisfaction of physicians has been associated with high levels of distress, leave direct patient care, burn - out and poor mental well - being. however, health care systems tend to overlook or ignore indicators of physician well - being. researchers have been studying factors associated with higher levels of satisfaction among physicians. in spain, several studies have evaluated the satisfaction of physicians among certain groups of specialists using different perspectives ; however, these studies are focused on the perspective of satisfaction with work. the satisfaction of physicians should have a multi - dimensional approach in order to evaluate their higher - order needs, like growth, life achievement, career development, autonomy, responsibility, creativity, self - esteem, and self - actualization, within maslow s hierarchy of needs. the perspective of career satisfaction of physicians has been conceptualized into lower and higher order needs, where inherent and performance satisfaction capture higher level needs, and personal and professional dimensions capture lower level needs. the questionnaire, developed in canada, to measure career satisfaction of physicians was adapted and validated with good psychometric measures among physicians in andalusia, spain ; in - depth analyses of the andalusian study are presented in this paper. subsequently, the objectives of this study were to : i) measure the career satisfaction of physicians working in hospitals of andalusia, using the adapted instrument ; ii) identify differences in the dimensions of career satisfaction ; and iii) test factors affecting higher and lower order needs of the career satisfaction. data were drawn from a cross - sectional study conducted among physicians working in six hospitals of andalusia, spain, from november 2009 to february 2010. in total, 299 physicians were eligible to participate in the study, excluding residents and medical students. eligible physicians were invited by email to complete an on - line questionnaire in spanish, asking about their career satisfaction, work conditions, incentives, and demographics. the study received the ethics approval from the ethics commission for research of the university of granada and the ethics board of the college of medicine of the university of granada, as well as from the chair of the participant hospitals. the selected instrument to measure career satisfaction has seventeen items ; 16 specific items and one global item, all scored on 6 point scales from very dissatisfied to very satisfied. the items of the questionnaire, in english and spanish, are presented in the appendix. a standardized mean score for the full 16 items was calculated by totalizing the items and dividing by 16, yielding an interval measure with a minimum of score of 1.00 to a maximum score of 6.00. the friedman s anova test was used to determine rank differences across the four dimensions of career satisfaction, and the wilcoxon matched - pairs signed rank test was used as post - hoc analysis to identify differences between the dimensions. a logistic regression model was built to identify associated factors with the probability that a physician reports a higher level of career satisfaction. the dependent variable was 1=score higher than the group median, and 0=score equal or lower than the group median. the independent variables in the model were personal features (gender, age, and being a foreign physician), career features (speciality by broad groups, having a second speciality, and years of experience), work features (job stability, monthly salary, and having a management position), and incentive features (non - monetary or monetary incentives received during the last year). finally, comparisons of differences in the four dimensions of satisfaction according to the identified predictor factors were done using the mann - whitney u - test. response bias was checked with a brief on - line survey sent to physicians who had not participated, asking about their overall career satisfaction, incentives received, age, gender and specialization. of the 299 eligible physicians, 121 completed the questionnaire (40.7% response rate). the mean age was 41.08 (sd=6.68), with a median of 41 years - old. the 21 specialties represented were grouped in four categories : 33.1% family practitioners, 35.5% medical and clinical specialists (internal medicine, gastroenterology, pediatrics, intensive care medicine, etc.), 24% surgical and procedural specialists (anesthesiology, orthopedics, obstetrics and gynecology, general surgery, etc.), and 7.4% laboratory and technical specialists (clinical laboratory and radiology) ; in addition, 17% of the physicians reported a second speciality, with family medicine as the most common one. in relation to incentive features, 47.9% of the physicians reported that they received from their hospitals monetary incentives during the last year (such as extra payments and bonus), while 33.1% physicians received non - monetary incentives in the last twelve months (such as training courses, research grants, academic and courses grants, participation in academic and research groups). furthermore, 35 physicians completed the brief online questionnaire and response bias was not detected. the mean career satisfaction among the participant physicians was 4.36 (sd=0.76), with a median of 4.56 ; in other words, the mean satisfaction was 72.7% (4.36 over 6.00) and median represents that half of the physicians have a satisfaction below or equal to 76% (4.56 over 6.00). the mean satisfaction by dimensions (table 1) were : professional, 4.53 (sd=0.92) ; inherent, 4.45 (sd=0.74) ; performance, 4.39 (sd=0.77) ; and personal, 4.06 (sd=0.98). the reliability of the questionnaire was verified through the cronbach s alpha, obtaining good internal consistency reliabilities for the 16-item scale (=0.92) and the four dimensions (>0.77). since the normality (shapiro - wilk tests with p<0.001) and equality of variances (levene s test=4.31, p=0.005) assumptions were rejected, non - parametric tests were used to identify significant differences among the dimensions of career satisfaction. thus, the friedman s anova test identified significant differences across the four dimensions of career satisfaction, (3)=55.18, p<0.001. table 1 shows rating scores by dimension (median, interquartile range, and mean rank), and in figure 1, box - plots of the four dimensions are compared. post - hoc tests of significance for pair - wise comparisons, using the wilcoxon matched - pairs signed rank tests, identified higher medians of professional, inherent and performance satisfaction in comparison to personal satisfaction (p<0.001). in contrast, no significant differences were detected between the inherent and the professional (p=0.05), and between the inherent and the performance (p=0.34) dimensions. the logistic regression model identified two significant predictors of higher levels of career satisfaction (table 2). physicians who received non - monetary incentives during the last year and foreign physicians were 3.11 (c.i. 95%=1.19 - 8.13) and 7.81 (c.i. 95%=1.40 - 43.48) times more likely to report a higher level of career satisfaction, respectively. the mann - whitney u test identified significant median differences in personal (u=306, z=-3.04, p=0.002, r=-0.21) and performance (u=394, z=-2.28, p=0.02, r=-0.21) dimensions of satisfaction when comparing foreign and locally trained physicians (table 3). between those who received non - monetary incentives and physicians who did not receive them during the last year, differences were identified in the professional (u=1167, z=-2.52, p=0.01, r=-0.23) and the performance (u=1239.5, z=-2.12, p=0.03, r=-0.19) dimensions (table 3). previous studies in andalusia have reported comparable levels of satisfaction among physicians : the study of the andalusian society of internal medicine identified that 73.1% of the participants were satisfied with the work content ; the study developed in the axarquia health district of malaga, using the font - roja questionnaire, found a global mean job satisfaction of 64.4% (77.22 over 120) ; and, among pediatric surgeons, a mean job satisfaction of 58.7% (70.46 over 120) was reported using also the font - roja questionnaire. in the primary health care district of majorca, similar mean levels of job satisfaction have been identified among family practitioners and pediatricians, also assessed with the font - roja scale (65 over 120). for instance in switzerland and the netherlands, an overall work satisfaction of 72.86% (5.1 over 7) and 75.71% (5.3 over 7) were reported, respectively. nevertheless, comparisons of these results with our study are limited due to differences in the perspective and scales used. physicians in canada appeared to be less satisfied (4.13) than in andalusia (4.36). by dimensions, practitioners in canada were more satisfied with the inherent dimension (4.75) than in andalusia (4.45) ; however, participant physicians in andalusia were more satisfied with personal (4.06 vs. 3.84), professional (4.53 vs. 4.10) and performance dimensions (4.39 vs. 3.87) than physicians in canada. in relation to the differences identified among the dimensions of the career satisfaction, the personal dimension was the lowest, suggesting that participant physicians of andalusia are having problems to keep work from intruding in their personal lives, sustain satisfying activities in the community, control their work schedule, and/or to be content with the way that administrative aspects of medical practice are handled. similarly, this dimension was also reported as the lowest among the canadian doctors. on the other hand, the professional dimension scored high levels of satisfaction, which could be promoting some balance between the two dimensions of the basic - order needs. this is a relevant discrepancy among the dimensions of career satisfaction but, as keeton. identified, physicians could remain satisfied with their careers despite having to struggle with a work - life balance. notwithstanding, a disproportion in the dimensions of the career satisfaction is an imbalance in the satisfaction of physicians which could lead to distress, leave direct patient care, burn - out, and leave medicine. physicians, generally, work longer hours than the general population and under emotionally - charged situations ; they also face increasing workloads, patient demands, and growing bureaucracy, that could negatively tip the effort - reward balance. this critical situation is especially susceptible in the current economic spanish environment, which might be negatively affecting the well - being of the health human resources. this threatening environment is especially relevant, since physicians are making personal sacrifices to practice medicine, temporally compensated with professional contentment. nonetheless, two relevant factors were identified with a higher level of career satisfaction : receiving non - monetary incentives and being a foreign physician. the fact that physicians were more likely to report a higher level of career satisfaction when receiving non - monetary incentives, has organizational implications on how physicians practice behaviors can be influenced to improve the quality of healthcare. in contrast, monetary incentives could increase physicians perception of burden and negatively affect satisfaction of patients. in fact, monetary incentives have shown modest results, like the pay - per - performance program in ontario, canada. healthcare policies are usually focused on monetary incentives and the non - monetary ones are neglected and underestimated. given that motivation is influenced by both financial and non - financial incentives, non - financial alternatives should be more explored. this kind of alternative incentives need to be acknowledged because it has been found that the satisfaction of physicians can be enhanced when career growth and autonomy are increased, along with time for continuing medical education. moreover, the career satisfaction and commitment of physicians could be positively influenced by providing recognition, respect, empathy, and appreciation. as it was identified in this study, non - monetary incentives could positively affect high- and lower - order needs of the physicians which could increase the probability of long - term positive effects among these professionals and health services provision. physicians are more likely to stay in practice, especially in rural and underserved areas, when they have personal satisfaction. medical students consider the impact of a speciality on their personal life as a significant variable when choosing a career specialty ; new physicians are more attracted to choose a speciality which is offering them a controllable lifestyle. personal satisfaction is a critical factor to promote professional satisfaction and it is important to create a limit between work and personal spheres ; however, this limit is difficult to create, especially among female phyisicians. physicians are aware of the required personal efforts that medicine has, accepting some trade - offs in their career - personal balance. healthcare policies that recognize their personal desires need to be promoted to develop healthcare providers well - being, looking above monetary rewards only. the gain strategy (greater access and independence now), developed in the mayo medical school and the mayo clinic, is an example of how physicians satisfaction and productivity can be increased with non - financial incentives, while improving the patients access to health services. the evidence of foreign physicians being more likely to report a higher level of career satisfaction than their local counterparts could be related to their career achievements and self - evaluation in a foreign country. foreign doctors use to evaluate their careers in the host country, taking into account previous experiences and opportunities from their countries of origin. a considerable number of foreign physicians from latin america work in spain ; in fact, before the european economic crisis, physicians from latin america, africa, and eastern europe often considered spain as a green pasture for their career development. however, economic downturns have directly affected the healthcare systems and working conditions of healthcare professionals in several european countries. countries that traditionally were welcoming international professionals, like spain, could be exposing their local and foreign physicians to important push factors to emigrate due to the anti - crisis policies. first, the results can not be extrapolated to the entire population of physicians in spain. second, it is recognized that not all hospitals in andalusia participated in the study. third, since this was a cross - sectional study, a cause and effect relation and sequence of events can not be determined. finally, despite the obtained response rate, it is important to consider that response bias was tested and found to be negligible. future studies are recommended with bigger and stratified samples by specialty, rural and urban areas, and perhaps by autonomous communities. furthermore, longitudinal studies evaluating the impact of monetary and non - monetary incentives on career satisfaction are recommended, as well as comparing the satisfaction of local and foreign trained physicians. career satisfaction has been measured among a sample of physicians from andalusian hospitals, identifying that they scored the lowest levels in the personal dimension. this finding suggests that these physicians in andalusian hospitals are making great personal sacrifices and are compensated by experiencing high levels of professional satisfaction in practicing medicine. this is an imbalance that, in the long term, could severely affect the well - being of these healthcare professionals and, subsequently, the quality of care that they are providing. furthermore, non - monetary incentives for physicians demonstrated a significant impact on their satisfaction, influencing the professional and performance dimensions of career satisfaction. in addition, foreign physicians have reported higher levels of satisfaction than their local counterparts, especially in their personal and performance satisfaction. this is a finding that deserves further investigation, particularly in times of crisis and transition where motivating factors are changing. | the satisfaction of physicians is a worldwide issue linked with the quality of health services ; their satisfaction needs to be studied from a multi - dimensional perspective, considering lower- and higher - order needs. the objectives of this study were to : i) measure the career satisfaction of physicians ; ii) identify differences in the dimensions of career satisfaction ; and iii) test factors that affect higher- and lower - order needs of satisfaction among physicians working in andalusian hospitals (spain). forty - one percent of 299 eligible physicians participated in a study conducted in six selected hospitals. physicians reported higher professional, inherent, and performance satisfaction than personal satisfaction. foreign physicians reported higher levels of personal and performance satisfaction than local physicians, and those who received non - monetary incentives had higher professional and performance satisfaction. in conclusion, physicians in the selected andalusian hospitals reported low levels of personal satisfaction. non - monetary incentives were more relevant to influence their career satisfaction. further investigations are recommended to study differences in the career satisfaction between foreign and local physicians. |
mental disorders, including dementia, can impair competence, but a diagnosis of dementia does not imply a complete loss of competence. there is a wide consensus on considering competence as the capacity of a person to make a specific decision. voting is a decision of particular interest since a consensus does not exist on which abilities the patient with dementia should retain to express a reliable choice. thus, identifying patients who, despite the presence of dementia, maintain the capacity to vote and increasing their chance to take part in a ballot (e.g., allowing their caregivers to have a role in facilitating this) would be of crucial importance. participation in the electoral process by citizens with dementia has become especially important in recent years, both for the growing number of individuals suffering from alzheimer disease (ad) or other progressive cognitive disorders, and in light of the fact that in at least two cases (2000 us presidential elections and 2006 italian elections for the prime minister designation), a small number of votes had a decisive effect on the results. it is especially in long - term facilities that inappropriate assumptions about the absence of voting capacity may deprive still capable and willing residents of the right to vote [4, 5 ]. recently, a novel test to assess the capacity to vote has been proposed : the competence assessment tool for voting (cat - v), which evaluates an individual 's performance on four decision - making abilities : understanding the nature and effect of voting, appreciating the reality of voting situation, making a choice, and reasoning about voting choices. in this paper, we report the results of a study that applied a modified version of the cat - v to individuals with mild - moderate ad who were temporarily residents in a long - term care facility before 2006 italian elections for designating the prime minister. our primary hypothesis was that although voting capacity would be inversely associated with dementia severity, the single decision - making abilities evaluated by the cat - v would be affected unequally by the dementing process. the subjects included in the present study (n = 38) represent all the patients with mild - moderate dementia (mini - mental state examination (mmse) 11) and a clinical diagnosis of probable ad (according to the national institute of neurological and communicative disorders and stroke (nincds) and the alzheimer disease and related disorders association (adrda) criteria) who were admitted into the alzheimer centre of the ospedale gazzaniga (bergamo, italy) from sixty to thirty days before 2006 italian general elections. although, in some respects, our centre has several characteristics of a long - term care facility, no patient is a permanent resident. the primary requirement for a patient 's admission into our centre is the presence of behavioural abnormalities or psychopathologic symptoms in the context of a dementing syndrome but, once these features are significantly relieved, the patient is discharged. the instrument we used to evaluate the capacity to vote was a modified version of the cat - v, an instrument that measures a person 's ability to understand the nature and effect of voting, make a choice, appreciate, and reason through a voting decision. these criteria were operationalized into five questions preceded by an introduction reminding each person that soon he / she would have the opportunity to take part in a ballot for the election of the prime minister. thus, as opposed to the original us version of the cat - v, in which subjects are asked to imagine that two candidates are running for governor and that the day of the interview is the election day, the scenario we proposed was real rather than hypothetical. furthermore, in order to shorten the time of interview, unlike the original, our version of the cat - v did not include a question evaluating subjects ' appreciation of the significance of voting. for each cat - v item, the scores assigned to each person ranged from 2 (correct response reflecting adequate performance) to 0 (inadequate performance). every participant was enrolled after an initial contact with his / her principal caregiver. once informed about the characteristics of the study and made sure that its results would be used exclusively for research purposes, each participant (or his / her caregiver) provided a written informed consent. all of the 38 participants were interviewed and rated by one investigator (m. sala), who was blinded to their mmse score. thirty of them were also interviewed and rated by another investigator (e. chit). weighted kappa and kendall tau - b twenty - nine subjects were again interviewed by m. sala two weeks later, to evaluate the test - retest reliability. the scores included in the main data analysis are those assigned to all participants at baseline by m. sala the spearman correlation coefficient was used to examine the association of the capacity to vote (as expressed by the score on each of the cat - v items) with severity of both cognitive impairment (as expressed by the mmse score) and behavioural and psychopathological symptoms (as expressed by the neuropsychiatric inventory (npi) score). each participant was administered the cat - v, the mmse, and the npi during the same session. all the subjects who were asked to participate in the project (n = 38) did complete the interview. behavioural and psychopathological symptoms were moderately severe at baseline but were significantly relieved prior to discharge. subjects ' performance on cat - v is shown in table 2. over a half of the subjects appeared to fully understand the nature of the vote, but only approximately a third was entirely able to understand its effect. however, the great majority of participants (~90%) was deemed to be completely able to make a choice. conversely, subjects ' ability to reason about voting by comparing the choices at disposal and, above all, by evaluating the possible consequences of the preference for a candidate on their life was considerably more impaired. in fact, only about 16% of the participants had a completely adequate performance on the latter measure. as shown in table 3, which relates subjects ' combined performance on understanding and choice to their performance on reasoning, only three of the 38 participants (8%) scored the maximum on all items. as emerges from table 4, there were better test - retest and interrater reliabilities for scores on understanding and choice than for scores on reasoning. there was no relation of cat - v scores to severity of behavioural and psychopathological symptoms (r = 0.14, p = 0.41). conversely, as expected, lower cat - v scores were associated with lower mmse scores (figures 1, 2, and 3). however, a great variability in subjects ' performance was noted at any stage of disease. on questions evaluating understanding and choice (figure 2), for example, only 58% of subjects with mild ad (mmse 20) obtained the maximum score but, remarkably, over one - third of those who scored the maximum was beyond mild - stage disease (mmse understanding > choice). on the basis of these data, the use of a structured interview, such as the cat - v, may offer advantages over unstructured or clinical assessments, especially in light of the fact that global measures of cognitive functioning, such as the mmse, do not appear to be strong predictors of the capacity to vote. further studies are needed to refine the clinicians ' approaches to identifying demented people who are still capable to vote from those who are no longer capable. nevertheless, a tool like the cat - v can adequately assist in this distinction (table 5). if you do not understand something of what i am saying or asking, please let me know and i will repeat it. some of the questions might seem very simple to you, but do not worry about that. two candidates are running for prime minister (make the patient name the candidates or, if he / she does not remember, remind him / her of their names). two candidates are running for prime minister (make the patient name the candidates or, if he / she does not remember, remind him / her of their names). understanding the nature of votingwhat will you do to pick the prime minister on election day?(if patient gives an indirect answer, describing how he / she or people in general would choose between the candidates, for example watching tv, listening to their campaign issues, ask:well, that is how you might decide who you like to be the prime minister. but how would actually express your choice?)score of 2 : entirely correct response, for example, i will go to the polls and vote or i will cast my vote for one or the other, and so forth.score of 1 : ambiguous or partially correct response, for example, that is why we have election day, and so forth.score of 0 : incorrect response, for example, there is nothing you can do ; the tv guys decide, and so forth. (if patient gives an indirect answer, describing how he / she or people in general would choose between the candidates, for example watching tv, listening to their campaign issues, ask : well, that is how you might decide who you like to be the prime minister. but how would actually express your choice ?) score of 2 : entirely correct response, for example, i will go to the polls and vote or i will cast my vote for one or the other, and so forth. score of 1 : ambiguous or partially correct response, for example, that is why we have election day, and so forth. score of 0 : incorrect response, for example, there is nothing you can do ; the tv guys decide, and so forth. understanding the effect of votingonce the election for prime minister is over, how is it going to be decided who is the winner?score of 2 : entirely correct response, for example, the votes will be counted and the candidate with more votes will be the winner.score of 1 : ambiguous or partially correct response, for example, the better between the two candidates will be the winner once the election for prime minister is over, how is it going to be decided who is the winner ? score of 2 : entirely correct response, for example, the votes will be counted and the candidate with more votes will be the winner. score of 1 : ambiguous or partially correct response, for example, the better between the two candidates will be the winner choicehand patient a card with the information in the following paragraph in large print ; allow to retain and consult this card for the remainder of the interview.for the sake of simplicity, the first candidate (of the right party) is willing to lower taxes by decreasing the burden of bureaucracy and public administration, in order to make people spend more as a result of higher income. the second candidate (of the left party) is willing to either raise taxes or, by fighting tax elusion, keep them unchanged getting every citizen to pay, so that the rights to education and welfare remain protected. based on either what i have just told you or what you already knew about the candidates, do you think you are able to choose between the two ? mind that i do not want to know from you which candidate you would vote for, but only if you have made your choice?score of 2 : the patient clearly indicates the choice, including a reasoned choice not to vote or a manifestation of indetermination (i still do not know which candidate to vote for).score of 0 : no choice is stated because the patient is unable to choose, does not understand what is asked, and so forth. hand patient a card with the information in the following paragraph in large print ; allow to retain and consult this card for the remainder of the interview. for the sake of simplicity, the first candidate (of the right party) is willing to lower taxes by decreasing the burden of bureaucracy and public administration, in order to make people spend more as a result of higher income. the second candidate (of the left party) is willing to either raise taxes or, by fighting tax elusion, keep them unchanged getting every citizen to pay, so that the rights to education and welfare remain protected. based on either what i have just told you or what you already knew about the candidates, do you think you are able to choose between the two ? mind that i do not want to know from you which candidate you would vote for, but only if you have made your choice ? score of 2 : the patient clearly indicates the choice, including a reasoned choice not to vote or a manifestation of indetermination (i still do not know which candidate to vote for). score of 0 : no choice is stated because the patient is unable to choose, does not understand what is asked, and so forth. reasoning comparative reasoningif the patient identifies a choice, ask : why do you think that it is worth voting for either of the candidates ? or why do you think neither of the candidates deserves to be voted for?score of 2 : entirely correct response, for example, because it is right to maintain the welfare state, because it is right that everybody pays taxes, because the state should not empty the citizen 's pockets with too high taxes, because, despite different views, neither of the candidates will fulfill the promises made before election day, and so forth.score of 1 : ambiguous or partially correct response, for example, healthcare, it is better to spend more than spend less, and so forth.score of 0 : the patient fails to mention a comparative attribute of the respective candidates. reasoning on consequencesif the patient is able to make his / her choice for either of the candidates or even in the case he / she wants to abstain from voting, ask : in your opinion, should the first candidate, that one who wants to lower taxes by decreasing the burden of bureaucracy, or second candidate, that one who wants to maintain the welfare state by fighting against tax elusion, be elected, how could that affect your life?score of 2 : entirely correct response, for example, i will have more money to spend,, i can not predict what will happen, because they wo n't do what they promised to do, and so forth.score of 1 : ambiguous or partially correct response, for example, health will improve, and so forth.score of 0 : the patient does not give a consequence for his / her life or a reason for saying that there are no personally relevant consequences. comparative reasoningif the patient identifies a choice, ask : why do you think that it is worth voting for either of the candidates ? or why do you think neither of the candidates deserves to be voted for?score of 2 : entirely correct response, for example, because it is right to maintain the welfare state, because it is right that everybody pays taxes, because the state should not empty the citizen 's pockets with too high taxes, because, despite different views, neither of the candidates will fulfill the promises made before election day, and so forth.score of 1 : ambiguous or partially correct response, for example, healthcare, it is better to spend more than spend less, and so forth.score of 0 : the patient fails to mention a comparative attribute of the respective candidates. reasoning on consequencesif the patient is able to make his / her choice for either of the candidates or even in the case he / she wants to abstain from voting, ask : in your opinion, should the first candidate, that one who wants to lower taxes by decreasing the burden of bureaucracy, or second candidate, that one who wants to maintain the welfare state by fighting against tax elusion, be elected, how could that affect your life?score of 2 : entirely correct response, for example, i will have more money to spend,, i can not predict what will happen, because they wo n't do what they promised to do, and so forth.score of 1 : ambiguous or partially correct response, for example, health will improve, and so forth.score of 0 : the patient does not give a consequence for his / her life or a reason for saying that there are no personally relevant consequences. if the patient identifies a choice, ask : why do you think that it is worth voting for either of the candidates ? or why do you think neither of the candidates deserves to be voted for ? score of 2 : entirely correct response, for example, because it is right to maintain the welfare state, because it is right that everybody pays taxes, because the state should not empty the citizen 's pockets with too high taxes, because, despite different views, neither of the candidates will fulfill the promises made before election day, and so forth. score of 1 : ambiguous or partially correct response, for example, healthcare, it is better to spend more than spend less, and so forth. score of 0 : the patient fails to mention a comparative attribute of the respective candidates. if the patient is able to make his / her choice for either of the candidates or even in the case he / she wants to abstain from voting, ask : in your opinion, should the first candidate, that one who wants to lower taxes by decreasing the burden of bureaucracy, or second candidate, that one who wants to maintain the welfare state by fighting against tax elusion, be elected, how could that affect your life ? score of 2 : entirely correct response, for example, i will have more money to spend,, i can not predict what will happen, because they wo n't do what they promised to do, and so forth. score of 1 : ambiguous or partially correct response, for example, health will improve, and so forth. score of 0 : the patient does not give a consequence for his / her life or a reason for saying that there are no personally relevant consequences. | voting by persons with dementia raises questions about their decision - making capacity. methods specifically addressing voting capacity of demented people have been proposed in the us, but never tested elsewhere. we translated and adapted the us competence assessment tool for voting (cat - v) to the italian context, using it before 2006 elections for prime minister. consisting of a brief questionnaire, this tool evaluates the following decision - making abilities : understanding nature and effect of voting, expressing a choice, and reasoning about voting choices. subjects ' performance was examined in relation to dementia severity. of 38 subjects with alzheimer 's disease (ad) enrolled in the study, only three scored the maximum on all cat - v items. mmse and cat - v scores correlated only moderately (r = 0.59 ; p < 0.0001) with one another, reflecting the variability of subjects ' performance at any disease stage. most participants (90%), although performing poorly on understanding and reasoning items, scored the maximum on the choice measure. our results imply that voting capacity in ad is only roughly predicted by mmse scores and may more accurately be measured by a structured questionnaire, such as the cat - v. among the decision - making abilities evaluated by the cat - v, expressing a choice was by far the least affected by the dementing process. |
bite marks left on foodstuff at the scene of crime offer a three - dimensional impression of the suspect 's dentition and the bite mark analysis can give useful clues about the perpetuator who caused it, leading to the implication or exclusion of the individual under investigation. the forensic value of bite marks on foodstuffs depends on the nature of the substrate and the ability of the perishable food substance to dehydrate and deform at room temperature, the precision of impression of the suspect 's dentition on the foodstuff and the time elapse in collecting and preserving the bite mark evidence. furthermore life size photographs of the bite mark taken with good quality and angulation is of extreme importance to the forensic odontologist for bite mark analysis and for identifying the victim. as photography is the safest means of obtaining a permanent record of the bite mark on perishable food substances such as apple, cheese, and chocolate, it must be of the highest standard for its forensic significance to be maximized. the process of comparing bite marks on a food substance with an individual 's dentition includes analysis of size, shape, and spatial orientation of the individual tooth. as bitten food substances are commonly left away at the scene of crime, an attempt was made to study the accuracy of bite mark analysis on three different food substances - apple, cheese and chocolate. the fact that human dentition is unique for each individual plays an important role in this study. the aims and objectives of the study were to compare the accuracy of bite mark analysis from three different food substances - apple, cheese, and chocolate using two techniques - the manual docking procedure and computer assisted overlay generation technique and to compare the accuracy of the two methods - the docking analysis and the computer assisted overlay generation technique for bite mark analysis on food substances. twenty - five individuals who participated in the study were made to bite on three food substances - apple, cheese and chocolate. the bitten food substance was then stored in a refrigerator in a sealed plastic bag within an hour. then, the impression of the bite mark on apple, cheese and chocolate was made using light body addition silicone (affins) by gently injecting from a center point to periphery using the plastic gun. it was then picked up with a heavy body silicone (affins) and poured with die stone (samit) to obtain the positive replica of the bitten surface on apple [figure 1 ], cheese [figure 2 ] and chocolate [figure 3 ]. the dental impressions were then made for the individuals using alginate impression material (velplast) and dental casts were obtained. showing the methodology to obtain positive replica of bitten surface on apple showing the methodology to obtain positive replica of bitten surface on cheese showing the methodology to obtain positive replica of bitten surface on chocolate in the manual docking (direct) analysis, the dental casts of each individual were docked to the die stone cast (positive replica) of the bite mark on apple, cheese [figure 4 ] and chocolate [figure 4 ] to check for matching of incisal edges of the anterior teeth with the bite mark pattern on the foodstuff. while doing the docking analysis for cheese and chocolate with mandibular casts, the positive replicas were reversed to check for matching. the scoring was then assigned as 0 for not matching, 1 for slight (consistent) matching, 2 for moderate (probable) matching and 3 for excellent (distinctive) matching respectively. the highest score was assigned to the correct match as per modified version of the american board of forensic odontology (abfo) scoring system for bite marks. showing manual docking analysis done on the die stone cast of bite mark on cheese and chocolate using maxillary and mandibular casts showing selection of incisal edges using the magic wand wizard tool in adobe photoshop software from the photograph of dental cast and the superimposition of computer generated overlays over the photograph of bite mark on apple, cheese and chocolate done to check for matching finally by the computer assisted overlay generation (indirect) technique, overlays of anterior dentition were obtained from life size photographs of dental casts using magic wand wizard tool in adobe photoshop cs4 software [figure 5 ]. then, the overlays were superimposed over the bite mark pattern in the life size photographs of the food stuff and placed in a preferred position until satisfactory matching could be established [figure 5 ]. the scoring was then assigned as 0 for not matching, 1 for slight (consistent) matching, 2 for moderate (probable) matching and 3 for excellent (distinctive) matching respectively. the highest score was assigned to the correct match as per modified version of the abfo scoring system for bite marks. the results were tabulated and the accuracy of bite mark analysis on the three food substances was analyzed by kuskall - wallis anova test. by manual docking analysis, excellent matching was observed in 24% of cases for apple, 56% of cases for cheese and 72% of cases for chocolate [table 1 ]. by computer assisted overlay generation technique, the excellent matching percentages of bite mark analysis were 32, 60, and 76, respectively for apple, cheese and chocolate [table 2 ]. the comparison of hand docking and computer assisted overlay generation technique was done by spearman 's correlation and the rho coefficient was found to be 0.896 for apple, 0.945 for cheese and 0.951 for chocolate [table 3 ]. comparison of accuracy of bite mark analysis for apple, cheese and chocolate by manual docking method using kruskall - wallis anova test comparison of accuracy of bite mark analysis for apple, cheese and chocolate by computer assisted overlay generation technique using kruskall - wallis anova test spearman 's correlation between the 2 techniques - manual docking analysis and computer assisted overlay generation technique for the three food stuffs - apple, cheese and chocolate a bite mark has been defined as a pattern produced by human or animal dentitions and associated structures in any substance capable of being marked by these means (clark 1992). bitemarks left away on a foodstuff offer a three - dimensional impression, which is superior to the two - dimensional impression often left away on the skin. a study conducted by macfarlane. supported the concept of dental uniqueness the physical characteristics of bite mark which contributes to uniqueness include the shape of the dental arch, distance between canines, presence of a tooth out of alignment, spacing between teeth, rotation of teeth, missing teeth, the curves of biting edges and wear patterns. hence, the premise of bite mark analysis is that human dentition is unique and that this asserted uniqueness is replicated on the bitten substrate in sufficient detail to enable a match to the individual under suspicion. the factors which influenced the accuracy of bite mark analysis in our study were physical nature of the foodstuff, the biting force with which bite mark was done, time elapse in the preservation of foodstuffs, proper impression technique, proper generation of dental casts and positive replica of bitten surface of the foodstuff and the quality and angulation of the bite mark photographs. in our study, bite mark impressions were made with addition silicone as this impression material has excellent dimensional stability and accurately duplicates the bite mark pattern on the foodstuff. on comparing the bite mark analysis on the three food stuffs by the two techniques - hand docking method and computer assisted overlay generation technique ; the spearman 's rho correlation coefficient was found to be 0.951 for chocolate, 0.946 for cheese and 0.896 for apple [table 3 ]. when meticulous steps were taken for preservation and handling of foodstuff after the bite mark was done by the individual, the accuracy of bite mark analysis was found to be slightly greater on chocolate than cheese and poor on apple in our study. the poor accuracy of bite mark on apple was because of its firm substrate and its dehydration and decomposition at room temperature and the good accuracy on chocolate and cheese could be because of fine bite registration due to homogeneity of the substrate of the two foodstuffs. on observing the bite mark pattern on chocolate and cheese, both chocolate and cheese showed a fine registration of incisal edges of maxillary and mandibular anterior teeth, but the bitten edges on cheese showed some irregularities and cracks and this could be the reason for a slightly greater accuracy of bite mark on chocolate than cheese in our study. in a comparative study conducted by bernitz. on accuracy of bite marks on cheese, butter and cooked potato, it was found that the examiners correctly identified all the true matches of bite marks on the three foodstuffs with their respective study models. rai. conducted a comparative study on bite mark analysis on cheese and clay and found that for bite marks on clay, the match by docking analysis and overlay method was positive in 95% of cases, while for bite marks on cheese, it was positive in 81% of cases. in our study, on comparing the hand docking method and computer assisted overlay generation technique, hand docking method showed an excellent matching in 24% of cases for apple, while for cheese in 56% of cases and for chocolate in 72% of cases [table 1 ]. computer assisted overlay generation technique showed an excellent matching in 32% of cases for apple, while for cheese in 60% of cases and for chocolate in 76% cases [table 2 ]. the hand docking (direct) method was found to be an objective technique, but the technique was difficult and cumbersome. generation of good dental casts and a proper positive replica of bite mark on foodstuff played a critical role in this study for bite mark analysis by hand docking method. the positive replica of bitten surface was not accurate when excessive pressure was applied on the bite mark pattern on the foodstuff during impression making. the computer assisted overlay generation (indirect) technique was relatively easier and less time consuming than hand docking method. however, the computer generated overlay technique was found to be a subjective analysis, so it was found to be more accurate when the analysis was done on bite marks caused by a dentition displaying some unique features like rotation, spacing, crowding, etc., sweet and bowers compared computer generated overlay method with other overlay generation techniques and concluded that computer generated overlay technique is the most accurate and the gold standard technique for bite mark analysis. in our study, the computer assisted overlay technique had a greater accuracy, when bite mark analysis was done on life size photographs of dental casts and the bite mark pattern on the three foodstuffs and on photographs with good quality and angulation and in cases of dentition showing some unique features. the results of our study were consistent with the study conducted by stavrianos., who concluded that computer assisted overlay generation technique for bite mark analysis was as accurate as hand docking method in cases of bite mark on apple and may be useful in a variety of substrates. rai. compared the direct (docking analysis) and indirect method (overlay method) of bite mark analysis on cheese and clay and concluded that when the comparison by indirect method remains inconclusive, the direct comparison method tends to match. mckenna. reported a case of bite marks on chocolates, which were recovered from the scene of theft in a chocolate factory and it was found that both direct and photomicrographic comparisons between the casts of chocolate and of the suspect 's dentition revealed correspondence between their unique characteristics and this led to the conviction of chocolate thieves. aboshi. have reported a case of fire in a snackbar in mount gambier, south australia in which the suspect was identified by comparing computer generated images of biting surfaces of the victim 's cast with that of bite marks on cakes obtained from the crime scene. the drawbacks of our study were there was no matching of the dentition with bite mark pattern when there were an excessive delay in the preservation of the bitten food stuff, poor cast generation and in cases of poor quality and angulation of photographs of bite mark on foodstuffs and of dental casts. on the basis of the results of our study, among the three perishable food substances apple, cheese and chocolate, bite marks on chocolates and cheese may serve as valuable, reliable and accurate evidences for identifying a victim because of the fine bite registration on these two foodstuffs than apple. computer assisted overlay generation method may serve as a reliable, easier, less expensive, and less time consuming technique for bite mark analysis, but further research is needed to develop sophisticated software with greater specificity for bite mark analysis to avoid wrong implication of crime due to subjective errors. | aims and objectives : the aims and objectives of the study were to compare the accuracy of bite mark analysis from three different food substances - apple, cheese and chocolate using two techniques - the manual docking procedure and computer assisted overlay generation technique and to compare the accuracy of the two techniques for bite mark analysis on food substances.materials and methods : the individuals who participated in the study were made to bite on three food substances - apple, cheese, and chocolate. dentate individuals were included in the study. edentulous individuals and individuals having a missing anterior tooth were excluded from the study. the dental casts of the individual were applied to the positive cast of the bitten food substance to determine docking or matching. then, computer generated overlays were compared with bite mark pattern on the foodstuff.results:the results were tabulated and the comparison of bite mark analysis on the three different food substances was analyzed by kruskall - wallis anova test and the comparison of the two techniques was analyzed by spearman 's rho correlation coefficient.conclusion:on comparing the bite marks analysis from the three food substances - apple, cheese and chocolate, the accuracy was found to be greater for chocolate and cheese than apple. |
the recent development of molecularly targeted cancer therapeutics has been accompanied by renewed interest in technologies for the tumor / cell - selective delivery of potent but intrinsically nonselective cytotoxic agents. these technologies include antibody drug conjugates (adcs) that recognize cell - surface antigens and tumor - activated prodrugs (taps) that exploit nutrient transporters or differences in hypoxia associated with the tumor microenvironment. accumulating evidence suggests that an increase in reactive, labile intracellular iron is another metabolic signature of cancer, as recently reviewed. tumor targeting strategies designed to exploit changes in iron homeostasis remain largely unexplored however, despite clinical precedent for iron - dependent pharmacology in antimalarial therapy with artemisinins. redox cycling of iron in enzyme cofactors is essential for cellular processes ranging from de novo nucleotide synthesis to the maintenance of genomic stability, cell cycle regulation, and mitochondrial respiration. however, when unbound and unregulated, redox active iron promotes the disproportionation of hydrogen peroxide (fenton reaction) to produce hydroxyl and hydroperoxyl radicals, reactive oxygen species (ros) that confer cellular damage and can lead to apoptosis or ferroptosis. iron homeostasis is therefore highly regulated to ensure sufficient labile iron is available to support essential enzyme function while limiting exposure to unbound, redox active iron species. rapidly proliferating cells have increased requirements for dna synthesis, repair, and mitochondrial respiration and therefore have increased demands for iron cofactor biosynthesis. accordingly, iron acquisition and export pathways are altered in many cancers so as to increase the labile iron pool. furthermore, iron has been shown to contribute to tumor initiation and growth and epidemiological evidence has established links between tumor iron metabolism and clinical outcomes in breast cancer patients. given that labile fe(ii) promotes fenton chemistry, we sought to develop a tumor - targeting strategy in which fenton reaction of a peroxidic prodrug was coupled to release of drug payloads. recognizing that antimalarial agents such as arterolane exhibit finely tuned iron(ii) reactivity, we subsequently developed an arterolane - inspired small molecule platform (denoted trx herein) for fe(ii)-dependent drug delivery. these trx - drug conjugates function via initial fe(ii)-promoted fragmentation of a 1,2,4-trioxolane ring to afford a cyclohexanone intermediate, followed by spontaneous -elminiation and decarboxylation to release the drug payload (figure 1 and supporting information figure s1). in previous studies, we demonstrated the utility of the trx scaffold by efficiently and selectively delivering antimalarial payloads to ferrous iron / heme rich compartments of the malaria parasite, both in vitro and in vivo. iron(ii)-promoted fenton - type reduction of the trioxolane ring affords the cyclohexanone species shown which then undergoes spontaneous -elimination and decarboxylation to release free payload. this process occurs with high efficiency and ferrous - iron selectivity in mammalian cancer cell lines, as described recently. while the iron - dependent pharmacology of 1,2,4-trioxanes and 1,2,4-trioxolanes in malaria is widely accepted, extending this concept to taps for oncology applications required a means to assess whether sufficient labile iron is present in cancer cells to efficiently and selectively activate trx - based conjugates. to address this question, we synthesized a trx conjugate of the aminonucleoside antibiotic puromycin (i.e., trx - puro) as a probe of intracellular labile iron (figure 1 where r nh2 = puromycin). we found that puromycin release from trx - puro in cells was dependent on ferrous iron as expected and was not affected by the addition of other biologically relevant metal ions and reducing agents. these initial studies with trx - puro also confirmed that labile iron pools are generally augmented in cancer cells when compared to nontumorigenic cells. our findings thus suggested that the trx scaffold might indeed be applied to produce a novel class of taps for cancer chemotherapy. here we describe prototypical trx - drug conjugates designed to confer tumor - selective delivery of a microtubule toxin (a combretastatin analog) or dna - alkylating agent (a duocarmycin analog) in an fe(ii)-dependent fashion. we show that the intrinsic cytotoxicity of these agents can be ablated in tap forms and then restored following activation in cancer cells. we show that cancer cell lines of diverse origins are generally susceptible to trx - based taps but that a nontumorigenic cell line (mcf10a) is highly resistant. we further show that the resistant mcf10a cell line can be measurably sensitized to the tap when transformed with the oncogene cmyc and that this likely derives from oncogenic changes to iron metabolism. finally, we show that the tap of a duocarmycin analog is tolerated in mice at doses up to 50-fold higher than the parent cytotoxin and this, combined with targeted toxin release within tumor, translates to superior efficacy in pc-3 and mda - mb-231 xenograft models. these studies provide the first evidence that reactive iron in tumor cells and/or the tumor microenvironment can be exploited to afford improved selectivity in the delivery of cancer chemotherapeutics. to explore ferrous iron - dependent drug delivery in cancer cells we synthesized a known small molecule microtubule toxin (1) and its trioxolane conjugate 2 (figure 2a and supporting information figure s2). a nonperoxidic dioxolane conjugate (3) was also prepared to confirm that the cytotoxicity of 1 is ablated in conjugated forms and that intracellular release of active 1 from 2 is peroxide - dependent (figure 2a and figure s2). finally, trioxolane analog 4(29) lacking the microtubule toxin was prepared to control for intrinsic cytotoxicity of the trx moiety (figure 2a). the cytotoxicity of the trioxolane - conjugate 2 and control compounds 1, 3, and 4 was then assessed across a small panel of cell lines. trioxolane - conjugate 2 displayed activity in the low nm range (ec50 = 21 nm), 3 orders of magnitude more potent than either of the negative controls (3 or 4) and nearly as potent as the free toxin (1) applied directly (ec50 = 11 nm) (figure 2b). these results confirm that release of 1 from 2 in these cells is both efficient and peroxide - dependent. moreover, the lack of measurable toxicity exhibited by 3 demonstrates that the intrinsic cytotoxicity of 1 is effectively blocked in tap form. exploiting augmented ferrous iron pools for selective drug delivery. (a) chemical structure of microtubule inhibitor 1 and corresponding conjugates and controls 24. (b) cytotoxicity of compounds 14 in mda - mb-231 cells after 24 h of exposure as determined by cell counting (n = 3 ; error bar, mean sem). (c) cytotoxicity of 1 and its trioxolane - conjugate 2 after 24 h of exposure in mda - mb-231 and mcf10a cells as determined by cell counting (n = 3 ; error bar, mean sem). dividing the ec50 value for 1 by that for 2 in a given cell line produces an ec50 ratio that is used to compare the efficiency of drug release across cell lines. this ratio is 0.54 for sensitive mda - mb-231 cells (2-fold ec50 shift) and 0.04 for relatively resistant, nontumorigenic mcf10a cells (25-fold ec50 shift). (d) ec50 ratios calculated as described in (c) for a small panel of cell lines. error bars represent sem from three individual experiments each conducted in biological triplicates. the ec50 values for 1 and 2 were determined after a 72 h incubation using the celltiter - glo cell viability assay. normalizing the activity of conjugate 2 to that of its cytotoxic payload (1) provided a convenient metric (ec50 ratio) to compare efficiency of payload release from 2 across different cell lines. in mda - mb-231 cells, this ratio was found to be 0.54 (ec50 = 11 nm and 21 nm for 1 and 2, respectively). among the other cell lines examined, u2os and rko cells were nearly as susceptible to 2 as mda - mb-231 cells (ec50 ratio of 0.46) while pc-3 and hela cells were somewhat less sensitive (ec50 ratio of 0.300.32), though 2 was still effective in these cells at therapeutically relevant, low - nm concentrations (figure 2d and supporting information table s1). consistent with our previous finding that nontumorigenic cells possess a smaller reactive iron pool, mcf10a cells were highly resistant to the trioxolane - conjugate 2 with an ec50 ratio of just 0.04 (figure 2c, d). encouraged by these initial findings, we further explored the cytotoxicity of 1 and 2 across a larger panel of cancer cell lines from diverse origins using a celltiter - glo assay to assess cell viability (figure 2e). we found ec50 ratios varied by about 9-fold across the different cell lines, from 0.89 for the most susceptible cells (pc-3) cells to 0.11 for the least susceptible cells (ekvx). despite the range of responses, tap 2 produced ec50 values in the nm range for even the least responsive cell lines (e.g., ekvx ec50 = 43 nm, supporting information table s2). as expected, the ec50 values for trioxolane control 4 across this panel were in the m regime and typically 2 log units less potent than 2 (supporting information table s2). this confirms that the cytotoxicity of tap 2 derives from release of 1 and not significantly from the trioxolane moiety itself. having found that nontumorigenic mcf10a cells were highly resistant to trx - conjugates, we explored whether oncogenic transformation of these cells would increase their reactive iron pools and thus sensitize them to trioxolane conjugates. bandyopadhyay and co - workers recently reported the generation of a panel of cell lines expressing single oncogenes in mcf10a cells. using these well - characterized cell lines, we examined the effects of the oncogenes ras and myc, whose effects on iron metabolism have been studied previously. first, we used qrt - pcr to evaluate the transcriptional profile of a panel of iron regulatory proteins in the mcf10a cells constitutively expressing hras. in previous work in different cell types, oncogenic hras has been variously reported to decrease ferritin mrna levels and increase labile iron or to have no effect on ferritin levels or labile iron. in the mcf10a cells expressing hras we observed no significant transcriptional changes to ferritin, transferrin receptor, or any other of the iron regulatory transcripts probed (figure 3 and supporting information figure s3). it was therefore unsurprising that the hras - expressing mcf10a cells were no more susceptible to 2 than mcf10a cells transformed with the empty vector (figure 3b). in contrast, mcf10a cells transformed with cmyc showed substantial down regulation of the iron exporter ferroportin and up regulation of the ferrireductase steap3 (figure 3a). interestingly, we did not observe an effect of cmyc on ferritin heavy chain mrna, as has been reported previously. nevertheless, the changes observed predict for increased labile iron in the cmyc - transformed mcf10a cells, and indeed, these cells were found to be more sensitive to trioxolane - conjugate 2 than cells transformed with the empty vector, as evidenced by a significant 3.2-fold shift in the ec50 ratio (figure 3b). these results indicate that oncogene induced changes to the reactive iron pool can sensitize cells to the delivery of potent cytotoxins from trx - based taps in an otherwise isogenic background. profiling the effects of oncogenic transformation on the ferrous iron pool of mcf10a cells. (a) relative mrna levels for ferroportin, ferritin, and the ferrireductase steap3 in mcf10a cells stably transfected with empty vector (control) or with vectors expressing the oncogenes myc or hras (n = 3 ; error bar, mean sem ; () p 0.01, () p 0.0001, two - way anova with dunnett s multiple comparisons test). (b) ec50 ratio (1/2) for mcf10a cells stably transfected with hras or cmyc oncogenes or the empty vector (control) as assessed by cell counting after 24 h of compound exposure. error bars represent sem from three independent experiments each conducted in biological triplicates (() p 0.01, one - way anova with dunnett s multiple comparisons test). the highly cytotoxic cyclopropylbenzindoline (cbi) class of natural products like cc-1065 and duocarmycin sa function by alkylating adenine bases in duplex dna. seco-cbi analogs are latent cytotoxins that can undergo spontaneous winstein - type spirocylization to form the active cyclopropylbenzindoline (cbi) species (i.e., seco-5 5, figure 4a and supporting information figure s4). this activation step can be prevented by acylation or carbamoylation of the aniline or phenolic function, and the ability to cage seco - cbi derivatives in this way has made these compounds popular as cytotoxic effectors in antibody we synthesized a known seco - cbi analog and further converted the material into the desired tap 6 and the 1,3-dioxolane - cbi conjugate 7 (dxl - cbi), a nonperoxidic control (figure 4a). (a) structure of duocarmycin type dna - alkylator seco-5 (latent form), the corresponding activated form 5 that reacts with nucleophilic bases of dna (: nu), trioxolane duocarmycin conjugate 6, and negative control dioxolane - conjugate 7. (b) cytotoxicity of compounds 5 and 6 in pc-3 cells after 72 h of exposure as determined by cell counting (n = 3 ; error bar, mean sem). (c) cytotoxicity of compounds 5 and 6 in mda - mb-231 cells after 72 h of exposure as determined by cell counting (n = 3 ; error bar, mean sem). we evaluated the cytotoxicity of seco-5, tap 6, and control 7 following 72 h of exposure in mda - mb-231 and pc-3 cells (figure 4b, c). as expected, seco-5 and its tap 6 exhibited potent cytotoxic effects in cells, while the dioxolane conjugate 7 was noncytotoxic at all concentrations examined. the inactivity of 7 confirms that conjugation of seco-5 via its aniline function effectively blocks formation of the activated (and toxic) cbi electrophile. the potent effects of 6 in cells are thus due to peroxide - dependent release of free seco-5 as desired. we further found that conversion of seco-5 to the tap 6 dramatically improves its chemical stability in the extracellular environment. thus, while free seco-5 degraded over several hours upon thawing from a dmso stock solution, trx conjugate 6 (and dxl conjugate 7) were chemically stable for more than a year at room temperature in dmso (supporting information figure s5). stability toward premature hydrolysis likely explains why tap 6 was measurably more potent than seco-5 itself in both cell lines examined (figure 4b, c). control experiments confirmed that 6 is stable for more than a week in cell culture media at 37 c, indicating that its cellular toxicity in the 72 h assays (figure 4b, c) results from intracellular release of seco-5 as desired. the cell culture studies of 57 above revealed the effective ablation of 5-mediated toxicity by chemical conjugation at an aniline function (figure 4a c). we next asked whether this caging effect would translate to reduced in vivo toxicity for tap 6 as compared to 5. to determine a maximally tolerated dose (mtd), nontumor bearing female nsg mice were administered three ip doses of either seco-5 or 6 at 4-day intervals (q4d). these studies revealed that seco-5 is highly toxic to mice (mtd 0.3 mg / kg), consistent with previous observations for related duocarmycin analogs. by contrast, tap 6 could be administered at significantly higher doses, with an mtd of 7.5 mg / kg, using the same three dose q4d regimen. a subsequent mtd study of 6 with q7d dosing returned a somewhat higher mtd of 10 mg / kg. previous work with duocarmycin analogs related to 5 has shown dose - limiting hepatotoxicity in mice and insufficient therapeutic index leading to failures in human clinical trials. we were therefore interested in exploring the toxic effects of 5 in mice and whether administration in the trx - conjugated form 6 could protect from these toxicities. mg / kg seco-5 showed substantially (10-fold) higher concentrations of liver transaminase enzymes (alt and ast) than did mice treated with 7.5 mg / kg of 6 (figure 5c). since hepatotoxicity of duocarmycin analogs has previously been observed to occur with a delayed onset, mice receiving escalating doses of 5 or 6 were observed for 50 days postdosing, then sacrificed and their livers collected and assessed for altered morphology and signs of toxicity. even at well - tolerated doses of 5, mice grossly showed darkened and roughened capsular and parenchymal changes indicative of hepatotoxicity in addition to enlarged intestines and serosanguinous ascites near the site of administration. the liver pathology showed increased hepatic lobular lymphohistiocytic infiltrates, reactive cellular changes, and minimal periportal fibrosis. in contrast, the livers of mice treated with 7.5 mg / kg of 6 appeared normal, and the dose limiting toxicity appeared instead to be localized toxicity at the site of administration as evidenced by enlarged intestines. the pathology in these was free of significant inflammation or fibrosis (figure 5d). (a) plasma concentrations of 5 in female nsg mice following a single 0.3 mg / kg ip dose. (b) plasma concentrations of 6, released 5, and the retro - michael intermediate 16 in female nsg mice following a single 7.5 mg / kg ip dose of 6. the study design and numbers of mice per group were the same as in (a). (c) measured levels of alkaline phosphatase (alk. phosp.), serum alanine aminotransferases (alt), aspartate aminotransferase (ast), and creatine phosphokinase (cpk) in blood samples from mice treated with either 5 (0.3 mg / kg) or trioxolane - conjugate 6 (7.5 mg / kg). (d) microscopic histology stains of representative mouse liver samples show only mildly increased reactive changes in the group receiving 0.3 mg / kg 5, including minimally increased lobular lymphohistiocytic infiltrates. in mice receiving the higher 7.5 mg / kg dose of 6 (right column), top two rows of images show representative h&e sections of centrizonal or periportal areas. bottom row of images show representative trichrome stains for evaluation of relative fibrosis, which shows no significant fibrosis ; scale bars are in m as indicated. to compare the pharmacokinetic properties of seco-5 and its trx conjugate 6, healthy nsg mice were administered a single ip dose of seco-5 (0.3 mg / kg) or conjugate 6 (7.5 mg / kg), representing the respective mtd values. analysis of blood samples showed seco-5 to have a reasonably long half - life (t1/2 = 3.8 h), moderate clearance (cl / f = 20 ml min kg) and a high volume of distribution (vz / f = 6.7 l / kg) (figure 5a, b, supporting information table s3). conjugate 6 exhibited a significantly longer half - life (t1/2 = 20.4 h), moderate - high clearance (cl / f = 31.3 ml min kg) and a very high volume of distribution (vz / f = 55 l / kg). in animals receiving 6, total exposure to 6 (auc = 5050 hng / ml) exceeded that of released seco-5 by approximately 8-fold, indicating a small degree of drug release in normal tissues of healthy mice. maximum plasma concentrations of free seco-5 in mice receiving 7.5 mg / kg of 6 were still lower than those in mice treated with 0.3 mg / kg of seco-5 directly (cmax = 48 ng / ml vs 108 ng / ml). most importantly, the total systemic exposure of 6 was 20-fold greater than for seco-5 when both agents were administered at their respective mtd (auc024h = 5050 and 246 hng / ml, respectively). the higher in vivo exposure achievable with tap 6 was thus expected to result in superior efficacy in tumor bearing mice, where 6 would be converted to the active agent (5) selectively in the tumor. to see whether the higher in vivo exposure levels achievable with conjugate 6 translated to improved in vivo efficacy, we treated pc-3 and mda - mb-231 xenograft - bearing mice with 6 or seco-5 and compared body weights and rates of tumor growth under different dosing regimens (figure 6). in mda - mb-231 xenograft mice, three 0.3 mg / kg doses of seco-5 given q4d produced only a minor reduction in the rate of tumor growth (figure 6a). significantly, the mice in this group showed substantial weight loss over the course of the study, presumably reflecting compound - related toxicity (figure 6b). comparatively, mice treated under the same regimen with 2.5 mg / kg of 6 exhibited a similar reduction in tumor growth rate but unlike 5-treated mice had negligible weight loss, suggesting reduced toxicity under a comparably efficacious regimen (figure 6a). using the same regimen but increasing the dose of 6 to its mtd of 7.5 mg / kg produced a substantially improved effect on tumor growth and with much less severe weight loss than was observed with a 25-fold lower dose of seco-5 (figure 6a, b). these findings were replicated in a follow - up study that also included the evaluation of dioxolane conjugate 7 (supporting information figure s6). as expected, compound 7 at 10 mg / kg had no effect on tumor growth or mouse weight, indicating that the beneficial in vivo effects of 6 result from peroxide - dependent activation and release of 5 in tumor. (a) changes in tumor volume over time as determined by calipers in mda - mb-231 xenograft bearing female scid - beige mice treated with the indicated doses of 5 or 6 via ip administration on a q4d dosing schedule (3 total doses) as compared to mice treated with vehicle. (b) changes in mouse weight over time for the mice in (a). (c) changes in tumor volume over time as determined by calipers in pc-3 xenograft bearing female nude mice (ncr nu / nu, taconic) treated with the indicated doses of 5 or 6 via ip administration on a q4d dosing schedule (3 total doses) as compared to mice treated with vehicle. mice treated with the highest dose of 6 (15 mg / kg, orange line) received three ip doses on a q7d schedule. (d) changes in mouse weight over time for the mice in (c). the results described above for mda - mb-231 xenograft mice were qualitatively replicated in pc-3 xenograft bearing mice. thus, administration of 6 at 7.5 mg / kg (3, q4d, ip) produced comparable if not superior effects on tumor growth rate without the weight loss observed in mice receiving seco-5 at 0.3 mg / kg (figure 6c, d). since the mtd and pathology studies had suggested that the dose - limiting toxicity of tap 6 was localized gi toxicity near the site of administration, we considered that an altered dosing regimen might mitigate this toxicity and enable a higher dose. indeed, by increasing the dose of 6 to 15 mg / kg but extending the dosing interval from q4d to q7d (once weekly), we observed robust tumor regression (figure 6c, orange line), albeit with weight loss that was similar to the animals receiving the 0.3 mg / kg q4d regimen of 5. the tumor regression produced with 15 mg / kg q7d dosing of 6 was found to be quite durable, with no evidence of further tumor growth observed for the remainder of the study, several weeks after the third and final dose. the search for more effective and better tolerated cancer therapies has yielded the first molecularly targeted agents (e.g., kinase inhibitors, parp inhibitors) and new technologies to more selectively deliver potent cytotoxins to tumors (e.g., antibody drug conjugates). herein we have described a new class of tumor activated prodrugs engineered to release a drug payload upon encountering reactive iron(ii) in the tumor microenvironment. the trioxolane - based scaffold employed in these studies is the same one we used previously to study intracellular labile ferrous iron in cells, revealing larger iron(ii) pools in cancer - derived cell lines compared to nontumorigenic lines. here we exploited this knowledge and the tools we developed to deliver cytotoxic payloads to diverse cancer cell lines and to target tumors in two different mouse xenograft models. the trioxolane tap scaffold used here was engineered to confer traceless release of drug payloads, thereby enabling a broad scope of potential applications encompassing molecularly targeted and generally cytotoxic agents possessing suitable amine or alcohol functionality for conjugation. when this strategy is employed, the site of drug conjugation is selected so that the intrinsic activity / toxicity of tethered drug is ablated, thereby preventing or minimizing exposure of active drug in nontargeted tissues. to exemplify this approach, we prepared amine - linked tap conjugates 2 and 6 from the microtubule inhibitor 1 and duocarmycin analog seco-5, respectively. we confirmed that the activity / toxicity of these agents was ablated in the tap form by preparing dioxolane - drug conjugates 3 and 7, which proved inactive at the highest concentrations tested (figure 2b and figure 4b, c). the lack of measurable activity for 3 and 7 thus confirms that drug release from 2 and 6 is peroxide dependent. consistent with the expected mechanism of iron(ii)-dependent drug release, the relative sensitivity of cells to 2 (figure 2d) largely mirrored the response of the trx - puro probe to intracellular labile iron in the same cell lines. we found that sensitivity to trx - cmb conjugate 2 varied by about 9-fold across a panel of cancer cell lines from diverse origins (figure 2e). further interrogation of these data may provide insight into the specific alterations of iron metabolism that predict for increased tumor susceptibility toward trx - drug conjugates. in our preliminary study of oncogenic changes and related effects on iron metabolism, we observed that myc - driven changes in mcf10a cells produced increased sensitivity to trioxolane conjugate 2. consistent with these observations, several of the most 2-susceptible cell lines examined here, such as rko and mda - mb-231, are known to overexpress myc. thus, iron(ii)-dependent drug delivery could find utility in targeting myc - driven tumors indirectly via the alteration of iron metabolism induced by this prevalent oncogene. these findings are particularly relevant given how intractable myc driven tumors have been toward other targeted therapies. to examine the utility of trioxolane - mediated drug delivery in vivo, we prepared trioxolane tap 6 in which a potent duocarmycin - class cytotoxin (seco-5) significantly, conjugate 6 was tolerated in mice at doses up to 50-fold higher than seco-5 and produced superior efficacy in two different xenograft models when administered at or near its mtd. a pharmacokinetic study in healthy mice revealed that administration of seco-5 in the form 6 not only limited exposure to free drug (to 15% of the total dose) but also significantly improved distribution to tissues and the total duration of drug exposure. these properties of 6 enabled safe administration at a relatively high dose of 15 mg / kg once weekly, and this dosing regimen produced a particularly robust and durable tumor regression in a pc-3 xenograft model. overall, our studies indicate that trioxolane - based taps release their drug payload in proportion to the concentration of labile iron(ii) encountered in different cell / tissue types. while intracellular iron(ii) pools are likely implicated in tap activation, it is possible that other aspects of tumor biology in vivo (e.g., hypoxia, macrophage infiltration, tumor necrosis) contribute to the presence of excess reactive iron(ii) in the tumor microenvironment. in conclusion, trioxolane - mediated iron(ii)-dependent drug delivery is a new approach for cell / tissue selective drug targeting that leverages elevated reactive iron(ii) concentrations in tumor cells and in the tumor microenvironment. here we described two prototypical trioxolane - drug conjugates bearing cytotoxins with distinct mechanisms of cellular toxicity. we confirmed that the intrinsic toxicity of these agents could be ablated in conjugated forms and yet fully realized following cell- or tumor - selective release at their intended site of action. these results should encourage further study of this concept to identify drugs and tumor types that best leverage this new drug delivery approach. these compounds were coupled to known trioxolane and dioxolane intermediates via activated nitrophenyl carbonate or isocyanate intermediates as we have described previously and as further detailed in the supporting information. all compounds tested in cells or animals were judged to be of > 95% purity as determined using a waters micromass zqtm, equipped with waters 2795 separation module, waters 2996 photodiode array detector (254 nm), and waters 2424 els detector. separations were carried out with an xterra ms c18, 5 m, 4.6 mm 50 mm column, at ambient temperature (unregulated) using a mobile phase of water methanol containing a constant 0.10% formic acid. mammalian cell lines were maintained in an atmosphere of 5% co2 in rpmi 1640 media purchased from hyclone supplemented with 10% fbs (gibco), pen / strep (1 final concentration, gemini bio - products), and nonessential amino acids (ucsf cell culture facility). unless otherwise noted, cell lines were obtained from atcc and verified by str profiling. graphing and analysis of data were done using graphpad prism 6 software and microsoft excel 2010. when three or more mean values were compared, one- or two - way anova tests were applied as required with dunnett s multiple comparisons tests used to determine significance. statistical significance is indicated as follows : = p 0.05, = p 0.01, = p 0.001, = p 0.0001. cells were plated in 96-well greiner black clear tissue culture plates at 30006000 cells per well in rpmi 1640 cell culture media (or the appropriate growth medium as specified) and incubated at 37 c in 5% co2 incubators for at least 16 h prior to exposure to compounds. cells were then treated in triplicate with escalating concentrations of compounds performed in medium containing 0.1% dmso (100 l of media, per well). 2472 h after treatment (as specified), medium was removed and cells were washed with 100 l of pbs and then fixed in 4% paraformaldehyde for 10 min at rt and stained with hoechst nuclear stain at a final concentration of 10 g / ml in pbs for 10 min at rt. after fixing, the cells were stored in 100 l of pbs for imaging. wells were imaged with an in cell 2000 automated cell imager at 10 magnification with 9 images per well (complete coverage) in bright field and dapi channel fluorescence, and images were analyzed for nuclei count by in cell developer software. cells were harvested, resuspended, and plated with a wellmate liquid handler (thermo scientific) into 384-well plates and cultured for 24 h before dosing. master compound plates were made with a janus (perkinelmer), then further diluted to achieve uniform final concentrations of dmso of 0.1% in media for all treatment conditions. compound treatments in media were added to the cell plates with a matrix platemate (thermo scientific). cell viabilities were determined 72 h after treatment by cell - titer glo assay (promega) on the envision multilabel plate reader (perkinelmer). relative luminescent units (rlu) were plotted against corresponding drug concentrations and fitted with a standard four - parameter sigmoidal curve with graphpad prism 6. data were further fit for ec50 shift parameters in graphpad prism 6 to determine ec50 ratios for 1/2. data are reported as the ec50 ratio, and error bars represent sem (n = 3). data for cell lines in which ec50 ratio fits were ambiguous, r values were less than 0.9, or response to the free drug was less than 40% were not reported. cells were seeded at 300 000 cells per well in 6-well plates and grown to confluence and then collected with trypsin (0.05%), washed with pbs, and snap - frozen in liquid n2 and then stored at 78 c. cell pellets were processed for mrna isolation using qiagen rneasy mini kit with qiashredder lysate homogenizers and on column dna digest. isolated mrna was analyzed for concentration and purity on a thermoscientific nanodrop 2000c spectrophotometer, and 1000 ng of mrna from each sample was translated to cdna using invitrogen superscript iii first - strand synthesis system. the resulting cdna was used for qrt - pcr analysis (10 ng / reaction) with ssoadvanced universal sybr green supermix in a roche lightcycler 480. gapdh was used as an endogenous control, and relative mrna levels were calculated from a standard curve of pooled samples with lightcycler 480 software used for second derivative maximum analysis and standard curve fitting. samples were prepared and run in biological triplicates, and error bars represent sem. a list of the gene specific primers used is provided in the supporting information. to evaluate the tolerability of the experimental agents, groups of three nsg (nod scid gamma) mice were treated with three ip injections of test article (q4d or q7d in separate studies) in a formulation comprising 50:40:10 peg 400/20% 2-hydroxypropylcylcodextrine in water / dmso. individual groups (n = 3) received doses that increased in 2- or 3-fold steps until a given dose caused one or more mice in the group to reach protocol limits for tolerance (> 20% weight loss) at any point post dosing. to evaluate the in vivo pharmacokinetic properties of the experimental agents, female nsg mice were treated with a single ip injection of test compounds formulated in 50:40:10 peg 400/20% 2-hydroxypropylcylcodextrine in water / dmso. blood samples were collected 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h after dosing (3 mice were sampled per time point ; each group of mice was sampled 3 times over 24 h) and analyzed for plasma concentrations of each compound via ms / ms analysis conducted by integrated analytical solutions, inc. the resulting data were analyzed with winnonlin software to calculate standard pk parameters. to evaluate the in vivo properties of the experimental agents, we used the heterotopic indirect tumor xenograft model in nude mice (ncr nu / nu, taconic) and scid - beige mice. early passage pc-3 cells were harvested, and a cell suspension (1:1 serum free dmem / matrigel) was injected subcutaneously (sc) into the right flank of anesthetized donor nude mice (10 cells / mouse in 0.1 ml). for mda - mb-231 xenografts, cells were injected into the mammary fat pad of anesthetized female scid - beige mice (10 cells / mouse in 0.1 ml of pbs). when the mean tumor volume was 250400 mm, tumor - bearing mice were treated with the indicated doses of compounds formulated in 50:40:10 peg 400/20% 2-hydroxypropylcylcodextrine in water / dmso via ip administration with the indicated frequency. | here we describe a new approach for tumor targeting in which augmented concentrations of fe(ii) in cancer cells and/or the tumor microenvironment triggers drug release from an fe(ii)-reactive prodrug conjugate. the 1,2,4-trioxolane scaffold developed to enable this approach can in principle be applied to a broad range of cancer therapeutics and is illustrated here with fe(ii)-targeted forms of a microtubule toxin and a duocarmycin - class dna - alkylating agent. we show that the intrinsic reactivity / toxicity of the duocarmycin analog is masked in the conjugated form and this greatly reduced toxicity in mice. this in turn permitted elevated dosing levels, leading to higher systemic exposure and a significantly improved response in tumor xenograft models. overall our results suggest that fe(ii)-dependent drug delivery via trioxolane conjugates could have significant utility in expanding the therapeutic index of a range of clinical and preclinical stage cancer chemotherapeutics. |
temperament is defined as the quality resulting from reciprocal action of the four contrary principal qualities (hot / cold / moist / dry) (1, 2) domiciled within the elements (air, earth, water, fire) (3, 4). there are four humors in the body : blood (dam) ; phlegm (balgham) ; yellow bile (safra) ; and black bile (sauda) (5), each of which is associated with a pair of qualities : hot and moist, cold and moist, hot and dry, and cold and dry, respectively. black bile) humors (akhlat) in the body (4, 6). each of these temperaments is dominant upon a part of the body : head, chest, kidneys, abdomen, etc. blood is dominant upon the ears, eyes, nose, and brain ; phlegm is dominant upon the chest ; safra is dominant upon the kidneys ; soda is dominant upon abdomen, intestine, gastric (7, 8). the dominant temperament is the one that is determined in the person prone to which disease who is diagnosed the unique treatment for that individual (9). there are two types of temperaments : motadil and gheire motadil (out of balance) (10). each of the four temperaments is intermingled with two qualities : safra (hot and dry), soda (cold and dry) (7), phlegm (cold and moist), blood (hot and moist). physicians found nine types of temperament in humans : hot, cold, moist, dry, which are single (mofrad) temperaments, and hot and moist, hot and dry, cold and moist, cold and dry, and motadil (compound temperaments) (11). all of these humors exist in each individual s body but given to the point that each individual has a dominant and subdominant temperament (12), one may be phlegmatic - dominant, bilious - dominant, sanguineous - dominant, or melancholic - dominant in the case of temperament (4, 13). sue mezaj or dystemprament is the deficiency and decrease in the physiologic action and reaction of body, in a way that disturbance and dullness happen in its chemical action (11). there are 16 kinds of disorders of temperament (called by names such as sue mizaj, inequable temperament, intemprament, dyscrasias), which cause sue mizaj gheire motadil or imbalance in the body (14). a) the simple types are those deviating from the normal equipoise only regarding to one contrary, including active contrary (hotter than it should be, not moister or drier : hot temperament ; colder than it should be, not moister or drier : cold temperament), passive contrary (drier than it should be, but not hotter nor colder : dry temperament ; moister than it should be, but not hotter nor colder : moist temperament). b) the compound types are those deviating from the normal equipoise or equability regarding to two contraries at once. thus, the temperament may be simultaneously hotter and moister than it should be, hotter and drier than it should be, colder and moister than it should be, colder and drier than it should be (4). in addition, there are different classification - related temperaments based on the individual s sex (male, female) (13), age or periods of life [adolescent (up to 30), period of beauty (up to 35 or 40), period of decline (up to 60), senility (60 to the end of life) ] (4), seasons (spring, summer, autumn, winter), temperament of different parts of the body (temperament of the eye, liver, brain, heart...) (13), general temperament of individuals (2, 1518). besides, temperament of anything entered into the body is of two kinds : hot / cold. hot gives heat to the body and cold gives coldness to the body (19). based on the unani system of medicine, it is believed that a particular illness in the patient has taken place due to disharmony in her / his temperament, which is a departure from its equilibrium status ; therefore, the goal of treatment is to remove the disturbance of temperament (20). treatment is, therefore, aimed directly at restoring balance or equilibrium of various elements, humors and faculties to the patient s temperament or humors (6, 2123). to treat disease, it should be kept in mind that it is fully individualized and relies on the psychophysiological physique of the patient (24). besides, it is said that only one drug should not be used to treat one and the same disease (25). the treatment must include the six factors to ensure that not only are the symptoms treated but the causes of illnesses are also focused upon (26). it is worth mentioning that while modern medicine aims at controlling symptoms and managing illnesses, tibb aims at curing illnesses and managing health (26). it is of high significance that traditional medicine should keep in mind all six factors if its purpose is to cure disease. these six factors are atmospheric air, food and drinks, rest and physical activity, psychological activity, sleep and wakefulness, elimination and retention (2527). different ideas about treatment or ilaj were introduced in traditional medicine that are ilaj - bil - tadbeer (regimental therapy), ilaj- bil -ghiza (dietotherapy), ilaj- bil -dawa (pharmacotherapy) (28), ilaj- bil -yad (surgery) (29). the aim of this study is to overview the relationship between dystemprament (sue mezaj) and treatment of diseases. this systematic review was carried out from 1965 to 2016 by searching studies in pubmed, medline, web of science, and iranmedex databases. the initial search strategy identified about 119 references. in this study, 48 studies were accepted for further screening and our inclusion criteria [in english, full text, dystemprament, diseases, sue mizaj, temperament, treatment, management ]. inclusion criteria included the following keywords used to search for the relevant articles published from august 1930 to june 2016 ; their full text should be available and in english. articles included consisted of clinical trials, in vitro, in vivo, review, and meta - analysis studies. those articles not matching our inclusion criteria (being in languages other than english and between the timeline of the study) were excluded. to assess the quality of articles, all sections of articles were checked with the strobe checklist (29). in the screening and selection of article, nevertheless, 14 studies were excluded from the review, leaving 48 articles for further analysis. this systematic review was carried out from 1965 to 2016 by searching studies in pubmed, medline, web of science, and iranmedex databases. the initial search strategy identified about 119 references. in this study, 48 studies were accepted for further screening and our inclusion criteria [in english, full text, dystemprament, diseases, sue mizaj, temperament, treatment, management ]. inclusion criteria included the following keywords used to search for the relevant articles published from august 1930 to june 2016 ; their full text should be available and in english. articles included consisted of clinical trials, in vitro, in vivo, review, and meta - analysis studies. those articles not matching our inclusion criteria (being in languages other than english and between the timeline of the study) were excluded. to assess the quality of articles, all sections of articles were checked with the strobe checklist (29). in the screening and selection of article, the presence of inclusion and exclusion criteria also were checked. nevertheless, 14 studies were excluded from the review, leaving 48 articles for further analysis. ilaj - bil - zid is a medical treatment based on the rule of opposites, i.e., the administration of drugs having contrary actions to etiological changes (11, 30), i.e., if a disease results from heat, it must be treated with cold ; if from moisture, then with dryness, etc. if the cause is fear or grief, then it must deal with tranquility and confidence ; but first the etiology of the disease must be diagnosed (31). to begin treatment, first the habits of that person should be distinguished and their benefits and harms be considered. if it is beneficial, the habits should be changed. a unani physician discovered that one and every person at the same time may have a hot organ and a cold organ. in this situation, eating both types of foods, i.e., hot and cold foods triggers incidence of diseases (11). the admixture of hot and cold drugs should be boiled and ask the patient to take it in the case that disorder resulted from different temperaments of two organs of body (one is hot and another one is cold). intake of drug causing these two contradicting states was decreased and gradually improved (11). avicenna, in his book canon of medicine, stated that whenever cold and hot drugs were given together, the nature or temperament of the person itself distinguished that where the hotness is required, and where the coldness is requited and guide them to the appropriate place (4). whenever the hot and cold temperament becomes dominant, thus, vomiting and juice of cold and moist fruits are proposed to treat this condition (13). in the case of hot and moist temperament, the treatment is as follow : lots of exercise, taking a bath before meals and after exercise, urinating, gargling, labs, avoidance of tadbeer, which increases hotness and moisture (4). in the case of cold and dry temperament, which is the worst of all among temperaments (in that it is similar to that of elderly), treatments are those tadbeer that increase hotness and moisture (enriched sherbet, message in bath, oil immersion, eat hot and moist and latif meal (4) ; for cold and moist temperament, treatment consists of lots of exercise, dry food, less sleep and hammam, and oil immersion before hammam. some of them were placed under the headings of ilaj - bil - tadbeer (regimental therapy) ; others were of ilaj - bil - ghiza (dietotherapy) or ilaj - bil - dawa (pharmacotherapy) and ilaj bil yad (surgery). states of the patient, and his organ should be first examined and the rate of his tolerance should be diagnosed. thus, it is necessary to first prescribe different kinds of herbal medicine to distinguish his temperament (3235). then his body should be purged with drugs that are in harmony with his temperament and will cause the intestines to start to work and gain complete knowledge about liver and kidneys, then find the root of the disease and prescribe the proper drugs and medications (11). sometimes nature finds a way to remove the toxin or the element of imbalance (11). sometimes it happens that the form of sue mizaj change (the movement of element [phlegm, blood, bile, black bile ], for example, from kidneys to legs) (11). whenever the physician intends to return the element to the state of balance, he should first soothe the pains, because pain itself attracts the troublesome element. there are general principles of ilaj as follows : 1) whenever the phlegmatic khilt is mixed with blood, the fasd is prioritized ; 2) whenever one or two khilt rose, it should be decreased first by eshal then by fasd ; 3) whenever the safra is dominant, vomiting is prioritized to eshal ; 4) in winter, laxatives are better for obese people, while vomiting is better for thin people. in the summer, it is reverse (13) : 5) cold dystemprement is never the reason for pain and disorder, but it is the dry dystemprement that is the cause of disease ; 6) cold and hot temperaments are fundamental, but dry and moist are subsidiary, and the two latter are the consequence of the two first. thus cold dystemprament is the subsidiary cause of pain because cold temperament involves the organ, and its approximate loss of contiguity (tafarrogh - e ettesal) will happen ; thus, this pain is innately due to tafarrogh - e ettesal and secondarily due to cold intemperament (13). literally, tadbeer is an arabic word meaning regimen or systemic plan (25). ilaj bil tadbeer, which is a synonym to panchkarma in ayurveda (26), is to cure via regimen. it is a method through which care of the sick person and maintenance of general health is performed by means of certain procedures using various tools and equipment (27), i.e., through modulation or modification in asbaabe sitta zarooriya (24) ; in other words, regimental therapies are mostly nonmedicinal procedures by which we modulate the lifestyle, dietary habits, and habitat of the patient and practice some other therapeutic regimens for the treatment of various diseases (25). the regimental therapy includes procedures such as exercise (physical activity) (36, 37), diaphoresis, diuresis, turkish bath, massage, cautery, purgation, emesis, blood - letting in the form of venesection, leech therapy, and cupping with scarification (28, 29, 30, 38). unani scholars have been practicing this therapy for prophylactic purposes for the maintenance of health as well as for therapeutic purposes for the treatment of diseases since ancient times (24,3841). moreover, ibn sina has described 36 regimes, including irsale araq (taleeq) (leeching), fasd (iranian traditional medicine, tehran university of medical sciences students attitudes towards it) (venesection), ishal (purgation), qai (emesis), idrar (diuresis), huqna (enema), hijamat (cupping), dalk (massage), riyazat (exercise), hammam (bathing), tareeq (diaphoresis), amle kai (cauterization), nutool (irrigation), inkebaab (inhalation), tanafis (expectoration), takmeed (fomentation), imala (diversion of morbid material), ilam (counter - irritation), aabzan (hydration therapy), zimaadwatila (ointment and liniment), etc. ilaj - bil - ghiza (dietotherapy) in unani medicine has great importance on treating certain diseases by administrating specific diets or by regulating the quality and quantity of food (42). as dietotherapy is the use of food as an agent in effecting recovery from illness, it is concerned with those receiving normal diet as well as those for whom modified diet has been prescribed. modified diets are the principal therapeutic agents in some metabolic diseases and chronic diseases (27). diet should be selected that is ghizae lateef, jaiyyadul kaimus, and sariul hazm. besides, general principles of diet therapy are abstinence (tarke ghiza, fasting), reduction in intake of diet (taqleele giza) (27). temperament of foods are of four kinds : hot / cold / moist / dry (43). the purpose of dietotherapy is to maintain good nutritional status, to correct deficiencies that may have occurred, and to afford rest to the whole body or to certain organs, which may have been affected as well as to adjust the food intake to the body s ability to metabolize the nutrients and to bring about changes in body weight whenever required. one kind of dietotherapy is tadbir - e - latif, which is eating less instead of doing fasd. in this way, the rest of the food would not return to vessels. as a consequence, less blood will produce, and there is no need to do fasd (11). before pharmacotherapy, unani physicians advise restriction or alteration in daily diet, adjusted according to disease, and wait for a few days because some diseases can be cured even with diet. during treatment, specific diets are advised according to disease. ilaj - bil - dawa (pharmacotherapy) deals with the use of naturally derived drugs that are recommended in conditions when health by natural ways and means such as change in lifestyle does not work (26). the drugs are classified into groups according to their origin, i.e., nabati (plant origin), haiwani (an animal origin), and madni advia (mineral origin) (23). temperament has two broad categories : one is concerned with the temperament of humans ; the other is about temperament of advia (drugs) (44). saying that the drug is hot or cold does not mean that the substance of the drug is hot or cold or that its substance is hotter or colder than the human body, but it means that this drug induces heat or cold in the body, exceeding heat or cold of the human body (23). treatment with drugs, as said by avicenna, must take into account the following rules : choice of drugs by their quality, selection of drugs by their quantity, and this rule includes change in weight, potency, and properties and the time of administration of drugs (45). drugs are classified into two kinds : single drugs, compound drugs (31). some single drugs are beneficial to reduce complications and are helpful in order to trigger protective mechanisms and to delay catabolic and tissue degrading pathways, which may be due to antioxidant, immuno - modulatory, neuroprotective, hepatoprotective, anti - inflammatory, and vitalizing (iksir - e - badan / rasayana / elixir of life) effects (46, 47). this kind of ilaj is helpful in dealing with disease such as those of the liver, peptic ulcers, hypertension, migraines, menorrhagia, diarrhea, diabetes mellitus, and constipation (table 1). ilaj - bil - zid is a medical treatment based on the rule of opposites, i.e., the administration of drugs having contrary actions to etiological changes (11, 30), i.e., if a disease results from heat, it must be treated with cold ; if from moisture, then with dryness, etc. if the cause is fear or grief, then it must deal with tranquility and confidence ; but first the etiology of the disease must be diagnosed (31). to begin treatment, first the habits of that person should be distinguished and their benefits and harms be considered. if it is beneficial, the habits should be changed. a unani physician discovered that one and every person at the same time may have a hot organ and a cold organ. in this situation, eating both types of foods, i.e., hot and cold foods triggers incidence of diseases (11). the admixture of hot and cold drugs should be boiled and ask the patient to take it in the case that disorder resulted from different temperaments of two organs of body (one is hot and another one is cold). intake of drug causing these two contradicting states was decreased and gradually improved (11). avicenna, in his book canon of medicine, stated that whenever cold and hot drugs were given together, the nature or temperament of the person itself distinguished that where the hotness is required, and where the coldness is requited and guide them to the appropriate place (4). whenever the hot and cold temperament becomes dominant, thus, vomiting and juice of cold and moist fruits are proposed to treat this condition (13). in the case of hot and moist temperament, the treatment is as follow : lots of exercise, taking a bath before meals and after exercise, urinating, gargling, labs, avoidance of tadbeer, which increases hotness and moisture (4). in the case of cold and dry temperament, which is the worst of all among temperaments (in that it is similar to that of elderly), treatments are those tadbeer that increase hotness and moisture (enriched sherbet, message in bath, oil immersion, eat hot and moist and latif meal (4) ; for cold and moist temperament, treatment consists of lots of exercise, dry food, less sleep and hammam, and oil immersion before hammam. some of them were placed under the headings of ilaj - bil - tadbeer (regimental therapy) ; others were of ilaj - bil - ghiza (dietotherapy) or ilaj - bil - dawa (pharmacotherapy) and ilaj bil yad (surgery). states of the patient, and his organ should be first examined and the rate of his tolerance should be diagnosed. thus, it is necessary to first prescribe different kinds of herbal medicine to distinguish his temperament (3235). then his body should be purged with drugs that are in harmony with his temperament and will cause the intestines to start to work and gain complete knowledge about liver and kidneys, then find the root of the disease and prescribe the proper drugs and medications (11). sometimes nature finds a way to remove the toxin or the element of imbalance (11). sometimes it happens that the form of sue mizaj change (the movement of element [phlegm, blood, bile, black bile ], for example, from kidneys to legs) (11). whenever the physician intends to return the element to the state of balance, he should first soothe the pains, because pain itself attracts the troublesome element. there are general principles of ilaj as follows : 1) whenever the phlegmatic khilt is mixed with blood, the fasd is prioritized ; 2) whenever one or two khilt rose, it should be decreased first by eshal then by fasd ; 3) whenever the safra is dominant, vomiting is prioritized to eshal ; 4) in winter, laxatives are better for obese people, while vomiting is better for thin people. in the summer, it is reverse (13) : 5) cold dystemprement is never the reason for pain and disorder, but it is the dry dystemprement that is the cause of disease ; 6) cold and hot temperaments are fundamental, but dry and moist are subsidiary, and the two latter are the consequence of the two first. thus cold dystemprament is the subsidiary cause of pain because cold temperament involves the organ, and its approximate loss of contiguity (tafarrogh - e ettesal) will happen ; thus, this pain is innately due to tafarrogh - e ettesal and secondarily due to cold intemperament (13). literally, tadbeer is an arabic word meaning regimen or systemic plan (25). ilaj bil tadbeer, which is a synonym to panchkarma in ayurveda (26), is to cure via regimen. it is a method through which care of the sick person and maintenance of general health is performed by means of certain procedures using various tools and equipment (27), i.e., through modulation or modification in asbaabe sitta zarooriya (24) ; in other words, regimental therapies are mostly nonmedicinal procedures by which we modulate the lifestyle, dietary habits, and habitat of the patient and practice some other therapeutic regimens for the treatment of various diseases (25). the regimental therapy includes procedures such as exercise (physical activity) (36, 37), diaphoresis, diuresis, turkish bath, massage, cautery, purgation, emesis, blood - letting in the form of venesection, leech therapy, and cupping with scarification (28, 29, 30, 38). unani scholars have been practicing this therapy for prophylactic purposes for the maintenance of health as well as for therapeutic purposes for the treatment of diseases since ancient times (24,3841). moreover, ibn sina has described 36 regimes, including irsale araq (taleeq) (leeching), fasd (iranian traditional medicine, tehran university of medical sciences students attitudes towards it) (venesection), ishal (purgation), qai (emesis), idrar (diuresis), huqna (enema), hijamat (cupping), dalk (massage), riyazat (exercise), hammam (bathing), tareeq (diaphoresis), amle kai (cauterization), nutool (irrigation), inkebaab (inhalation), tanafis (expectoration), takmeed (fomentation), imala (diversion of morbid material), ilam (counter - irritation), aabzan (hydration therapy), zimaadwatila (ointment and liniment), etc. (4). ilaj - bil - ghiza (dietotherapy) in unani medicine has great importance on treating certain diseases by administrating specific diets or by regulating the quality and quantity of food (42). as dietotherapy is the use of food as an agent in effecting recovery from illness, it is concerned with those receiving normal diet as well as those for whom modified diet has been prescribed. modified diets are the principal therapeutic agents in some metabolic diseases and chronic diseases (27). diet should be selected that is ghizae lateef, jaiyyadul kaimus, and sariul hazm. besides, general principles of diet therapy are abstinence (tarke ghiza, fasting), reduction in intake of diet (taqleele giza) (27). temperament of foods are of four kinds : hot / cold / moist / dry (43). the purpose of dietotherapy is to maintain good nutritional status, to correct deficiencies that may have occurred, and to afford rest to the whole body or to certain organs, which may have been affected as well as to adjust the food intake to the body s ability to metabolize the nutrients and to bring about changes in body weight whenever required. one kind of dietotherapy is tadbir - e - latif, which is eating less instead of doing fasd. in this way, the rest of the food would not return to vessels. as a consequence, less blood will produce, and there is no need to do fasd (11). before pharmacotherapy, unani physicians advise restriction or alteration in daily diet, adjusted according to disease, and wait for a few days because some diseases can be cured even with diet. during treatment, specific diets ilaj - bil - dawa (pharmacotherapy) deals with the use of naturally derived drugs that are recommended in conditions when health by natural ways and means such as change in lifestyle does not work (26). the drugs are classified into groups according to their origin, i.e., nabati (plant origin), haiwani (an animal origin), and madni advia (mineral origin) (23). drugs used may be in single or compound formulation. temperament has two broad categories : one is concerned with the temperament of humans ; the other is about temperament of advia (drugs) (44). saying that the drug is hot or cold does not mean that the substance of the drug is hot or cold or that its substance is hotter or colder than the human body, but it means that this drug induces heat or cold in the body, exceeding heat or cold of the human body (23). treatment with drugs, as said by avicenna, must take into account the following rules : choice of drugs by their quality, selection of drugs by their quantity, and this rule includes change in weight, potency, and properties and the time of administration of drugs (45). drugs are classified into two kinds : single drugs, compound drugs (31). some single drugs are beneficial to reduce complications and are helpful in order to trigger protective mechanisms and to delay catabolic and tissue degrading pathways, which may be due to antioxidant, immuno - modulatory, neuroprotective, hepatoprotective, anti - inflammatory, and vitalizing (iksir - e - badan / rasayana / elixir of life) effects (46, 47). this kind of ilaj is helpful in dealing with disease such as those of the liver, peptic ulcers, hypertension, migraines, menorrhagia, diarrhea, diabetes mellitus, and constipation (table 1). some of them were placed under the headings of ilaj - bil - tadbeer (regimental therapy) ; others were of ilaj - bil - ghiza (dietotherapy) or ilaj - bil - dawa (pharmacotherapy) and ilaj bil yad (surgery). states of the patient, and his organ should be first examined and the rate of his tolerance should be diagnosed. thus, it is necessary to first prescribe different kinds of herbal medicine to distinguish his temperament (3235). then his body should be purged with drugs that are in harmony with his temperament and will cause the intestines to start to work and gain complete knowledge about liver and kidneys, then find the root of the disease and prescribe the proper drugs and medications (11). sometimes nature finds a way to remove the toxin or the element of imbalance (11). sometimes it happens that the form of sue mizaj change (the movement of element [phlegm, blood, bile, black bile ], for example, from kidneys to legs) (11). whenever the physician intends to return the element to the state of balance, he should first soothe the pains, because pain itself attracts the troublesome element. there are general principles of ilaj as follows : 1) whenever the phlegmatic khilt is mixed with blood, the fasd is prioritized ; 2) whenever one or two khilt rose, it should be decreased first by eshal then by fasd ; 3) whenever the safra is dominant, vomiting is prioritized to eshal ; 4) in winter, laxatives are better for obese people, while vomiting is better for thin people. in the summer, it is reverse (13) : 5) cold dystemprement is never the reason for pain and disorder, but it is the dry dystemprement that is the cause of disease ; 6) cold and hot temperaments are fundamental, but dry and moist are subsidiary, and the two latter are the consequence of the two first. thus cold dystemprament is the subsidiary cause of pain because cold temperament involves the organ, and its approximate loss of contiguity (tafarrogh - e ettesal) will happen ; thus, this pain is innately due to tafarrogh - e ettesal and secondarily due to cold intemperament (13). literally, tadbeer is an arabic word meaning regimen or systemic plan (25). ilaj bil tadbeer, which is a synonym to panchkarma in ayurveda (26), is to cure via regimen. it is a method through which care of the sick person and maintenance of general health is performed by means of certain procedures using various tools and equipment (27), i.e., through modulation or modification in asbaabe sitta zarooriya (24) ; in other words, regimental therapies are mostly nonmedicinal procedures by which we modulate the lifestyle, dietary habits, and habitat of the patient and practice some other therapeutic regimens for the treatment of various diseases (25). the regimental therapy includes procedures such as exercise (physical activity) (36, 37), diaphoresis, diuresis, turkish bath, massage, cautery, purgation, emesis, blood - letting in the form of venesection, leech therapy, and cupping with scarification (28, 29, 30, 38). unani scholars have been practicing this therapy for prophylactic purposes for the maintenance of health as well as for therapeutic purposes for the treatment of diseases since ancient times (24,3841). moreover, ibn sina has described 36 regimes, including irsale araq (taleeq) (leeching), fasd (iranian traditional medicine, tehran university of medical sciences students attitudes towards it) (venesection), ishal (purgation), qai (emesis), idrar (diuresis), huqna (enema), hijamat (cupping), dalk (massage), riyazat (exercise), hammam (bathing), tareeq (diaphoresis), amle kai (cauterization), nutool (irrigation), inkebaab (inhalation), tanafis (expectoration), takmeed (fomentation), imala (diversion of morbid material), ilam (counter - irritation), aabzan (hydration therapy), zimaadwatila (ointment and liniment), etc. ilaj - bil - ghiza (dietotherapy) in unani medicine has great importance on treating certain diseases by administrating specific diets or by regulating the quality and quantity of food (42). as dietotherapy is the use of food as an agent in effecting recovery from illness, it is concerned with those receiving normal diet as well as those for whom modified diet has been prescribed. modified diets are the principal therapeutic agents in some metabolic diseases and chronic diseases (27). diet should be selected that is ghizae lateef, jaiyyadul kaimus, and sariul hazm. besides, general principles of diet therapy are abstinence (tarke ghiza, fasting), reduction in intake of diet (taqleele giza) (27). temperament of foods are of four kinds : hot / cold / moist / dry (43). the purpose of dietotherapy is to maintain good nutritional status, to correct deficiencies that may have occurred, and to afford rest to the whole body or to certain organs, which may have been affected as well as to adjust the food intake to the body s ability to metabolize the nutrients and to bring about changes in body weight whenever required. one kind of dietotherapy is tadbir - e - latif, which is eating less instead of doing fasd. in this way, the rest of the food would not return to vessels. as a consequence, less blood will produce, and there is no need to do fasd (11). before pharmacotherapy, unani physicians advise restriction or alteration in daily diet, adjusted according to disease, and wait for a few days because some diseases can be cured even with diet. during treatment, specific diets are advised according to disease. ilaj - bil - dawa (pharmacotherapy) deals with the use of naturally derived drugs that are recommended in conditions when health by natural ways and means such as change in lifestyle does not work (26). the drugs are classified into groups according to their origin, i.e., nabati (plant origin), haiwani (an animal origin), and madni advia (mineral origin) (23). temperament has two broad categories : one is concerned with the temperament of humans ; the other is about temperament of advia (drugs) (44). saying that the drug is hot or cold does not mean that the substance of the drug is hot or cold or that its substance is hotter or colder than the human body, but it means that this drug induces heat or cold in the body, exceeding heat or cold of the human body (23). treatment with drugs, as said by avicenna, must take into account the following rules : choice of drugs by their quality, selection of drugs by their quantity, and this rule includes change in weight, potency, and properties and the time of administration of drugs (45). drugs are classified into two kinds : single drugs, compound drugs (31). some single drugs are beneficial to reduce complications and are helpful in order to trigger protective mechanisms and to delay catabolic and tissue degrading pathways, which may be due to antioxidant, immuno - modulatory, neuroprotective, hepatoprotective, anti - inflammatory, and vitalizing (iksir - e - badan / rasayana / elixir of life) effects (46, 47). this kind of ilaj is helpful in dealing with disease such as those of the liver, peptic ulcers, hypertension, migraines, menorrhagia, diarrhea, diabetes mellitus, and constipation (table 1). while traditional medicine contains many useful, less expensive, and less risky remedies for lots of morbidities, modern medicine makes them appear not beneficial. this study gathers some of these remedies to remind one that it is possible to apply them in our daily lives. the findings show three kinds of treatment based on traditional medicine : regimental therapy, dietotherapy, and drug therapy. it is suggested that modern medicine gets more help from the achievements of traditional medicine in the treatment of patients. besides, it is recommended that more research can be done to elaborate upon these treatments to other and perhaps newly borne diseases. | backgroundtemperament refers to four different humors differentiating in individuals and, as a result, proposes specific therapy for diseases as well as special types of management (avoidance).objectivethe aim of this study was to overview the relationship between dystemprament and treatment and management of diseases.methodsa computerized search of published articles was performed using pubmed, scopus, web of science, and medline databases as well as local sources from 1965 to 2016. additional sources were identified through cross - referencing. original and translated books were also used. of the whole 105 articles, 40 of them were selected as our database. the search terms used were as follows : temperament, dystemprament, diseases, sue mizaj, treatments, management.resultsthe findings of this study indicated that many remedies are used based on traditional medicine to cure disorders derived from dystemprament such as different kinds of regimen, diet, and drugs. the result of this study shows that regimental therapy contributes to the treatment of some disorders such as muscular dystrophy ; alzheimer s ; ms ; epilepsy ; falij ; convulsion ; depression ; eye diseases ; ear disease ; mouth, tongue, teeth disease ; common cold (nazle) ; asthma ; polyphagia or anorexia ; heart diseases ; esophagus ; peptic ulcer ; herpes simplex ; liver ; colic ; jaundice ; spleen ; kidney and bladder diseases ; hemorrhoid ; stomach worm ; hyperlipidemia. further, the findings suggest that dietotherapy is beneficial to treat and manage some disease such as sinusitis, lung, asthma, fever, muscular dystrophy, esophagus, peptic ulcer, liver, mouth, tongue, teeth disease, heart disease, polyphagia or anorexia, kidney and bladder diseases, ms, insomnia, piles, acne, permanent ejaculation, anemia, angina and heart attack, sore throat (tonsillitis), osteo - arthritis, rheumatoid arthritis, gout, and impotency.conclusionwhile traditional medicine contains many useful, less expensive, and even cheap and less risky remedies for lots of morbidities, modern medicine makes them appear nonrelevant. this study gathers some of these remedies to remind one about applying them in our daily lives. |
the following online material is available for this article : mutations identified by direct sequencing of the mtdna d - loop region in 93 individuals (generations f1 and f3). mutations observed in the mtdna of 120 clones from 12 grandmothers. | the accumulation of somatic mutations in mtdna is correlated with aging. in this work, we sought to identify somatic mutations in the hvs-1 region (d - loop) of mtdna that might be associated with aging. for this, we compared 31 grandmothers (mean age : 63 2.3 years) and their 62 grandchildren (mean age : 15 4.1 years), the offspring of their daughters. direct dna sequencing showed that mutations absent in the grandchildren were detected in a presumably homoplasmic state in three grandmothers and in a heteroplasmic state in an additional 13 grandmothers ; no mutations were detected in the remaining 15 grandmothers. however, cloning followed by dna sequencing in 12 grandmothers confirmed homoplasia in only one of the three mutations previously considered to be homoplasmic and did not confirm heteroplasmy in three out of nine grandmothers found to be heteroplasmic by direct sequencing. thus, of 12 grandmothers in whom mtdna was analyzed by cloning, eight were heteroplasmic for mutations not detected in their grandchildren. in this study, the use of genetically related subjects allowed us to demonstrate the occurrence of age - related (> 60 years old) mutations (homoplasia and heteroplasmy). it is possible that both of these situations (homoplasia and heteroplasmy) were a long - term consequence of mitochondrial oxidative phosphorylation that can lead to the accumulation of mtdna mutations throughout life. |
but the arecoline and flavonoid, components of areca nut when exposed to buccal mucosal fibroblast results in the accumulation of collagen. reduced collagenase activity and increased cross - linking of the fibers results in decreased degradation of collagen. this evidence implies that osf may be considered a collagen - metabolic disorder resulting from exposure to areca nut. the possible molecular alteration - taking place in epithelium and connective tissue has been explained in figure 1. the important histopathological characteristic of osf is the deposition of collagen in the oral submucosa. structural changes of epithelial and connective tissue in osf have been studied in detail both at the light and electron microscopic levels. the atrophic epithelium may exhibit loss of rete pegs, hyperkeratinization, intercellular edema, signet cells and focal dysplasia. it may also show vacuolization of prickle cell layer and increased mitotic activities in few cases. the connective tissue changes were graded based on presence or absence of edema, nature of the collagen bundles, overall fibroblastic response, state of the blood vessels and predominant cell type in the inflammatory exudates. several classifications based on clinical, histological and combined feature have been put forth by several researchers at several time periods is shown in table 1. criterias included by different authors the higher prevalence of leukoplakia as well as epithelial atypia in osf leading to oral carcinoma was observed. one third of the patient with this disease has developed slow growing squamous cell carcinoma. the precancerous nature of this disease has been proved by, higher occurrence of osf in oral cancer patients, higher incidence of squamous cell carcinoma in patients with osf, histological diagnosis of cancer without any clinical suspicion in osf, high frequency of epithelial dysplasia and higher prevalence of leukoplakia among osf. initiation of malignancy in osf occurs because of epithelium or connective tissue is still in debate. but, it might be that the pathology develops within the epithelium due to intraoral trauma and various factors may play a significant role in oral cancer. the various factors causing oral trauma include, irritation from jagged teeth, ill - fitting denture, sharp overhanging restoration, jacket crowns, prolong use of tobacco and poor oral hygiene. the higher prevalence of leukoplakia as well as epithelial atypia in osf leading to oral carcinoma was observed. one third of the patient with this disease has developed slow growing squamous cell carcinoma. the precancerous nature of this disease has been proved by, higher occurrence of osf in oral cancer patients, higher incidence of squamous cell carcinoma in patients with osf, histological diagnosis of cancer without any clinical suspicion in osf, high frequency of epithelial dysplasia and higher prevalence of leukoplakia among osf. initiation of malignancy in osf occurs because of epithelium or connective tissue is still in debate. but, it might be that the pathology develops within the epithelium due to intraoral trauma and various factors may play a significant role in oral cancer. the various factors causing oral trauma include, irritation from jagged teeth, ill - fitting denture, sharp overhanging restoration, jacket crowns, prolong use of tobacco and poor oral hygiene. the main problems plaguing the patients with osf are the burning sensation and progressive trismus which impedes normal function. the treatment should aim at alleviating the symptoms as well as try to stop the progression of fibrosis. the treatment modalities which are currently being used can be broadly divided into three main categories, viz. but, whatever the treatment method may be, the first step of preventive measure should be in discontinuation of habit, which can be encouraged through education, counseling and advocacy. several studies have been performed to study the effects of various drugs in the management of osf. the main non - surgical methods of managing osf include : corticosteroids are immunosuppressive agents, they inhibit action of inflammatory products released by sensitized lymphocytes. they may be able to partly relieve patients of their symptoms, at an early stage, but less useful in reversing the abnormal deposition of fibrotic tissues and recovering the suppleness of the mucosa.proteolytic enzymes such as hyaluronidase, collagenase and chymotrypsin are often used for the treatment of osf. hyaluronidase breaks down hyaluronic acid, and also decreases collagen formation. a combination of triamcinolone 10 mg / ml + hyaluronidase 1500 iu for 4 weeks or biweekly submucosal injections of a combination of dexamethasone (4 mg / ml) and two parts of hyaluronidase (200 usp. unit / ml) diluted in 1.0 ml of 2% xylocaine have proven effective in the past.vitamins, antioxidants and minerals : lycopene is a such powerful antioxidant obtained from tomatoes, which given at a dose of 16 mg twice daily for 2 months showed significant improvement.levamisole : immuno - modulatory drug, given at a dose of 50 mg tab thrice in a day (tid) showed significant improvement in signs and symptoms. it modifies both cellular and humoral immunity, has an anti - inflammatory effects and its ability to modulate inflammatory cytokines reduces burning sensation.[1922]placental extract : they were found to have anti - oxidant and anti - inflammatory activity and brought about reduction of bands, improvement in mouth opening and tongue protrusion. but recent regulations in india allows usage of placental extracts only for topical wound healing and pelvic inflammatory diseases.interferon- : has a known anti - fibrotic effect. when given in a dosage of 50 g (0.25 ml) intralesionally twice a week over 8 weeks, recombinant human inf- showed improvement in both mouth opening and burning sensation. adverse effects included simple headache, flu - like symptoms and myalgia.peripheral vasodilators : pentoxifylline has a vasodilating property, which when given at dosage of 400 mg, 3 times a day for 7 months showed better results when compared to a control group which was on only antioxidants. it blocks tnf- induced synthesis of fibroblast collagen.immune milk : immune milk is a kind of skimmed, produced from cows immunized with multiple human intestinal bacteria. corticosteroids are immunosuppressive agents, they inhibit action of inflammatory products released by sensitized lymphocytes. they may be able to partly relieve patients of their symptoms, at an early stage, but less useful in reversing the abnormal deposition of fibrotic tissues and recovering the suppleness of the mucosa. proteolytic enzymes such as hyaluronidase, collagenase and chymotrypsin are often used for the treatment of osf. hyaluronidase breaks down hyaluronic acid, and also decreases collagen formation. a combination of triamcinolone 10 mg / ml + hyaluronidase 1500 iu for 4 weeks or biweekly submucosal injections of a combination of dexamethasone (4 mg / ml) and two parts of hyaluronidase (200 usp. unit / ml) diluted in 1.0 ml of 2% xylocaine have proven effective in the past. vitamins, antioxidants and minerals : lycopene is a such powerful antioxidant obtained from tomatoes, which given at a dose of 16 mg twice daily for 2 months showed significant improvement. levamisole : immuno - modulatory drug, given at a dose of 50 mg tab thrice in a day (tid) showed significant improvement in signs and symptoms. it modifies both cellular and humoral immunity, has an anti - inflammatory effects and its ability to modulate inflammatory cytokines reduces burning sensation.[1922 ] placental extract : they were found to have anti - oxidant and anti - inflammatory activity and brought about reduction of bands, improvement in mouth opening and tongue protrusion. but recent regulations in india allows usage of placental extracts only for topical wound healing and pelvic inflammatory diseases. interferon- : has a known anti - fibrotic effect. when given in a dosage of 50 g (0.25 ml) intralesionally twice a week over 8 weeks, recombinant human inf- showed improvement in both mouth opening and burning sensation. adverse effects included simple headache, flu - like symptoms and myalgia. peripheral vasodilators : pentoxifylline has a vasodilating property, which when given at dosage of 400 mg, 3 times a day for 7 months showed better results when compared to a control group which was on only antioxidants. immune milk : immune milk is a kind of skimmed, produced from cows immunized with multiple human intestinal bacteria. immune milk contains an anti - inflammatory component that may suppress the inflammatory reaction and modulate cytokine production. 45 g of milk powder prepared from an immunized cow, given twice a day for 3 months has found to show some improvement. ayurvedic medicine : in india, turmeric has been used as a common household spice and has been used as a remedy for wound healing for centuries. in one clinical trial alcoholic extracts of turmeric 3 g, turmeric oil 600 mg and turmeric oleoresin 600 mg, when consumed orally, decreased the number of micronucleated cells both in exfoliated oral mucosal cells and in circulating lymphocytes in osf patients. ayurvedic medicine : in india, turmeric has been used as a common household spice and has been used as a remedy for wound healing for centuries. in one clinical trial alcoholic extracts of turmeric 3 g, turmeric oil 600 mg and turmeric oleoresin 600 mg, when consumed orally, decreased the number of micronucleated cells both in exfoliated oral mucosal cells and in circulating lymphocytes in osf patients. surgical therapy aims at relieving severe trismus by incising the fibrous bands, which has led to further fibrosis in the past. muscle stretching exercises like ballooning of mouth, forceful mouth opening using splints and sticks have been tried in the past as a supportive therapy post - surgically. it is one of the most poorly understood and unsatisfactorily treated oral diseases because of its multifactorial etiology. no single drug has provided a complete relief and this is mainly due to the fact that the etiology of the disease is not fully understood. the better approach in demystifying the pathogenesis of osf could provide a new and successful therapeutic direction in near future. | oral submucous fibrosis (osf) is a chronic, progressive, potentially malignant condition affecting the oral cavity and frequently involving the upper part of the aerodigestive tract including the oropharynx and the upper part of the esophagus. it is characterized by juxtaepithelial inflammatory reaction and progressive fibrosis of lamina propria, leading to stiffening of the oral mucosa eventually causing trismus. this condition is associated with significant morbidity and high risk of malignancy. over the years, several drugs and combinations have been tried for the treatment of submucous fibrosis, but with limited success, because of its unclear molecular pathogenesis. till date, there are no known effective treatments for osf. the aim of this article is to emphasize on the molecular changes taking place in osf and possible therapeutic interventions. |
rhabdomyoma is the most common cardiac tumor in fetal life, though a rare condition. cardiac rhabdomyomas (cr) are closely associated with tuberous sclerosis complex (tsc). tuberous sclerosus (ts) is an autosomal dominant condition involving multiple organs such as heart, brain, eye, kidney and skin. crs may precede the skin, neurological and radiological signs of tsc by months or even years. however, association of craniofacial malformation such as pierre robin sequence (prs), clubfoot and intestinal obstruction with ts has not been described earlier. this is probably the first case where ts and prs have been described in the same patient and that too in a neonate. the present case report is about a 3-day - old late preterm (36 weeks of gestational age at birth) male neonate, who was referred to us from an outside institution with complaints of bilious vomiting and antenatally detected multiple cardiac tumors. the baby was born out of a non - consanguineous marriage to a 29-year - old primigravida. two homogenous echogenic tumors of size 7 mm and 4.5 mm were noted in the ventricular septum. other locations were medial papillary muscle of mitral valve and right ventricular free wall. the tumors were non - obstructive. however, feeds could not be started as he developed abdominal distension and bilious vomiting on day 1. physical examination was remarkable for the presence of prs (micrognathia, glossoptosis and cleft palate). clinically, bradycardia and irregularly irregular heart rate was present (heart rate varying between 80 and 110/min). x - ray erect abdomen showed multiple air - fluid levels suggestive of lower small bowel obstruction and a barium enema study revealed microcolon probably indicating an atretic lesion in the distal small bowel. nature of convulsions became mixed on day 6 with predominantly myoclonic type and was controlled with phenobarbitone and levetiracetam. computed tomography scan - brain showed subependymal nodules and cortical tubers [figure 2b ]. a diagnosis of ts was made in view of cr and cortical tubers in brain. the parents did not give consent for explorative laparotomy and took discharge against medical advice on day 10 of life. lack of facilities and logistic reasons precluded the genetic work - up of our patient. bilateral club foot and bilious gastric aspirates apical four chamber view of two - dimensional echocardiogram showing multiple rhabdomyomas of heart in the mid and apical interventricular septum (a). multiple subependymal nodules and cortical tubers (shown by black arrows) in computed tomography scan of brain (b) fetal cr is a rare condition, but the most common cardiac tumor in fetal life accounting for 60 - 86% of fetal cardiac tumors. though crs have a natural history of spontaneous regression, prognosis is guarded as it is very frequently associated with ts which is inherited as an autosomal dominant trait with variable penetrance and prevalence of 1/6,000 people. clinical manifestations include seizures, skin lesions and hamartomas affecting multiple organs such as heart, brain, eye and kidneys. the first signs of ts can be picked up on routine prenatal ultrasound screening where lesions are found in heart (rhabdomyomas) and brain (cortical tubers, subependymal nodules and subependymal giant cell astrocytoma). with advancement in fetal echo and magnetic resonance imaging, prenatal diagnosis a recent meta - analysis showed that the incidence of ts was 64% in fetuses with crs. furthermore, a positive family history of ts and multiple cardiac tumors were more likely to be associated with ts. a 42 year retrospective review of 70 patients consisting of 43 fetuses and 27 neonates with ts showed that crs comprised 33% of pathological findings, those of central nervous system origin 47% and renal cystic disease 13%. skeletal anomalies like clubfoot and cleft palate are not usually associated with tsc and have been reported in isolated cases. our case is unusual in having bilateral congenital talipes equino varus along with prs and small bowel obstruction. prs has been associated with nearly 40 varieties of syndromes and chromosomal anomalies, but the underlying pathogenesis of this disease has still remained enigmatic. recently a genetic association has been identified between a neurocutaneous syndrome (neurofibromatosis type 2) and prs. the present case is also a neurocutaneous syndrome with prs as an additional finding, but genetic work - up could not be done. when ts is diagnosed in the perinatal period, it is associated with high incidence of morbidity and mortality. however, it is observed that survival rates of patients diagnosed antenatally was practically the same as for those after birth it has been reported that more than 80% of crs have complete resolution within infancy and early childhood. however unlike crs, cerebral lesions do not regress, but instead progressively increase in size and number. our knowledge of natural history of crs in utero is limited and meta analytical approach to the prenatal management of ts is lacking. the age - dependent nature of the characteristic features of tsc has presented challenges for the diagnosis in the first year of life. hypomelanotic macules may be present in infancy, but facial angiofibromas and shagreen patches usually do not occur until puberty and renal angiomyolipomas occur in adults. crs may be the earliest finding of tsc in utero and may precede the detection of the brain or kidney lesions and can be symptomatic in the fetus and newborn. | we report a case of a neonate who presented to us with multiple rhabdomyomas of heart, cortical tubers in the brain and skeletal anomalies such as pierre robin sequence, bilateral clubfoot and lower small bowel obstruction. though a diagnosis of neonatal tuberous sclerosis was made, the association of skeletal anomalies and intestinal obstruction was a rare and unusual finding. |
gibberellin (ga) is considered to control diverse growth and developmental processes, including seed germination, stem elongation, and flower development (1). despite its complexity, the ga biosynthetic pathway has been well characterized by using biochemical techniques as well as by studying mutants defective in biosynthesis (2). on the other hand, genetic and cell biological studies have revealed key components in the ga response pathway (3). however, additional ga signaling components and downstream cellular and biochemical events need to be investigated further to better understand the molecular nature of ga response. the genes for most of the enzymes involved in ga biosynthesis have been isolated and characterized (3). the dwarf1 (d1) mutant in rice is characterized by a ga - insensitive semi - dwarf phenotype, and cloning of the d1 locus has revealed that it encodes the putative -subunit of the heterotrimeric g protein (4). the della proteins function as negative regulators of ga signaling, and their degradation through the ubiquitin / proteasome pathway is considered as a key event in the regulation of ga - stimulated processes (5). the gid2 gene of ga - insensitive dwarf phenotype, gid2, encodes a putative f - box protein, and is expected to form a skpl - cullin - f - box complex and to function as e3 ubiquitin ligase (5). recently, gibberellin insensitive dwarf1 (gid1) has been characterized to show similarity to hormone sensitive lipase and it serves as a soluble receptor for ga (6). complete genome sequences of arabidopsis and rice have yielded a wealth of information about plants 7., 8.. these accomplishments promise to provide detailed insights into the understanding of plant physiology and the molecular mechanisms of different signal transduction pathways. however, knowing the exact sequence and location of all genes of a given organism is only the first step towards understanding how all parts of a biological system work together. although 25,426 genes have been identified in arabidopsis thaliana, less than 10% have been documented experimentally (9). significant progress has been made in annotating the genomes of a. thaliana and rice during the past few years and by now, most of the predicted genes are supported by full - length cdnas 10., 11.. to assign function to unknown genes, different functional genomic methodologies are currently being developed and used. dna microarray technology uses hundreds and thousands of dna probes arrayed on a solid surface to examine the abundance and/or binding ability of dna or rna target molecules. depending on the dna probes used, dna microarrays are categorized into cdna microarrays and dna oligonucleotide probe microarrays (12). because of a high - throughput manner analysis of thousands of genes, dna microarrays have proved to be a powerful tool for the analysis of global gene expression patterns. moreover, gene functions can be inferred by comparing and making association of expression patterns of different samples for a particular trait (13), which can be exploited for plant improvement (14). promoter microarrays with chromatin immunoprecipitation have been used to identify target genes and their regulatory domains on a genome scale 15., 16.. similarly, tiling microarrays using tilling probes of the entire genome have been used to discover new transcript types (17). therefore, it can be strongly argued that dna microarrays hold tremendous promise for dissecting the regulatory mechanisms and networks of genes and consequently their products that govern plant phenotype. effects of ga on plant growth and development are mediated through gene expression modulation as rna and protein synthesis inhibitors interfere with these processes. to further understand the molecular mechanism by which ga regulates the growth and development of plants, it is necessary to identify and analyze more genes that are controlled by ga. microarrays provide high - throughput, simultaneous analysis of mrna for hundreds and thousands of genes (18) ; however, there are only few reports on the microarray analysis of ga - regulated gene expression in arabidopsis and rice 19., 20., 21., 22.. a throughput analysis of transcript profiles in ga - regulated gene expression using different plant tissues and organs remains pertinent, and a further characterization of the individual genes will help in understanding how ga regulates the growth and development of plants. in this review, we discuss the progress of identifying new members of genes involved in ga - regulated rice leaf sheath growth using microarray system. although fine progress has been made in the study of the biosynthesis and metabolism of ga (23) using biochemical techniques with the characterization of its biosynthetic mutants, not much is known about how it regulates a wide variety of physiological processes at the molecular level. progress has been made towards an understanding of the mechanism of ga action in the cereal aleurone, where ga induces the synthesis and secretion of a number of hydrolytic enzymes (24). although some other ga - regulated genes have been identified in shoot (25), leaf (26), flower (27), and stem (28) in various plants, how ga regulates the growth and development of these organs is still not clear. ga plays an important role in regulating many physiological processes in the growth and development of plants, including seed germination, shoot and stem elongation, and flower development (29). it is known that ga regulates shoot elongation by affecting cell division and elongation, though its precise mode of action in shoot growth is not yet clear. cell elongation is controlled by the turgor pressure and cell wall extensibility in a particular direction, which is in turn regulated by the orientation of both cellulose microfibrils and the cell wall matrix containing polysaccharides and proteins 30., 31.. similarly, the process of cell elongation in plants requires loosening of the cell wall structure and the deposition of new materials to maintain cell wall integrity. auxin, ga, and brassinosteroid promote stem elongation, whereas cytokinin, ethylene, and abscisic acid have a growth - inhibiting effect (32). although researchers have provided information on the signal mediators transmitting signals from plant hormones for cell elongation, the mechanism for regulating cell elongation is still poorly understood at the molecular level. while rapid progress has been made in the study of the biosynthesis and metabolism of ga (23), in contrast, much remains to be learned about the ga signal transduction pathways that lead to stem elongation and other ga - regulated processes. the d1 mutant in rice is characterized by a ga - insensitive semi - dwarf phenotype, and cloning of the d1 locus revealed that it encodes the putative -subunit of the heterotrimeric g protein (4). genetic analysis of ga - response mutants of rice and arabidopsis and cloning of the respective genes revealed that della proteins function as negative regulators of the ga signaling pathway 33. gid1 has been identified as a soluble ga - receptor in rice (6). efforts have been made to determine precisely where the bioactive ga is synthesized in plants, and which cells / tissues are the targets to initiate ga - mediated biological actions. combined gas chromatography mass spectrometry analysis and bioassays with dwarf plants have revealed that ga is mainly present in actively growing and elongating tissues, such as shoot apices, young leaves, and flowers 36., 38.. contradictorily, there is evidence for the presence of ga in xylem and phloem exudates 39. however, the expression analysis of some genes involved in ga biosynthesis and ga signaling has confirmed that ga is synthesized at the site of their action (41). rice leaf sheath is an important part where considerable critical metabolic and regulatory activities take place, which eventually control rice height and robustness. rice leaf sheath elongates rapidly with the treatment of ga (42). to understand the mechanism by which ga regulates rice leaf sheath growth, it is necessary to identify more genes involved in it. the use of cdna microarrays for monitoring gene expression provides an efficient high - throughput approach to assessing the possible functions of large numbers of genes. there are only few reports on the microarray analysis of ga - regulated gene expression in a. thaliana 19., 20. and rice 22., 43., this might be due to the differences in the experimental conditions and materials they used. these microarrays were either affymetrix genechips or were made of ests or oligonucleotides representing the gene expression profiles during normal growth conditions (44). in the study of yang. (21), a rice cdna library was prepared from ga3-treated rice seedlings with the aim to enrich it for novel ga - regulated genes. the original cdna microarray containing 4,000 clones was constructed from this enriched ga - regulated cdna library and was analyzed for expression differences in rice seedlings that had been treated with ga3. the results indicated that 2.2% of the 4,000 randomly selected clones were affected by treatment with exogenous ga3 (21). a total of 29 unique cdna clones were identified as being up - regulated, while 33 unique cdna clones were identified as down - regulated by ga3. a total of 62 unique genes were identified as ga3 responsive, of which 37 genes had potential functions in signal transduction, transcription, metabolism, cellular organization, and defense or anti - stress responses based on blast homology searches. these results indicate that ga3 is involved in regulating a wide range of growth and development processes. ten clones with high induction ratio were further analyzed by northern blot analysis and they were found to be up - regulated by ga3, which confirmed the microarray results. using an original cdna microarray, yang. (21) identified three new ga - regulated genes involved in rice seedlings, which displayed increased expression in response to ga3 treatment. using dna microarray, yamauchi. (20) identified a subset of ga up - regulated ga biosynthesis genes, such as atga3ox1 and atga20ox1, and analyzed ga deficient mutants and cold stress response in a. thaliana. genes involved in ga biosynthesis (45), which might be subject to feedback regulation, were not identified in the original cdna microarray analysis by yang. (21), which perhaps because these genes were not included in the original cdna microarray. in order to identify ga - regulated genes in rice, the use of microarrays containing more genes, with detailed analysis on ga deficient and insensitive mutants, and on the timing and tissue specificity of expression are required (21). despite the number of unique genes in the original cdna microarray in yang. (21) is less than 4,000, many genes that were not identified previously 22., 43. were identified using the original cdna microarray (21). among them, three genes, namely xyloglucan endotransglu - cosylase / hydrolase 8 (osxth8) (46), pyruvate dehydrogenase kinase 1 (ospdk1) (47), and a novel ga - enhanced gene 1 (osgae1) (48), showed clearly ga - differential expression when analyzed by northern blot analysis. the three genes were selected for further characterization in order to elucidate their functions in rice growth and development. four clones representing a single xth gene were induced by ga3, implying a role in regulating cell elongation and cell wall organization. xth catalyzes the endo cleavage of xyloglucan polymers and the subsequent transfer of the newly generated reducing ends to other polymeric or oligomeric xyloglucan molecules 49., 50.. the existence of a family of 29 xth genes in rice suggests that individual xth may exhibit distinct patterns of expression in terms of tissue specificity and responses to hormonal and environmental stimuli (51). the osxth8 gene identified in the original microarray was specifically up - regulated by ga3 and not by any other hormones (46). computer analysis using the place signal scan program (52) also revealed the presence of three potential ga response elements in the 2-kb sequence of osxth8. northern blot analysis showed that the level of osxth8 mrna in tanginbozu, a ga - deficient semi - dwarf mutant, was lower than that in its wild type. the expression of osxth8 in the mutant was induced to exceed wild - type level following treatment with ga3 for 24 h, while osxth8 expression was quite high in the slender rice 1, which is a ga - insensitive mutant growing 2 to 3 times more than the wild type (33). rnai osxth8 expressed under the control of camv 35s promoter produced plants with repressed growth caused by stunted growth of the second, third, and fourth internode (50). these observations demonstrate that osxth8 is a unique gene that can be used to modify rice plant growth (figure 1). ospdk1 was identified as a gene up - regulated by ga3 using the cdna microarray (47). pdk is a negative regulator of mitochondrial pyruvate dehydrogenase (mtpdh), and plays a pivotal role in controlling mitochondrial pyruvate dehydrogenase complex (mtpdc) activity, and hence, in the tricarboxylic acid (tca) cycle and cell respiration (53). jan. (47) provided the first report of transcriptional up - regulation of plant pdk by ga3, whereas transcriptional down - regulation of ospdk1 gene expression by abscisic acid (aba) using microarray has been observed by yazaki. (43). considering the antagonistic effects of ga and aba (54), it is reasonable that ga3 up - regulates ospdk1 identified in the original microarray (47). further characterization of ospdk1 showed that ga modulates the activity of mtpdc by regulating ospdk1 expression and subsequently controlling plant growth. rnai transgenics developed normally, but were almost 10% to 30% shorter in height compared to control. the possible explanation for the reduced vegetative growth is that the reduction in ospdk1 expression causes increased mtpdh activity that allows enhanced conversion of pyruvate to acetyl - coa and hence an increase in the respiration. tissue - specific repression of atpdk increased the oil content in seeds (55). in rice, there is no significant effect of rnai ospdk1 on reproductive growth traits like flowering time or the time to reach maturity. the effect of rnai ospdk1 on the seed content in rice has yet to be examined, but may lead to insights on how the plant balances metabolic demands between developing seed grains and other tissues when primary metabolism is challenged at the entry point of tca cycle. this study demonstrated that the ospdk1 gene can be exploited to challenged primary metabolism at the entry point of tca cycle, which will not only result in shaping the rice plant but also in the efficient use and conversion of different metabolite resources in different organs (figure 1). in the study by jan. (48), a novel gene of unknown function that was up - regulated by ga3 was identified and analyzed, which expressed highly in callus and at a moderate level in leaf sheath. the gene from this clone was found to be a novel ga - enhanced gene and hence was designated as osgae1 (48). analysis of the osgae1 amino acid sequence revealed some similarity to the atpdf1 and wm5 protein 56., however, the osgae1 gene was unique in the sense that it was hormonally regulated. in situ hybridization and promoter - gus analysis revealed that osgae1 was predominantly expressed in stem, shoot apex meristem, and young leaves. computer analysis using the place signal scan program (52) also revealed the presence of three potential ga response elements in the 1.5-kb promoter region of osgae1. osgae1 antisense transgenic plants were repressed in growth and the plants were almost 55% to 70% shorter than the control upon maturity. the typical phenotype of osgae1 antisense transgenics resembled that of ga - deficient mutants. the complete ga signaling cascade is not yet fully understood and it is believed that gid1 is a soluble ga receptor (6) whereas the semi - dwarf stature of tanginbozu phenotype is caused by a defective early step of ga biosynthesis, which is catalyzed by ent - kaurene oxidase (58). exogenous application of ga3 restores tanginbozu leaf sheath growth whereas there is no significant effect of ga3 on gid1. the repressed leaf sheath growth of rice plants expressing antisense osgae1 was not completely reversed by application of ga3. these observations indicate that osgae1 is not involved in regulating a basic reaction shared by ga biosynthesis or signaling cascade rather than it is a downstream gene playing a vital function in the ga - mediated rice leaf sheath elongation (figure 1). four clones representing a single xth gene were induced by ga3, implying a role in regulating cell elongation and cell wall organization. xth catalyzes the endo cleavage of xyloglucan polymers and the subsequent transfer of the newly generated reducing ends to other polymeric or oligomeric xyloglucan molecules 49., 50.. the existence of a family of 29 xth genes in rice suggests that individual xth may exhibit distinct patterns of expression in terms of tissue specificity and responses to hormonal and environmental stimuli (51). the osxth8 gene identified in the original microarray was specifically up - regulated by ga3 and not by any other hormones (46). computer analysis using the place signal scan program (52) also revealed the presence of three potential ga response elements in the 2-kb sequence of osxth8. northern blot analysis showed that the level of osxth8 mrna in tanginbozu, a ga - deficient semi - dwarf mutant, was lower than that in its wild type. the expression of osxth8 in the mutant was induced to exceed wild - type level following treatment with ga3 for 24 h, while osxth8 expression was quite high in the slender rice 1, which is a ga - insensitive mutant growing 2 to 3 times more than the wild type (33). rnai osxth8 expressed under the control of camv 35s promoter produced plants with repressed growth caused by stunted growth of the second, third, and fourth internode (50). these observations demonstrate that osxth8 is a unique gene that can be used to modify rice plant growth (figure 1). ospdk1 was identified as a gene up - regulated by ga3 using the cdna microarray (47). pdk is a negative regulator of mitochondrial pyruvate dehydrogenase (mtpdh), and plays a pivotal role in controlling mitochondrial pyruvate dehydrogenase complex (mtpdc) activity, and hence, in the tricarboxylic acid (tca) cycle and cell respiration (53). jan. (47) provided the first report of transcriptional up - regulation of plant pdk by ga3, whereas transcriptional down - regulation of ospdk1 gene expression by abscisic acid (aba) using microarray has been observed by yazaki., it is reasonable that ga3 up - regulates ospdk1 identified in the original microarray (47). further characterization of ospdk1 showed that ga modulates the activity of mtpdc by regulating ospdk1 expression and subsequently controlling plant growth. rnai transgenics developed normally, but were almost 10% to 30% shorter in height compared to control. the possible explanation for the reduced vegetative growth is that the reduction in ospdk1 expression causes increased mtpdh activity that allows enhanced conversion of pyruvate to acetyl - coa and hence an increase in the respiration. tissue - specific repression of atpdk increased the oil content in seeds (55). in rice, there is no significant effect of rnai ospdk1 on reproductive growth traits like flowering time or the time to reach maturity. the effect of rnai ospdk1 on the seed content in rice has yet to be examined, but may lead to insights on how the plant balances metabolic demands between developing seed grains and other tissues when primary metabolism is challenged at the entry point of tca cycle. this study demonstrated that the ospdk1 gene can be exploited to challenged primary metabolism at the entry point of tca cycle, which will not only result in shaping the rice plant but also in the efficient use and conversion of different metabolite resources in different organs (figure 1). (48), a novel gene of unknown function that was up - regulated by ga3 was identified and analyzed, which expressed highly in callus and at a moderate level in leaf sheath. the gene from this clone was found to be a novel ga - enhanced gene and hence was designated as osgae1 (48). analysis of the osgae1 amino acid sequence revealed some similarity to the atpdf1 and wm5 protein 56., however, the osgae1 gene was unique in the sense that it was hormonally regulated. in situ hybridization and promoter - gus analysis revealed that osgae1 was predominantly expressed in stem, shoot apex meristem, and young leaves. computer analysis using the place signal scan program (52) also revealed the presence of three potential ga response elements in the 1.5-kb promoter region of osgae1. osgae1 antisense transgenic plants were repressed in growth and the plants were almost 55% to 70% shorter than the control upon maturity. the typical phenotype of osgae1 antisense transgenics resembled that of ga - deficient mutants. the complete ga signaling cascade is not yet fully understood and it is believed that gid1 is a soluble ga receptor (6) whereas the semi - dwarf stature of tanginbozu phenotype is caused by a defective early step of ga biosynthesis, which is catalyzed by ent - kaurene oxidase (58). exogenous application of ga3 restores tanginbozu leaf sheath growth whereas there is no significant effect of ga3 on gid1. the repressed leaf sheath growth of rice plants expressing antisense osgae1 was not completely reversed by application of ga3. these observations indicate that osgae1 is not involved in regulating a basic reaction shared by ga biosynthesis or signaling cascade rather than it is a downstream gene playing a vital function in the ga - mediated rice leaf sheath elongation (figure 1). current researches have indicated that suitable rice morphogenesis can be achieved by cleverly tailoring ga - regulated genes. sakamoto. (59) modified the level of ga by overproduction of a ga catabolic enzyme, ga 2-oxidase. when the gene encoding ga 2-oxidase, osga2ox1, was constitutively expressed by the actin promoter, transgenic rice showed severe dwarfism and the plants failed in seed setting because ga is involved in both shoot elongation and reproductive development. in contrast, osga2ox1 ectopic expression at the site of bioactive ga synthesis in shoots under the control of the promoter of a ga biosynthesis gene, osga3ox2 (d18), resulted in a semi - dwarf phenotype that was normal in flowering and grain development (59). in molecular studies of rice and wheat varieties, the phytohormone ga has been identified as a key player in controlling crop plant architecture (60). however, along controlling plant architecture, grain numbers and grain quality are also parameters of prime importance. recently it has been demonstrated that cytokinin metabolism also contributes to crop productivity. as cytokinin controls cell division and lateral meristem activity, its accumulation in the inflorescence meristem can cause higher grain numbers (61). 48. showed that ga regulates plant growth and development by regulating important genes of different functions. identification of agronomically important genes and pyramiding of such genes presents a useful strategy for efficient crop development. wise tailoring of such genes of different check points will greatly facilitate artificially controlling the morphogenesis of rice plant, which will result in the development of next generation of rice plant with ideal grass type having high yield and improved grain quality. | gibberellin (ga) is collectively referred to a group of diterpenoid acids, some of which act as plant hormones and are essential for normal plant growth and development. dna microarray technology has become the standard tool for the parallel quantification of large numbers of messenger rna transcripts. the power of this approach has been demonstrated in dissecting plant physiology and development, and in unraveling the underlying cellular signaling pathways. to understand the molecular mechanism by which ga regulates the growth and development of plants, with reference to the monocot model plant rice, it is essential to identify and analyze more genes and their products at the transcription and translation levels that are regulated by ga. with the availability of draft sequences of two major rice types, indica and japonica rice, it has become possible to analyze global expression profiles of genes on a genome scale. in this review, the progress made in finding new genes in rice leaf sheath using microarray system and their characterization is discussed. it is believed that the findings made in this regard have important implications for understanding the mechanism by which ga regulates the growth and development of rice. |
major depression is a highly prevalent condition as well as a debilitating psychiatric illness that has a negative impact on individual and community health.1 antidepressant drugs are the mainstay of the treatment of depression ; however, their efficacy is still lower than desired, and many patients present subsyndromal residual symptoms after the initial clinical response. up to 15% of cases will remain significantly depressed even after multiple pharmacological approaches,2 with a negative impact on clinical course.3 since a partial response to treatment represents a widespread condition among depressed patients and should be readily identified to decide the most appropriate treatment plan, many helpful systems for staging levels of treatment resistance have been proposed for major depression.4 in this way, rush described difficult - to - treat depression as a clinical condition which includes both those patients who do not respond adequately to one or more established treatments and those in whom a successful treatment is prevented by concomitant conditions, precluding the optimal delivery of potentially effective treatments. such circumstances mainly involve limited adherence to treatment, adverse side effects preventing an adequate dose or duration of treatment, and comorbid axis i, ii, or iii conditions.5 hence, difficult - to - treat depressed (dtd) patients form a clinical population of vulnerable subjects, representing a common medical challenge for clinicians who often have to develop individualized therapeutic decisions based on limited scientific evidence. many augmentation strategies have been developed to increase the efficacy of antidepressant treatments, and alternative strategies based on multidisciplinary competences are probably essential. among safe and less - demanding treatment strategies, a growing number of evidence supports the usefulness of chronotherapeutic interventions, such as bright light therapy (blt), in the management of patients suffering from mood disorders. when combined with antidepressants, blt hastens recovery with improvements since the first week of treatment.6,7 clinical benefits are well known for unipolar depressed patients, but despite the amount of data provided by benedetti8 on combined chronotherapeutic intervention, only few trials have looked at blt alone as an augmentation of pharmacotherapy for bipolar depression.9,10 because of brief latency of action, blt alone11,12 has been little studied in the vulnerable clinical population of dtd patients. hence, the main aim of our study was to explore the effects of blt augmentation on clinical outcome of these patients, and compare unipolar and bipolar subjects. we tested blt combined with ongoing medication as a first - line choice for both more vulnerable patients (with concomitant medical and/or psychiatric comorbidity that preclude the optimal delivery of the ongoing treatment) and patients with stage i treatment - resistant depression (trd) (those who failed to achieve a remission after the first pharmacological trial at an adequate dose). finally, we observed the effectiveness of such an approach both in the acute phase and after blt discontinuation over 8 weeks. thirty - one depressed outpatients (16 unipolar and 15 bipolar without seasonal pattern) were enrolled in the study. a total of 110 outpatients referred for the treatment of depressive episode at the institute of psychiatry of the agostino gemelli the population consisted of 18 women and 15 men with a mean age of 47.6 years. subjects were included if they 1) met diagnostic criteria for a major depressive episode according to diagnostic and statistical manual of mental disorders, fourth edition, text revision (dsm - iv - tr), 2) had a baseline 21-item hamilton depression rating scale (hdrs-21) score of 15 or higher,13 and 3) had a condition of difficult - to - treat depression that, as determined by rush,5 included both patients partially responding to at least a 5-week open - label trial of an adequate dose of antidepressant monotherapy and those in whom a successful treatment is prevented by concomitant medical or psychiatric conditions. subjects who did not respond to two or more different classes of adequate antidepressant trials according to the classical definition of stage ii trd were excluded. other exclusion criteria were 1) a diagnosis of non - affective psychotic disorder, 2) a diagnosis of substance use disorders within the last year, 3) a diagnosis of dementia or other cognitive disorders, 4) electroconvulsive therapy (ect) within 1 year prior to enrollment, 5) recent history of suicide attempts, and 6) ocular disease (glaucoma, cataracts, retinal detachment, retinopathy). subjects having general medical conditions or personality disorders affecting treatment adherence or tolerability were not excluded. the local ethics committee approved the study, and a written informed consent was obtained from all subjects. the psychopathological status was assessed by the same experienced rater blind to the week of study and the study design. at baseline, severity of mood disorder was evaluated with hdrs-21 and mania rating scale (mrs);14 in order to identify a specific antidepressant effect on depression core items, a secondary outcome measure, the six - item hamilton depression rating scale (hdrs-6), was used.15 the snaith hamilton pleasure scale (shaps)16 was used to assess anhedonia, whereas the depression retardation rating scale (drrs)17 was used for psychomotor retardation. in order to evaluate treatment outcomes, hdrs-21, hdrs-6, and mrs were repeated at week 1, week 3, week 4, and week 8 after onset of blt. responders were defined by a decrease of at least 50% in the hdrs-21 total score from baseline. autumn / winter and spring / summer were taken together as the two seasons with a shorter and longer photoperiod, respectively. blt was added to the ongoing medication treatment after recruitment, and nothing was altered in psychopharmacotherapy during the experiment. at baseline, all subjects were required to be on a stable dose of their psychotropic medication for at least 4 weeks prior to enrollment. unipolar patients were taking antidepressant drugs (ademetionine, escitalopram, duloxetine, agomelatine, clomipramine, fluoxetine, citalopram, sertraline, amisulpride, nortriptyline, amitriptyline, and trazodone). bipolar patients were required to take at least one mood stabilizer, with the exception of a patient who was pregnant (lithium carbonate, lamotrigine, and valproic acid). patients on lithium or valproic acid had plasma levels higher than 0.4 meq / l and 50 mg / ml, respectively. six bipolar patients were taking antidepressants (duloxetine, agomelatine, nortriptyline, sertraline, venlafaxine, clomipramine). once enrolled, no change in the dosage of any psychotropic medication was allowed, and any photosensitizing medication was prohibited. ten patients took benzodiazepines (up to 2 mg of lorazepam - equivalents per day) or sleeping drugs (zolpidem 510 mg or zaleplon 510 mg) for insomnia. patients were instructed to undergo daily treatment in their own homes between 5.45 am and 8.15 am, for 3 weeks, and the regimen was carefully explained to each subject. the exact time schedule for light therapy sessions was defined following a predictive algorithm based on morningness eveningness questionnaire scores.1822 according to most studies, we chose an exposure of 30 minutes at approximately 10,000 lx for 3 weeks. after the first week of treatment, partial or no responders were instructed to increase the exposure to 45 minutes / day for the next 2 weeks.23,24 light therapy was administered by means of the daylight simulator day - light classic model (uplift technologies inc, nova scotia, canada). this device measures 31.8 cm 40.6 cm 7.6 cm, with a lens material of high - impact polycarbonate, 99.3% uv filter, and three 36 w fluorescent light tubes of 4,000 k color temperature, with height - adjustable legs and two light intensity settings of 10,000 lx and 7,000 lx at 12 in (30.5 cm). subjects were instructed to place the box on a desk or tabletop at an angle of 15, adjust the height so that the center of the box would be at eye level, and use the 10,000 lx. descriptive analyses were carried out using student s t - test, test, and the one - way or repeated - measures analysis of variance (anova). assessment scale scores before and after treatment were compared using paired samples t - test or wilcoxon signed - ranks test. the trends in the mean scores in individual assessment scales and the influence of factor affecting course of symptoms were determined using repeated measures anova with multiple comparison correction (bonferroni adjustment). comparisons of response rates between unipolar and bipolar patients and between seasons of treatment have been estimated using a 22 crosstab and test. all tests were two - tailed, and significance was set with an alpha value of 0.05. data were analyzed using spss 15.0 (spss 15.0, chicago, il, usa). blt was added to the ongoing medication treatment after recruitment, and nothing was altered in psychopharmacotherapy during the experiment. at baseline, all subjects were required to be on a stable dose of their psychotropic medication for at least 4 weeks prior to enrollment. unipolar patients were taking antidepressant drugs (ademetionine, escitalopram, duloxetine, agomelatine, clomipramine, fluoxetine, citalopram, sertraline, amisulpride, nortriptyline, amitriptyline, and trazodone). bipolar patients were required to take at least one mood stabilizer, with the exception of a patient who was pregnant (lithium carbonate, lamotrigine, and valproic acid). patients on lithium or valproic acid had plasma levels higher than 0.4 meq / l and 50 mg / ml, respectively. six bipolar patients were taking antidepressants (duloxetine, agomelatine, nortriptyline, sertraline, venlafaxine, clomipramine). once enrolled, no change in the dosage of any psychotropic medication was allowed, and any photosensitizing medication was prohibited. ten patients took benzodiazepines (up to 2 mg of lorazepam - equivalents per day) or sleeping drugs (zolpidem 510 mg or zaleplon 510 mg) for insomnia. patients were instructed to undergo daily treatment in their own homes between 5.45 am and 8.15 am, for 3 weeks, and the regimen was carefully explained to each subject. the exact time schedule for light therapy sessions was defined following a predictive algorithm based on morningness eveningness questionnaire scores.1822 according to most studies, we chose an exposure of 30 minutes at approximately 10,000 lx for 3 weeks. after the first week of treatment, partial or no responders were instructed to increase the exposure to 45 minutes / day for the next 2 weeks.23,24 light therapy was administered by means of the daylight simulator day - light classic model (uplift technologies inc, nova scotia, canada). this device measures 31.8 cm 40.6 cm 7.6 cm, with a lens material of high - impact polycarbonate, 99.3% uv filter, and three 36 w fluorescent light tubes of 4,000 k color temperature, with height - adjustable legs and two light intensity settings of 10,000 lx and 7,000 lx at 12 in (30.5 cm). subjects were instructed to place the box on a desk or tabletop at an angle of 15, adjust the height so that the center of the box would be at eye level, and use the 10,000 lx. descriptive analyses were carried out using student s t - test, test, and the one - way or repeated - measures analysis of variance (anova). assessment scale scores before and after treatment were compared using paired samples t - test or wilcoxon signed - ranks test. the trends in the mean scores in individual assessment scales and the influence of factor affecting course of symptoms were determined using repeated measures anova with multiple comparison correction (bonferroni adjustment). comparisons of response rates between unipolar and bipolar patients and between seasons of treatment have been estimated using a 22 crosstab and test. all tests were two - tailed, and significance was set with an alpha value of 0.05. data were analyzed using spss 15.0 (spss 15.0, chicago, il, usa). two unipolar and four bipolar subjects dropped out (19% of total sample) : one (unipolar) patient had an incoming adverse event not related to treatment (patient decided to stop blt after starting an antibiotic therapy for a toothache) and five (one unipolar and four bipolar) patients did not comply with the treatment procedures (not properly respecting wake - up time or instructions on light exposure). twenty - five patients (14 unipolar and eleven bipolar) completed the 3 weeks of treatment (figure 1)., patients showed a moderate burden of depressive symptoms (mean hdrs-21 standard deviation = 19.54.4), relevant psychomotor retardation (score > 10 on the drrs), and/or anhedonic features. no significant differences were observed at basal evaluation between unipolar and bipolar patients (table 2). depressive symptoms improved significantly during adjunctive blt in the overall study sample. a repeated- measures anova determined that mean hdrs-21 total scores significantly differed between time points (f[4, 80 ] = 16.306 ; p<0.001) (figure 2). a bonferroni correction showed a statistically significant reduction in hdrs-21 scores since the first week of therapy (p=0.001). a statistically significant effect of time was observed since the first week on the hdrs-6 scores, as well (f[4, 80 ] = 14.875 ; p<0.001 ; bonferroni correction, p=0.009) (figure 2). twenty - three patients received extra daily exposure of light (shifting from 30 minutes to 45 minutes) after first week of treatment, but this did not have an independent effect over the course of depressive symptoms (f[2.719, 62.538 ] = 0.964 ; p=0.409). during the overall treatment period, no significant changes were reported in mrs scores, and no patient presented clinical signs of a (hypo)manic switch. a repeated - measures anova determined that mean mrs total scores did not significantly change between time points (f[2.804, 56.087 ] = 2.025 ; p=0.125). although mrs scores increased in bipolar group, no statistically significant difference was found between time points (wilcoxon signed - ranks test : z=0.4921 ; p=0.623). after 1 week from blt discontinuation (at week 4 of the study), seven patients (28%) showed a treatment response. after another month (at week 8 of the study), they were still responders, and two more patients achieved this treatment outcome (in total nine patients ; 36%). as reported by hdrs-21 scores, we observed a substantial clinical improvement, in both unipolar and bipolar groups (figure 3). although bipolar patients showed a more severe psychopathological burden throughout the treatment period, bipolarity did not seem to have an independent effect over the course of depressive symptoms (f[4, 76 ] = 14.850 ; p=0.826). however, when considering differences between mean hdrs-21 scores, unipolar patients showed a significantly better outcome as from week 3 until the end of the study (figure 3). at the end of the study, unipolar patients also showed a significantly better response rate than bipolar patients (8 and 1, respectively ; =6.173 ; p=0.013). in order to determine the role of other demographic, clinical, and treatment variables in the course of depressive symptoms concerning season of treatment, at the end of follow - up, patients in the spring / summer season had a significantly higher remission rate (hdrs score < 8 ; =4.427 ; p=0.035). we checked for confounding between type of depression and season of treatment, but no significant differences in uni-/bipolarity were found in the distribution of patients over spring summer and autumn winter (=1.924 ; p=0.165) (figure 4). no significant changes in anhedonic features were observed at the end of the study in the overall sample (wilcoxon signed - ranks test : z=0.514 ; p=0.607). conversely, comparison of drrs scores at baseline and at the end of the study (14.59.3 and 8.16.8, respectively) showed a statistically significant improvement of psychomotor retardation (wilcoxon signed - ranks test : z=3.327 ; p=0.001). no statistically significant differences in shaps or drrs scores were found in comparing unipolar and bipolar patients (table 2). previous trials on the efficacy of chronotherapeutic interventions in treatment - resistant depressed patients mainly focused on the combined chronotherapeutic intervention25,26 or were mostly conducted during hospitalization.27 to the best of our knowledge, this is the first study addressing the blt augmentation of psychopharmacotherapy, not combined to other chronotherapeutics, in the management of dtd outpatients, including both bipolar and unipolar subjects. in our experience, this therapeutic approach appeared not only helpful but also simple and safe for such a vulnerable, still not well - studied, clinical population. our study also seems to confirm an improvement of depressive core symptoms during blt, as measured by hdrs-6 subscale, supporting the hypothesis of its genuine antidepressant effect.7 significant changes in depressive symptoms were observed since the first week of therapy in keeping with previous findings on seasonal and nonseasonal depression.6,7,11 this improvement endured throughout the continuation phase, and all patients who responded to blt augmentation still maintained the favorable outcome after 5 weeks from blt discontinuation ; therefore, we supposed that blt augmentation in our patients could boost the antidepressant effect of ongoing treatment. according to previous observations in trd, more than one - third of our sample (36%) still showed a clinical response at the end of the study.25,26 although both unipolar and bipolar patients showed a substantial clinical improvement, the outcome and the maintenance of therapeutic benefits were significantly better in the unipolar group. up to date, only few trials on blt, not in combination with other chronotherapeutics, have specifically looked at patients with bipolar depression,9,10 and their findings suggested that blt might also be helpful in nonseasonal bipolar depression.28 these results did not clearly emerge in our study, since unipolar patients showed a greater improvement (as from week 3) and a significantly better response rate after week 8 (eight unipolar vs one bipolar). more in general, blt appeared to be useful and safe for both unipolar and bipolar subjects, since none of our patients experienced side effects or (hypo)manic switches or significant changes in mrs scores. considering the effect of season of treatment on the course of illness, patients treated during spring summer period appeared to have a significantly better remission rate (hdrs-21 score < 8) at the end of follow - up (figure 4). previous studies focusing on the role of the season in blt augmentation reported that during autumn and winter, blt timing early in the morning was associated with a better response in unipolar depressed patients, while during spring and summer, the clinical response was unrelated to blt timing during the day.18 according to our results, we hypothesized that the differences reported might have been influenced by seasonal changes of the photoperiod, with an increased daylight exposure during spring and summer extending the therapeutic benefit of blt. although our findings are in line with the previous observations in nonseasonal major depressed patients,2931 they must be interpreted with caution because of the limitations of our study. first, the open - label design and the lack of a placebo or active control group might bias our results. therefore, we can not exclude that high response rates observed in our sample were partially driven by the placebo response, even if more severe depressed patients are less likely to show a placebo effect.3235 we tried to reduce observer bias and eventual halo effects by keeping the patient rater blind to diagnosis and treatment.36 another major limitation is the relatively small number of subjects recruited. finally, our results might also have been influenced in an unpredictable way by the psychometric evaluation that has not yet been specifically validated for trd.37 according to different psychopathological dimension analyses, anhedonic symptoms persisted after blt augmentation, regardless of clinical remission. this observation is in keeping with previous findings on anhedonic symptoms that are particularly difficult to treat38 and might predict a poor treatment outcome39 due to some possible dysfunctions in the neurobiological mechanisms of hedonic response and reward processing that may persist, irrespective of mood state40 and clinical changes.41 on the contrary, in our sample, blt augmentation led to a significant improvement of psychomotor retardation as reported by drrs score changes. this is in agreement with martiny who found that the retardation score (item 8 of hdrs) was the most sensitive item in discriminating the effect of blt compared to the placebo treatment group.7 accordingly, recent literature on parkinson s disease also shows a positive effect of blt not only on mood but also on motor function.4244 basal ganglia through a dysfunctional dopaminergic neurotransmission probably play a crucial role in pathophysiology of psychomotor changes of mood disorders.4548 striatal dopamine metabolism seems to be regulated by clock proteins, and stimulation of dopamine receptors affects the rhythm of expression of clock genes in the striatum.43,4951 dopamine also regulates the rhythmic expression of melanopsin in retinal ganglion cells, thereby influencing the entrainment of the circadian rhythm by light.52 although the mechanism of action is still unknown, our evidence might suggest a specific dopamine - increasing activity of blt. in conclusion, in our experience, blt augmentation appeared to be a simple, safe, well - tolerated, and low - cost strategy in outpatient settings, for both unipolar and bipolar patients. blt augmentation was particularly helpful for unipolar dtd patients adding the assumption that during autumn and winter, it might be necessary to increase the intensity of blt (ie, longer exposure or more lux) in order to achieve satisfying results. this augmentation strategy might also have a clinically significant influence on depressive core symptoms and on psychomotor retardation, a key feature of depression suggesting a specific dopamine - increasing activity of blt. these preliminary findings deserve further investigation in this complex clinical population who requires tailored combined treatment approaches. when considering the major limitations of our study, these results need to be confirmed in placebo - controlled, randomized, double - blind clinical trial on a larger sample. | objectiveswe investigated the clinical benefits of bright light therapy (blt) as an adjunct treatment to ongoing psychopharmacotherapy, both in unipolar and bipolar difficult - to - treat depressed (dtd) outpatients.methodsin an open - label study, 31 depressed outpatients (16 unipolar and 15 bipolar) were included to undergo 3 weeks of blt. twenty - five completed the treatment and 5-week follow-up.main outcome measuresclinical outcomes were evaluated by the hamilton depression rating scale (hdrs). the snaith hamilton pleasure scale and the depression retardation rating scale were used to assess changes in anhedonia and psychomotor retardation, respectively.resultsthe adjunctive blt seemed to influence the course of the depressive episode, and a statistically significant reduction in hdrs scores was reported since the first week of therapy. the treatment was well - tolerated, and no patients presented clinical signs of (hypo)manic switch during the overall treatment period. at the end of the study (after 5 weeks from blt discontinuation), nine patients (36%, eight unipolar and one bipolar) still showed a treatment response. blt augmentation also led to a significant improvement of psychomotor retardation.conclusionblt combined with the ongoing pharmacological treatment offers a simple approach, and it might be effective in rapidly ameliorating depressive core symptoms of vulnerable dtd outpatients. these preliminary results need to be confirmed in placebo - controlled, randomized, double - blind clinical trial on larger samples. |
they are of high ecological importance due to their high abundance in oceans, accounting for up to 25% of the worldwide primary production (for review see falciatore and bowler, 2002). despite a different thylakoid membrane organization in bands of three thylakoids each, the light reactions of photosynthesis in diatoms resemble those of higher plants. diatoms possess membrane - intrinsic light - harvesting complexes (lhc), called fucoxanthin - chlorophyll proteins (fcps), which belong to the same protein family as the light - harvesting proteins of higher plants (green and pichersky, 1994). however, the pigmentation is different with chlorophyll (chl) c replacing chl b and fucoxanthin (fx) being the main carotenoid. the carotenoid to chl ratio of 4:5 is much higher in fcp complexes (fcps) than the 4:14 ratio in plant lhcs, which also highlights the light - harvesting function of carotenoids in these antenna polypeptides (papagiannakis., 2005 ;, 2010a, b). by means of stark spectroscopy on isolated fcps (premvardhan., 2008), and electrochromic shift experiments using whole cells (szab., 2010), it was proven that the binding of up to eight fx molecules to fcp results in different bathochromic shifts. thus, fx molecules absorbing at slightly longer or shorter wavelengths, i.e. red, the overall arrangement of the fcp polypeptides in supercomplexes seems to differ, too. whereas trimeric complexes have been proven for several diatoms, higher oligomers have also been described. in cyclotella meneghiniana, two complexes make up the main antenna pool (bchel, 2003), the trimeric fcpa and the oligomeric fcpb, which differ in polypeptide composition. fcp1/2/3, belonging to the group of major fcp polypeptides, also called lhcf, and fcp6/7/8, belonging to the group of lhcx proteins, were identified in fcpa. fcpb consists of one polypeptide, most probably fcp5, a member of the lhcf group (beer., 2006). these fcps not only function in light harvesting by each having all spectral forms of fx (red, blue) bound, but they also act in protection, due to their ability to bind pigments of the so - called xanthophyll cycle. the main cycle in diatoms consists of diadinoxanthin (dd) and diatoxanthin (dt), whereby dd is converted to dt under high light conditions. the appearance of high amounts of dt under high light correlates with a high non - photochemical quenching (lavaud., 2002), a mechanism to dissipate excess energy via heat. recently it could be shown that dt is reducing the fluorescence yield of one of the isolated fcps, fcpa, thus identifying this complex as one possible quenching site (gundermann and bchel, 2008). in contrast to the main fcps, much less is known about fcp proteins that are specifically associated with one of the photosystems. for photosystem i (psi), genetic analyses point to lhcr genes, which were annotated due to their similarity with red algal genes coding for psi antenna proteins (oudot - le secq., 2007). psi has been demonstrated to be a monomer, like in higher plants (veith and bchel, 2007), allowing for fcp proteins to be associated with it in a similar manner like lhci. for psi of c. meneghiniana, fcp4, an lhcr protein, could lately be identified as a specific antenna protein for psi, whereas fcp6/7/8 as well as fcp1/2/3 were completely missing in the psi - fcp (veith., 2009). later lhcr gene products have been reported as constituents of psi in the pennate diatom phaeodactylum tricornutum (lepetit., 2010) and in the centric diatom thallassiosira pseudonana (grouneva., 2011), a close relative to c. meneghiniana. xanthophyll cycle pigments are located close to the photosystems as well, e.g. in a mixed photosystem fraction in p. tricornutum, where they were shown to be interconvertible depending on the light intensity (lepetit., 2007), but also in pure psi - fcps of c. meneghiniana (veith., 2009 ; lepetit. in order to get more insight into the antenna composition of psi and the characteristics of the fcps bound, this study compared psi complexes isolated from c. meneghiniana cells cultivated under different conditions. in a former publication (veith., 2009), the basic composition of psi from cells grown under high light conditions was studied. since already a slight reduction of the iron supply in the growth medium resulted in differences in chloroplast size and influenced the polypeptide composition of the fcpa complex (beer., 2011), this study used cells cultured with a reduced iron supply under high light (hl) or low light (ll) conditions. in addition, psi complexes from a ll culture fully supplemented with iron were tested. the diatom cyclotella meneghiniana (culture collection gttingen, strain 1020 - 1a) was grown under ll (40 mol photons m s) or hl (140 mol photons m s) regimes in culture medium according to provasoli. (1957), but modified so that it contained only 1 m iron (fe). usually this medium contains 12 m iron (+) and also (+) cultures were grown under ll. cells were kept in the different conditions by inoculating every fortnight for at least a year before experiments were started. cells were harvested in the early light phase by centrifugation after 1014 days of culture. thylakoid membranes were isolated by several centrifugation steps after breaking the cells in a bead mill according to bchel (2003). solubilization of thylakoids was done at 0.25 mg / ml total chl with 15 mm -dodecyl maltoside (1:54, mol / mol) on ice. the purity of the complexes was checked by sds - polyacrylamide gel electrophoresis (page) as described in schgger and von jagow (1987), using separating gels with 15% acryl amide (w / v). preparation of the samples, silver staining of the gels and immunodetection were carried out according to beer., 2006. the antibody used, -cmfcp, was created to detect all fcps of c. meneghiniana. to this end, the antenna complexes fcpa and fcpb were purified from ll(+) cells according to beer. (2006), pooled and used for commercial antibody production (biotrend, germany) in rabbits. a chemically synthesized peptide, gdfrngyidfgwdsfd, with a sequence identical in the lhcf1 - 11 sequences of p. tricornutum and also found in fcp5 of c. meneghiniana, was used for the commercial production (eurogentec, belgium) of -lhcf1 - 11. according to sequence comparisons this antibody should also detect fcp1/2/3 of c. meneghiniana and lhcf1 - 5, 8 and 9 of t. pseudonana, but none of the lhcr or lhcx proteins. the intensities of the fcp bands of the different psi complexes after immunodecoration with -cmfcp and using enhanced chemoluminescence for detection were analysed using the program imagej. in order to compare the different culture conditions, equal amounts of chl a (5 g) were loaded and samples were probed on the same blotting membrane. pigment stoichiometries of isolated psi complexes were determined by analytical hplc (elite lachrom, l-2130/l-2450, merck). samples were centrifuged shortly at maximal speed in an eppendorf centrifuge, and the supernatants were loaded onto the column. pigments were separated and quantified using a rp18 column and a photo - diode array detector as described in papagiannakis., 2005. absorbance of isolated psi complexes was recorded in the qy maximum of chl a absorption and complexes were then adjusted to an absorbance of 0.03 (corresponding to roughly 0.3 10 g / ml chl a) with a buffer containing 60% glycerol as described in veith. samples prepared accordingly were used to record fluorescence spectra at 77 k with a jasco fluorometer (fp-6500). emission spectra were taken upon excitation at ex = 440 nm for preferential excitation of chl a and measured from em = 600 to 800 nm in 0.1 nm steps. excitation spectra of the same samples were recorded from ex = 400 to 600 nm for em = 674, 686, and 717 nm. band passes of 3 nm were used both on the emission and the excitation side. a rhodamine b spectrum served as a reference for the correction of the excitation side and the photomultiplier was corrected using a calibrated lamp spectrum. the diatom cyclotella meneghiniana (culture collection gttingen, strain 1020 - 1a) was grown under ll (40 mol photons m s) or hl (140 mol photons m s) regimes in culture medium according to provasoli. (1957), but modified so that it contained only 1 m iron (fe). usually this medium contains 12 m iron (+) and also (+) cultures were grown under ll. cells were kept in the different conditions by inoculating every fortnight for at least a year before experiments were started. cells were harvested in the early light phase by centrifugation after 1014 days of culture. thylakoid membranes were isolated by several centrifugation steps after breaking the cells in a bead mill according to bchel (2003). solubilization of thylakoids was done at 0.25 mg / ml total chl with 15 mm -dodecyl maltoside (1:54, mol / mol) on ice. the purity of the complexes was checked by sds - polyacrylamide gel electrophoresis (page) as described in schgger and von jagow (1987), using separating gels with 15% acryl amide (w / v). preparation of the samples, silver staining of the gels and immunodetection were carried out according to beer., 2006. the antibody used, -cmfcp, was created to detect all fcps of c. meneghiniana. to this end, the antenna complexes fcpa and fcpb were purified from ll(+) cells according to beer. (2006), pooled and used for commercial antibody production (biotrend, germany) in rabbits. a chemically synthesized peptide, gdfrngyidfgwdsfd, with a sequence identical in the lhcf1 - 11 sequences of p. tricornutum and also found in fcp5 of c. meneghiniana, was used for the commercial production (eurogentec, belgium) of -lhcf1 - 11. according to sequence comparisons this antibody should also detect fcp1/2/3 of c. meneghiniana and lhcf1 - 5, 8 and 9 of t. pseudonana, but none of the lhcr or lhcx proteins. the intensities of the fcp bands of the different psi complexes after immunodecoration with -cmfcp and using enhanced chemoluminescence for detection were analysed using the program imagej. in order to compare the different culture conditions, equal amounts of chl a (5 g) pigment stoichiometries of isolated psi complexes were determined by analytical hplc (elite lachrom, l-2130/l-2450, merck). samples were centrifuged shortly at maximal speed in an eppendorf centrifuge, and the supernatants were loaded onto the column. pigments were separated and quantified using a rp18 column and a photo - diode array detector as described in papagiannakis., 2005. absorbance of isolated psi complexes was recorded in the qy maximum of chl a absorption and complexes were then adjusted to an absorbance of 0.03 (corresponding to roughly 0.3 10 g / ml chl a) with a buffer containing 60% glycerol as described in veith. samples prepared accordingly were used to record fluorescence spectra at 77 k with a jasco fluorometer (fp-6500). emission spectra were taken upon excitation at ex = 440 nm for preferential excitation of chl a and measured from em = 600 to 800 nm in 0.1 nm steps. excitation spectra of the same samples were recorded from ex = 400 to 600 nm for em = 674, 686, and 717 nm. band passes of 3 nm were used both on the emission and the excitation side. a rhodamine b spectrum served as a reference for the correction of the excitation side and the photomultiplier was corrected using a calibrated lamp spectrum. cyclotella meneghiniana cells were grown under three different conditions : either in ll with full supplement of iron (+) or under slightly reduced iron concentrations (), and in addition hl cells cultured under reduced iron were used as well. from the different cultures, psi complexes were prepared using sucrose density centrifugation after a mild solubilization of the thylakoid membranes as reported in veith. no changes in band patterns of the gradients were observed concerning the different light conditions (fig. the lower green bands formerly attributed to psi complexes having fcps bound were harvested from the gradients and characterized further. (a) results of separating complexes from the differently grown cultures using sucrose density centrifugation [low light plus 12 mol iron (ll(+)), ll plus 1 mol iron (ll()), and high light plus 1 mol iron (hl()) ] : for attribution of bands see veith. (b) signal intensities from immunoblots of complexes from cells grown under ll(+) (black), ll() (grey), and hl() (white), developed using -cmfcp : identical amounts of chlorophyll a (5 g each) were loaded and preparations from all three cultures were run on two blots each. (c) one example of the original blots and the results using the -lhcf1 - 11 antibody. in order to determine the fcp composition of the psi complexes, western blots were carried out using an antibody directed against all fcp polypeptides of c. meneghiniana, and the relative intensities of the bands were quantified using two different blots from two preparations each (fig. same amounts of chl were loaded in each case, but due to the differences in absolute intensities of the different blots, standard deviations were high. nevertheless, this study abstained from normalizing to one band since no data existed about a constant amount of any of the proteins recognized by the fcp antibody. as reported before for psi from hl(+) cells (veith., 2009), several fcp proteins are found in these psi preparations. three main bands, at 19, 18, and 17 kda, could again be distinguished (veith., 2009). since the sensitivity of the antibody used might differ between the different polypeptides, results do not represent absolute values for the distribution of different proteins inside one sample. on the other hand, whereas three bands were detected in psi complexes from (+) cells as already reported for hl(+) psi, the 19-kda band was severely reduced under () conditions. psi complexes from ll(+) seemed to have the highest amounts of each of the polypeptides, but taking into account the high variations of the blots, the 18- and 17-kda bands showed rather unchanged amounts under all conditions. 19- and 18-kda bands are found in fcpa complexes as well and these molecular masses are thus very common to fcps of c. meneghiniana. 17-kda bands were sometimes also visible in fcpa, but only under hl conditions (beer., 2006), making it unlikely that they are identical to those found here under ll as well as hl conditions. some subunits of the psi core have similar molecular weights, so cross - reactions of the antibody can not be ruled out, but the 17-kda polypeptide was detected with another antibody made from fcp proteins prepared in a different way as well (veith., 2009). using a more specific antibody, which was raised against a small peptide specific for fcps encoded by lhcf1 - 11 from p. tricornutum and fcp5 as well as fcp1/2/3 of c. meneghiniana, only the 19-kda band far more genes are described for fcp polypeptides in c. meneghiniana (eppard and rhiel, 2000) and another centric diatom, t. pseudonana (armbrust., 2004) meneghiniana, identification, for example by mass spectrometric analysis, is further hampered by the fact that the genome is not sequenced, in contrast to the related species t. pseudonana and the pennate diatom p. tricornutum (armbrust. thus, although altogether only five distinctive fcp proteins (fcp1/2/3, fcp4, fcp5, fcp6/7/8, and fcp12) have been identified in c. meneghiniana so far, each of the bands in psi is most probably composed of several proteins. for the 19-kda band, only three proteins identified in c. meneghiniana (fcp5, fcp6/7/8, and fcp12) come into question. two of them (fcp6/7/8 and fcp12) belong to the lhcx family, whereas fcp5 is a lhcf protein. 2011) identified mainly lhcr proteins, but also several lhcx and lhcf proteins when analysing psi complexes isolated by blue native (bn) page using mass spectrometry. fcp6/7/8 can be ruled out, since a specific antibody did not react with the 19-kda band (veith., surprisingly, the homologous protein, lhcx1, was found in psi of t. pseudonana (grouneva., 2011). fcp12 might be present in psi as well, although its homologue, lhcx5, was not found in psi of t. pseudonana (grouneva., 2011). other members of the lhcx family have been detected as well, but no identical sequences are known from c. meneghiniana so far. the 19-kda proteins were earlier shown to be more loosely bound to psi than the 18- or 17-kda polypeptides (veith., 2009), which might explain an absence after bn - page. since the lhcf antibody reacted with the 19-kda band, fcp5 might be one of the constituents, although none of the t. pseudonana proteins which should be detected by this antibody were found in psi, including the fcp5 homologue lhcf8 (grouneva., allen. (2008) tested the expression levels of some of the fcp genes under iron limitation in p. tricornutum and also reported on t. pseudonana genes in their supplementary material. one of the latter, annotated lhcr6 (jgi : 3815), was downregulated under iron limitation, but again not found in psi by grouneva. on the other hand lhcr2 was downregulated in p. tricornutum and the most similar sequence in t. pseudonana belongs to lhcr7, a protein of higher molecular mass and a constituent of psi according to grouneva. none of these lhcr proteins have been identified so far in c. meneghiniana. in summary, the lhcf protein fcp5, the lhcx protein fcp12, and/or as - yet unknown lhcx proteins of c. meneghiniana and/or homologues of lhcr6 and lhcr7 of t. pseudonana may be constituents of the 19-kda band. the severe decrease in the amount of this protein seen here under iron reduction and the reduced expression of lhcr2 in p. tricornutum under iron limitation make a similar protein the most likely candidate. concerning the 18-kda proteins, it is only known from mass spectrometric analyses that fcp4 is one of the constituents of this band (veith 2009). (2011) in t. pseudonana, and in p. tricornutum this gene is downregulated during hl (nymark., 2009), fitting to the biochemical analysis shown here. the current study could not identify polypeptides in higher abundance in hl, although the genes for lhcr6, 7, 8, and 10 were reported to be significantly upregulated in cells of p. tricornutum under hl conditions (nymark., moreover, the proteins were detected exclusively under hl conditions by lepetit. (it has to be emphasized that none of these p. tricornutum proteins has close homologues in t. pseudonana. most probably, some of the other lhcr polypeptides of similar size as fcp4 contribute to the 18-kda band in c. meneghiniana. when predicting proteins from gene sequences, another difficulty for these nuclear - encoded plastidic proteins is the lack of data about the precise start of the mature protein. diatoms possess secondary plastids, and nuclear - encoded proteins like fcps have thus to be transferred across four envelope membranes. the signal peptide responsible for the first part of this transfer is known, but for the transit peptide, needed for further transport, no typical sequence has been identified so far, making it difficult to predict the molecular mass of the mature protein. in many cases, the calculated molecular mass still including the target signal is about 20 kda or below, and thus the proteins with an apparent mass of 17 kda most probably belong to the group of proteins discussed above. the ratio of these typical pigments, chl c / fx was constant (fig. 2), and the amount of xanthophyll cycle pigments, dd plus dt, was only slightly decreased per fx in psi complexes from ll() cells compared to the two other growth conditions. these results point to a similar pigmentation of the various fcp polypeptides despite the different composition of fcps associated with psi seen in the western blots. in contrast, the de - epoxidation ratio expressed as dt/(dd + dt) differed. here the fcps in psi complexes followed the same trends as reported for the major fcps before (beer. psi from hl cells had a high de - epoxidation ratio whereas reduced iron led to a decreased de - epoxidation ratio. there are several reports in the literature also indicating a light - dependent increase in xanthophyll cycle pigments and de - epoxidation of dd to dt in psi complexes (lepetit., 2007 ; (2010) also demonstrated that this dt is protein bound and not associated with the lipids present in the preparations as well. the presence of dt in fair amounts inside the psi complexes has already been discussed with regard to protection, especially as a scavenger of chl a and o2 (lepetit., 2010), although this is a general feature of carotenoids and not restricted to dt. here it is important to note that neither the ratio of xanthophyll cycle pigments to fx nor the chl c / fx ratio depend on the amount of the 19-kda polypeptide, the only polypeptide for which the amount changed significantly. since further solubilization led to removal of the 19-kda proteins with similar pigment stoichiometries as reported here (veith., 2009), the current results point to all fcp polypeptides of psi having similar pigmentation. thus, the binding of xanthophyll pigments and the de - epoxidation are located in the 19-kda as well as in the other fcp polypeptides. ratios of the pigments specific for fucoxanthin - chlorophyll protein polypeptides [chlorophyll (chl) c, fucoxanthin (fx), diadinoxanthin and diatoxanthin (dd + dt) ] are shown for complexes isolated from cells grown under low light plus 12 mol iron [ll(+), black ], ll plus 1 mol iron [ll(), grey ], and high light plus 1 mol iron [hl(), white ]. 3 demonstrates the low - temperature fluorescence emission spectra obtained from the different psi preparations. the spectra were recorded at very low chl concentrations (0.3 ng / ml) to avoid reabsorption artefacts as much as possible. as reported several times before, the core of psi of diatoms is characterized by a long wavelength emission at around 710717 nm at 77 k (berkaloff., 1990 ; veith and bchel, 2007 ; ikeda., 2008 ; yamagishi., in addition, two additional peaks became obvious, especially in psi from ll() grown cultures, with maxima around 674 and 685 nm as reported before for psi from hl(+) cells (veith., 2009). however, at 77 k, theoretically all energy should end up on the chls of the core emitting at long wavelength, in case a full coupling is achieved. thus, both short wavelength emissions have to be attributed to chls decoupled from the core to various extents. in principle this could be due to complete uncoupling, i.e. broken fcps, or functional fcps having partly lost the functional connection to the core. to examine whether the fcps themselves are intact, i.e. whether chl c and fx are transferring energy to the different emitters, low - temperature excitation spectra were recorded at 674, 686, and 717 nm, respectively (fig. since these spectra were almost identical (data not shown), this study only reports on psi from ll() here. from the excitation spectrum at 674 nm, it is pretty obvious that those emitting chls can be identified as fully decoupled, since no other pigments are transferring energy to them. the 685-nm spectrum shows coupling of fx (around 530 nm) and chl c (at 465 nm), albeit to a lesser extent than in the spectrum recorded at 717 nm. a higher energy transfer from, for example, fx to the final 717-nm emitter than to the 685-nm emitters can only be explained if fx molecules other than those connected to the 685-nm emitter transfer their energy to the 717-nm chls as well. these could either be fx molecules bound directly to the core, or fx molecules coupled to another emitter inside the fcps, which is transferring most of the energy to the core and is thus not visible in the emission spectra. the cores containing fx molecules are unlikely since the polypeptides show high similarities to the core proteins of higher plants and cyanobacteria, which do not bind accessory pigments. thus, a better coupling of some of the fx to an emitter inside the fcps other than the 685-nm chl a is highly likely. the same reasoning holds for chl c. (a) fluorescence emission spectra at 77 k of photosystem i complexes from low light plus 12 mol iron [(ll(+), dotted line ], ll plus 1 mol iron [ll(), solid line ], and high light plus 1 mol iron [hl(), dashed line ] cells excited at 440 nm and normalized to identical 717-nm emission. (b) excitation spectra at 77 k of complexes from ll() cells, recorded at 674 nm (dashed line), 686 nm (dotted line), and 717 nm (solid line). (c) a fit of the emission spectra with five gaussians is shown exemplarily for complexes from ll() : the original spectrum is shown with circles, the fit is demonstrated as a solid line, and the single gaussians as dotted lines. residuals (lower panel) were plotted on the same axis but shifted by 0.1. the fx molecules bound to the complexes and actively transferring energy to chl a molecules of the cores showed an extended absorption in the long wavelength range up to 550 nm (fig. red fx molecules like found in both major fcps of c. meneghiniana. since the spectra of psi complexes from different cultures were indistinguishable this preferential excitation energy transfer occurred to a similar extent under hl and ll conditions, albeit different amounts of red fx molecules bound in the vicinity of psi. in order to identify the different chl a emitters and to get a better insight into the various contributions seen in the different samples, the spectra were fitted using gaussians. five gaussians, with maxima at 674, 685, 697, 717, and 750 nm, were needed to optimize the fits as shown exemplarily for the ll() spectrum measured upon excitation at 440 nm in fig. 1b. using more gaussians did nothing to improve the fit (data not shown). it has to be emphasized that all spectra could be fitted using the same parameters, including spectra recorded using other excitation wavelengths (data not shown). as to be expected from the excitation spectra, one additional emitter with a tight coupling was identified, emitting at 697 nm. whereas the 750-nm band is certainly a mixed band containing the vibrational bands of all others, the emission around 717 nm was earlier attributed to the psi core as stated above (berkaloff., 1990 ; veith and bchel, 2007 ; ikeda., 2008 ; yamagishi., these chl a species probably represent the red chls with energy levels below p700 found in all psi complexes, like those from higher plants or cyanobacteria (mullet., 1980 ; gobets., 2001 ; mangels., 2002) in order to compare the complexes from algae of different growth regimes, the current study assumed that the number of long - wavelength - emitting chl a per core is fixed for a species, as shown for different cyanobacteria (e.g. shubin., 1991 ; 4a, the summarized areas of the fluorescence bands at 674, 685, and 697 nm normalized to the 717-nm emissions are compared. the values were obtained using two independent preparations each and thus demonstrate the reproducibility of the complexes obtained. as already seen from the emission spectra, psi from ll(+) was characterized by high emission at short wavelengths, whereas complexes from ll() and hl() cells showed a lower, but similar amount. the higher emission can be either due to a stronger decoupling or to fcps equally decoupled but more abundant. in fig. 4b the relative contributions of the 674-, 685-, and 697-nm bands to the short wavelength emission are depicted. the figure reveals that the contribution is similar for ll(+) and ll() psi complexes, but that hl() complexes showed significantly higher emission at 674 nm. taking the excitation spectra into account, the emission at 674 nm is due to stronger uncoupling of chl a : that is, psi from hl() cells showed a higher sensitivity against the isolation procedure. this is in line with the observation that an additional gel filtration step did remove this emission from the psi preparations from hl(+) cells (veith., 2009). the other two bands, at 685 and 697 nm, changed in intensity in parallel in all psi complexes. this became more obvious when setting the sum of both emissions to one and then comparing the relative contributions (fig. the very obvious enrichment in 19-kda polypeptides as seen in ll(+) psi has thus no counterpart in the fluorescence yield of one single band. furthermore, since the 685- and 697-nm emissions changed in parallel, no attribution of certain fluorescence maxima to special polypeptides is possible. wavelengths at peak maximum and half width at half peak height of the gaussians used to fit the 77 k fluorescence emission spectra of photosystem i complexes data are means standard deviations of two spectra from three culture conditions each. areas under the single gaussians, calculated using the fits shown in fig. 3 and the parameters given in table 1, depicted for photosystem i complexes from low light plus 12 mol iron [ll(+), black ], ll plus 1 mol iron [ll(), grey ], and high light plus 1 mol iron [hl(), white ] cultures. (a) sum of the areas of the 674-, 685-, and 697-nm bands normalized to the area of the 717-nm band. (b) relative contribution of the three emitters to the short wavelength emission. (c) relative contribution of the 685- and 697-nm bands : sum of the areas set to one. the 674-nm emission is due to decoupled chl a, and the 685- and 697-nm emissions are due to chl a molecules that have different pools of fx and chl c molecules bound, which are active in energy transfer. thereby the chl a emitting at 697 nm are far better coupled to the cores than the 685-nm chls. the parallel change of the fluorescence yield of the two emitters poses the question whether these emitters work in parallel or in series. in the latter case the excitation harvested by fx and chl c would first be transferred into the 685-nm emitter, where most of it would get lost in the preparations, and then into the 697-nm emitting species before reaching the core. this is in contradiction to the excitation spectra, since in this case no better coupling of fx and chl c to the core (717-nm emission) than to the 685-nm emitter would be measurable. the excitation spectra also rule out the possibility that the 697-nm emission is an artefact of the fitting (see above). on the other hand, if the two emitters work independently, then each of them will have fx and chl c bound, which is active in excitation energy transfer, explaining the excitation spectra. however, since the 685- and 697-nm emissions were always changing in parallel, both seem to be equally sensitive against the isolation procedure. for another diatom, chaetoceros gracilis, ikeda. (2008) concluded from time - resolved spectra that two emitters are located in the core. here, ll(+) psi has the highest absolute value of short wavelength emission per core, in accordance with the highest amount of fcp protein found. in addition, from sequence analyses, no hints exist for a binding of accessory pigments to the core proteins as stated above. since both emitters are served by accessory pigments independently, it is much more likely that both emitters are located in fcps. the comparative analysis of psi complexes from differently grown alga cultures identified two groups of inner antenna proteins, having molecular weights of 18 and 17 kda. only for the 18-kda protein group, one constituent, fcp4, has been identified so far. the 19-kda proteins have been demonstrated to be peripherally bound earlier and are not found in psi from algae grown with lower iron. thus, in addition to reducing the total amount of psi (allen., 2008), iron stress seems to lead to smaller psi complexes. under iron reduction, the hl antenna composition is similar to the one under ll, at least within the resolution of the current blots. the xanthophyll cycle pigments are found in all fcps, with psi of hl() cultures having significantly more dt per dd. the psi complexes from the double - stressed cultures (hl()) were in addition slightly more sensitive against destruction by the detergent treatment as judged from a higher 674-nm emission at 77 k. two major emission bands of fcps could be identified, whereby those chl a fluorescing at 697 nm were more strongly coupled to the core than those fluorescing at 685 nm. as already known for long, a higher dt / dd ratio helps to protect cells from a surplus of light, thus explaining the higher levels of dt found in psi - fcps from hl cells. when comparing the sucrose gradients, displayed in fig. 1a, it also seems that ll cells did contain more psi - fcp compared to hl, in line with a higher reduction of the plastoquinone pool found in the latter (beer., 2011). under lowered iron supply, not only the amount of psi is reduced (allen., 2008), but also the antenna size is decreased, helping in avoiding over - excitation of the existing complexes. thus, under hl, protection is increased by converting dd to dt and the overall amount of psi - fcps is decreased. the different components of the psi antenna enable, therefore, variable reactions to several stress factors commonly experienced by the cells in vivo. | analysis of photosystem i (psi) complexes from cyclotella meneghiniana cultured under different growth conditions led to the identification of three groups of antenna proteins, having molecular weights of around 19, 18, and 17 kda. the 19-kda proteins have earlier been demonstrated to be more peripherally bound to psi, and their amount in the psi complexes was significantly reduced when the iron supply in the growth medium was lowered. this polypeptide was almost missing, and thus the total amount of fucoxanthin - chlorophyll proteins (fcps) bound to psi was reduced as well. when treating cells with high light in addition, no further changes in antenna polypeptide composition were detected. xanthophyll cycle pigments were found to be bound to all fcps of psi. however, psi of high light cultures had a significantly higher diatoxanthin to diadinoxanthin ratio, which is assumed to protect against a surplus of excitation energy. psi complexes from the double - stressed cultures (high light plus reduced iron supply) were slightly more sensitive against destruction by the detergent treatment. this could be seen as a higher 674-nm emission at 77 k in comparison to the psi complexes isolated from other growth conditions. two major emission bands of the fcps bound to psi at 77 k could be identified, whereby chlorophyll a fluorescing at 697 nm was more strongly coupled to the psi core than those fluorescing at 685 nm. thus, the build up of the psi antenna of several fcp components enables variable reactions to several stress factors commonly experienced by the diatoms in vivo, in particular diatoxanthin enrichment under high light and reduction of antenna size under reduced iron conditions. |
genome - wide association for fasting plasma glucose was performed among 5,089 nondiabetic indian asians (fasting glucose levels 95% for 99.7% of snps. call rate was > 95% for 99.1% of people ; 46 people were excluded for call rates 95% for 99.7% of snps. call rate was > 95% for 99.1% of people ; 46 people were excluded for call rates 0.5 ; fig. the most closely associated snp was rs2166706 ; when conditioned on rs2166706, there was no evidence for a separate effect of rs3847554 or rs1387153 on glucose levels. the associations of rs2166706, rs3847554, and rs1387153 with glucose levels were confirmed among indian asians in the hap300 sample, in whom risk allele of rs2166706 was associated with 0.05 mmol / l higher glucose levels than that for the alternate allele (table 2). genomic context of melatonin receptor mtnr1b region showing pairwise ld between snps with minor allele frequency 0.05 directly genotyped in both indian asians (a) and european caucasians genomic context and association test results for the three snps associated with fasting blood glucose levels among indian asians in the hap610 sample at p 99% in the three study samples. alt, alternate allele ; chr, chromosome ; ec, european caucasian ; ia, indian asian. the risk allele of snp rs2166706 was associated with elevated a1c and reduced homa - b among nondiabetic indian asians (table 3) in the hap300 sample. in contrast, there was no association of rs2166706 with weight, bmi, insulin levels, or homa - s (table 3). risk allele frequency of rs2166706 was higher among indian asians with type 2 diabetes than among nondiabetic indian asians in the hap300 sample. the age- and sex - adjusted odds ratio (or) for type 2 diabetes was 1.21 (95% ci 1.061.38) per copy of risk allele of rs2166706 among indian asians (p = 0.006). after additional adjustment for homa - b, or for type 2 diabetes was reduced and no longer statistically significant (1.12 [0.971.30 ] ; p = 0.14). associations of rs2166706 with secondary phenotypes among indian asians in the hap300 sample and european caucasians effects are unit change (initial to final quantity) or odds ratio (95% ci) per allele copy, under additive genetic model, adjusted for sex (and age among indian asians). in meta - analysis of the indian asian hap610 and hap300 samples, 29 snps reached p 0.5 ; fig. the most closely associated snp was rs2166706 ; when conditioned on rs2166706, there was no evidence for a separate effect of rs3847554 or rs1387153 on glucose levels. the associations of rs2166706, rs3847554, and rs1387153 with glucose levels were confirmed among indian asians in the hap300 sample, in whom risk allele of rs2166706 was associated with 0.05 mmol / l higher glucose levels than that for the alternate allele (table 2). genomic context of melatonin receptor mtnr1b region showing pairwise ld between snps with minor allele frequency 0.05 directly genotyped in both indian asians (a) and european caucasians (b) using haploview 's standard color scheme. genomic context and association test results for the three snps associated with fasting blood glucose levels among indian asians in the hap610 sample at p 99% in the three study samples. alt, alternate allele ; chr, chromosome ; ec, european caucasian ; ia, indian asian. the risk allele of snp rs2166706 was associated with elevated a1c and reduced homa - b among nondiabetic indian asians (table 3) in the hap300 sample. in contrast, there was no association of rs2166706 with weight, bmi, insulin levels, or homa - s (table 3). risk allele frequency of rs2166706 was higher among indian asians with type 2 diabetes than among nondiabetic indian asians in the hap300 sample. the age- and sex - adjusted odds ratio (or) for type 2 diabetes was 1.21 (95% ci 1.061.38) per copy of risk allele of rs2166706 among indian asians (p = 0.006). after additional adjustment for homa - b, or for type 2 diabetes was reduced and no longer statistically significant (1.12 [0.971.30 ] ; p = 0.14). associations of rs2166706 with secondary phenotypes among indian asians in the hap300 sample and european caucasians effects are unit change (initial to final quantity) or odds ratio (95% ci) per allele copy, under additive genetic model, adjusted for sex (and age among indian asians). in meta - analysis of the indian asian hap610 and hap300 samples, 29 snps reached p < 5 10. these were distributed in the mtnr1b, gck, and g6pc2 loci (supplemental table s2 and fig. the ld structure of the gck and g6pc2 loci were similar among indian asians and european caucasians (supplemental figs. s3 and s4). genetic variants near gckr that have been associated with glucose levels in european populations (9,10) were also associated with fasting glucose levels among indian asians, although not at a genome - wide significance level (gckr : rs1260326, p = 3.3 10). snp rs2166706 was associated with glucose levels in european caucasians (table 2). based on data from the present study and on the hapmap ceu population (hapmap phaseiii / rel#1 sept 2008, ncbi b36 assembly, dbsnp b126), snp rs2166706 is in moderate ld with both rs1387153 and rs10830963 (supplemental table s1), snps that have been reported to be associated with glucose in european caucasians (7,8). frequencies for risk allele of rs2166706 were similar among indian asians and european caucasians (46.2 vs. 45.0%, respectively ; p = 0.44) with no evidence for heterogeneity of effect on glucose (p = 0.84 ; table 2). results for association of rs2166706 with secondary phenotypes were similar in indian asians and european caucasians (table 3) except for homa - b, where the relationship with rs2166706 was weaker and less statistically significant among european caucasians. risk allele frequencies of rs1260326 (gckr) and rs560887 (g6pc2) were higher, and of rs2166706 (mtnr1b) and rs4607517 (gck) similar, among indian asians and european caucasians (table 2 and supplemental table s3). effect sizes on glucose levels at the mtnr1b, gckr, g6pc2, and gck loci were similar among indian asians and european caucasians, with no evidence for heterogeneity between the populations (table 2 and supplemental table s3). a score comprising number of risk alleles at these four loci was closely related to glucose levels in both indian asians and european caucasians (change in glucose per unit of allele score : indian asians 0.06 0.01 mmol / l, p = 1.1 10 ; european caucasians 0.04 0.01 mmol / l, p = 4.6 10 ; fig. 2). risk allele score was greater among indian asians than european caucasians (4.3 1.2 vs. 3.8 1.3, respectively ; p = 2.3 10 ; fig. 2) and accounted for 1.2% of population variance in glucose levels in both populations. however, at each level of risk allele score, indian asians had higher glucose levels than european caucasians (p = 3.3 10 ; fig. 2). distribution of risk allele score (number of risk alleles at the mtnr1b, gckr, gck, and g6pc2 loci) among indian asians (blue squares) and european caucasians (red circles) as well as the relationship of risk allele score to glucose levels in the two populations. we have identified and replicated an association of snp rs2166706 near mtnr1b with plasma glucose levels and show that rs2166706 is also associated with increased risk of type 2 diabetes among indian asians. we also replicate associations of the gck, gckr, and g6pc2 loci with glucose levels in indian asians. our findings extend the results of recent studies in european caucasian populations that identify genetic variation near mtnr1b as an important determinant of glucose levels (7,8). mtnr1b encodes a high - affinity receptor for melatonin, a circulating hormone released by the pineal gland (18). secretion of melatonin is tightly regulated by the hypothalamic suprachiasmatic nucleus, the anatomic center of the mammalian circadian clock (19,20). melatonin release is one of the key mechanisms by which the circadian clock maintains synchronization and regulates the biologic activities of peripheral tissues (18). melatonin receptors (including mtnr1b) are highly expressed in the hypothalamus, where they contribute to melatonin - induced negative feedback and phase timing of circadian activity (21). mtnr1b is also expressed in human pancreatic islet cells and may mediate the effects of melatonin on basal and glucose - induced insulin release (22). our findings of an association between snp rs2166706 near mtnr1b and raised glucose, reduced pancreatic -cell function, and increased risk of type 2 diabetes are consistent with accumulating evidence that circadian clocks play a key role in the regulation of carbohydrate and energy metabolism. clock / clock null mice develop hyperglycemia, and in humans sleep loss and depression are associated with circadian desynchronization and increased risk of type 2 diabetes (2325). although further functional studies will be required to clarify the contribution of peripheral and central melatonin signaling pathways to glucose metabolism and type 2 diabetes, our observations suggest that mtnr1b signaling may be a therapeutic target for the development of agents to improve glucose regulation and to prevent or treat type 2 diabetes. we also confirm association of gck, gckr, and g6pc2 loci with glucose levels in indian asians. effect sizes were similar among indian asians and european caucasians, and a risk allele score was associated with glucose similarly in the two populations. however, risk allele frequencies were higher among indian asians than european caucasians. these findings suggest that genetic variation at these loci makes an important contribution to raised glucose levels among indian asians. these are the first data identifying genetic variants that influence glucose metabolism among indian asians, a population at high risk for type 2 diabetes and related metabolic disorders. the 21% of indian asians homozygous for the g allele of rs2166706 of mtnr1b have 0.1 mmol / l higher fasting glucose levels and 45% higher odds of type 2 diabetes compared with people with aa genotype. our findings give new insight into the genetic mechanisms underlying abnormalities of glucose metabolism among high - risk indian asians and provide the basis for future studies to investigate the role of circadian rhythms and melatonin signaling in the etiology of type 2 diabetes. | objectivefasting plasma glucose and risk of type 2 diabetes are higher among indian asians than among european and north american caucasians. few studies have investigated genetic factors influencing glucose metabolism among indian asians.research design and methodswe carried out genome - wide association studies for fasting glucose in 5,089 nondiabetic indian asians genotyped with the illumina hap610 beadchip and 2,385 indian asians (698 with type 2 diabetes) genotyped with the illumina 300 beadchip. results were compared with findings in 4,462 european caucasians.resultswe identified three single nucleotide polymorphisms (snps) associated with glucose among indian asians at p < 5 108, all near melatonin receptor mtnr1b. the most closely associated was rs2166706 (combined p = 2.1 109), which is in moderate linkage disequilibrium with rs1387153 (r2 = 0.60) and rs10830963 (r2 = 0.45), both previously associated with glucose in european caucasians. risk allele frequency and effect sizes for rs2166706 were similar among indian asians and european caucasians : frequency 46.2 versus 45.0%, respectively (p = 0.44) ; effect 0.05 (95% ci 0.010.08) versus 0.05 (0.030.07 mmol / l), respectively, higher glucose per allele copy (p = 0.84). snp rs2166706 was associated with type 2 diabetes in indian asians (odds ratio 1.21 [95% ci 1.061.38 ] per copy of risk allele ; p = 0.006). snps at the gck, gckr, and g6pc2 loci were also associated with glucose among indian asians. risk allele frequencies of rs1260326 (gckr) and rs560887 (g6pc2) were higher among indian asians compared with european caucasians.conclusionscommon genetic variation near mtnr1b influences blood glucose and risk of type 2 diabetes in indian asians. genetic variation at the mtnr1b, gck, gckr, and g6pc2 loci may contribute to abnormal glucose metabolism and related metabolic disturbances among indian asians. |
the clinical laboratory has become an essential component in meeting high - priority consumer need and a vital patient resource, occupying an increasingly important part of mainstream medicine. knowledge of a utilization pattern of laboratory investigations can provide health planners with basic information that can help in resource allocation.1 for a long time, obtaining the history of a patient and physical examination have been the most important elements of patients assessment.2 however, in recent years, the care of patients has become increasingly dependent on the results of laboratory investigations as well, and it follows that clinical laboratories have become a major component in the delivery of health care. laboratory investigations have always been and will continue to be an integral part of clinical practice in primary health care centers.3 the advancement of medical technology has made laboratory tests easily obtainable and virtually indispensable to physicians in diagnoses from the most common to the rarest disease. there would be less concern regarding the over - utilization of diagnostic tests if the use of more tests led to an improvement in health, but there is evidence to suggest that part of the increase in test ordering is unnecessary. several studies have found that test results were often ignored by physicians, implying that the tests were not needed to begin with.4 in the usa, between 1975 and 1978 the total number of clinical laboratory investigations performed increased at an annual rate of 8%,5 even though the per capita visits to physicians decreased from 5% to 4.8%. between 1972 and 1978, out - of hospital laboratory tests (excluding x - rays and radiation therapy) increased from 850 tests per 1000 visits to 1510 tests per 1000 visits.4 the aim of this research was to study the utilization of laboratory investigations in primary health care centers in the al - khobar area by studying the family health record checklist of patients. all the 9 primary health care centers were taken as population under study. before selection of a sample, al - khobar government hospital (one of the phc centers) was included in the sample as a special case as it was a primary health care center well - equipped with both personnel and facilities. moreover, it had a sophisticated laboratory set - up that acted as a referral laboratory for other centers. a two - stage sampling design was used. at the first stage, 4 primary health care centers were selected by means of a simple random sampling design and at the second stage, family health records of the patients were selected using a systematic sampling design within each selected first stage primary health care center. in all, 5 primary health care centers were selected. two primary health care centers were selected at random, by means of a simple random sampling design. accordingly, the sample size was calculated as 659 from the total records of 179,272. the sample was distributed proportionately among all selected primary health care centers : south khobar, al - doha, albaornia, south west thouqba and al - khobar government hospital 's primary health care centers. each family health record was studied according to the patient 's complaint, and the diagnosis at the particular visit subjected to the check list. this was used to collect information retrospectively about patient 's age, gender, nationality, diagnosis and the laboratory investigations which had been requested. a standard list of the most common health problems encountered in primary health care centers and the laboratory investigations that should be done for each disease was compiled to determine whether the diagnosis was legitimate. these laboratory investigations were established by using references, such as the quality assurance guidance in primary health care published by the ministry of health67 as well as laboratory medicine, primary care and family medicine textbooks89 and who guidance for basic laboratory medicine.1011 moreover, for validity, the standard list was verified and re - evaluated by 5 faculty members at the level of consultants in the department of family and community medicine, college of medicine, king faisal university (kfu) to enumerate the laboratory tests required for a proper diagnosis of a health problem. the investigations were considered inappropriate when (i) more or fewer tests than those listed for a particular disease in the standard list were requested or (ii) if non - relevant tests were requested for a case. they were considered appropriate (a) when for chronic diseases such as diabetes mellitus or hypertension, all follow - up visits during the year showed that laboratory tests. which needed to be performed at intervals, were ordered according to the standard list (b) if the test was carried out once a year. chi - square test, t - test, anova (one - way) and kruskal - wallis (h - test) tests were used for data analysis. the mean and standard deviation of their ages were 30.3 17.9 and 28.4 19.1 years respectively. the combined mean and standard deviation were 29.4 18.4 years. a total of 789 laboratory tests were carried out among the sample, with a mean and standard deviation of 1.2 1.15 tests per patient. the utilization of laboratory investigations according to the age of the patients along with means (sd) are given in table 1. on the average, the laboratory test utilization for patients under 21 years was less than one test per patient, whereas for patients between 21 and 40 years, the average was about 1.2 tests per patient. utilization of lab investigations per age with advancing age, more laboratory tests were utilized with a maximum of 2.1 tests for patients above the age of 40 years. the kruskal - wallis method was used to test the difference in utilization of laboratory investigations in different age groups and it was found that there were highly significant differences statistically among the age groups with respect to utilization of laboratory investigations (p = 0.001). these data were further divided into two groups to examine the pattern of utilization between age groups 40 and under and the age groups over 40. it was found that in the first set of groups, the utilization of laboratory tests varied in different age groups (kruskal - wallis, p = 0.002), where as in the second group it was found that there was no statistically significant difference in the use of laboratory tests in different age groups (p=0.3410 - anova one way). again, all age groups 40 and under were combined in one group, and all those over 40 were combined in another group. it was found that there were statistically significant differences in the utilization of laboratory tests (t - test ; p < 0.001). utilization was greater on the average for patients older than 40 years and greater for females than males. the mean and standard deviation for males and females were 1.16 1.62 and 1.24 1.57 tests respectively but there was no statistically significant difference among males and females (t = 0.64 ; p = 0.525). on the average, for both males and females for the 496 (73.3%) saudi and 163 (24.7%) non - saudi patients, it was found that on the average, more tests were undertaken for non - saudi than for saudi patients. their respective means and standard deviation were 1.27 1.87 and 1.17 1.49 tests but the difference between saudi and non - saudi was not statistically significant (t=0.59, p = 0.558). the proportion and the utilization of laboratory tests according to diagnostic categories is given in table 2. there were large variations in the number of tests used for different diagnostic categories, sometimes amounting to more than tenfold. respiratory diseases accounted for more than a quarter (29.1%) of the health problems in primary health care centers. chronic diseases, mainly diabetes and hypertension, accounted for another quarter (24.5%). musculo - skeletal diseases were 10.8%, and gastro - intestinal tract (git) disease and urinary tract infection 10.2% and 10%, respectively. other diseases such as those of the eye, ear and skin totaled 15.4%. even for some diseases such as diabetes, hypertension and urinary tract infection that were investigated in detail, the utilization of laboratory tests was improper. respiratory infection, bronchial asthma, eye and ear diseases achieved a high rate of proper utilization laboratory use according to diagnosis a variety of tests was carried out and the distribution of the utilization of specific laboratory tests has been described in table 3. they are grouped into four categories, i.e., hematology, biochemistry, microbiology and serology. microbiology was the most heavily used service, comprising 43% of all the investigations followed by biochemistry (36.1%), hematology (19.5%) and serology (1.4%). urinalysis was the most frequently requested test making up 30.7% of all tests, followed by blood glucose(19.9%), and complete blood count(14.8%).(table 3) the distribution of the utilization of specific laboratory tests there was no statistically significant difference among the primary health care centers regarding the proper and improper utilization of laboratory tests (chi - square = 4.13 p = 0.389) (table 4). neither was there any significant difference in the two categories of over- and under - utilization, both of which are improper (kruskal - wallis = 4.13 ; p = 0.389). moreover, there was no statistically significant difference between males and females regarding proper and improper utilization of laboratory tests (chi - square = 0.68 ; p = 0.411). the utilization pattern for laboratory tests among phc centers of the 323 (40%) cases of proper utilization of laboratory tests, 168 (52%) were for males and 155 (48%) for females. of the 336 cases of improper utilization, 164 (48.8%) were for males and 172 (51.2%) for females. however, no statistical difference was noted on the overall requests for male and female patients. of all those studied, 496 (75.3%) were saudi, and of these 47.8% were identified with improper utilization. of the 163 (24.7%) who were non - saudi, improper utilization was 47.2%. there was no statistically significant difference with regard to patients of a particular nationality (chi - square = 1.22 ; p=0.269). the results of this study provide some information on the utilization of laboratory investigations in the primary health care centers in the al - khobar area. the tests were ordered for 49% of the sampled population, with an average of 1.2 tests per patient. this result may be compared with the national ambulatory medical care survey in united states.12 it was reported that general practitioners used laboratory tests for 34% of patients. hartley et a113 found in the greater london general practice that two - thirds of all the tests requested during the year were ordered for less than 10% of the patients. mills and reilly,14 however, reported that an average of only 12% of the patients seen in four general practices in belfast underwent laboratory testing. beaulieu et a115 studied the utilization of laboratory tests in a random sample of 1029 visits occurring over a one - year period in a family medicine service. they found that no test was ordered in 62.5% of the visits, and that the average was 1.04 tests per visit. at a community health center in oslo staffed by three general practitioners, hjortdahl16 found that laboratory tests were ordered in 50% of all consultations, and an average of one test was ordered per consultation. therefore, we can conclude that physicians are using laboratory services more frequently in recent years than before. the utilization of laboratory services was more noticeable for patients above 40 years of age in this study, where on average 2.1 laboratory tests per patient were used. patients under the age 20 were less likely to be investigated, where an average 0.7 laboratory tests per patient were used. this may be due to the fact that there may be an under - utilization of the services. the utilization of laboratory tests was almost equal with respect to gender and nationality, but among diagnostic categories variation was found which was sometimes more than tenfold. for example, of the total number of laboratory tests carried out, 26.4% were for diabetes mellitus (dm) and 2.4% were for diarrhea. the variation between them was 11-fold, (diarrhea got a higher percentage of proper utilization than diabetes mellitus). this variation may be compared with that of hartley and his colleagues13 who found more than a ten - fold variation in test use among diagnostic categories. an actual use of diagnostic tests was measured and it was found that the pattern of practice was truly representative of the actual practice behavior of the physicians. the utilization of laboratory investigations in primary health care centers was inappropriate in 51% of the cases, 37.8% of which was under - utilization and 13.2% over - utilization. this is proportionally close to the finding of wheeler.17 they studied 258 cases of anemia in a san francisco hospital, and found about 11% over - use and 24% under - use of laboratory investigations. microbiology was the most heavily used service, comprising 43% of all investigations, followed by biochemistry (36.1%) and hematology (19.5%). urinalysis was the most frequently requested test, accounting for 30.7% of all tests, followed by blood glucose (19.9%) and cbc (14.8%). in the mills and reilly14 study, hematology tests accounted for more than 30% of laboratory requests, followed by microbiology (25%), and biochemistry (20%). kelly and barber18 found that hematology was the most heavily used service (33.3%), followed by microbiology (22%) and biochemistry (18.8%). they also found that cbc was the most frequently requested test, representing 23.6% of all the investigations, followed by urinalysis (16.1%) and cervical smear (13.9%). the high percentage of microbiology in this study compared to other studies, may be due to over - utilization of urinalysis and stool analysis. we found that the rate of inappropriate use of laboratory tests was 51% ; 37.8% of which were cases of under utilization and 13.2% of over utilization. this may be due to the fact that the problem of over - utilization does not seem to be as acute in primary health care centers as has been observed in hospitals.19 laboratory tests were utilized for only half of all the patients and 84% of them had a maximum of 3 tests. the utilization was inappropriate in 51% of the cases, 13.2% of which cases were of over utilization, and 37.8% of under utilization. as age of patients increased, laboratory facilities were used more frequently, and this almost doubled after the age of 40 years. there was no difference between primary health care centers with regard to the pattern of utilization of lab tests for males and females and for saudi and non - saudi patients. respiratory diseases accounted for the majority of the health problems in primary health care centers, followed by diabetes mellitus and hypertension. microbiology was the most heavily used mode of investigation, followed by biochemistry and hematology. test selections and interpretation of results should be adequately taught in medical schools. this has already been proved very effective at the university of minnesota.20a handbook on appropriate test selection should be issued to encourage the proper use of laboratory testing, and a regular audit should be established to enable physicians to review their activities.continuing medical education should be directed towards investigation, indication, and rational, cost conscious utilization through a variety of approaches such as close interaction between clinical chemist and clinician, circulating laboratory newsletters, and through the organization of discussions on specific topics.undergraduate curricula of medical colleges should include courses on health economics and cost effective decision making.family health records in primary health centers need evaluation and improvement on documentation. test selections and interpretation of results should be adequately taught in medical schools. this has already been proved very effective at the university of minnesota.20 a handbook on appropriate test selection should be issued to encourage the proper use of laboratory testing, and a regular audit should be established to enable physicians to review their activities. continuing medical education should be directed towards investigation, indication, and rational, cost conscious utilization through a variety of approaches such as close interaction between clinical chemist and clinician, circulating laboratory newsletters, and through the organization of discussions on specific topics. undergraduate curricula of medical colleges should include courses on health economics and cost effective decision making. | objective : the objective of this study was to determine the pattern of utilization of laboratory investigations in the al - khobar area of saudi arabia.material and methods : a two - stage sampling design was used to select a family health records checklist. at the first stage, 5 primary health care centers were selected out of 9 using a random sampling method. a family health records checklist was selected using a systematic sampling design from each selected primary health care center at the first stage.results:the results showed that laboratory investigations were used for 49% of the sample population tested. of these, 84% recorded a maximum of 3 laboratory tests. in over half of the cases, the tests were inappropriately utilized, 37.8% were underutilized and 13.2% were over - utilized. there was no significant difference in the pattern of utilization between males and females and between saudi and non - saudi patients. however, laboratory services were utilized more for patients above the age of 40 years, where an average of 2.1 tests per patient was recorded.conclusion:there was a significant difference between primary health care centers regarding pattern of laboratory utilization. respiratory disease accounted for the majority of the health problems, followed by diabetes mellitus and hypertension. microbiology was the most heavily used investigation followed by biochemistry and hematology. urinalysis was the most frequently requested test followed by blood glucose and complete blood count (cbc). this study highlighted the problems in the utilization of laboratory investigations and led to a number of solutions and recommendations. |
a 49-year - old male has been managed in the intensive care unit for 5 days after a large left diaphragmatic hernia repair and is currently being weaned from mechanical ventilation. he suddenly has significant hematemesis and becomes hemodynamically unstable, with alteration to his coagulation. you start to resuscitate him with fluid, blood and plasma, in order to reverse the hemorrhagic shock and correct the coagulopathy. you are aware that factor viia (fviia) has been used in acute traumatic hemorrhage to stop bleeding. paola pieri and deborah m stein fviia (novoseven) was developed by novo nordisk for use in patients with congenital and acquired hemophilia and inhibitors of factor viii or ix. since it was licensed in europe in the 1990s and in the usa in 1999 it has been utilized off - label in an increasing number of nonhemophiliac patients with severe bleeding, such as the patient described in the scenario above. at present the precise role of fviia in treating life - threatening hemorrhage has not been determined. however, numerous studies have demonstrated benefit from off - label use. several case series have been published that describe successful use of fviia in severely injured patients [1 - 4 ]. additionally, in a recently published prospective randomized placebo - controlled double blind trial, a reduction in transfusion requirement was observed in trauma patients, as was a decrease in overall morbidity and mortality when early deaths were excluded from the analysis. there are numerous other reports of successful use of fviia in the noninjured patient with acute hemorrhage, such as that secondary to esophageal varices, hemorrhagic pancreatitis, and hemorrhage occurring during cardiac surgery and liver transplantation. case reports of fviia use to treat patients with resistant coagulopathies that developed in the intensive care unit setting have demonstrated efficacy in restoring hemostasis, with subsequent survival largely dependent on the underlying disease process. prospective randomized trials have demonstrated successful use of fviia in other patient populations, including those with acute intracerebral hemorrhage and those undergoing elective radical prostatectomy. with fviia use, the potential complications of pathological and inappropriate thrombus formation is present but thought to be low. a recently published us food and drug administration medwatch database described 151 complications associated with off - label use of fviia, the majority occurring in trauma patients. however, medwatch is a database for voluntary reporting of observed complications, and therefore the incidence of complications can not be calculated from it. randomized studies have found the frequency of adverse events associated with administration of fviia to be similar to those with placebo. the patient presented above is a relatively healthy male, with no assumed underlying significant medical conditions, who undergoes an elective surgical procedure and subsequently develops stress gastritis and life - threatening upper gastrointestinal bleeding. despite adequate and aggressive resuscitation and medical management, he continues to hemorrhage. life - threatening hemorrhage and coagulopathy in critical care patients carries significant morbidity and mortality, with increased incidence of respiratory failure and renal failure as well as multiple organ dysfunction. additionally, early administration before the development of acidosis, hypothermia, and subsequent additional coagulopathy is likely to be more efficacious. the risk for adverse events after fviia administration is low, and in this case, although the patient is in extremis, the potentially life - saving benefit of correcting the patient 's coagulopathy and ceasing his hemorrhage clearly takes precedent. furthermore, the only other option for arresting hemorrhage in this patient in whom coagulopathy can not be reversed is a total gastrectomy, which is a procedure with unacceptably high morbidity, both in the short and long term. therefore, off - label use of fviia in this setting is not only warranted but also potentially beneficial and life saving. sandro scarpelini and sandro rizoli we understand the clinical scenario and debate question as whether one should administer a drug outside its licensed indications (off - label) to treat a condition (upper gastrointestinal bleeding) without any evidence for the drug 's efficacy and safety. the first issue is the off - label use. after a single successful report of off - label use of recombinant fviia (rfviia) in 1999 retrospective case reports followed, almost invariably describing remarkable results and further stimulating unlicensed use of rfviia. more recently, many of the expectations raised by retrospective reports are being revised as more balanced results from randomized controlled trials (rcts) are published [8,9,11 - 15 ]. particularly in gastrointestinal bleeding, rcts have contradicted many initial expectations and demonstrated no clinical benefit of rfviia. in 2004 romero - castro and coworkers reported that all 10 patients with cirrhosis and bleeding varices stopped bleeding after a single bolus of rfviia. however, a subsequent 245-patient european rct conducted in this same population demonstrated that rfviia had no effect. the same occurred in liver transplantation, in which a pilot six - patient study reported 100% efficacy in reducing bleeding but an 86-patient rct concluded that rfviia had no impact on perioperative blood loss or need for blood transfusion. one more caveat comes from a multicenter survey of off - label use of rfviia in american academic hospitals, which reported that only 52% of the patients stopped bleeding within 6 hours of rfviia administration and 9% suffered adverse events. the latter finding is worse than any case report or series and is curiously similar to the 7% complication rate reported in a 399-patient rct on intracerebral hemorrhage. in conclusion, off - label use might be inappropriate, wasteful, and cause adverse effects more often than is currently estimated. there are no reports of rfviia use in diffuse gastric erosions, with the arguable exception of a sketchy report. in other gastrointestinal bleeds, there are two rcts (discussed above and found no clinical benefit) and case reports / series in crohn 's disease, peptic ulcer, cancer / hematological diseases, anticoagulant use, pancreatitis, and cirrhosis. the case reports / series are dismally small (two patients with crohn 's disease, one with peptic ulcer, one receiving anticoagulant, and two with pancreatitis) ; case reports are typically biased toward positive results, and the diseases reported are different from the case in question. in conclusion a third consideration is that the moral / ethical implications of administering a drug with unknown efficacy / safety, for unlicensed indications. there is reasonable evidence to justify its off - label use in trauma, intracerebral hemorrhage, cardiovascular surgery, large perioperative hemorrhage, obstetrics, and anticoagulant - induced hemorrhage. however, there is also evidence disproving any benefit in liver transplant, gastrointestinal bleeding (cirrhosis), liver resection, and pelvic / acetabular surgery [11 - 13 ]. rfviia is not a panacea for all bleeding, and inappropriate use should be as much a concern as not using it when it could be beneficial. since most indications for rfviia remain largely untested, we should struggle to enroll patients into clinical trials that will eventually define which patients do benefit from rfviia, rather than promoting off - label use. paola pieri and deborah m stein dr rizoli presents accurate and important information concerning the off - label use of rfviia. the clinical situation presented, however, is more akin to the coagulopathy seen in trauma, in which rfviia has demonstrated efficacy without increased complications, than to a patient with a chronic disease. although some rcts have shown no benefit in patients with cirrhosis, varices, or transplants, this is not necessarily relevant in the patient who was previously healthy and developed life - threatening hemorrhage. although rfviia is certainly not a ' panacea ', the potentially life - saving benefit should outweigh concern that administration may fail, and should not preclude its use. sandro scarpelini and sandro rizoli many believe that rfviia cures all bleeding, which is an unsustainable conviction considering the current evidence. rcts have questioned the near perfect efficacy described by many case reports, particularly in upper gastrointestinal bleeding. the reasons to avoid rfviia in this patient are as follows : it has not previously been used for a similar indication ; rcts have shown no benefit in upper gastrointestinal bleeding (cirrhotic) ; and use of rfviia as ' last resort ' is futile. the colleagues incorrectly stated that the trauma rct demonstrated that rfviia decreases mortality in trauma. apart from ongoing bleeding, even off - label use demands reasonable expectation of benefit. ongoing bleeding and reasonable expectation of benefit are mandatory even for compassionate off - label use of rfviia. sr is a member of the niastase / novoseven international scientific advisory board and has received consultancy fees from novonordisk a / s. | perhaps it is not surprising that in the critical care environment, where lives are frequently on the line, off - label use of certain drugs is relatively common. in general, there are two camps of opinion on this type of utilization. one camp would suggest that potentially life saving products can not ethically be withheld from patients who may benefit. the other camp would counter that it is inappropriate to administer products if the risk / benefit ratio has not been clearly defined in clinical trials. off - label use of factor vii is debated in this issue of critical care for a patient with uncontrolled nontraumatic hemorrhage. perhaps this product promotes additional discussion given that its ability to control bleeding can be dramatic, yet its costs and potential for complications high. |
calcifying aponeurotic fibroma was first described and referred to as juvenile aponeurotic fibroma by keasbey in 1953 (1). it is a rare, benign, locally aggressive fibroblastic soft tissue tumor that typically occurs in the palm of the hand and in the sole of the feet in children and adolescents (1 - 3). the tumor has a predilection for local recurrence after surgical resection (2, 3). to our knowledge, there have been only few radiology studies describing the mr findings of calcifying aponeurotic fibroma. furthermore, previous articles mainly investigated radiologically non - calcified masses in young children (3, 4). we report a very rare case of aponeurotic fibroma in a 67-year - old man with a 20-year clinical history that presented with a relatively densely calcified mass affecting his dorsal wrist. the purposes of this article are to report the radiographic and mr findings of calcifying aponeurotic fibroma and to provide useful information for the differential diagnosis of the lesion. a 67-year - old healthy man presented with a 20-year history of a painless, slow growing mass in the dorsum of his right wrist. physical examinations showed a firm, non - tender, palpable mass, measuring 1.5-cm, and overlying the carpal bones in the dorsum of his wrist. radiographs demonstrated a relatively ill - defined soft tissue mass with speckled calcifications on the dorsal ulnar aspect of the wrist. mri showed an ill - defined rounded subcutaneous soft tissue mass partially encasing the tendons of the extensor carpi ulnaris (ecu) and the extensor digiti minimi (edm) on the dorsum of the wrist. mri revealed low to intermediate signal intensity on the t1-weighted image (t1wi) and heterogeneous mixtures of low and high signal intensity on the t2-weighted image (t2wi). the lesion had a poorly demarcated margin and was associated with edema like signal changes in the adjacent soft tissues on mr images (fig. in view of the radiographic and mr findings, the preoperative differential diagnosis included soft tissue chondroma, bizarre paraosteal osteochondromatous proliferation (bpop), giant cell tumor (gct) of the tendon sheath, fibroma of the tendon sheath, nodular fasciitis and a small sized synovial sarcoma. the mass was excised and was revealed to be an off - white colored, firm lesion. the mass adhered to and involved the tendon sheaths of ecu and edm, the adjacent extensor retinaculum, and the wrist joint capsule with the intracapsular extension. however, the mass could be excised without any resection of the bones or tendons. microscopically, an ill - defined tumor mass showed multifocal calcifications surrounded by dense fibrous stroma. the fibrous stroma revealed mixed cellular areas near the calcification and lesser cellular fibromatosis - like areas in the background. multilayers of fibroblasts with small round nuclei, histiocytes and multinucleated giant cells in the cellular areas were adjacent to the calcification. lesser cellular and densely collagenous areas showed early chondoid metaplasia - like cellular changes such as small round nuclei and perinucler clear spaces in the hyalinized stroma. the tumor usually appears in the first to second decade of life, although cases have been reported for ages ranging from birth to 67 years. calcifying aponeurotic fibroma typically occurs in the distal extremities, most commonly in the fingers, palms and soles (1 - 3). however, this lesion has been observed at other sites, including the neck, mandible, forearm, elbow, knee and thigh (3). the tumor has been typically described as a firm, non - tender, slow growing mass, measuring less than 3 cm in diameter. it has a tendency to infiltrate the surrounding tissue, and has a predilection for local recurrence after surgical resection. (5) made a pathologic confirmation of a juvenile aponeurotic fibroma that had metastasized to the lung and bone five years after its secondary excision. enzinger and weiss (6) stated that malignant transformation of aponeurotic fibroma was not found in the registry of the armed forces institute of pathology but that they had seen one case in consultation. histologically, the lesion reveals characteristic scattered chondroid or calcific nodular foci surrounded by rounded, chondrocyte - like cells arranged in a linear or palisade pattern. there is less cellular, spindle fibroblastic component with a fascicular pattern between the coalescent calcified or chondroid nodules and emanating into the surrounding soft tissue. mitotic figures are rare (7). according to a pathologic analysis of 22 cases by fetsch and miettinen (7), chondroid foci and mineralization were demonstrated in all but one case, regardless of radiographic evidence of calcification. enzinger and weiss (6) suggested the existence of biphasic development of the tumor, consisting of the initial and late phases. in the initial phase, which is seen more often in the young, the tumor has infiltrative and destructive growth and often lacks calcification. in the late phase, the tumor is more compact and nodular, and shows more prominent calcification and cartilage formation. therefore, imaging appearances of calcifying aponeurotic fibroma can vary depending on the patient 's age, presence of calcifications, and osseous involvement (6). base on the literature, the mass seen in this case is considered to clinically and radiologically correspond to the late phase of calcifying aponeurotic fibroma, because the mass had a nodular appearance with relatively dense calcifications in our advanced aged patient with a long clinical history. radiographic features of calcifying aponeurotic fibroma include an ill - defined or poorly demarcated margin of the tumor and a variable extent of fine, stippled calcifications within the lesion. all these radiographic features with relatively dense calcifications were evident in our case (fig. 1a - c). computed tomography scan is optimal for depicting the calcified area of the lesion, with other lesions demonstrating non - specific soft tissue attenuation. mr imaging features of the mass have rarely been described in the radiology literature. (4) reported a case of radiologically non - calcified calcifying aponeurotic fibroma in a young child, and described the mri findings of the mass showing lower signal intensity than that of muscle on t1wi and t2wi, and intense heterogeneous enhancement after gadolinium administration. they stated that the low signal intensity on t2wi was attributed to the fibrous component and little cellularity of the mass. the dense calcifications of the mass in our case are considered to be also responsible for the low signal intensity on t1wi and t2wi, in addition to the fibrous component and little cellularity of the mass. when a small, slow growing, calcified or non - calcified soft tissue mass is identified in the wrist or hand, the differential diagnosis should include gct of the tendon sheath, soft tissue chondroma, florid reactive periostitis or bpop, fibroma of tendon sheath, and nodular fasciitis (8, 9). gct of the tendon sheath is the second most common mass affecting the hand and wrist, after ganglion. clinically, patients report asymptomatic, slowly growing masses attached to the tendon sheath and/or joint capsule. radiographs may depict pressure erosion, and less commonly, periosteal reaction. typical signal void artifacts can be seen on all sequences, particularly on gradient - echo images, and a more heterogeneous and predominantly low signal is found on t2wi. the lesion typically enhances after gadolinium administration (8). soft tissue chondroma is a very rare well - defined cartilaginous mass. clinical presentation is of a slow growing mass of the hand or wrist with deep attachment to the tendon, tendon sheath, joint capsule, or periosteum. radiographs may be negative, may demonstrate intralesional calcifications in 30% to 70% of cases, or may show extrinsic cortex erosion of the underlying bone (8, 9). mr imaging is highly specific and depicts a cartilaginous mass that demonstrates low signal intensity on t1wi and very high si on t2wi. after contrast administration, small lesions show peripheral enhancement and larger lesions show more central enhancement. florid reactive periostitis and bpop are considered to depict different phases of development of the same posttraumatic proliferative reaction (8). it usually involves the small bones of the hands and feet and usually occurs in young patients, between 20 and 30 years of age. the lesion is depicted as an ill - defined paraosseous mass with spare calcifications with florid lamellar or compact periosteal reaction on radiographs, which is an unusual feature for calcifying aponeurotic fibroma. bpop also has a predilection for the small bone of the hand, and may widely occur in patients ranging from first to sixth decade. radiograph shows a well - defined lobulated ossifying mass in the hand, usually less than 3 cm. mr imaging demonstrates low signal intensity on t1wi, varying high to low signal intensity on t2wi depending on the degree of its ossification (8). fibroma of the tendon sheath presents as a slow growing, hard mass, firmly attached to the tendon. it shares imaging features and clinical presentations with gct of the tendon sheath, but occurs much less commonly in the hand and wrist. the lesion may appear as a well - defined, focal nodular mass with decreased signal intensity on all mr sequences and little or no contrast enhancement. mr signal intensity of the mass may vary depending on the degree of cellularity and myxoid component (8, 9).. the mass may be mistaken for sarcoma due to its clinical presentation as a rapidly growing mass. radiograph may demonstrate a soft tissue mass, which is rarely calcified (8, 9). mr imaging is non - specific, showing equal to or slightly higher signal intensity to muscle on t1wi, intermediate to hyperintensity on t2wi, and diffuse or peripheral enhancement after gadolinium administration. features that may suggest the diagnosis of the lesion are linear extensions along the superficial fascia and mild surrounding edema (9). malignant soft tissue tumor of the hand and wrist are extremely rare. because they tend to be rather slowly growing, and are generally small at presentation, the lesion may be misdiagnosed as a benign tumor (8). hence, calcifying aponeurotic fibroma should also be distinguished from the most common malignant soft tissue tumors in the hand and wrist, particularly in the presence of intratumoral calcifications, including epithelioid sarcoma, synovial sarcoma, and undifferentiated pleomorphic sarcoma (ups) (8 - 10). epitheliod sarcoma usually occurs in the flexor aspect of the upper extremity, and commonly affects young adults in the second to fourth decade of life. there can be a prominent hemorrhage component resulting in a heterogeneous signal on t1wi and fluid - fluid levels. two third of the tumor occurs in the lower extremities, particular near large joints. the tumor has relatively well - defined margins and can demonstrate fluid - fluid levels due to hemorrhage. mr signal of the tumor is mostly isointense to muscles with areas of high signal intensity consistent with hemorrhage on t1wi, and markedly heterogeneous signal intensity on t2wi (9, 10). ups is a tumor formerly known as malignant fibrous histiocytoma, and is the most common type of soft tissue sarcoma in adults, but is not common in the hand and wrist (10). ups typically presents as a painless, circumscribed, multinodular, lobulated tumor with degeneration. calcifications or ossifications within the tumor can be seen in 5 - 20% of cases, usually curvilinear or punctuate, at the periphery of the lesion. mri signal intensity is nonspecific, depending on the cellularity and myxoid content of the lesion and on the presence of hemorrhage, necrosis, and/or calcification.. the main locations of extraskeletal myxoid chondrosarcoma are the deep soft tissues of the proximal extremities. the most common location of extraskeletal osteosarcoma is the thigh (10). in summary, we report a rare case of relatively densely calcified calcifying aponeurotic fibroma in an elderly patient with radiographic and mri features which have rarely have been described in the radiology literature. because the recurrence rate of this tumor is high, accurate preoperative diagnosis and complete excision are important to prevent the recurrence of the tumor after surgical removal. clinical, radiographic and mr imaging features of calcifying aponeurotic fibroma can be helpful in the preoperative differential diagnosis, and mr imaging is also helpful to evaluate the extension of the mass. | calcifying aponeurotic fibroma is a rare, benign fibroblastic tumor. the lesion has a propensity for local invasion and a high recurrent rate. therefore, accurate preoperative diagnosis and complete excision are important to prevent the recurrence of the tumor after surgical removal. however, radiographic and magnetic resonance imaging findings of calcifying aponeurotic fibroma have been extremely rarely described in the radiology literature. thus, we report a rare case of calcifying aponeurotic fibroma affecting the dorsal wrist in a 67-year - old man, describe radiographic and mr findings, and discuss the differential diagnosis of the tumor. |
their aggressiveness added to their high tendency to recur, provoke real diagnostic and therapeutic challenges for the dental practitioner and make a postoperative follow - up over the years indispensable. in this report, we present a case of a seven - year - old girl presented with a swelling in the face at the upper right maxillary region. after clinical, radiological, and histopathological examinations the lesion was surgically excised and followed up for two years with no evidence of recurrence. juvenile ossifying fibromas (jofs) are rare benign fibro - osseous tumors (1). they usually appear under the age of fifteen (2), and concern the facial bones with possible intracranial and orbital extensions (3). generally, jofs are asymptomatic, aggressive and of osteolytic nature (4). due to their high recurrence rate, early diagnosis, suitable treatment and a long - term follow - up are essential (5). histologically, and like in all the ossifying fibromas, the normal bone is replaced by a fibrous cellular stroma containing mineralized bone trabeculae and cementum - like material (4, 6). according to their prototype of mineralization, jofs have been classified into two types : the psammomatous characterized by small uniform spherical ossicles resembling psammoma bodies located mainly around paranasal sinuses and orbits, and the trabecular distinguished by fibrous trabeculae and usually affecting the jaws (4). this report describes a case of a maxillary trabecular jof of a seven - year - old girl removed surgically and followed up for two years without evidence of recurrence. a seven - year - old girl presented to our specialized pediatric dentistry office referred from her dentist for a persistent firm swelling localized at the upper right maxillary region. medical and physical examinations revealed a healthy girl with no extra - oral findings except the facial asymmetry. intra - orally, a painless tumefaction of hard consistency obliterating the vestibule of the right maxilla and extending from the tooth # 53 to the distal of the tooth # 16 was detected (figure 1). a well - defined swelling obliterating the vestibule and extending from the tooth # 53 to # 16. the panoramic radiograph, done previously by her dentist, showed a well - defined radiolucent lesion in the right maxilla and absence of the tooth # 54 which was extracted as an attempt of treatment for her condition (figure 2). a panoramic radiograph showing a well - defined radiolucent lesion in the right maxilla and absence of tooth # 54. in order to assess the bone and to evaluate the extension of the lesion, a cone - beam computed tomography (cbct) radiography was requested. the cbct radiograph showed a well circumscribed mixed image, starting distal to the tooth # 53 till the mesial of the # 55, and from the crestal edge till the germ of the permanent canine with a diffuse border containing fine radio - opaque trabeculae with a buccal, palatal, and crestal expansion of the bone ; a disruption of the buccal bone was also noticed. a cbct radiograph showing a mixed image extending from # 53 to # 55 and from the crestal edge till the germ of the permanent canine ; a diffuse border contains fine radio - opaque trabeculae with a buccal, palatal, and crestal expansion of the bone with disruption of the buccal bone. microscopic examination showed a proliferation of fusiform cells with regular nuclei arranged in bundles and storiform pattern associated with multinucleated giant cells. within these irregular strands, mineralized pieces in the form of trabeculae the tumor measuring approximately 1.8 x 2.5 cm was surgically excised under general anesthesia via intra - oral approach. during the procedure, post - operative recovery was without complications, and the patient was discharged from the hospital 48 hours following the surgery. a panoramic radiograph showing new positions of the germs of the teeth # 13, # 14, and # 15. juvenile ossifying fibromas are rare aggressive fibro - osseous tumor arising in the craniofacial bones (2). usually, jofs occur under the age of fifteen without any significant gender predilection (5, 7). clinically, jofs are mostly asymptomatic with slow or rapid development leading to facial asymmetry (8). as for the radiological aspect, jofs can be seen as uni- or multilocular radiolucencies / mixed radiolucencies (9) ; rare root resorption can be observed (4). cone - beam computed tomography assessment may show well - defined sclerotic borders with a variable amount of calcifications (10). histologically, jofs are lesions characterized by cell - rich fibrous tissue with giant cells and bands of cellular osteoid trabeculae (11). one is characterized by spherical ossicles, the psammomatoid, and the second less commonly reported, the trabecular, distinguished by trabeculae osteoid and woven bone as seen in our case (4). many bone pathologies usually found in the region may constitute a differential diagnosis challenge for jof. fibrous dysplasia remains the most prominent condition (1, 2, 12) ; it can be ruled out as it typically shows normal marginal bone with less cellular stroma and an important amount of lamellar bone instead of woven one (13). cemento - ossifying fibroma, a histological variant of jof, can also be considered ; however, giant cells which are evidently found in jofs are not present in cement - ossifying fibromas (14). cementoblastoma, osteoblastoma, osteosarcoma, and others entities must be separated as well when diagnosing jofs (1, 12) ; this type of separation must be made with the aid of clinical, radiological and histological examinations. complete resection, conservative local excision, or enucleation with curettage, are among the treatment techniques (15 - 17). abuzinad and alyamani (2) suggested that conservative management with less aggressive approach must be considered the treatment of choice of jofs. the same was proposed by slootweg and mller (18) who recommended conservative surgery ; for them, there are no differences in the results between the jofs that had limited surgical excision and those removed with major surgery. on the other hand, waldron (19) stated that the local excision / curettage of the lesion is the best treatment of jofs. however, many other authors reported a high recurrence rate after conservative or mini - invasive treatment (in 3056% of cases) (15, 20, 21), and thus, a complete surgical resection remains the preferred line of treatment (1, 5, 12, 22). it is to be noted that whatever the surgical technique was, a long - term postoperative surveillance is essential (1, 2, 7, 12, 23). in our case, we have presented a trabecular jof localized in the right maxilla of a seven - year - old girl. after histological and radiological confirmations, a complete surgical resection was performed with no recurrence till date. juvenile trabecular ossifying fibroma is a rare tumor with a rather high risk of recurrence. a careful evaluation of the clinical, radiological, and histological components of this lesion is needed to surmount the diagnostic and therapeutic challenges connected with it. furthermore, its early diagnosis and adequate treatment consisting in a complete surgical excision followed by long - term follow - up of the patient is indispensable. | introduction : juvenile ossifying fibromas are uncommon benign tumors. their aggressiveness added to their high tendency to recur, provoke real diagnostic and therapeutic challenges for the dental practitioner and make a postoperative follow - up over the years indispensable.case report : in this report, we present a case of a seven - year - old girl presented with a swelling in the face at the upper right maxillary region. after clinical, radiological, and histopathological examinations the diagnosis of trabecular juvenile ossifying fibroma was made. the lesion was surgically excised and followed up for two years with no evidence of recurrence. |
forkhead box m1, foxm1, is a transcription factor of the forkhead family, which is overexpressed in a large number of human cancer 1. foxm1 regulates the expression of genes involved in the cell cycle progression and the proper execution of mitosis. the genes that are activated by foxm1 initiate the g1/s and g2/m transition and s and m phase execution. it was identified as one of the most commonly overexpressed proteins in solid tumors based on microarray data 2. it is also shown to be involved in the metastatic and angiogenic progression of cancer. the expression of foxm1 is increased by oncogenic proteins such as c - myc, akt, h - ras, etc. conversely, the expresion of foxm1 is downregulated by tumor - suprressor such as pten, rb, p53, etc. as a result nucleophosphomin (npm)1 is a universally expressed chaperone phosphoprotein associated with the cell cycle regulation. it shuttles between the cytoplasm and the nucleus, however it mostly exists in the nucleolus 4. the expression of npm increases following mitogenic stimuli and it is involoved in proliferation and regulation of growth of many cancers. it can interact with a several cellular proteins in various parts of cells, such as p53, foxm1, etc. affecting their stabilty and activity5, 6. arf is a tumor suppressor protein transcribed from the alternate reading frame of the ink4a / arf locus (cdkn2a). the expression of arf leads to inhibition of aberrant cell cycle progression by direct inhibition of mdm2 (negative regulator of p53). arf mediated inhibition of mdm2 leads to activation of the p53 transcription factor resulting in cell cycle arrest or apoptosis following dna damage and or oncogenic activation. it is also been demonstrated that arf can initiate tumor suppressive function independent of the p53 status. arf is inactivated in many types of cancer and as a result the physiological expression level of arf (p14) is usually very low in tumor cells 8. here, we will provide evidence that all these three proteins stabilize each other in cancer cells and suppression of arf and npm1 could be partially explained as a result of foxm1 initial suppression by proteasome inhibitors. the cells were maintained in dmem medium supplemented with 10% fetal bovine serum (atlanta biologicals) and 1% penicillin - streptomycin (gibco). hela cells were transiently transfected with a control (aacaguc g cguuugcgacugguu) small interfering rna (sirna) and sirna specific to foxm1 (ggaccacuuucccuacuuuuu) or arf (cgcggaaggucccucagac) synthesized by sigma. 100 nm of sirna duplexes were transfected into cells using lipofectamine 2000 (invitrogen) according to the manufacturer 's recommendation for 48 hours, following which the expression of foxm1, npm and arf were analyzed by western blotting. transient transfection with 5 g of plko1 or shrna#70 from the trc (thermo scientific library) 5 was carried out for the transient npm knockdown. the cells were harvested and lysed following the transient transfection by using ip buffer (20 mm hepes, 1% triton x-100, 150 mm nacl, 1 mm edta, 1 mm egta, 100 mm naf, 10 mm na4p2o7, 1 mm sodium orthovanadate, 0.2 mm pmsf supplemented with protease inhibitor tablet (roche applied sciences)). protein concentration was determined by the bio - rad protein assay reagent (bio - rad). isolated proteins were separated on sds - page and transferred to pvdf membrane (millipore). immunoblotting was carried out with antibodies specific for foxm1, npm and arf purchased from santa cruz biotechnology. the gel analysis tool was used to obtain the absolute intensity (ai) for each experimental band of foxm1, npm and arf followed by transient knockdown of arf, npm and foxm1. the relative intensity (ri) was calculated for each experimental band by normalizing the experimental ai to the corresponding loading control (actin). the cells were maintained in dmem medium supplemented with 10% fetal bovine serum (atlanta biologicals) and 1% penicillin - streptomycin (gibco). hela cells were transiently transfected with a control (aacaguc g cguuugcgacugguu) small interfering rna (sirna) and sirna specific to foxm1 (ggaccacuuucccuacuuuuu) or arf (cgcggaaggucccucagac) synthesized by sigma. 100 nm of sirna duplexes were transfected into cells using lipofectamine 2000 (invitrogen) according to the manufacturer 's recommendation for 48 hours, following which the expression of foxm1, npm and arf were analyzed by western blotting. transient transfection with 5 g of plko1 or shrna#70 from the trc (thermo scientific library) 5 was carried out for the transient npm knockdown. the cells were harvested and lysed following the transient transfection by using ip buffer (20 mm hepes, 1% triton x-100, 150 mm nacl, 1 mm edta, 1 mm egta, 100 mm naf, 10 mm na4p2o7, 1 mm sodium orthovanadate, 0.2 mm pmsf supplemented with protease inhibitor tablet (roche applied sciences)). protein concentration was determined by the bio - rad protein assay reagent (bio - rad). isolated proteins were separated on sds - page and transferred to pvdf membrane (millipore). immunoblotting was carried out with antibodies specific for foxm1, npm and arf purchased from santa cruz biotechnology. the gel analysis tool was used to obtain the absolute intensity (ai) for each experimental band of foxm1, npm and arf followed by transient knockdown of arf, npm and foxm1. the relative intensity (ri) was calculated for each experimental band by normalizing the experimental ai to the corresponding loading control (actin). it has previously been shown that foxm1 interacts with npm1 5 and arf 9 and that arf interacts with npm1 10. additionally, it has also been demonstrated that the knockdown of npm leads to a down - regulation of foxm1 (bhat, 2011 } and arf 11 in various cell types. in the previously published studies we have demonstrated that proteasome inhibitors that usually stabilize majority of the cellular proteins by inhibiting their degradation paradoxically suppress expression of foxm1, npm1 and arf proteins 12, 13. we have also shown that proteasome inhibitors to some extent suppressed the mrna of npm1 and arf 13. in order to evaluate the role of the interaction between foxm1, npm and arf proteins on their stability in hela cells we used rna interference. the use of hela cells was carried out in this study as it is known to express all three proteins 13. first, we depleted the cells of foxm1 using foxm1 sirna and evaluated the expression of npm and arf. we observed that transient foxm1 knockdown induced suppression of arf and npm protein levels (fig 1a). in order to validate this observation we repeated this study in three separate experiments. a 70% inihibition of npm and almost 90% inhibition of arf protein was observed following treatment with 100 nm foxm1 sirna for a 48 hour treatment period (fig 1b). a greater suppression of arf as compared to npm1 could be explained by the fact that npm is abundantly expressed in most cancer cells. the qualitative and quantitative measurement validated the observation that suppression of foxm1 by sirna leads to downregulation of npm1 and arf proteins. we found that the transient knockdown of npm1 by shrna in hela cells for 48 hours led to the suppression of foxm1 and arf proteins (fig 2a) as has been shown in different cell types previously 5, 11. we also measured the percent of supresssion of foxm1 and arf proteins by densitometric analysis. we found that transient knockdown of npm1 led to inhibtion of up to 80% of arf and foxm1 expression as compared to control (plko1) transfected cells (figure 2b). finally we decided to examine how the suppression of arf by rna interference could affect foxm1 and npm1 protein expression. the hela cells were transiently transfected with 100 nm arf sirna for 48 hours following which the expression of foxm1 and npm proteins were investigated. we observed that a transient inhibition by arf - sirna led to the inhibition of npm and foxm1 proteins (fig 3a). it was seen that suppression of arf by rnai inhibited up to 65% of npm and up to 85% of foxm1 expression (fig 3b). interestingly, we found that a tumor suppressor protein arf stabilizes two proteins with oncogenic properties, npm1 and foxm1. our data suggest that, in cancer cells, depletion of any one of foxm1/npm1/arf protein leads to down - regulation of the two other proteins. | arf, npm and foxm1 proteins interact with each other in mammalian cells. we showed previously that proteasome inhibitors suppress not only foxm1 expression, but also the expression of arf and npm proteins. using rna interference we found that the depletion of each of these proteins by rnai in human cancer hela cells leads to down - regulation of the two other partners, suggesting that these proteins stabilize each other in human cancer cells. since the suppression of foxm1 is one of hallmarks of proteasome inhibition, suppression of arf and npm by proteasome inhibitors may be explained in part as a secondary effect of downregulation of foxm1 that modulate stability of arf and npm1 proteins. |
since the introduction of laparoscopy, surgeons have been intrigued by the idea of reduced port placement and smaller scars for their patients. the potential gain for the patient it was thus inevitable for surgeons performing robot - assisted laparoscopic operations to attempt to attain that goal, and single - port surgery was born. nevertheless, robotic single - port surgery requires the use of a novel robotic platform which increases cost and still needs to prove its efficacy and safety. in the following, we describe a novel technique, in which both bedside surgical instruments are introduced simultaneously through the valveless airseal system trocar (surgiquest, milford connecticut, usa). this novel technique may provide a substitute for the 5-port configuration classically used during robot - assisted laparoscopic surgery on the upper urinary tract. from february 2015 through june 2015, 11 robot - assisted laparoscopic partial nephrectomies were performed with this novel technique. the patient was positioned in a modified flank position with the diseased side up and then flexed using the table break at the level of anterior superior iliac crest. the outward - facing arm was tucked to the side. the robotic platform used was davinci xi (intuitive surgical inc., the first one was placed lateral to the rectus sheath and 8 cm cranial to the camera port. a 12-mm assistant port was placed on the midline, 5 cm cranial to the umbilicus. a suction device and the grasping forceps were both used through the airseal system trocar, either alone or simultaneously in all cases, obviating the need for an additional trocar for the fourth robotic arm. a long suction - and - irrigation device was preferred, to minimize instrument collision with the atraumatic grasping forceps (figure 2, 3). there was no decrease in intra - abdominal pressure during insertion of both instruments through the airseal trocar. of the 11 patients enrolled in the study, 8 were male and 3 female. mean age and body mass index (bmi) of the patients was 55 y (14.6) and 29.2 (6.85), respectively. operative time was 132.2 minutes (37.17) with an estimated blood loss and ischemia time of 103.6 ml (65.92) and 16.7 minutes (9.52), respectively. intra - abdominal pressure did not decrease by passing the instruments through the airseal trocar and was stable at 10 mm hg. the surgical experience with laparoscopy increased over time, and one of the new goals of laparoscopic surgery became to accomplish the same good results through fewer ports and incisions. this challenge led to the establishment of the laparoendoscopic single - site surgery (less), in which a novel multichannel access system is used that allows for simultaneous passage of several laparoscopic instruments through 1 incision. the size of the fascial incision is determined by the number and size of instruments that the port can accommodate and varies, depending on the specific operative indication. even though this technique is promising and its results are not inferior to standard laparoscopic surgery, challenging ergonomics and instrument clashing make this technique demanding, even for the experienced laparoscopic surgeon. robotic technology responded to this demand with a newly modified surgical platform developed to fulfill the need of robotic (r)less procedures. results of the initial experience with this system were promising, but its safety and its feasibility are yet to be established. r - less procedures require a new robotic surgical system and novel instruments, further increasing cost. there are no clear proven major advantages over the conventional robotic procedures, at least up to date. one less incision could potentially decrease postoperative pain (even though proving this assumption would be challenging) and provide slightly improved cosmesis. inspired by this notion, we developed a technique that uses 4 ports only (3 robotic and 1 assistant). from the assistant port the role of the fourth robotic arm is to assist during renal hilar dissection and manipulation of the kidney during tumor resection. in the present technique, the assistant managed to help the console surgeon successfully during these steps of the procedure, and hence the requirement for the fourth robotic arm was obviated. use of a long suction device was the key to preventing instruments from clashing as they passed through the same port. an interesting observation with the 4-port technique was that pneumoperitoneum was always stable, even with two 5-mm instruments inside the airseal trocar. although one 10-mm instrument through the airseal port compromises pneumoperitoneum, simultaneous introduction of two 5-mm instruments did not have the same impact. our median operative time of 132 minutes and warm ischemia time of 16.72 minutes are parallel to those reported in the literature (surgical time, 83265 minutes ; warm ischemia time, 2131minutes). the present technique was not associated with any complication (clavien - dindo grade 2), proving it to be potentially safe. it would appear to be easily reproducible, because it involves the standard triangular placement of the ports, an arrangement familiar to most upper urinary tract surgeons. there were no upper pole tumors among the 11 patients ; surgical excision of an upper pole kidney tumor may require an addition fourth robotic arm. this technique is also associated with some compromise of the console surgeon 's autonomy, because he controls one less robotic arm. finally, no pain score was recorded, and hence no conclusion may be drawn about the potential positive impact of fewer ports on postoperative pain. the goal of this study was mainly to assess the feasibility and safety profile of this technique. further studies comparing results with the standard 5-port approach would be necessary to prove whether it has any clinical advantages. reducing the number of ports by using the suction device and grasping forceps through the airseal system and its valveless trocar may provide a substitute for the 5-port configuration normally used during robot - assisted laparoscopic surgery on the upper urinary tract. the technique appears to be feasible and safe and thus may be used in selected cases of robotic - assisted partial nephrectomy. | background and objective : robotic upper urinary tract surgery is in most of the cases performed utilizing a standard 5 port configuration. fewer ports can potentially produce a less invasive operation. taking in consideration the above we report a novel technique for robot assisted laparoscopic partial nephrectomy utilizing fewer ports and we test its feasibility and safety profile.methods:data on 11 robot - assisted laparoscopic partial nephrectomies performed by using our technique from february 2015 through june 2015 were retrospectively analyzed. the robotic platform used was davinci xi (intuitive surgical, inc., sunnyvale, california, usa) with a 3-arm setup. the airseal system (surgiquest, milford, connecticut, usa) was used as a port allowing simultaneous introduction of 2 instruments for the bedside surgeon, obviating the need for an additional (fourth) robotic arm. a long suction - and - irrigation device and atraumatic grasping forceps were used. both instruments were introduced through the trocar of the airseal system, making simultaneous introduction and use possible. we preferred the long suction - and - irrigation device, because it minimizes collision of the instruments.results:mean age and bmi of the patients were 55 14.6 y and 29.18 6.85, respectively. seven tumors were on the right side and 4 were on the left. the mean size of the tumors was 32.45 mm (11.31). surgical time was 132.2 minutes (37.17), with an estimated blood loss and ischemia time of 103.63 ml (65.92) and 16.72 minutes (9.52), respectively. one patient had postoperative bleeding that was resolved without transfusion. the median hospitalization period was 3.9 d (0.53). loss of intra - abdominal pressure was not observed, and pressure was stable at 10 mm hg.conclusion:the airseal system and its valveless trocar eliminated the need for an additional port placement in our series. the technique is feasible, safe, and reproducible ; therefore, it may be implemented in selected cases of robot - assisted partial nephrectomies. |
hepatocellular carcinoma (hcc) is the sixth most common cancer worldwide and the third most common cause of cancer - related mortality, with 746,000 deaths each year ; the estimated survival of patients with advanced hcc is < 10%.1,2 nearly 80% of hcc cases occur in the developing world ; however, the incidence of hcc in the usa has increased over the last several decades with the incidence rising from 3.1 per 100,000 persons in the 1990s to 5.1 per 100,000 in the 2000s.3 the increased incidence of hcc in the western hemisphere is most directly attributable to the hepatitis c virus (hcv) infection, which accounts for approximately one - half of hcc cases.4 while some data suggest that the incidence of hcc will continue to rise, based on the increased prevalence of hcv in the usa, other data imply that the peak of the hcv epidemic in the west has been reached.5,6 the metabolic risk factors for hcc, such as diabetes and obesity, have also resulted in an increased incidence of nonalcoholic fatty liver disease and, in turn, nash has been associated with the increased incidence of primary liver cancer in the usa.6 in the eastern hemisphere, hepatitis b virus (hbv) continues to be a main cause of hcc, due to the endemicity observed in adults of this geographic region and the paucity of vaccination programs before the 1980s.4,7 however, the institution of vaccination campaigns has begun to decrease the incidence of hbv infection seen in children.7 similarly, the ubiquitous spread of hbv and hcv continues to drive the incidence of hcc in eastern asia and sub - saharan africa.4 while resection, ablation, or transplantation is indicated for patients with early - stage disease, the treatment options for patients with advanced hcc are limited. patients with early - stage disease who are treated with resection or transplantation can expect a 5-year survival of 40%75% ; in contrast, survival among patients with locally advanced disease is dismal, with a 5-year survival ranging from 5%13%.3,8 chemotherapeutic options are available ; however, they provide limited treatment response.9 for example, studies using cytotoxic agents, such as doxorubicin or gemcitabine plus oxaliplatin, show a response rate as low as 18% and combination therapy with cisplatin, interferon - alpha-2b, doxorubicin, and 5-fluorouracil (piaf) report response rates of 26%.9 targeting signaling pathways for therapeutic use in oncology has begun to take root over the last decade. biologic agents, such as sorafenib, bevacizumab, and erlotinib, add more to the clinicians armamentarium ; however, more options are needed for patients.9 in a recent phase iii study, the biologic agent sorafenib was shown to increase median survival and the time to radiologic progression by nearly 3 months for patients treated with sorafenib than for those given a placebo.10 given these data, a better and more fundamental understanding of the genetic and epigenetic mechanisms of hepatocarcinogenesis is needed. this, in turn, may help lead to the discovery of novel treatment regimens for patients with unresectable disease. several groups have reported that the dysregulation of the hippo (hpo) signaling pathway can lead to hcc formation.1117 yap, the downstream effector of the hpo pathway, controls a myriad of protein targets that influence gene expression.16,18 the emergence of the hpo - signaling pathway in hcc development will hopefully further delineate the molecular drivers of hcc while providing the clinician avenues for risk stratification and novel therapeutic strategies. the hpo signaling cascade regulates the expression of genes favorable to cell cycle progression, proliferation, differentiation, and survival.18 the canonical pathway members hpo, warts (wrts), and salvador (sav) were discovered through genetic screens in drosophila melanogaster, while searching for suppressors of tissue growth.16 the origin of the pathway names arises from the hippopotamus - like phenotype observed in d. melanogaster when the members of the pathway are mutated. the pathway plays an integral role in determining organ size and the growth control regulation.16,19,20 core components of the pathway are conserved in mammals and act in a kinase cascade that exhibits a complex network of crosstalk with other important signaling pathways, including the tgf/smad, wnt/-catenin, pi3-kinase / akt, jnk, hedgehog, jak / stat, notch, and apoptotic pathways.18 ultimately, yap is the effector of the hpo pathway and coordinates interactions with these signaling pathways by the induction of gene expression.16,18 the hpo pathway also mediates intercellular signaling responding to external cellular signals, leading to cell contact - driven proliferation and growth.16,18 the individual components of hpo signaling were linked to pivotal intracellular processes prior to the delineation of the pathway. for example, yap was identified as the first ww domain containing protein and was reported to enhance the transcription of genes, serving a constellation of cellular functions while the transcriptional coactivator with pdz - binding motif (taz) was linked to cell differentiation.2126 mammalian sterile 20-like kinase 1/2 (mst1/2) was identified as a promoter of apoptosis via the histone modification of h2b and via foxo - mediated apoptosis following oxidative stress.27,28 concurrently, there are reports that implicate the scaffolding protein sav or ww45 as having a role in cell cycle entry and exit.29 finally, the large tumor suppressor homolog 1/2 (lats1/2) was observed to regulate mitosis and cytokinesis by modulating cdc2, limk1, and zyxin.3032 the hpo core signal cascade commences with the mst1/2-mediated phosphorylation of the lats1/2 kinases (figure 1). the adaptor protein ww45 couples the mst1/2lats1/2 interaction, increasing the efficiency of the reaction, while the mps one binder kinase activator - like 1a and 1b (collectively, mob1) enhances the kinase activity of lats1/2. these series of events culminate in yap phosphorylation, inactivating the oncoprotein s ability to induce target gene expression. the protein phosphorylation is a central tenant of transcriptional regulation acting to sequester proteins inside the cytoplasm via coupling reactions by the 14 - 3 - 3 proteins.33 phosphorylated yap is competent to bind these 14 - 3 - 3 proteins, which prevents its intranuclear transport, thereby rendering it powerless over its transcriptional targets.25,34 yap acts as a transcriptional coactivator, which partners with transcription factor tead to initiate the transcription on a multitude of genes involved in cell proliferation, cell contact, and apoptosis, including c - myc, survivin, sox4, cyclin d, and ctgf.18,35 alternatively, yap phosphorylation results in proteasomal degradation, eliminating its ability to bind protein targets and hindering its transcriptional coactivator role.36 neurofibromatosis 2/merlin (nf2/mer) and kidney- and brain - expressed protein are upstream components of the hpo pathway and act by translating signals from the cell membrane to mst1/2.16,37 the nf2/mer inactivation results in carcinogenesis, behaving antagonistically to yap as yap activation leads to the development of cancer.37 importantly, yap appears to be an effector of nf2/mer as deletion of yap in nf2-deficient mice abrogates the uncontrolled cell growth typically observed in the nf2-deficient phenotype.37 the genetic alterations and epigenetic events in the hpo kinase cassette drive tumor progression in cancer cell lines, animal models, and human cancers. while the genetics of the tumor suppressor members of hpo signaling play a role, the epigenetic changes through the cpg island methylation and microrna (mir) also prove to be the prevalent mechanisms of tumorigenesis.16,38,39 the hpo pathway is pivotal in the control of cell growth and dysregulation of the signaling cascade in mammals, leading to carcinogenesis. the mammalian hpo orthologs epigenetic silencing with the hypermethylation of the cpg island promoter of mst1/2 is observed in human soft tissue sarcomas, resulting in the alteration of the sav rassf1hpo tumor suppressor pathway.40 in colorectal cancer, the cytoplasmic loss of mst1/2 is predictive of more aggressive tumor biology and is associated with a higher t stage and n stage, as well as vascular invasion and worse overall survival (p<0.05).41 the mechanism of this phenomena in hcc remains unknown as downregulation of mst1/2 was not found to be related to promoter methylation.41 interestingly, in prostate cancer, the attenuation of the mst1/2 expression as manifested through the activation of the pi3k / akt / mtor pathway has been associated with the more advanced stages of disease.42 ww45, the human sav ortholog, helps stabilize hpo, promoting the phosphorylation of lats1/2, thereby regulating yap / taz and promoting cell cycle exit and apoptosis.16 ww45 maps to the chromosomal region 14q1314q23, a locus that is frequently deleted in human renal cell carcinoma, ovarian cancers, and mesothelioma, thereby substantiating its importance in regulating carcinogenesis.43 along with the large multigenic deletions of ww45, various human cancer cell lines, including those of colorectal, renal, and ovarian lineage harbor single base substitutions in the ww45 gene.29 tumorigenesis is driven by the mutations in sav, leading to elevated cyclin e and drosophila inhibitor of apoptosis 1 levels, resulting in delayed cell cycle exit and impaired apoptosis in d. melanogaster.29 lats1/2, the mammalian ortholog of wrts, operates as the molecular off switch to yap, as its phosphorylation by lats1/2 downregulates gene expression signatures associated with proliferation and survival.16 two distinct lats1/2-mediated mechanisms result in yap inactivation via ser-127 phosphorylation - mediated spatial regulation and the ser-381 phosphorylation - mediated temporal regulation.36 the first mechanism precipitates yap phosphorylation at the ser-127 residue, leading to the 14 - 3 - 3-protein binding, sequestration from the nucleus, and the inhibition of gene expression. moreover, yap function is alternatively mitigated via ser-381 phosphorylation by casein kinase 1 that ushers the polyubiquitination of yap, followed by the e3 ubiquitin ligase degradation.36 the epigenetic modifications are implicated in the dysregulation of lats1/2 activity. for example, the hypermethylation of the lats1/2 promoter induces carcinogenesis and is identified in human astrocytomas and breast cancers.44,45 in astrocytomas, the promoter hypermethylation of lats1/2 occurred in 63.6% and 71.5% of cpg islands, respectively, with a corresponding decrease in the messenger rna (mrna) expression, demonstrating the critical role that lats1/2 plays in cell cycle modulation.44 breast cancers also exhibit this hypermethylation pattern of the lats1/2 promoter regions (56.7% ; 50.0%) and correlate to a biologically aggressive phenotype.45 recent reports indicate the novel microrna - mediated silencing of lats1/2 by mir-372 and mir-373 leads to the development of human testicular germ cell tumors by regulating the p53 pathway.46 the hpo pathway member mob1 functions to strengthen the kinase activity of lats1/2 following phosphorylation by mst1/2. this increased kinase activity serves as another decelerating force in yap / taz target gene expression thereby inducing growth inhibition and tumor supression.47 genetic variants in mob1 were identified in mammary- and melanocyte - derived carcinomas resulting in increased cell proliferation and defective apoptosis.47 similarly, evaluation of the colorectal cancer patients revealed significantly lower mrna expression of mob1 and clinicopathologic variables, such as tumor size, lymphatic invasion, and metastasis were more frequent as the mob1 expression declined (p<0.05).48 this same decrement in the mrna expression is reported in non - small - cell lung cancer ; however, the decrease was more pronounced in early stage tumors, indicating an early phase phenomenon in lung carcinoma.49 protein phosphatase-1, a recently identified activator of yap, acts to dephosphorylate the oncoprotein, allowing its entry into the nucleus and transcriptional acceleration of key proliferative genes.50 investigators noted that protein phosphatase-1 inhibition occurred following the administration of okadaic acid, thereby increasing the percentage of phosphorylated yap and abrogating its transcriptional coactivator potential ; this may serve as a possible target for modulating hpo signaling.50 the regulation of yap / taz activation is pivotal to cellular homeostasis as they are the final downstream effectors of the hpo kinase pathway. in their active form, yap / taz possess a strong transcriptional coactivation domain and interact with the dna - binding proteins that influence gene transcription of merlin, smad7, ctnnb1, erbb4, src, abl, cdh1, ppar, runxs, p73, pax3, pax8, ttf-1, and many more (figure 2).18,22,25 in healthy tissue, yap / taz phosphorylation acts a molecular brake triggered by cell cell contact. in the functional cells, this cell density - dependent activation of the hpo kinase cascade is required for contact inhibition in mammalian cell lines, corroborating its role in controlling proliferation and, ultimately, organ size.51 important to invasive cancers, the taz induction was reported to promote the epithelial mesenchymal transition by forming spindle - shaped cells in the culture and upregulation of vimentin, n - cadherin, and fibronectin ; the epithelial mesenchymal transition was mitigated by the hpo pathway tumor suppressor lats1/2.34 the activation of the yap / taz - tead transcription factor complex initiates carcinogenesis ; in addition, amplification of the yap gene has been reported in a plethora of human and murine tumors.16 for example, the chromosomal amplification of the 11q22 region has been implicated in various tumor types and is home to the yap gene locus ; examination of yap expression in resected colonic adenocarcinoma, lung adenocarcinoma, and ovarian serous exhibited intense and diffuse signals in both the nuclear and cytoplasmic compartments.21,52 in the hcc cell lines, there is a marked increase in yap expression as compared to the human hepatocyte lines and the short hairpin ribonucleic acid - mediated knockdown of yap resulted in a 50% decrease in cell viability.53 in addition, yap has been reported to be linked to initiation and recurrence in medulloblastomas, with expression upregulated in cells following irradiation especially in the perivascular niche.54 furthermore, yap is known to be overexpressed in the areas of the brain responsible for harboring progenitor cells ; in contrast, little immunolabeling of yap has been observed in the cerebral cortex, thereby suggesting that yap may be important in brain development.55 yap expression is enhanced in brain cancer and loss of function results in decreased cell growth in vitro.55 the association between elevated bile acids and carcinogenesis is well - known, and recent reports indicate that this process is yap mediated.56 bile acids act as upstream regulators of yap via the induction of the scaffold protein iqgap1, and patients with known biliary stasis exhibit increased expression of yap and iqgap1.56 interestingly, sonic hedgehog induces yap expression and promotes its nuclear transport in neuronal stem cells, representing a novel effector for sonic hedgehog signaling.54 investigators have also observed that coamplification of the birc3 gene, an inhibitor of apoptosis and a mammalian homolog of the drosophila inhibitor of apoptosis 1 located adjacent to yap, confers a selective advantage to primary lung carcinomas, suggesting synergistic effect on tumorigenesis as seen in d. melanogaster.57 consonant with the fundamental roles of hpo signaling, alterations of mst1/2, sav, lats1/2, and nf2/mer lead to excess proliferation and in some cases carcinogenesis in the mammalian liver. across the globe, hbv is the predominant etiology precipitating hcc, and yap activation may play a role. yap expression is markedly elevated in samples of hbv - induced hcc, hbv - infected cell lines, and hbv x protein (hbx) transgenic mice.58 hbx upregulates yap by the cre - dependent hbx - binding of the yap promoter with subsequent activation of yap target genes. these data further confirm the importance of the hpo pathway in cancer and, possibly, offer novel methods of treating patients with known hbv infection to prevent progression to hcc. for example, the transgenic mst1/2 double - knockout mice exhibit abrogation of yap phosphorylation and a profound nuclear accumulation of yap.59 other studies have shown that the specific deletion of mst1/2 leads to the development of multiple foci of liver tumors in mice by 56 months.13 the importance of deleting both mst1/2 has been demonstrated, as deleting either mst1 or mst2 alone did not potentiate liver size or the number of tumor foci in the liver.13 these data strongly suggest a pivotal role of mst1/2 in yap activity. in fact, nearly 30% of human hccs have a loss of yap phosphorylation, due to the defective mst1/2 activity culminating in carcinogenesis.59 introducing the active form of mst1/2 or using upstream effectors of mst1/2 activation may decrease the gene expression of the target genes that can lead to uncontrolled cell growth, culminating in hcc formation. the attenuation of the hpo pathway also results after the liver - specific knockout of ww45. this leads to progressive liver enlargement secondary to the aberrant expansion of hepatic progenitor cells resulting in the development of hcc.13,60 these data indicate the importance that sav plays critical role in differentiation of progenitor cells. delayed proliferation arrest is mechanism, not accelerated proliferation.61 there is, however, a requirement for mst1/2 deletion concurrent with sav1 deletion to see the downstream attenuation of the hpo pathway. the levels of phosphorylated yap and lats1/2 are not affected in the sav1 mutants alone, suggesting that the mst1/2 kinase activity may be a more prominent driver of carcinogenesis.13 the sav1-deficient mice do develop tumors ; however, the course of tumor development is more indolent with a mean tumor onset of 14 months.13 the liver - specific inactivation of nf2/mer results in a dramatic expansion of hepatic stem cells without changing differentiated hepatocytes.62 the analysis of lats1/2 and yap / taz phosphorylation in mst1/2-or sav - livers has exhibited mixed results concerning the need for mst1/2 or sav in lats1/2 phosphorylation.59,61 the pathway also appears to be specific to hepatic tissues, as mst1/2 are not required for yap phosphorylation in mouse fibroblasts.59 this tissue specific divergence from the defined pathway points to the presence of noncanonical members of the cascade that remain to be defined. the biologic importance of hpo signaling in hepatocellular carcinogenesis is reinforced by studies of yap in transgenic murine models. the harmonious orchestration of cell proliferation and apoptosis is crucial in attaining proper organ size and maintaining cellular homeostasis. in a transgenic murine model, yap induction resulted in uniform liver expansion and after 8 weeks of yap induction, numerous nodules appeared throughout the hepatic parenchyma (figure 3).12 the nodules were composed of discrete areas of proliferative hepatocytes that compressed the adjacent parenchyma and displayed the pathognomonic features of hcc. similarly, the liver - specific deletion of both mst1/2 in hepatocytes results in significant liver growth due to uncontrolled cell proliferation.13 after 6 months, mst1/2 conditional mutant livers exhibited numerous liver tumors and an increase in the fraction of progenitor cells, demonstrating that the combined activities of mst1/2 act as redundant tumor suppressors in hepatocytes.13,14 these data prove that the transgenic overexpression of yap leads to the dysregulation of organ size and eventual hepatocellular carcinoma.12 the clinical significance of yap / taz overexpression was investigated in 177 patients with hcc.63 yap was expressed in 62.1% of the hcc tumor specimens compared with 9.0% in matched normal specimens (p<0.0001). most accumulation was noted in the nucleus of tumor cells and overexpression of yap was significantly associated with a higher serum -fetoprotein level (p<0.001) and worsening tumor grade (p=0.021).63 nuclear yap was also an independent predictor of hcc - specific disease - free survival (hazard ratio, 1.653 ; 95% confidence interval 1.0812.528 ; p=0.02), implicating yap expression in early disease recurrence.63 the increased yap induction was also predictive of the overall survival (hazard ratio, 2.148 ; 95% confidence interval 1.2553.677 ; p=0.005) with yap - negative tumors demonstrating a 5-year survival of 58% versus 36% for patients with yap - positive tumors.63 survivin is a member of the antiapoptotic protein family and also has been shown to be correlated with a poor prognosis among patients with hcc.64 recently, yap expression has been shown to be increased along with survivin in patients with hcc. it appears that yap actually potentiates oncogenic effects on the cell via survivin upregulation resulting in carcinogenesis.64 many patients with hcc present with advanced disease. unfortunately, hcc is highly resistant to most systemic cytotoxic chemotherapy agents, leaving the oncologist with limited treatment options.65 resistance to doxorubicin is observed in many patients with hcc, and the yap connection to drug resistance has recently been elucidated.66 yap induction in hcc cell lines leads to increased resistance to doxorubicin - induced apoptosis ; whereas, suppression of yap expression via rna interference has been noted to reverse yap - augmented chemoresistance.66 these effects are mediated through the map kinase pathway activation rather than an akt - driven mechanism of chemoresistance.66 this mechanism of chemoresistance is supported after treatment with the mek1/2 inhibitor u0126 prior to doxorubicin administration, which significantly decreases cell viability in yap overexpressing hcc lines.66 identifying patients with yap - induced chemoresistance may alter and enhance the efficacy of current drug regimens, leading to improved clinical outcomes in hcc. the pharmacologic targeting of aberrant yap induction may further elucidate the hpo - signaling network and lead to the development of novel drugs against yap - driven human cancers.67 previous studies demonstrate that yap s oncogenic capacity is mediated by tead factors, rendering it pharmacologically targetable.35 transgenic murine models that overexpress yap do not develop hcc when crossed with mice featuring a truncated form of tead2 that lacks its dna - binding domain.67 the abrogation of yap hyperactivity also resulted in mice with nf2/merlin - deficient livers after crossing with mice harboring the defective tead2 gene, further strengthening the therapeutic role of targeting the tead / yap complex.67 using a drug - screening library, the small molecule inhibitor verteporfin was discovered to prevent the physical association between yap / tead by selectively binding yap and changing its conformational structure to preclude any interaction with tead.67 verteporfin administration has been shown to decrease liver size in yap transgenic mice and has no effect on the tissue homeostasis of wild - type mice, highlighting the compound s potential for therapeutic use in patients with hcc.67 verteporfin exhibits several key features that make its clinical implementation for hcc promising. although it was principally designed for use in phototherapy, it does not require photoactivation to bind and to inhibit yap activity. moreover, verteporfin use is safe as it has been successful in mouse models, and the human phase i / phase ii clinical trials reported that verteporfin use is feasible and safe with minimal phototoxicity noted.68,69 these results stress the need for future clinical trials in patients with hcc. hcc remains a disease with a poor prognosis, due to the ineffective therapies for advanced disease. recent studies have further elucidated the inner workings of the hpo - signaling cascade and unmasked this critical pathway s role in developmental and cancer biology. the hpo pathway drives hepatocarcinogenesis, and yap serves as a prognostic marker in hcc. the seminal discoveries of the hpo pathway and yap have opened up a new field of study focused on implementation of biologically active molecules that can alter the aberrant hpo cascade and, ultimately, restore yap target genes to wild - type expression levels. the modulation of the hpo pathway offers hope for future therapies to improve the survival of this devastating disease. one of its targets may serve as a diagnostic marker for early detection in high - risk individuals, as well as a target for improved therapeutics. | hepatocellular carcinoma (hcc) is the sixth most common cancer and the third most common cause of cancer - related mortality worldwide. due to the poor prognosis and limited therapeutic options, there is great interest in further understanding better the molecular underpinnings and potential molecular targets associated with hcc. the hippo (hpo) signaling pathway and yap, its principal downstream effector, represent an innovative area of research in hcc. pioneered in drosophila melanogaster, the hpo cascade controls tissue homeostasis including organ size, cell proliferation, apoptosis, as well as cell - cycle regulation and differentiation. this conserved kinase cascade in mammals depends on central control by the tumor suppressor mammalian sterile 20-like kinase 1/2 (mst1/2). the mst1/2 commences the downstream kinase cascade, ultimately activating the oncoprotein yap and allowing its physical association with downstream targets to enhance the gene expression signatures that are involved in proliferation and survival. alterations in yap expression and defective regulation of other key hpo pathway members, such as mst1/2, salvador, neurofibromatosis and mer (nf2/mer), large tumor suppressor homolog 1/2 (lats1/2), and mps one binder kinase activator - like 1a and 1b (mob1) drive carcinogenesis in animal models. the dysregulation of the hpo pathway resulting in an unchecked activation of yap culminates in the development of a broad range of human tumor types, including hcc. the abrogation of mst1/2-mediated yap phosphorylation permits yap entry into the nucleus in murine models and functions similarly in human hccs. chemoresistance mechanisms displayed by hcc tumors occur in a yap - dependent manner. the hcc specimens exhibit yap overexpression, and yap serves as an independent prognostic marker for disease - free survival and overall survival in patients with hcc. recently, the small molecule inhibitor, verteporfin has been shown to attenuate yap activity in murine models, perhaps offering a novel therapeutic approach for patients with advanced hcc. |
the field of exercise - oncology has burgeoned dramatically in scope and impact since publication of the first scientific papers in the mid to late 1980s.1 several systematic reviews and meta - analyses have evaluated the efficacy of structured exercise treatment in cancer.26 for example, speck.7 identified a total of 66 studies reported as high - quality that examined the impact of exercise treatment in a broad array of oncology scenarios (e.g. differences in histological primary site, stage and treatment) on a total of 60 different primary end points in adults with cancer. intriguingly, despite the degree of heterogeneity in how exercise was being utilized to manipulate physiological adaptation, the nature of the exercise prescription in the vast majority of studies was similar. more specifically, almost all prescriptions followed traditional guidelines consisting of either supervised or home - based endurance (aerobic) training or endurance training combined with resistance training, prescribed at a moderate - intensity (5075% of a predetermined physiological parameter, typically age - predicted heart rate maximum or reserve), for two to three sessions per week, for 10 to 60 min per exercise session, for 12 to 15 weeks. despite the adoption of a relatively homogeneous prescription approach, exercise training was, for the most part, associated with benefit across a diverse range of end points, largely irrespective of the oncology setting.7 on this evidence, it could be argued that a standardized, largely homogeneous exercise prescription that adopts a conventional approach is safe, efficacious, and therefore sufficient. this has resulted in a limited perceived need to elucidate the optimal dose, sequencing, or combination of different training stresses to specifically alter a desired physiological end point in any clinical population, including oncology. however, the significant benefit of generically dosed exercise treatment on heterogeneous end points rather reflects the remarkable pleiotropic physiologic impact of exercise. moreover, the use of generic exercise prescriptions (irrespective of clinical population or primary end point) is masking the full therapeutic promise of exercise treatment. indeed, for more than half a century, exercise training has been continually refined, precisely dosed, and scheduled to minimize injury and optimize human / athletic performance ; the basis of all training prescriptions in this arena adheres to fundamental tenets of human exercise physiology known as the principles of training. these tenets are rarely applied or even considered when designing exercise trials in clinical populations.8,9 accordingly, the purpose of this opinion paper is to provide an overview of the application of these principles in the design and conduct of clinical trials in exercise - oncology research. this paper will focus primarily on application of these principles to aerobic - based training, although the concepts also apply to resistance training or combination training programs. stage 1 objective assessment of response to exercise : an essential prerequisite in the design of all exercise training trials is the objective assessment of baseline physiological functioning of the systems that will be primarily targeted. this generally refers to either the cardiopulmonary responses to exercise (in the context of aerobic training) or the assessment of maximal muscular strength or endurance (in the context of resistance training). the relative strengths, weaknesses, and conduct of the various different assessment tools available to researchers have been reviewed previously.1012 in the context of aerobic training studies, the preferred assessment tool is a cardiopulmonary exercise test (cpet). the use of a cpet offers a number of distinct advantages compared with other assessments. of these, arguably the most important is that cpet provides specific information on patients ' peak rate of oxygen consumption (vo2peak) as well as their submaximal cardiopulmonary responses to exercise, permitting precise tailoring of training to the individual patient. in settings in which cpet is not available, investigators can also use a maximal incremental exercise tolerance test (ett) (without direct vo2 measurement) to determine peak workload and peak exercise heart rate.10,13 in addition, both cpets and etts are performed with 12-lead ecg and blood pressure monitoring, thus, they also provide important pre - exercise training clearance information on the detection of any exercise - induced impairments (e.g. ischemia) or symptom limitations at both submaximal and maximal exercise tolerance.14 the latter information is of critical importance when considering the inclusion of high - intensity (75% of vo2peak) aerobic training sessions in the prescription. stage 2 application of the principles of training : perhaps the most essential principles of training are individualization, specificity, progressive overload, and rest / recovery (figure 1). individualization : the concept of individualization is defined as the customized application of training towards the physiological status of the patient/ subject.15,16 clearly, even within carefully selected homogenous clinical trial cohorts, considerable heterogeneity likely still exists in cardiopulmonary function, lifestyle behaviour, age, prior treatment, concomitant comorbidities, and, of course, genetic predisposition. thus, application of a generic prescription that fails to consider such parameters will most often result in either an under - dosing or over - dosing of exercise treatment. fortunately, there is a broad array of parameters available on which to individualize training. the preferred approach is individualization on the basis of workloads (e.g. treadmill speed and power output) corresponding to a specific percent of vo2peak or peak workload (e.g. 55%, 65%) elicited during each individual patient 's pre - randomization cpet or ett. the corresponding heart rate, blood pressure, and rating of perceived exertion (rpe) directly measured at each percent workload is used to ensure that subjects are training at, or close to, the prescribed intensity during subsequent exercise sessions (figure 2).17 it is important to note that although the absolute training intensity may be different between individual patients, the relative intensity of training is similar, ensuring standardization across patients a critical aspect of clinical trial conduct.specificity : it is widely acknowledged that the physiological adaptations elicited by aerobic training are unique from those caused by resistance training. lesser appreciated, however, is that even within the same exercise modality, there is enormous capacity to manipulate the nature and configuration of the elements of exercise prescription (i.e. frequency, intensity, duration, and time) to confer remarkably distinct physiological adaptations.18,19 the concept of specificity addresses the notion that the selected exercise stress must be specific and targeted to the primary underlying system(s) or pathway(s) known or postulated to underpin the primary end point of interest. for example, in a trial designed to examine the impact of aerobic training on vo2peak in a particular cancer cohort, a critical prerequisite for the design of the optimal exercise prescription is determination of the primary limitation (determinant) to vo2peak ; in other words, is there a cardiovascular, ventilatory, and/or skeletal muscle limitation to exercise?20 for example, if the primary limitation is cardiovascular exercise training emphasizing moderate intensity, aerobic training (i.e. 50 to 70% of vo2peak) for longer duration (45 min) is indicated to initially enhance plasma volume and structural changes in the heart and blood vessels.21 in contrast, if more general deconditioning is observed and skeletal muscle adaptation is the desired outcome, aerobic training at a higher relative intensity (i.e. > 70% of vo2peak) that induces enzymatic adaptation and increased capillarization and mitochondrial biogenesis may be emphasized (after a period of more general training that acclimatizes the patient to exercise).2224 progressive overload : aerobic training provides a potent physiological stimulus that perturbs the equilibrium of multiple organ systems.18 perturbations in the cellular and systemic environments create biological stress, which challenges homeostasis. chronic (repeated) disturbance of homeostasis triggers a highly preserved, multi - organ stress - response leading to physiologic adaptation (i.e. the concept of hormesis25 or supercompensation), wherein the host can withstand greater future system perturbations, meaning that a greater stress stimulus is required to further perturb homeostasis. by definition therefore, a requirement of effective exercise prescriptions is optimization and progressive increase in stress to confer continued physiological adaptation.26 of equal importance is recognition that increasing exercise stress pushed beyond the homeostasis will result in chronic overreaching or overtraining, leading to fatigue, maladaptation, or even illness and injury.27 accordingly, optimization of progressive overload requires appropriate increase in training stress across the training prescription and monitoring of the individual 's physiological response or readiness to receive the increased exercise load. rest and recovery : the tenet that is most frequently underemphasized or commonly neglected in the design of exercise prescriptions is the principle of rest and recovery. central to this principle is the availability of nutrients and rest (or reduced training load) in order to permit necessary biological resynthesis to replace the required constituents of the impacted system(s). quantifying optimal recovery is challenging28 because of the multidimensional application of the exercise stress.29 conversely, an extended period without adequate stress will result in a loss of adaptation and a down - regulation of the entire system.30 clearly, a balance between the principles of progressive overload and recovery are necessary to elicit optimal physiological adaptations.31 a training stress must perturb homeostasis with enough impulse to lead to chronic supercompensatory adaptations ; however, the appropriate volume of recovery must be prescribed in concert with this perturbation in order to optimize adaptation. there are many techniques in the prescription of an exercise training program that ensure adequate and optimal rest and recovery ; this is most commonly achieved by altering the frequency and duration of training (while maintaining intensity) or sequencing the training stress across each week in order to reduce the accumulated fatigue that may amass through repeated, consecutive high - intensity bouts of training.32,33 this is especially critical in clinical populations in which demographic and medical characteristics (e.g. cardiopulmonary function, immune status, comorbidities, and current therapies) further complicate optimal adaptation. with appropriate balance, positive adaptations will continue, permitting increases in exercise stresses to be applied and further physiologic conditioning towards the targeted end point. individualization : the concept of individualization is defined as the customized application of training towards the physiological status of the patient/ subject.15,16 clearly, even within carefully selected homogenous clinical trial cohorts, considerable heterogeneity likely still exists in cardiopulmonary function, lifestyle behaviour, age, prior treatment, concomitant comorbidities, and, of course, genetic predisposition. thus, application of a generic prescription that fails to consider such parameters will most often result in either an under - dosing or over - dosing of exercise treatment. fortunately, there is a broad array of parameters available on which to individualize training. the preferred approach is individualization on the basis of workloads (e.g. treadmill speed and power output) corresponding to a specific percent of vo2peak or peak workload (e.g. 55%, 65%) elicited during each individual patient 's pre - randomization cpet or ett. the corresponding heart rate, blood pressure, and rating of perceived exertion (rpe) directly measured at each percent workload is used to ensure that subjects are training at, or close to, the prescribed intensity during subsequent exercise sessions (figure 2).17 it is important to note that although the absolute training intensity may be different between individual patients, the relative intensity of training is similar, ensuring standardization across patients a critical aspect of clinical trial conduct. specificity : it is widely acknowledged that the physiological adaptations elicited by aerobic training are unique from those caused by resistance training. lesser appreciated, however, is that even within the same exercise modality, there is enormous capacity to manipulate the nature and configuration of the elements of exercise prescription (i.e. frequency, intensity, duration, and time) to confer remarkably distinct physiological adaptations.18,19 the concept of specificity addresses the notion that the selected exercise stress must be specific and targeted to the primary underlying system(s) or pathway(s) known or postulated to underpin the primary end point of interest. for example, in a trial designed to examine the impact of aerobic training on vo2peak in a particular cancer cohort, a critical prerequisite for the design of the optimal exercise prescription is determination of the primary limitation (determinant) to vo2peak ; in other words, is there a cardiovascular, ventilatory, and/or skeletal muscle limitation to exercise?20 for example, if the primary limitation is cardiovascular exercise training emphasizing moderate intensity, aerobic training (i.e. 50 to 70% of vo2peak) for longer duration (45 min) is indicated to initially enhance plasma volume and structural changes in the heart and blood vessels.21 in contrast, if more general deconditioning is observed and skeletal muscle adaptation is the desired outcome, aerobic training at a higher relative intensity (i.e. > 70% of vo2peak) that induces enzymatic adaptation and increased capillarization and mitochondrial biogenesis may be emphasized (after a period of more general training that acclimatizes the patient to exercise).2224 progressive overload : aerobic training provides a potent physiological stimulus that perturbs the equilibrium of multiple organ systems.18 perturbations in the cellular and systemic environments create biological stress, which challenges homeostasis. chronic (repeated) disturbance of homeostasis triggers a highly preserved, multi - organ stress - response leading to physiologic adaptation (i.e. the concept of hormesis25 or supercompensation), wherein the host can withstand greater future system perturbations, meaning that a greater stress stimulus is required to further perturb homeostasis. by definition therefore, a requirement of effective exercise prescriptions is optimization and progressive increase in stress to confer continued physiological adaptation.26 of equal importance is recognition that increasing exercise stress pushed beyond the homeostasis will result in chronic overreaching or overtraining, leading to fatigue, maladaptation, or even illness and injury.27 accordingly, optimization of progressive overload requires appropriate increase in training stress across the training prescription and monitoring of the individual 's physiological response or readiness to receive the increased exercise load. rest and recovery : the tenet that is most frequently underemphasized or commonly neglected in the design of exercise prescriptions is the principle of rest and recovery. central to this principle is the availability of nutrients and rest (or reduced training load) in order to permit necessary biological resynthesis to replace the required constituents of the impacted system(s). quantifying optimal recovery is challenging28 because of the multidimensional application of the exercise stress.29 conversely, an extended period without adequate stress will result in a loss of adaptation and a down - regulation of the entire system.30 clearly, a balance between the principles of progressive overload and recovery are necessary to elicit optimal physiological adaptations.31 a training stress must perturb homeostasis with enough impulse to lead to chronic supercompensatory adaptations ; however, the appropriate volume of recovery must be prescribed in concert with this perturbation in order to optimize adaptation. there are many techniques in the prescription of an exercise training program that ensure adequate and optimal rest and recovery ; this is most commonly achieved by altering the frequency and duration of training (while maintaining intensity) or sequencing the training stress across each week in order to reduce the accumulated fatigue that may amass through repeated, consecutive high - intensity bouts of training.32,33 this is especially critical in clinical populations in which demographic and medical characteristics (e.g. cardiopulmonary function, immune status, comorbidities, and current therapies) further complicate optimal adaptation. with appropriate balance, positive adaptations will continue, permitting increases in exercise stresses to be applied and further physiologic conditioning towards the targeted end point. stage 3 design of the exercise prescription : exercise prescriptions are most often operationalized using the following parameters : frequency (sessions per week), intensity (how hard per session), time (session duration), and type (modality) or f.i.t.t.34 in the next section, we will outline the design of an exercise prescription (using f.i.t.t) that adopts either a conventional approach (figure 3a) or an alternative approach that systematically adheres to the principles of training (figure 3b). to facilitate practical understanding, mock clinical trial vignette : a randomized trial to determine the efficacy of supervised exercise training on cardiac function in older (65 years) women following completion of anthracycline - containing adjuvant therapy for early - stage breast cancer. the principles of training. oxygen consumption and ventilatory responses to incremental treadmill exercise in a 65 year - old woman with early - stage breast cancer. (a) increasing workloads during the cardiopulmonary exercise test causes linear increases in oxygen consumption (vo2 in ml / kg / min) to the point of volitional fatigue at a vo2peak of 18.9 ml / kg / min. (b) a graphical representation of alveolar ventilation (ve in l / min) demonstrate two exponential breakpoints in ventilation corresponding to ventilatory threshold 1 (vt1) and ventilatory threshold 2 (vt2). these thresholds demarcate the transition of low, medium, and high exercise intensity, and correspond to specific parameters that may be used for identification of relative intensity for exercise prescription and monitoring. these intensities and the corresponding ranges of physiological identification thereof (heart rate, blood pressure, and rating (rtg) of perceived exertion on a 620 scale) each bar represents a training session at prescribed workloads based on the absolute or relative intensity. (a) the conventional (linear) approach utilizes standard intensity, frequency, and duration parameters after an initial lead in period, with static increases in session duration (i.e. 20 to 45 min). (b) the alternative non - linear approach considers the principles of exercise training in order to optimize the adaptations to the exercise stimulus. sessions are tailored to an individual 's relative intensity, based on cardiopulmonary exercise testing or exercise tolerance testing, and specified to address a particular endpoint. sessions and weeks progress over the course of the prescription and vary between low intensity (e.g. 55% vo2peak ; white bars) and moderate (e.g. 75% ; grey bars) and high intensity (e.g. 100% vo2peak ; black bars) training in order to target various physiological systems involved in the cardiopulmonary response to exercise. session intensity is inversely related to session duration, that is, sessions involving high relative intensity workloads are conducted in shorter bouts through short duration training sessions and are less frequent to ensure recovery between sessions. vo2peak, peak rate of oxygen consumption. in the conventional approach (figure 3a), exercise training intensity is prescribed using the heart rate reserve technique (a method based on chronological age and resting heart rate) without consideration of the patients ' functional limitations or the primary study end point. this is the most common method of individualizing training in the oncology setting.7 such an approach is problematic, however, given the 10 to 12beat - per - minute variation in maximal heart rate in normal subjects.35,36 there may be even greater variation in cancer patients, given the documented impact of current or previous systemic therapy on autonomic function.37 this aerobic training intensity prescribed at 75% heart rate reserve (for example) may elicit very different physiologic adaptations between patients (because of the inaccuracy of age - predicted maximum heart rate and the decreased heart rate reserve due to elevated resting heart rate). further, the majority of sessions within this prescription are performed at the same intensity and duration, as determined by their initial cpet or ett ; the workload prescribed to correspond with their percent heart rate reserve will no longer be appropriate after an initial adaptation period. in this instance, training volume remains constant and does not progress across the entire intervention. this is problematic considering that as cardiorespiratory fitness improves, the adaptation from an identical exercise stimulus diminishes ; therefore, there is an insufficient stimulus to induce further physiologic adaptation. as such, this prescription fails to consider three important principles of training, namely individualization, specificity, and progressive overload. contrastingly, in the alternative exercise prescription approach, the use of appropriate baseline testing (i.e. cpet) permits aerobic training to be tailored to a patients baseline vo2peak (figure 2), and thereby adhering to the principle of individualization. second, the intensity, duration, and occasionally, the frequency of training sessions are sequenced in such a fashion that training volume is continually increased across the entire program (i.e. the principles of specificity and progressive overload). this approach also adds important variety to the prescription that not only continually alters the exercise third, training intensity is sequenced, such that higher intensity or higher volume training is followed by lower intensity (recovery) training and rest days to optimize adaptation (i.e. the principle of rest and recovery).38 finally, although not specifically outlined in figure 3b, the principle of reversibility may be a particularly important consideration in aerobic training trials conducted in oncology populations because patients may be forced to temporarily discontinue training because of therapy - induced toxicity and/or disease progression.39 detraining effects can occur rapidly (within days to weeks), thus, subsequent training sessions may need to be resumed at reduced duration and intensity than initially planned. despite the proven efficacy in the arena of sports / athletic performance, consideration of the principles of training has not been translated into the design of exercise prescriptions in clinical populations. indeed, to our knowledge, only one trial to date has compared the efficacy of an exercise prescription following a non - linear approach vs. a traditional linear approach in any clinical population. specifically, klijn. compared the efficacy of non - linear periodized training with that of traditionally - prescribed linear combined aerobic and resistance training in 110 patients with severe chronic obstructive pulmonary disease.43 exercise training in both arms was performed three times a week for 10 weeks. results indicated that non - linear exercise training was associated with superior improvements in cycling endurance and health - related quality of life compared with linearly prescribed training. it is important to state that a common perception is that studies examining the efficacy of high - intensity interval training (hitt) also adhere to the principles of training / non - linear approach. however, if studies exclusively test hitt (i.e. all exercise sessions are hitt) and proceed without appropriate progression, then these programs are also linear in design and do not adhere to the principles of training. in the oncology setting, approximately six studies to date have examined the safety, tolerability, and preliminary efficacy of non - linear aerobic training, compared with a usual care (no exercise training) control group. as presented in table 1, exercise prescriptions adhering to a non - linear approach appear to be safe (low adverse event rate), tolerable (mean adherence 75% of prescribed sessions both during and after primary adjuvant therapy), and efficacious, conferring favourable improvements in vo2peak, quality of life, and other physiological outcomes. on the basis of this data, our group is comparing the efficacy of either non - linear periodized training or traditionally prescribed linear aerobic training with an attention control group (i.e. supervised progressive stretching) in 174 women completing primary therapy for early - stage breast cancer. aerobic training in both arms is being performed four times a week for 16 weeks. the primary end point is vo2peak.44 exercise training studies adopting a non - linear approach in exercise - oncology research (chronological order) itt, intention to treat analysis ; ppa, per protocol analysis ; wmax, maximal work rate ; vo2peak, peak oxygen consumption ; ac, doxorubicin plus cyclophosphamide ; cpet, cardiopulmonary exercise test ; vt, ventilatory threshold ; mwd, minute walk distance. vt determined by a systematic increase in the ve / vo2 ratio, whereas ve / vco2 remained constant. the purpose of this commentary was to provide an overview of the application of the fundamental principles of training in the design and conduct of clinical trials in exercise - oncology research. it is hoped that attention to these issues will provide the platform for constructive dialogue with the view towards the development of best practice guidelines to optimize exercise training in the oncology setting. application of these guidelines will ensure continued progress in the field by producing the high - quality evidence base necessary to convince oncology professionals that exercise training is an integral aspect of the therapeutic armamentarium in the treatment and control of cancer. j.f.c. is supported by research grants from trygfonden, the novo nordic foundation, the danish cancer society, and the beckett foundation. l.w.j. is supported in part by research grants from the nci and aktiv against cancer. | the field of exercise - oncology has increased dramatically over the past two decades, with close to 100 published studies investigating the efficacy of structured exercise training interventions in patients with cancer. of interest, despite considerable differences in study population and primary study end point, the vast majority of studies have tested the efficacy of an exercise prescription that adhered to traditional guidelines consisting of either supervised or home - based endurance (aerobic) training or endurance training combined with resistance training, prescribed at a moderate intensity (5075% of a predetermined physiological parameter, typically age - predicted heart rate maximum or reserve), for two to three sessions per week, for 10 to 60 min per exercise session, for 12 to 15 weeks. the use of generic exercise prescriptions may, however, be masking the full therapeutic potential of exercise treatment in the oncology setting. against this background, this opinion paper provides an overview of the fundamental tenets of human exercise physiology known as the principles of training, with specific application of these principles in the design and conduct of clinical trials in exercise - oncology research. we contend that the application of these guidelines will ensure continued progress in the field while optimizing the safety and efficacy of exercise treatment following a cancer diagnosis. |
autophagy is the primary intracellular catabolic process responsible for long - lived proteins and organelles degradation and recycling, whereas the ubiquitin / proteosome system is the major cellular pathway responsible for short - lived proteins degradation. autophagy is an evolutionarily conserved mechanism throughout macromolecules, ribosomes, and organelles are degraded. initial steps include vesicle nucleation (isolation of the membrane), vesicle elongation, and completion of the double - membrane vesicle. in autophagy, the cytosolic elements that must be degraded are sequestrated by an isolating double - membrane vesicle of nonlysosomal origin that is sealed, creating an autophagic vacuole or autophagosome. the initial phagophores are formed from the endoplasmic reticulum, and they act surrounding and packing organelles to form autophagosomes (figure 1). recently, autophagy emerged as a multifunctional pathway activated in response to microenvironmental stress, intracellular damage caused by hypoxia, chemotherapeutic agents, virus infections, and toxins. autophagy may also have a role in cell death, as cancer cells often develop mutations that confer resistance to apoptosis. nonapoptotic forms of programmed cell death (pcd) might be targeted for novel approaches [5, 6 ]. autophagy is considered a physiological mechanism that may serve for temporary cell survival and is triggered by starvation, such as amino acid and nutrient deprivation, hypoxia, and metabolic stress. recent studies have demonstrated the existence of a nonapoptotic form of programmed death called autophagic cell death, which is now considered as programmed cell death (pcd ii). although autophagy was initially described as a protective mechanism allowing cell survival and generating nutrients and energy, other studies have demonstrated that continuous stress can also promote pcd ii. several works have proved that autophagy is implicated in normal mammary gland development. in mammalian, the cycle of proliferation - differentiation - regression is repeated at each gestation and can be reproduced in culture systems in vitro. a deeper understanding of how growth and differentiation of the mammary tissue are regulated can complement the knowledge of the developmental process as well as the treatment and prevention of mammary cancers. pcd is an essential physiological process operating at all stages of mammary gland remodeling. during mammary gland involution, also, the alveoli lose their structural integrity, and massive death of mammary epithelial cells is observed. pcd i (apoptosis) is responsible for cell loss during mammary gland involution [8, 9 ]. however, there is a lot of evidence suggesting that not only pcd i, but also pcd ii is observed in mammary epithelial cells. in vitro and in vivo studies of bovine mammary gland physiology have revealed that an enhanced process of autophagy is observed at the end of lactation and during dry periods [10, 11 ]. it is manifested by the increased expression of beclin 1 and the higher number of cells with typical morphological features of autophagy. furthermore, 3d model of bovine mammary epithelial cells grown on matrigel showed that during the development and differentiation of mammary acini, the level of membrane - bound microtubule - associated protein chain 3 (lc3) was increased [12, 13 ]. the development of targeted small molecule inhibitors, like those used for pi3k - akt - mtor pathway, has presented a molecular link between the disruption of this signaling cascade and the autophagy process. the cellular consequence of stimulating or inhibiting autophagy in cancer cells is not completely understood, so it is important that this process be monitored, along with antiproliferative and apoptotic biomarkers, in the preclinical setting. this molecule is cleaved to form lc3-i, and upon induction of autophagy, lc3-i is conjugated to the lipid phosphatidylethanolamine to form lc3-ii, which is tightly bound to the membrane of the autophagosome. immunoblotting assessment of lc3 expression is an easy method to predict autophagic activity of mammalian cells, because the amount of lc3-ii correlates with the number of autophagosomes [13, 1618 ]. the product of autophagic conversion of lc3, lc3-ii, tightly associates with the autophagosome membrane and migrates faster than lc3-i on sds - page. therefore, lc3 immunoblotting may detect two bands : lc3-i with an apparent mobility of 18 kda and lc3-ii with an apparent mobility of 16 kda. autophagy could be associated with various pathological conditions including, cardiomyopathy, muscular diseases, neurodegenerative disorders, and cancer. studies in different cells lines have shown that cancer cells express lower levels of the autophagy - related proteins lc3-ii and beclin 1 than normal epithelial cells [19, 20 ]. besides, while heterozygous disruption of becn1 gene promotes tumor development, the overexpression inhibits tumorigenesis, supporting the idea that defective autophagy or autophagy inhibition plays a role in malignant transformation. becn1 gene is deleted in about 50% of breast cancers [21, 22 ]. in addition, reduced expression of beclin1 has been reported in other types of cancers such as colon and brain tumors [23, 24 ]. overall, the data suggest that a defective autophagic process is clearly linked to cancer development. the most important evidence linking dysfunctional autophagy and cancer comes from studies demonstrating that autophagy inhibition in mice, by disruption of becn1, increases cellular proliferation as well as mammary hyperplasia and accelerates tumor development. in addition, transfection of mcf-7 breast cancer cells, that express low levels of beclin 1, with becn1 gene, inhibits growth and tumor formation. these results suggest that beclin 1 is a haploinsufficient tumor suppressor and defective autophagy may be critical for cells malignant transformation. in contrast to apoptosis, pcd ii, in general, is caspase independent, does not involve classic dna laddering, and is believed to be a result of an extensive autophagic degradation of intracellular content. studies also suggest that apoptosis and autophagy are linked by effectors proteins (e.g., bcl-2, bcl - xl, mcl-1, atg5, and p53) and common pathways (e.g., pi3k / akt / mtor, nfb, and erk) [5, 26, 27 ]. for example, p53 activation triggers starvation response in primary mouse embryonic fibroblasts, which is marked by activation of ampk (amp - activated kinase) that inhibits mtor pathway. in other tissues and cells, p53 may communicate with mtor pathway by the upregulation of the pten and tsc2 genes. there is evidence that autophagy may function as a pcd ii in cancer cells in which apoptosis is defective or hard to induce. therefore, it is reasonable to propose that the induction of autophagic cell death may be used as a therapeutic strategy for cancer treatment. the physiological function of autophagy is related to the maintenance of cellular homeostasis under cellular stress. utilizing autophagy as a survival mechanism in the severe tumor microenvironment, which is highly hypoxic and acidic, may favor the development of cancer cells. it was observed that a high number of antineoplastic therapies, radiation therapy, chemotherapy (e.g., doxorubicin, temozolomide, and etoposide), histone deacetylase inhibitors, arsenic trioxide, tnf, ifn, imatinib, rapamycin, and antiestrogen hormonal therapy (e.g., tamoxifen) induce autophagy, and this induction act as a protective and prosurvival mechanism in human cancer cell lines. in fact, the therapeutic efficacy of these agents can be increased if autophagy is inhibited [3035 ]. other studies have shown that a tumor necrosis factor (tnf) family ligand - tumor necrosis factor - related apoptosis - inducing ligand (trail), induces autophagy in epithelial cells and that trail inhibition promotes luminal filling, when it is combined with bcl - xl - mediated inhibition of apoptosis. altogether, disruption of autophagy is involved in diverse human diseases including cancer. in particular, the regulation of autophagy in cancer cells is complex, since it can enhance tumor cell survival in response to certain stresses, but it can also act to suppress the initiation of tumor growth. in contrast to its protective role, inhibition of autophagy through specific gene inactivation can promote tumorigenesis. autophagy, which could be either cytoprotective or cytotoxic, is often observed in tumor cells in response to chemotherapy (table 1). mutations or allelic loss of beclin 1 is frequently found in breast, ovarian, and prostate cancer [9, 10 ]. it has been suggested that autophagy plays an important role in chemoresistance of cancer to some therapeutic agents that typically induce an apoptotic response. endocrine therapy is administered as an antiestrogen (ae) like tamoxifen (tam) or fulvestrant (fas ; faslodex ; ici 182,780) or as aromatase inhibitor (ai) such as letrozole or exemestane. it is less toxic and potentially more effective therapy in management of hormone - dependent breast cancers. antiestrogens, and tam in particular, have been the gold standard first - line endocrine therapy for over 30 years. it is likely that the clinical experience with this drug exceeds 15 million patient years. moreover, tam is the only single agent with demonstrated efficacy in both premenopausal and postmenopausal women with invasive breast cancer. unfortunately, until nowadays, the inability of endocrine therapies to cure many women with er+ disease remains. the precise mechanism by which breast cancer cells die following estrogen withdrawal (or ai treatment) or ae treatment is unclear. for example breast cancer cells respond to aes and to estrogen withdrawal even if they have a mutated p53 [52, 53 ]. although cell death is one of the apoptosis endpoints, there are earlier events initiated by autophagy signals that could be explaining these treatment responses. autophagy has been implicated by the induction of this mechanism in response to endocrine therapy. recent studies showed that endocrine therapy modifies the number of autophagosomes, increases lc3 protein cleavage, and reduces expression of p62. consistent with other reports, pcd ii is associated with the growth inhibitory effects of endocrine therapy in breast cancer cells [32, 55, 56 ]. it remains unclear whether autophagy or apoptosis dominates as the cell - death mechanism or whether this varies among different breast cancer cells. while there is currently no definitive understanding of the primary cell - death mechanisms either in experimental models or breast tumors in women about the relative importance of endocrine therapy - induced changes in proliferation, there are potentially important implications for the underlying biology of the cancer cells. if the primary driver of response as seen in tumor shrinkage is a reduction in proliferation, this will leave many cells alive and still metabolically active. surviving cells have the ability to adapt to the endocrine - induced stress and eventually overcome the proliferative blockade and grow so that, they will become resistant [5759 ]. it is quite possible that autophagy allows breast cancer cells to adapt to endocrine - induced stress and survive. the 26s proteasomes are multicatalytic protease complexes consisting of a 20s catalytic core and a regulator 19s subunit responsible for most nonlysosomal intracellular degradation. the dipeptide boronic acid bortezomib is a selective and potent inhibitor of the 26s proteasome that reversibly inhibits the proteasomal chymotrypsin - like activity [60, 61 ]. the inhibition of the 26s proteasome by bortezomib may lead to the accumulation and aggregation of misfolded proteins in the endoplasmic reticulum lumen resulting in the activation of an unfolded protein response (upr) through the action of three key endoplasmic reticulum - resident transmembrane proteins, perk, ire1, and atf6 [6264 ]. the activated protein perk is a member of a family of protein kinases that phosphorylates the subunit of the cytosolic eukaryotic translation initiation factor eif2a, resulting in a reduced global protein synthesis and in a preferential translation of selected mrnas including activating transcription factor 4 (atf4) [63, 64 ]. some reports have identified endoplasmic reticulum stress and the eif2a / perk pathway as potent inducers of macroautophagy where it promotes cell survival [6567 ]. a recent study in mcf7 cell line showed that during bortezomib treatment, lc3b protein and mrna levels increased significantly in a dose and time - dependent manner. the increase of autophagy in bortezomib - treated cells was dependent on upregulation of lc3b by atf4. in addition, mcf7 cells transfected with rnai specific to lc3b, atf4, or perk were more sensitive to bortezomib treatment. furthermore, the loss of lc3b or atf4 was associated with a significant increase in dead cells staining for both annexin v and propidium iodide after 48 and 72 hours of treatment. from a clinical point of view, it would be an attractive possibility to target autophagy to enhance the response of breast cancer to bortezomib and sensitize to environmental stress that normally occurs in solid tumors. however, clinical experience with bortezomib has shown limited activity against breast cancer when used as a single agent. trastuzumab (tzb and herceptin) was the first immunotherapeutic drug for the treatment of breast carcinomas overexpressing the her2 (erbb-2) oncogene that was successful [6973 ] ; however, the mechanisms that could explain de novo and acquired resistance to anti - her2 monoclonal are not well understood. proposed mechanisms for innate or acquired resistance to tzb include steric inhibition of tzb binding to the extracellular domain (ecd) of the her2 tyrosine kinase receptor imposed by other extracellular factors such as the glycoprotein mucin 4 (muc-4) [74, 75 ]. recent work showed that tzb - resistant her2-positive breast cancer cells (skbr3 cell line) exhibit increased basal autophagy through an increase in lc3-ii expression compared to tzb - nave skbr3 parental cells, suggesting that acquired tzb autoresistance of tzb - conditioned cells is accompanied by increased autophagy. furthermore, inhibition of formation of preautophagosomal structure upon treatment with 3-methyladenime (3-ma), a pharmacological inhibitor of autophagy, notably reduced cell viability in tzb - resistant her2-positive breast cancer cells but not in tzb - nave skbr3 parental cells. to provide additional evidence that autophagy plays a critical survival role in enabling tzb - insensitive high - rates of cell proliferation in tzb - refractory cells, the potent and highly sequence - specific mechanism of rna interference (rnai) this assay avoided any off - target side effects that may confound interpretation of the results obtained with autophagy inhibitors, showed that tzbr cells were extremely fragile. these findings, altogether, clearly established that hyperactivation of basal autophagy plays an essential survival role in tzb - refractory tzbr cells rechallenged with tzb. therefore, the tzb combination with autophagy inhibitors may be a promising strategy in patients resistant to therapy with trastuzumab. arhi encodes a small gtp - binding protein belonging to the ras / rap superfamily, which has the characteristics of a tumor suppressor gene in ovarian and breast cancers, despite sharing 5459% homology with ras proto - oncogenes, arhi is expressed in normal breast epithelial cells, but in more than 70% of breast cancers, it is dramatically downregulated. loss of arhi expression has been linked to tumor progression from in situ to invasive cancer. paclitaxel, a cytotoxic drug, can inhibit cancer cell growth by inducing apoptosis and g2/m cell - cycle arrest. tsa, an hdac inhibitor, can activate several tumor suppressor genes and induces autophagy. skbr3 and mda - mb231 cells, expressing low levels of endogenous arhi transfected with arhi, had an increase of lc3 punctate number, which represent the accumulation of lc3 membrane - bound form on autophagic vesicles. furthermore, it has been observed that tsa treatment enhanced autophagy, but transfection with sirna - arhi blocked the effects of tsa, demonstrating that arhi is essential for autophagy induction. other results from the same group showed that tsa greatly enhanced the inhibitory effect of paclitaxel and tumors treated with a combination of arhi and paclitaxel grew significantly more slowly than controls, whereas the individual treatments did not significantly inhibit tumor growth. we can conclude that autophagy regulation may provide a useful tool to prevent cancer development, limit tumor progression, and increase the efficiency of cancer treatment. this autophagy regulation has to be context dependent, since an autophagy process increased may be necessary to prevent tumor development in individuals at high risk of cancer. but autophagy activity must be reduced when tumor is already established and subjected to the environmental stresses associated with limited angiogenesis, nutrient deprivation, and hypoxia. understanding the signaling pathways involved in autophagy regulation represents a new direction in the development of anticancer therapies however, the proteins and trafficking mechanisms involved in the autophagosomal maturation step are not completely understood. the effectiveness of chemotherapeutics is diminished by the fact that they induce toxicity to both normal and cancer cells. many targeted therapies studies have been conducted to new drugs development with higher therapeutic index. currently, signaling transduction pathways, tumor angiogenesis, and malignant stem cells are considered prime targets for new therapeutics development. | autophagy is a catabolic process responsible for the degradation and recycling of long - lived proteins and organelles by lysosomes. this degradative pathway sustains cell survival during nutrient deprivation, but in some circumstances, autophagy leads to cell death. thereby, autophagy can serve as tumor suppressor, as the reduction in autophagic capacity causes malignant transformation and spontaneous tumors. on the other hand, this process also functions as a protective cell - survival mechanism against environmental stress causing resistance to antineoplastic therapies. although autophagy inhibition, combined with anticancer agents, could be therapeutically beneficial in some cases, autophagy induction by itself could lead to cell death in some apoptosis - resistant cancers, indicating that autophagy induction may also be used as a therapy. this paper summarizes the most important findings described in the literature about autophagy and also discusses the importance of this process in clinical settings. |
cryptococcal meningitis (cm) is the most common cause of meningitis in adults in sub - saharan africa. first, paradoxical iris, in which a patient previously effectively treated for cryptococcosis has a recurrence of symptoms as the immune system is restored (e.g. by antiretroviral therapy in hiv - infected persons or by removal of immunosuppressive medications required for another medical condition). second, unmasking iris, where upon restoration of immune function, a previously subclinical infection is unmasked by a large inflammatory reaction by the now more potent immune system. these syndromes are most frequently described in the setting of hiv, where paradoxical iris has been noted in 1330% of patients with cryptococcosis who survive to receive hiv therapy, while unmasking iris has been described less frequently, in 0.41.7% of cm cases. cryptococcal iris has also been described as both unmasking and paradoxical iris in persons without hiv, due to withdrawal of immunosuppressive medications either in transplant recipients or conditions such as myasthenia gravis requiring immunosuppressive treatments [4, 5 ]. here, we describe the first reported case, to our knowledge, of unmasking iris due to treatment of chemotherapy - induced neutropenia with filgrastim (neupogen), a granulocyte colony - stimulating factor (g - csf) analog. a 56-year - old male visited his primary care physician to be evaluated for abdominal pain, and the subsequent workup revealed a large retroperitoneal mass measuring 21.8 17.5 11 cm. a diagnostic workup revealed a poorly differentiated germ cell neoplasm, most likely an embryonal carcinoma. two months after his initial presentation he began chemotherapy (bleomycin, etoposide and cisplatin). by day 10 of cycle 3 (approximately 4 months after initial presentation), imaging showed a drastic decrease in tumor size. six days after his most recent dose of chemotherapy, he presented to the emergency department noting intermittent fatigue, nausea, vomiting and diarrhea since his last chemotherapy treatment. he also noted headache and fever over the past day. in the emergency department (hospital day 0), he was found to be neutropenic with a total white blood cell (wbc) count of 0.6 10 cells / l and an absolute neutrophil count (anc) of 0.4 10 cells / l. he appeared to be in no acute distress but febrile with a temperature of 38.7c. no obvious signs of an infectious source were noted, including examination of his port - a - cath. the patient was started on cefepime and admitted to the inpatient oncology service for treatment of neutropenic fever. the admitting physician noted tachycardia, but the patient 's blood pressure was within normal limits. the only possible localizing sign of infection was a questionable cellulitis of the right lower leg. as the patient had previously been on prophylactic levofloxacin and had frequently been to infusion clinics, vancomycin was added for methicillin - resistant staphylococcus aureus coverage. the patient was also started on g - csf (480 g / day) for neutropenia. over the subsequent 2 days, his fever and tachycardia continued, and on hospital day 3 the patient became lethargic and confused. a chest radiograph (which showed no sign of pneumonia), blood and urine cultures and a ct scan of the head without contrast showed no acute changes. throughout this period day 4, the patient was unable to respond to commands and was transferred to our institution for further care ; his wbc count had risen to 1.8 10 cells / l (anc not measured) and he remained febrile. his vancomycin was continued while cefepime was changed to ceftazidime out of concern for cefepime - induced encephalopathy ; g - csf was continued. shortly after arrival, he became more lethargic, with periods of apnea, and was transferred to the intensive care unit for endotracheal intubation. an mri of the brain and mra of the brain and neck with and without contrast showed an acute punctate infarct in the right hippocampus and no major vascular findings. 10 cells / l with an anc of 7.0 10 cells / l. fig. 1 shows anc, wbc count, g - csf dosing and the patient 's symptoms over time. a lumbar puncture was performed on hospital day 5 and revealed 64 wbc/l, 30% neutrophils, 39% lymphocytes, glucose 37 mg / dl and protein 99 mg / dl. cerebrospinal fluid india ink stain was completed in the early evening of hospital day 5 and showed rare encapsulated yeast, and the cerebrospinal fluid cryptococcal antigen (latex agglutination, immy, inc., the infectious disease service was informed of the result, amphotericin b lipid complex was started at a dose of 5 mg / kg, and flucytosine 100 mg / kg / day was also ordered but was not but available until hospital day 7. on hospital day 6, the wbc count rose to 28.0 10 cells / l and g - csf, acyclovir, ceftazidime and vancomycin were stopped. throughout hospital days 6 and 7, the patient was minimally responsive despite no sedation since intubation ; however, he did withdraw from painful stimuli. no additional seizures occurred. on hospital day 8, the patient moved his lower extremities on command, and his neurological function rapidly improved. repeat lumbar puncture was performed on hospital day 9 with an opening pressure of 16 cm h20. by hospital day 10, the patient 's neurologic status had improved, and he was extubated. his culture showed cryptococcus neoformans resistant to flucytosine, and so flucytosine was replaced with fluconazole 800 mg daily. after 14 days total of amphotericin - based combination therapy, the patient continued on fluconazole 800 mg daily consolidation monotherapy. repeat lumbar puncture at the time of stopping amphotericin showed a decreased cryptococcal antigen titer of 1:4 and an opening pressure of 22 cm h20. on hospital day 21, the patient has continued to do well in terms of his cm, although he has required hospitalization for pericarditis. he is being maintained on fluconazole 200 mg daily for secondary prophylaxis, and further chemotherapy has been postponed pending improvement of his other health issues. we describe the first reported case, to our knowledge, of unmasking iris due to filgrastim, a g - csf analog. cryptococcal iris, both paradoxical and unmasking, has been described most frequently in patients with hiv. less often cases have been described in patients without hiv but with other conditions that require immune suppressive therapy. in these non - hiv cases, patients who require lessening of their immune suppressive medication (e.g. tacrolimus, mycophenolate, corticosteroids and cyclosporine) can subsequently develop iris [4, 5 ]. interestingly, a patient given alemtuzumab (campath) as treatment for t cell prolymphocytic leukemia was described as having paradoxical cryptococcal iris. this patient developed cm while lymphopenic and neutropenic due to alemtuzumab, was treated and improved but later developed recurrent meningitis symptoms without culture - positive infection, thereby representing paradoxical iris. iris in this case was attributed to the waning effect of alemtuzumab allowing for immune reconstitution. alemtuzumab frequently causes decreases in all cell lines but is most commonly seen to cause lymphopenia, in particular, depletion of t lymphocytes, making the patient 's immune system similar to that of a person with advanced hiv. our patient had a rapid increase of wbcs, primarily neutrophils, after g - csf administration. cm - iris has not, to our knowledge, been previously described in this setting. interestingly, autoantibodies to granulocyte - macrophage csf (gm - csf) were recently identified in cryptococcal patients without hiv, potentially implicating the clinical importance of gm - csf in the immune response to protection from cryptococcus. although csfs, such as g - csf and gm - csf, are well known to increase bone marrow production of neutrophils and monocytes, these csfs also have direct immunomodulatory properties. chiller. demonstrated enhanced killing of cryptococcus when g - csf or gm - csf was added in vitro to neutrophils, monocytes or monocyte - derived macrophages with or without azole antifungals, suggesting that g - csf and gm - csf do, in fact, stimulate existing innate cells to phagocytize and kill cryptococcus. additionally, hiv - infected persons with cm who go on to develop cryptococcal iris have a relative paucity of g - csf and gm - csf prior to initiation of hiv therapy as compared to those who do not go on to develop iris. we hypothesize that our patient 's leukopenia - induced anergy was reversed due to g - csf administration, which resulted in an unmasking iris event with an exaggerated immune response and rapid, profound clinical deterioration in the setting of minimal infectious burden (i.e. cryptococcal antigen titer 1:32). in summary, we describe the first case, to our knowledge, of unmasking cm - iris due to g - csf administration for neutropenia. this is a potentially life - threatening side effect of csf therapy that clinicians should consider in patients presenting with signs of meningitis or altered mental status. this work was supported by the national institute of allergy and infectious diseases and fogarty international center at the national institutes of health (k23ai073192, u01ai089244, r25tw009345). | cryptococcal meningitis immune reconstitution inflammatory syndrome (iris) is frequently seen in patients with hiv and less frequently in patients on immune suppressive medications for other conditions. here, we describe the first reported case of unmasking cryptococcal iris due to granulocyte colony - stimulating factor used in an hiv - negative patient with chemotherapy - induced neutropenia. |
its future rests on the development of innovative, integrated systems that improve quality, deliver safer care, are more affordable, and provide access to diverse patient populations. the current healthcare system is unable to define who owns the patient and their family as well as who is responsible for shepherding the patient and their family through a complex, fragmented system where quality is mediocre, costs are high, care is scattered, and services often duplicated. transformation of the current system relies on the implementation of pioneering models of care that address cost, access, and quality issues. as part of healthcare reform legislation, the federal government took a major step toward solving these issues beginning in march of 2010. the department of health and human resources authorized medicare programs to contract with accountable care organizations (acos) to create entities that share in medicare savings to achieve better outcomes at lower costs. this statute is built on a foundation to transform and improve patient care and place clinical outcomes at the forefront of healthcare. development of acos provides an opportunity for healthcare professionals, particularly nurses to redefine their roles within the current healthcare system. in line with a movement toward the development of acos, the robert wood johnson foundation and institute of medicine (rwj / iom) publication, the future of nursing : leading change, advancing health, outlines opportunities for nurses to fill new and expanding roles within a redesigned system of healthcare ; one that is safe, high quality, patient - centered, and equitable. the recommendations set forth in this report represent a blueprint of deliberate steps that must be taken for nurses to fully participate in a transformed healthcare system. transformation of the healthcare system mandates that issues surrounding quality, access, and value be on the forefront of the agenda. nursing is a key driver of constructing a safer, high - quality system that is value based and patient focused. of particular note is the rwj / iom 's position that nurses be full partners with physicians and other health professionals in transforming the current healthcare system. the committee further supports nurses assuming responsibility for identifying problems and implementing necessary changes to increase quality, access, and value and to provide care that is patient centered. this responsibility calls for the development of leaders through education, mentoring, and competency building. as a result, pioneering partnerships are forming that call for deliberate relationships and realignment of resources across academic institutions and healthcare systems. these unique academic - service partnerships enable parties to work together toward definitive tasks with shared risks, responsibilities, and resources. an academic - service partnership consists of a strategic alliance between an academic unit, such as a school of nursing, and a service entity for the purpose of advancing the service, education, and research missions of each. within the partnership, the strategic goals of each unit are aligned and the resulting synergy provides increased opportunities for interprofessional collaboration, curriculum reformation, improved resource utilization, and expanded models of clinical education [410 ]. the rwj / iom report clearly outlines nursing 's responsibility in driving changes that would transform current healthcare system to one designed to bridge the existing gaps in quality, safety, and value. de geest and colleagues describe five forces that drive the need for the development of academic - service partnerships, including (1) the shift toward elder and chronic care, (2) preparation and availability of the nursing workforce, (3) nursing 's imperative to demonstrate impact on outcomes, (4) the necessity to meet quality and safety standards, and (5) cost containment and efficiency enhancements. these driving forces lay the foundation for the development of academic - service partnerships and provide a framework for nursing 's contributions in addressing the rwj / iom recommendations. this article describes a distinct academic - service partnership formed to address issues of quality, access, and value within an academic medical center. the stony brook university medical center (sbumc) is an academic medical center in suburban new york and is composed of a university hospital and five professional schools, including a school of nursing (son). while the hospital and the son enjoyed a long - standing collegial relationship, collaboration focused mainly on clinical placement opportunities for students and a few joint appointments focused on clinical practice. recently, leadership from the hospital and the son capitalized on an opportunity to create an innovative academic - service partnership to meet the rwj / iom directives. this change in organizational structure provided a unique opportunity to align the mission and visions of hospital units with the school of nursing. as a result, a strong partnership formed whereby the individual units were able to align their visions and resources and brainstorm about initiatives that would transform patient care. the issues faced by sbumc mirror those of the broader healthcare system, including care of patients with complex, chronic conditions, an aging and diverse patient base, disparities in treatment among groups, and an increasing number of patients with limited english proficiency. for these reasons, it was mutually decided to focus the partnership on areas that would address some of the evolving healthcare challenges and primary concerns for healthcare reform outlined in the rwj / iom 's report. the goals of the partnership were to (1) respond to challenges inherent in the care of elderly and chronically ill patients, (2) develop systems improvements that increase quality and safety and reduce costs, and (3) increase the research capacity through the development of a collaborative research infrastructure. the academic - service partnership resulted in four key initiatives that were designed to support nursing 's role as a driver of healthcare system transformation. these initiatives exemplify the key role nursing plays in improving quality, safety, access, and value. one of the first initiatives of the partnership was to strengthen the alliance between the hospital and the son. as a result, leadership from both entities responded to a unique opportunity for nursing faculty to partner with clinical leaders to learn and practice quality, safety, and microsystem development and to advance nursing education and curriculum. the sbumc partnership was formalized through participation as academic and clinical partners in the jointly sponsored american association of colleges of nursing (aacn)/the dartmouth institute nursing faculty and clinical partners improving health care together : the dartmouth institute microsystem academy. a focus of the academy was to develop unique strategies for care process improvements and to strengthen and advance academic and clinical partnerships around a common vision. for the pilot project of the sbumc partnership, improving the process of patient and family centered care (pfcc) was selected as it was central to both the missions of the hospital and son. the partnership targeted improving the process of pfcc on an inpatient, medical oncology unit. an interprofessional team, representing multiple disciplines engaged in care / service on the unit, as well as individuals from the son, was formed. the primary aims were to (1) reduce patient falls, ; (2) decrease patient pressure ulcers, (3) decrease interruptions due to call lights, and (4) improve patient satisfaction related to communication between the healthcare team and patients and families. a secondary aim of this partnership was to strengthen quality and safety knowledge in the undergraduate and graduate curricula of the son. using the microsystems approach, a 5p (purpose, patients, professionals, processes, and patterns) assessment was conducted and current performance metrics (fall rates, unit - acquired pressure ulcer (uapu) incidence and prevalence rates, and satisfaction scores) were reviewed by the team. results indicated that opportunities for quality and safety improvement centered on patient and staff satisfaction with communication between healthcare team and patients and families and fall and pressure ulcer rates. the team brainstormed and selected the intervention of hourly rounding by registered nurses (rn) and nursing assistant (na) staff to target the change concepts of improved workflow, time management, designing systems to avoid mistakes, enhancement of patient and staff relationships, and improvement of overall patient and staff satisfaction. the intervention consisted of rns and nas making alternating rounds every hour (every two hours between the hours of 10 pm and 6 am) on each assigned patient. hourly rounding focused on assessment of pain, toileting, comfort needs, position changes and assessment of pressure points, identification and correction of fall hazards, and all items (phone, call bell, beverages, urinal, and bedside table) were in patients ' reach. finally, patients were reminded that a staff member would return in an hour to round again. the intervention was implemented using tests of change over a six - month period and results indicate improvements in fall and uapu rates, patient and staff satisfaction rates, enhanced patient and staff communication, and decreased interruptions to nursing 's workflow. additionally, the processes and outcomes of the project served to inform undergraduate and graduate curricular revisions in the son, including the development of an undergraduate elective on patient and family centered care and a reformation of the graduate core curriculum to included courses on quality improvement and safety and organizational leadership. the partnership 's initial success with this pilot project laid the foundation for additional initiatives aimed at improving quality, access, and value at the hospital and expanded the capacity of the son to educate nurse leaders capable of meeting current and future healthcare demands. a second initiative, called for the participation of faculty from the son to partner with nursing to improve hospital discharge processes, reduce the readmission rate, and streamline patient 's transition from acute - care to home - based care. consequently, a task force was formed which included hospital leaders and faculty from the son. this initiative began with meetings discussing the hospital 's current discharge process and specific outcomes related to discharge, namely, readmission rates. from these meetings it was concluded that an intervention aimed at identifying patients at risk for adverse events during transitions, improving patient and family preparation for discharge, and reducing readmission rates was needed. as a result key drivers from medicine, nursing, managed care, pharmacy, case management, clinical informatics, finance, planning, and other clinical and support areas, as well as faculty from the son were integral to the project 's implementation. project components, including identification of a high - risk group of patients, formalization of patient and family teaching, enhanced discharge preparation, medication reconciliation, improved transfer of patient information to community healthcare providers, appropriate referrals to case management, and a refined discharge follow - up process, were designed and implemented by the project team. a doctoral student from the son serves as the project coordinator and has assumed responsibility for creation and maintenance of the database for all project boost patients and analysis of indicators for project boost patients that are readmitted. early results indicate that the project improved identification of complex patients, increased patient and family readiness for discharge, streamlined the medication reconciliation process, and identified key issues and opportunities for improvement during patient transitions from acute care to community settings, including underutilization of palliative care. perhaps the most problematic issue identified during implementation of project boost was securing early, postdischarge provider access. two challenges central to this issue were finding a primary care provider (pcp) and obtaining a timely follow - up appointment. in a recent study of rehospitalizations among medicare patients, it was noted that 50 percent of patients readmitted within 30 days did not have a bill for an outpatient physician visit between the time of discharge and readmission. only 43% of project boost patients at sbumc identified a pcp with whom they would follow up after discharge. consequently, care was poorly coordinated and information sharing, including pending laboratory and diagnostic tests, changes to medication regimen, and disease - specific information, between the hospital and pcps was problematic. even for patients who identified a pcp, obtaining timely follow - up appointments was difficult. for the most complex patients, obtaining appointments with community providers in the very immediate patients who were scheduled to followup within the hospital 's network of providers also faced lengthy wait times for appointments. additionally, boost patients, who transition from the acute care environment of the hospital to a community setting, have multiple and complex acute and chronic healthcare needs and their management relies on a mix of different healthcare professionals in a variety of healthcare settings. frequently, systems of care are often fractured and disintegrated causing miscommunication among healthcare providers and a patient plan of care that is uncoordinated. the challenge for healthcare systems is to improve coordination of care across settings through enhanced communication among healthcare providers and increased access to a patient 's current information and plan of care across healthcare settings. managing effective transitions across care settings enables healthcare institutions to gain measurable improvements in quality and reduce costs. these are the building blocks of implementation of an aco model. as a result, the leadership in the son identified a unique opportunity to address the concerns of transitional care and implemented a model to assume care for patients transitioning from the acute care setting to the community pcps. a business plan for a transitional care center (tcc), developed with input from nursing leadership and faculty from the hospital and son, finance, managed care, planning, and physician champions, registered nurses, the tcc is designed to see patients in the immediate postdischarge period for the purpose of coordinating patient handoffs between acute - care providers and after discharge care providers. in the pilot phase, the tcc sees project boost patients within seven to ten days postdischarge, or earlier, as their condition warrants. nurse practitioners from the tcc communicate with the acute care team on all project boost patients that are discharged. the patient 's discharge summary including current plan of care, updated problem list, discharge medications, pending laboratory results, and further work - up orders are addressed. patients leave the acute care setting with an appointment to see a nurse practitioner in the tcc within the first week, after discharge. once the patient presents to the tcc, medications are reconciled, pending results are obtained, and further work - up arrangements are made, including appointments with specialists for further followup. patients are also taught how to recognize early warning signs of a change in their health status and whom to call or how to respond should such a situation warrant medical attention. a continuing plan of care, which incorporates the patient 's goals, preferences, and values, is developed and preparation for transition to the next site of care is completed. the nurse practitioner from the tcc explicitly communicates the patient 's plan of care to the pcp. following this eased transition, the patient continues care with their pcp. given the dearth of primary care physicians and the increasing complexity of healthcare, this initiative has provided an essential clinical service to the hospital and has supported its investment in care coordination and development of an aco. however, the interprofessional collaboration of nurse practitioners working in parallel with care providers across settings, particularly physicians, has fulfilled strategic goals within the son as well. organizing a component of the son into a separate pc, one which works alongside and within the aco being developed, is providing rich opportunities for the son to enhance faculty salaries and attract diverse, well - qualified faculty into academic positions. another area of collaboration that realigned the relationships between the son and hospital was in the realm of research scholarship. many nurses engaged in patient care lack the knowledge, skills or time necessary to initiate and engage in research activities. they do, however, possess the ability to identify areas of quality concern. the partnership was instrumental in facilitating the research missions of both the son and the division of nursing in the hospital. faculty is embedded in multiple projects and initiatives relevant to the mission of each institution and many of the projects are focused on improving patient care outcomes. as a preliminary step, son faculty and nursing staff were cross - appointed to each entity 's committee on research to provide mutual support for the conduct and dissemination of research. the son assists with developing scholarship skills of clinicians by identifying collaborators from the son who mentor clinicians interested in research and/or translational projects. there is an increased awareness of the members of research committees and their colleagues regarding research - related events (workshops, conferences) available within each setting. the partnership assists with identification of appropriate patients for ongoing studies and facilitates linkages between nurse scientists and interested clinicians in ongoing research - related activities. one of the highlights of the relationship is participation in workshops focusing on writing and publishing manuscripts related to the clinical and or research activity. another important focus of the collaboration is identification of appropriate funding mechanisms and subsequent development and submission of grants. an exemplar of particular relevance to the rwj / iom report is a study on limited english proficiency patient 's experience in the emergency department (ed). faculty also serve as mentors to clinicians on abstract, poster, and podium presentation development through direct support and a mock peer review process. academic - service partnerships offer unique opportunities for schools of nursing and service institutions to explore nontraditional relationships. partnering to improve quality, access, and value exemplifies mutual commitment and collaboration toward meeting the rwj / iom 's recommendation. the evolving academic - service partnership at sbumc characterizes shared goals, missions, and values ; and ; minimizes segregation of vision and resources. early results indicate that sbumc 's partnership will be an essential component of driving improvements in patient care and serve as a model for the unique contributions that can be expected from academic - service partnerships. | transformation of the current healthcare system is critical to achieve improved quality, safety, value, and access. patients with multiple, chronic health conditions require integrated care coordination yet the current health care system is fragmented and complex. nursing must play a key role in constructing a system that is value based and patient focused. the robert wood johnson / institute of medicine (rwj / iom) report on the future of nursing outlines strategic opportunities for nursing to take a lead role in this transformation. partnerships across academic institutions and health care systems have the potential to address issues through mutual goal setting, sharing of risks, responsibilities, and accountability, and realignment of resources. the purpose of this paper is to present stony brook university medical center 's (sbumc) academic - service partnership which implemented several of the rwj / iom recommendations. the partnership resulted in several initiatives that improved quality, safety, access, and value. it also characterized mutual goal setting, shared missions and values, and a united vision for health care. |
there are a few studies reporting the long term outcome of conservatively treated acetabular fractures. the present study aims to evaluate the quality of reduction, and radiological and functional outcome in displaced acetabular fractures treated conservatively. sixty - nine patients (55 men and 14 women) with 71 displaced acetabular fractures (mean age 38.6 years) managed conservatively were retrospectively evaluated. there were 11 posterior wall, 5 posterior column, 6 anterior column, 13 transverse, 2 posterior column with posterior wall, 9 transverse with posterior wall, 6 t - shaped, 1 anterior column with posterior hemi - transverse, and 18 both - column fractures. the follow - up radiographs were graded according to the criteria developed by matta j. functional outcome was assessed using harris hip score and merle daubigne and postel score at final followup. radiologic outcome in incongruent reduction (n=26) was good or excellent in 6 and fair or poor in 20 hips. the functional outcome in patients with incongruent reduction was good or excellent in 16 and satisfactory or poor in 10 hips. good to excellent radiologic and functional outcome was achieved in all patients with posterior wall fractures including four having more than 50% of broken wall. good to excellent functional outcome was observed in 88.8% of both - column fractures with secondary congruence despite medial subluxation. nonoperative treatment of acetabular fractures can give good radiological and functional outcome in congruent reduction. posterior wall fractures with a congruous joint without subluxation on computed tomography axial section, posterior column, anterior column, infratectal transverse or t - shaped, and both - column fractures may be managed conservatively. small osteochondral fragments in the cotyloid fossa or non weight - bearing part of the hip with a congruous joint do not seem to adversely affect the functional outcome. the operative treatment of acetabular fractures is technically challenging. the surgical approaches and reduction techniques must be thoroughly understood to properly manage this three - dimensional problem.1 the complicated anatomy of the acetabular region, frequent severe associated injuries, and long - term complications, all contribute to management difficulties.1 displaced fractures of the acetabulum are best treated with anatomical reduction and rigid internal fixation.26 good to excellent functional outcomes have been reported in 7188% patients after operative management of acetabular fractures.235710 the type of fracture and the quality of reduction influences on the functional outcome.51012 however ochs reported that despite changes in the chosen approaches and an increased surgical frequency, the operative treatment of the last 15 years did not lead to an increased reduction quality.13 conservative treatment continues to be the mainstay of treatment in most centers in the developing countries. lack of infrastructure, non - availability of skilled services, delayed referrals from peripheral units because of associated injuries, economic constraints, patient 's unwillingness to undergo surgery often make the conservative treatment inevitable.14 patients may even not be able to undergo the complete supervised conservative treatment at times because of reasons beyond surgeon 's control. there are several studies on the outcome of operative management of acetabular fractures.23712 however, only a few studies have reported the long - term outcome of acetabular fractures managed conservatively.1418 hence, we conducted a retrospective study to evaluate the radiologic and functional outcome of patients with displaced acetabular fractures, who were treated conservatively on traction. this retrospective study involves 318 patients of acetabular fractures treated between 1997 and 2007. of these patients, records were scrutinized for all those patients admitted with the diagnosis of acetabular fractures who were managed conservatively at author 's institute. the radiographs and computed tomography (ct) scan of the patients (if available) were studied as per the classification of judet. the patients involving sacro - iliac joint dislocation or associated pelvic injury were excluded from the study. the inclusion criteria of the study were displaced acetabular fracture with a minimum follow - up of 2 years. a fracture was considered displaced if any of the radiographs including judet views showed more than 3 mm intra - articular displacement (either a step or widening of fracture). from the 232 patients therefore, 69 patients with 71 displaced acetabular fractures fulfilled the inclusion criteria of the study. the patients were treated conservatively for reasons beyond surgeons control despite fractures being displaced, i.e. severe comminution, osteoporosis, associated abdominal, head and chest injury, local soft tissue problem, medical contraindications, patient 's unwillingness for surgery and occasional non - availability of skilled surgeons. the treatment protocol of nonoperative treatment for the acetabular fractures at the institute involved initial closed reduction maneuvers under sedation / anesthesia followed by skeletal (longitudinal and/or lateral) traction. most commonly used closed reduction maneuvers included reduction of posterior dislocation by stimson / bigelow 's methods and lateral traction in patients with central dislocation under anesthesia. skeletal traction was given for 68 weeks with weights equivalent to 1020% of body weight of the patient. this was followed by touch toe non - weight bearing for 34 weeks and full weight bearing after 1216 weeks. there were 55 men and 14 women with an average age of 38.6 years (range 2065 years). the patients presented to the author 's institute at 6.9 days (range 116 days) after injury. the right hip was involved in 36 patients and the left hip in 35 patients. fifty - one patients (74%) suffered injuries in road traffic accidents and 18 (26%) patients had fall from height. four patients had associated head injury, 3 had visceral, 3 had chest injury and 16 patients had associated injuries to the upper and lower extremities. the mode of injury, the type and duration of traction, were inquired and confirmed from the patients and noted. the complications, if any, of non - operative treatment, e.g. bed sore, lateral popliteal nerve palsy, joint stiffness, pin site infection, deep vein thrombosis, were also noted. all clinical data retrieved from old records were further confirmed from the patients directly at final follow - up. follow - up radiographs of the affected hip including the anteroposterior, iliac, and obturator views were assessed. patients were subjected to ct scan examination where possible at the time of final assessment. there were 11 posterior wall, 5 posterior column, 6 anterior column, 13 transverse, 2 posterior column with posterior wall, 9 transverse with posterior wall, 6 t - shaped, 1 anterior column with posterior hemi - transverse, and 18 both - column fractures. five patients (of both - column fractures with medial subluxation of femoral head) received additional lateral traction through trochanteric pin. six patients were treated with skin traction alone two patients did not receive any type of treatment. forty - one patients (59%) had follow - up of 5 years or more. roof arc angle measurements were done as described by matta.19 to quantify the intact acetabular weight - bearing dome (wbd). the roof arc angles were not measured in posterior wall fractures and both - column fractures. measurements of the percentage of remaining posterior acetabulum on ct scan in posterior wall fractures of acetabulum were evaluated according to the criteria of calkins.20 congruent reduction was defined as presence of parallelism between joint surfaces. the final follow - up radiographs were graded according to the criteria developed by matta.2 excellent denotes a normal - appearing hip joint, good as mild changes with minimal sclerosis and joint narrowing, fair indicates intermediate changes with moderate sclerosis and joint narrowing (< 50%), and poor signifies advanced changes.2 functional outcome was assessed using harris hip score and merle daubigne and postel score.21 the study was conducted in accordance with the ethical standards of the responsible committee on human experimentation (institutional or regional) and with the helsinki declaration of 1975, as revised in 2000. the fisher 's exact test (two - tailed) for categorical data and student 's t - test (unpaired two - tailed) for continuous data were used for statistical analysis. for the purpose of statistical analysis, the clinical outcome scores (merle daubigne and postel score) and radiologic outcomes were defined as excellent or good, and fair or poor. there were 11 cases of posterior wall fractures, all associated with posterior dislocation of hip which were reduced within 24 hours of injury. four patients demonstrated osteochondral fragments in the region of the cotyloid fossa, three of these had lateral subluxation of the head of the femur [figure 1 ]. the posterior wall fragment was displaced postero - superiorly, presenting as cap sign with a congruent joint [figure 2a d ]. all the four patients had more than 50% of the posterior wall broken as assessed on ct measurements20 (range 5668.5%) [figure 2 ]. axial ct section shows posterior wall fracture and intra - articular osteochondral fragment in cotyloid fossa. the patient had good functional outcome according to the merle daubigne and postel score (a) anteroposterior radiograph shows broken posterior wall fragment of right acetabulum seated over the head of femur like a cap (black arrow). (b) obturator and (c) oblique iliac radiographs also show congruous reduction between the head of femur and weight - bearing dome of acetabulum axial ct section shows more than 50% of broken posterior wall 3-d ct reconstruction shows displaced comminuted posterior wall fragments covering the femoral head posteriorly and superiorly in a patient with more than 50% of broken posterior wall (a) anteroposterior and (b) obturator radiographs of the same patient at 3 years followup shows good radiological outcome in all the patients, the displaced posterior wall fragments covered the femoral head posteriorly on coronal sections as well as 3-d reconstruction [figure 2 ]. the fracture gap showed new bone formation and fibrous tissue on axial ct examination [figure 2 ]. at final follow - up, the radiologic was graded as excellent in eight and good in three patients. functional outcome was assessed as excellent using harris hip score ; and good to excellent using merle daubigne and postel score in all patients. six patients were classified as transtectal fractures, two as juxtatectal and five as infratectal fractures. an average medial roof arc of 15.6, anterior roof arc of 11.6 and posterior roof arc of 10.4 were noted in transtectal transverse fractures, indicating inadequate wbd in these patients. one patient of transtectal transverse fractures had congruent reduction with good radiologic outcome and excellent functional outcome. medially in these five transtectal fractures with incongruent reduction constituting an area of stress concentration at the apex of the v [figure 3a c ]. the radiologic outcome in these five transtectal transverse fractures was graded as good in one, fair in one and poor in three patients. functional outcome was assessed as good in one patient and satisfactory or poor in four patients using merle daubigne and postel score. out of two patients, an average medial roof arc of 25, anterior roof arc of 18.3 and posterior roof arc of 18.3 were observed in juxtatectal fractures, indicating inadequate wbd in these two patients. an average medial roof arc of 67.3, anterior roof arc of 72.6 and posterior roof arc of 76.6 were noted in infratectal transverse fracture. the radiologic outcome was graded as excellent in two patients, good in two patients and fair in one patient. functional outcome was assessed as excellent using harris hip score, and good to excellent using merle daubigne and postel score in these patients. the radiologic outcome was graded as good in six and fair in one patient ; and functional outcome was graded as good to excellent in these seven patients using both scores. both the patients had transtectal transverse with posterior wall fracture and persistent subluxation of head of femur. the radiologic outcome was graded as poor and functional assessment was graded as fair in both patients using harris hip score and satisfactory in both patients using merle daubigne and postel score. two patients had osteochondral intra - articular fragments in the region of cotyloid fossa ; however, joint was congruent in wbd and functional outcome was assessed as excellent. in two patients, the posterior wall fragment was displaced postero - superiorly presenting as cap sign but with a congruent joint. (a) anteroposterior radiograph of the pelvis of a 45-year - old female shows v the apex is formed medially between the weight - bearing dome and head of femur constituting an area of stress concentration. (b) skeletal traction failed to reduce the transtectal fracture with v sign as seen in anteroposterior x - rays. (c) follow - up anteroposterior radiograph shows poor radiological outcome and the patient had fair functional outcome there were 18 cases of both - column fractures. joint was congruent in seven patients ; radiologic outcome was graded as excellent in one, good in five and fair in one patient. functional outcome was assessed as good to excellent in all seven patients using both scores. radiologic outcome was excellent in one, good in four and fair in six patients. functional assessment was good in nine and satisfactory in two patients using merle daubigne and postel score. function was good to excellent in six patients who had persistent medial subluxation of head of femur with fracture lines extending into the wbd. patients with medial subluxation of head of femur had incongruent reduction ; however, these patients developed secondary congruence later on follow - up. (a) anteroposterior, (b) obturator and (c) oblique iliac radiographs of both - column fracture shows medial subluxation of head of femur with secondary congruence. the patient had good functional outcome in spite of heterotopic bone formation all patients with posterior column (n=5) fractures had congruent reduction and good to excellent radiologic and functional outcome. two patients had incongruent reduction and had fair radiologic and good to excellent functional outcome. there were six patients with t - shaped fractures. two patients had incongruent reduction and had fair radiologic and poor functional outcome using merle daubigne and postel score. two patients of posterior column with posterior wall fractures had incongruent reduction and fair radiologic and good functional outcome. one patient of anterior column with posterior hemi - transverse fracture had congruent reduction with good radiologic and functional outcome [table 1 ]. quality of reduction, radiologic and functional outcome in fractures of acetabulum two patients had bilateral fractures occurring as separate event. one patient initially sustained fracture posterior column with posterior wall on right side and two years later sustained t - shaped fracture on left side. good or excellent radiological outcome was achieved in 43 of 45 hips with congruent reduction in comparison to 6 of 26 hips with incongruent reduction in our study (statistically significant, p < 0.0001, fisher 's exact test). good or excellent functional outcome was achieved in all hips (n = 45) with congruent reduction in comparison to 16 of 26 hips with incongruent reduction in our study using merle daubigne and postel score (statistically significant, p < 0.0001, fisher 's exact test). mean merle daubigne and postel score was 16.64 (range 1518 ; sd 1.17) in patients with congruent reduction in comparison to 14.88 (range 1118 ; sd 1.63) in patients with incongruent reduction (statistically significant, p < 0.0001, student 's t - test). wbd was adequate in 26 hips of whom radiologic outcome was good or excellent in 21 (had congruent reduction) and fair in 5 hips (had incongruent reduction), and functional outcome was good or excellent in 24 and fair in 2 hips. wbd was inadequate in 16 hips of whom radiologic outcome was good in 6, fair in 5 and poor in 5 hips, and functional outcome was good or excellent in 10 and fair or poor in 6 hips [table 2 ]. the functional outcome (according to merle daubigne and postel score) in relation to congruency and involvement of weight - bearing dome good or excellent functional outcome was achieved in 24 of 26 hips with adequate wbd in comparison to 10 of 16 hips with inadequate wbd in our study using merle daubigne and postel score (statistically significant, p = 0.037, fisher 's exact test). mean merle daubigne and postel score was 16.35 (range 1218 ; sd 1.62) in patients with adequate wbd in comparison to 15 (range 1118 ; sd 1.71) in patients with inadequate wbd (statistically significant, p = 0.014, student 's t - test). overall, the radiologic outcome was graded as excellent in 20 (28.1%), good in 29 (40.8%), fair in 17 (23.9%) and poor in 5 (7%) hips. the functional outcome according to merle daubigne and postel criteria was assessed as good (1517 points) or excellent (18 points) in 61 patients (85.9%) and satisfactory (1214 points) or poor (< 12 points) in 10 hips (14.1%). satisfactory or poor functions included four displaced transverse fractures, two both - column, two t - shaped and two transverse with posterior wall fractures [table 3 ]. clinical details of 71 fractures of acetabulum six patients had avascular necrosis of head of femur. five patients developed pin site infection which was treated with curettage and antiseptic dressing, two had knee joint stiffness, one patient developed bed sore and another one had permanent lateral popliteal nerve palsy which was treated with tendon transfers after 1 year. displaced fractures of the acetabulum are best treated with anatomical reduction and rigid internal fixation.26 in spite of advances made in the operative management, conservative treatment is being followed in most of the centers in developing countries. the present study is aimed to evaluate quality of reduction, and radiologic and functional outcome in patients of displaced acetabular fractures managed conservatively. all the patients with posterior wall fractures (n=11) had congruent reduction and good to excellent radiologic and functional outcome. epstein advocated conservative treatment for patients who have a fracture dislocation of the hip with a so - called insignificant acetabular fragment, but he did not specify what constituted an insignificant fragment.22 posterior wall fractures involving more than 50% of wall are considered unstable,2324 and therefore require osteosynthesis. however, we are of the opinion that if the joint is congruent, femoral head is well contained in the socket of the hip and not subluxating on ct examination, and head of femur is well covered with displaced posterior wall fragment on 3d reconstruction, conservative treatment may be considered even if more than 50% of the posterior wall is broken. congruent reduction was achieved in 20 of 22 (90.9%) patients with single column or single wall fracture, with good to excellent results in all patients in the present series. similar findings have been reported in various studies on conservative treatment.141625 the fracture line is hardly ever located on the weight - bearing surface of the acetabulum and the post - traumatic arthrosis is seldom found.141625 lovric. prefer operative treatment in anterior column fractures.26 good functional outcome was observed in patients with intra - articular osteochondral fragments located in the cotyloid fossa or in the inferior portion of the hip in six patients. the bony or cartilaginous fragments in the joint are considered indications for surgery.2728 rosenthal and coker concluded in their study that the presence of intra - articular bone fragments does not necessarily indicate open reduction unless the fragments are trapped in the weight - bearing area.29 the present study highlights that small osteochondral fragments in the cotyloid fossa or non weight - bearing part in a congruous joint do not adversely affect the functional outcome. we had good to excellent functional outcome in 88.8% of both - column fractures even though medial subluxation was seen in six cases. tipton. reported good to excellent functional outcome in 58.4% patients.18 we believe that secondary congruence is achieved even though femoral head remains subluxated medially. sen and veerappa observed good functional outcomes in patients with central fracture dislocations where fracture reduction could be achieved.14 displaced both - column fractures with secondary congruence gave better results than other displaced fractures.430 the v sign is formed due to (i) intimate contact of the head of the femur with the impacted wbd and (ii) tilting of the part of wbd to form an area of stress concentration on the head of the femur. the average medial, anterior and posterior roof arc measurements were 15.6, 11.6 and 10.4, respectively, in these patients. the study reveals that traction failed to reduce such a transtectal fracture showing the v sign [figure 3b and c ]. the identification of v sign on anteroposterior radiograph of pelvis is quite simple and does not require exact measurements of roof arc. v sign was observed in 6 of 10 transtectal (transverse, t - shaped ; and transverse with posterior wall) fractures and 5 of these had poor or fair functional outcome. we, therefore, believe that the presence of v sign is an indication for operative management to achieve a congruous joint. matta. depicted marked reduction in contact area between the femoral head and acetabulum in a diagram.30 operative management is advised in all acetabular fractures involving wbd or with insufficient roof arc.4171927 weise. observed that the larger the intact roof arc, the better the prognosis.31 good to excellent functional outcomes were achieved in 62.5% fractures despite involvement of wbd in the present series. observed good to functional outcome in 47.3% fractures involving wbd.15 good clinicoradiologic results are expected in fractures involving wbd, but with adequate or congruent fracture reduction.1415 lovric. preferred conservative treatment in transverse fractures.26 however, operative treatment has been recommended in high or displaced transverse fractures.152530 congruent reduction was achieved in 63.3% of fractures, which is comparable to findings (56.280.7%) reported in the literature.1415 good or excellent radiologic outcome was achieved in 95.5% of patients having congruent reduction in comparison to 23% of patients with incongruent reduction in our study (statistically significant). similarly, good or excellent radiologic outcome was reported in 77.8100% of patients with congruent reduction in other studies.1415 good or excellent functional outcome was achieved in all patients with congruent reduction in comparison to 61.5% patients with incongruent reduction in our study using merle daubigne and postel score (statistically significant). similarly, good or excellent functional outcome was reported in 83.3100% of the conservatively treated patients with congruent reduction in other studies.11415 these results emphasize that good functional outcome can be achieved in displaced acetabular fractures managed conservatively if congruent reduction could be achieved. the functional outcome was fair or poor in 38.4% patients with incongruent reduction using merle daubigne and postel score as compared to 78.581.8% reported in other studies.1415 the functional outcome in patients with incongruent reduction seems better because the number of both - column fractures achieving secondary congruence was more in our series. functional outcome after operative treatment in fractures of more than 3 weeks duration was reported as fair or poor in 38.440.6% patients.3233 despite changes in the chosen approaches and an increased surgical frequency, the operative treatment of the last 15 years did not lead to an increased reduction quality.13 conservative treatment also avoids complications related to operative treatment, such as iatrogenic nerve palsies, wound infections, deep vein thrombosis / pulmonary embolism. arthrosis, necrosis of the femoral head and heterotopic ossification tend to decline the outcome of acetabular fractures despite good fracture reduction achieved after surgery.734 overall, good to excellent functional result was achieved in 85.9% patients in the present series. therefore, the outcome of conservatively managed fractures is not bleak and operative treatment should be considered when absolute indications are there. in conclusion posterior wall fractures with a congruous joint without subluxation on ct axial section, posterior column, anterior column, infratectal transverse or t - shaped, and both - column fractures may be managed conservatively and good or excellent functional outcome can be expected in most of the patients. small osteochondral fragments in the cotyloid fossa or non weight - bearing part of the hip with a congruous joint do not seem to adversely affect the functional outcome. the transtectal transverse or t - shaped fractures presenting with v | background : there are a few studies reporting the long term outcome of conservatively treated acetabular fractures. the present study aims to evaluate the quality of reduction, and radiological and functional outcome in displaced acetabular fractures treated conservatively.materials and methods : sixty - nine patients (55 men and 14 women) with 71 displaced acetabular fractures (mean age 38.6 years) managed conservatively were retrospectively evaluated. there were 11 posterior wall, 5 posterior column, 6 anterior column, 13 transverse, 2 posterior column with posterior wall, 9 transverse with posterior wall, 6 t - shaped, 1 anterior column with posterior hemi - transverse, and 18 both - column fractures. the follow - up radiographs were graded according to the criteria developed by matta j. functional outcome was assessed using harris hip score and merle daubigne and postel score at final followup. average follow - up was 4.34 years (range 211 years).results : patients with congruent reduction (n=45) had good or excellent functional outcome. radiologic outcome in incongruent reduction (n=26) was good or excellent in 6 and fair or poor in 20 hips. the functional outcome in patients with incongruent reduction was good or excellent in 16 and satisfactory or poor in 10 hips. good to excellent radiologic and functional outcome was achieved in all patients with posterior wall fractures including four having more than 50% of broken wall. good to excellent functional outcome was observed in 88.8% of both - column fractures with secondary congruence despite medial subluxation.conclusions:nonoperative treatment of acetabular fractures can give good radiological and functional outcome in congruent reduction. posterior wall fractures with a congruous joint without subluxation on computed tomography axial section, posterior column, anterior column, infratectal transverse or t - shaped, and both - column fractures may be managed conservatively. small osteochondral fragments in the cotyloid fossa or non weight - bearing part of the hip with a congruous joint do not seem to adversely affect the functional outcome. displaced transverse fractures with v sign may require operative treatment. |
staphylococcus aureus (s. aureus) infections are a major cause of pneumonia and are especially implicated in hospital - acquired pneumonia (hap), ventilator - associated pneumonia (vap), and healthcare - associated pneumonia. the recent infectious diseases society of america guidelines recommend treating hap / vap patients with either vancomycin or linezolid. higher vancomycin minimum inhibitory concentrations (mics) have been associated with more frequent treatment failure and higher mortality rates [36 ]., george town, grand cayman, cayman islands) is a parenteral bactericidal lipoglycopeptide antibiotic that has a dual mechanism of action [710 ]. telavancin is approved in the us, canada, russia, and europe for treatment of hap, including vap that is caused by susceptible isolates of s. aureus (methicillin - resistant s. aureus [mrsa ] only in europe). in the us, russia, and europe, the indication is limited to infections in which alternative medicines are unsuitable [8, 11, 12 ]. in the assessment of telavancin for treatment of hospital - acquired pneumonia [attain (clinicaltrials.gov identifiers nct00107952 and nct00124020) ] studies, a total of 1503 patients with hap were treated with telavancin or vancomycin for up to 21 days. drug loss due to binding to plastics in the mic assay has been found with other lipoglycopeptides and is resolved with the addition of polysorbate 80 (p-80) [14, 15 ]. to improve the accuracy and precision of telavancin susceptibility testing, a revised broth microdilution method (rbmd) mic testing method for telavancin was approved by the us food and drug administration (fda) and published by clinical and laboratory standards institute (clsi) in 2014 to include dimethyl sulfoxide (dmso) in the diluent to improve solubility and p-80 in the broth microdilution assay cation - adjusted mueller hinton broth (camhb) to reduce drug loss due to binding to plastic [8, 16, 17 ]. this revised method has produced up to 8-fold lower telavancin mic results compared with the previous method and has been used to establish new values for telavancin activity against a variety of clinical isolates [18, 19 ]. the fda and clsi previously have approved 0.12 g / ml as the telavancin mic susceptibility breakpoint for s. aureus [both methicillin - susceptible s. aureus (mssa) and mrsa ] isolates ; intermediate or resistant breakpoints for telavancin have not been established as of 2016, due to the rarity of telavancin - resistant s. aureus isolates [8, 20 ]. the objective of this study was to determine the effect of the revised method on telavancin mic values for s. aureus clinical isolates obtained from the attain hap / vap patients and evaluate the clinical outcome by the revised telavancin mics. these results were part of the data package presented to breakpoint setting committees, including fda, clsi, and the european committee on antimicrobial susceptibility testing for the evaluation of the revised telavancin breakpoints. briefly, these were identical, randomized, double - blind, comparator - controlled phase 3 trials (clinicaltrials.gov identifiers nct00107952 and nct00124020) in which inpatients who developed pneumonia caused by gram - positive infections were randomized to receive telavancin (10 mg / kg every 24 h) or vancomycin (1 g every 12 h) for 7 to 21 days. the primary endpoint of the study was the investigator - assessed clinical response at a follow - up / test - of - cure visit between 7 and 14 days after the last dose of study medication. respiratory and blood culture specimens were collected at baseline, and isolates underwent susceptibility testing and confirmation at a central laboratory (covance laboratories, indianapolis, in, usa ; duke clinical research institute, durham, nc, usa). clinical cure rates by mic reported in the attain trials were reassessed using the mics obtained using the rbmd method. in the attain studies, the clinically evaluable study population consisted of patients who met study inclusion criteria and adhered to study protocol such that their clinical outcome could be considered to accurately reflect the effects of study medication. the microbiologically evaluable population in the attain studies included all clinically evaluable patients who had a gram - positive (s. aureus in this analysis) respiratory pathogen at baseline. presumed microbiological eradication was defined as failing to identify the baseline pathogen in the last postbaseline culture or if the patient was clinically cured and there were no follow - up cultures available. the analysis of the effects of telavancin on clinical isolates from the attain studies was performed using the rbmd method at jmi laboratories (north liberty, ia, usa). telavancin stock solutions of 1600 g / ml were prepared by dissolving dry powder in dmso in a glass vial ; stock solutions were further diluted in dmso to achieve intermediate concentrations ranging from 0.004 to 8 g / ml, per clsi recommendations. the telavancin / dmso solutions were further diluted 100 in camhb containing 0.002% (volume / volume) p-80 (tween 80 ; croda international, snaith, uk) to minimize adhesion to plastic surfaces. following dilution, 100-l aliquots of telavancin solutions ranging in concentration from 0.004 to 8 comparator agents tested against the same s. aureus isolates included linezolid (mic range 0.254 g / ml), ampicillin (mic range 0.2532 g / ml), and vancomycin (mic range 0.2516 g / ml). isolates were sent from the attain study central laboratories (covance laboratories) to jmi laboratories for retesting using the rbmd method. the isolates were stored at 80 c at covance, then were shipped to jmi laboratories on dry ice, where they were stored at 80 c. sample quality was assured via concurrent testing of clsi - recommended quality control (qc) reference strains (s. aureus american type culture collection [atcc ] 29213, enterococcus faecalis atcc 29212, and s. pneumoniae atcc 49619, manassas, va, usa). all the analyses were descriptive and performed using sas 9.4 software (cary, nc, usa). this article is based on previously conducted studies, and does not involve any new studies of human or animal subjects performed by any of the authors. briefly, these were identical, randomized, double - blind, comparator - controlled phase 3 trials (clinicaltrials.gov identifiers nct00107952 and nct00124020) in which inpatients who developed pneumonia caused by gram - positive infections were randomized to receive telavancin (10 mg / kg every 24 h) or vancomycin (1 g every 12 h) for 7 to 21 days. the primary endpoint of the study was the investigator - assessed clinical response at a follow - up / test - of - cure visit between 7 and 14 days after the last dose of study medication. respiratory and blood culture specimens were collected at baseline, and isolates underwent susceptibility testing and confirmation at a central laboratory (covance laboratories, indianapolis, in, usa ; duke clinical research institute, durham, nc, usa). clinical cure rates by mic reported in the attain trials were reassessed using the mics obtained using the rbmd method. in the attain studies, the clinically evaluable study population consisted of patients who met study inclusion criteria and adhered to study protocol such that their clinical outcome could be considered to accurately reflect the effects of study medication. the microbiologically evaluable population in the attain studies included all clinically evaluable patients who had a gram - positive (s. aureus in this analysis) respiratory pathogen at baseline. presumed microbiological eradication was defined as failing to identify the baseline pathogen in the last postbaseline culture or if the patient was clinically cured and there were no follow - up cultures available. the analysis of the effects of telavancin on clinical isolates from the attain studies was performed using the rbmd method at jmi laboratories (north liberty, ia, usa). telavancin stock solutions of 1600 g / ml were prepared by dissolving dry powder in dmso in a glass vial ; stock solutions were further diluted in dmso to achieve intermediate concentrations ranging from 0.004 to 8 g / ml, per clsi recommendations. the telavancin / dmso solutions were further diluted 100 in camhb containing 0.002% (volume / volume) p-80 (tween 80 ; croda international, snaith, uk) to minimize adhesion to plastic surfaces. following dilution, 100-l aliquots of telavancin solutions ranging in concentration from 0.004 to 8 comparator agents tested against the same s. aureus isolates included linezolid (mic range 0.254 g / ml), ampicillin (mic range 0.2532 g / ml), and vancomycin (mic range 0.2516 g / ml). isolates were sent from the attain study central laboratories (covance laboratories) to jmi laboratories for retesting using the rbmd method. the isolates were stored at 80 c at covance, then were shipped to jmi laboratories on dry ice, where they were stored at 80 c. sample quality was assured via concurrent testing of clsi - recommended quality control (qc) reference strains (s. aureus american type culture collection [atcc ] 29213, enterococcus faecalis atcc 29212, and s. pneumoniae atcc 49619, manassas, va, usa). all the analyses were descriptive and performed using sas 9.4 software (cary, nc, usa). this article is based on previously conducted studies, and does not involve any new studies of human or animal subjects performed by any of the authors. a total of 647 s. aureus isolates from patients with hap in the attain studies were analyzed for telavancin mics using the rbmd method (644 samples were tested using the original method). three isolates that did not have an initial central laboratory mic result and were not included in the original susceptibility analysis were included in this set of retested isolates. the mic values obtained using the rbmd method for telavancin and control agents tested against attain s. aureus isolates were within the clsi - approved and accepted qc ranges determined using s. aureus atcc 29213 (telavancin 0.030.12 g / ml, ampicillin 0.52.0 g / ml, linezolid 1.04.0 g / ml, vancomycin 0.52.0 g / ml). telavancin mics for all s. aureus isolates ranged from 0.015 to 0.12 g / ml, with a range of 0.015 to 0.12 g / ml for mssa isolates and 0.015 to 0.12 g / ml for mrsa isolates (table 1). for the overall set of s. aureus isolates and for mssa isolates, telavancin mic50/90 values were 0.06/0.06 g / ml ; for mrsa, the telavancin mic50/90 values were 0.06/0.12 g / ml. these results represent a substantial downward shift in telavancin mic values for s. aureus compared with the mics obtained using the original method (fig. 1), which produced higher values of mics for telavancin (mic50/90 of 0.25/0.50 g / ml for the total pool of s. aureus isolates).table 1distribution of telavancin mic values obtained using rbmd on hap isolates from attain trialsorganismmethodmic (g / ml) n 0.0150.030.060.120.250.51.0range (g / ml)mic50/90 (g / ml) s. aureus (all)revised647316741364 0.0150.120.06/0.06original644213346258250.0610.25/0.50mssarevised24019414410.0150.120.06/0.06original239101785010.1210.25/0.5mrsarevised407273269630.0150.120.06/0.12original40523168208240.0610.5/0.5 s. aureus vancomycin mic 1 g / mlrevised52894370613 0.030.250.06/0.12original4667229209210.1210.25/0.5mssa vancomycin mic 1 g / mlrevised1664811710.030.120.06/0.06original1375103290.120.50.25/0.5mrsa vancomycin mic 1 g / mlrevised362462536030.030.250.06/0.12original3292126180210.1210.5/0.5data presented as number of isolates except where indicated attain assessment of telavancin for treatment of hospital - acquired pneumonia ; hap hospital - acquired pneumonia ; mic minimum inhibitory concentration ; mrsa methicillin - resistant s. aureus ; mssa methicillin - susceptible s. aureus ; rbmd revised broth microdilution ; s. aureus staphylococcus aureus fig. 1distribution of telavancin mics obtained using the rbmd or original bmd method in a all s. aureus isolates, b mssa isolates, c mrsa isolates, and d s. aureus isolates with vancomycin mics mic minimum inhibitory concentration ; mrsa methicillin - resistant s. aureus ; mssa methicillin - susceptible s. aureus ; rbmd revised broth microdilution ; s. aureus staphylococcus aureus ; van vancomycin distribution of telavancin mic values obtained using rbmd on hap isolates from attain trials data presented as number of isolates except where indicated attain assessment of telavancin for treatment of hospital - acquired pneumonia ; hap hospital - acquired pneumonia ; mic minimum inhibitory concentration ; mrsa methicillin - resistant s. aureus ; mssa methicillin - susceptible s. aureus ; rbmd revised broth microdilution ; s. aureus staphylococcus aureus distribution of telavancin mics obtained using the rbmd or original bmd method in a all s. aureus isolates, b mssa isolates, c mrsa isolates, and d s. aureus isolates with vancomycin mics mic minimum inhibitory concentration ; mrsa methicillin - resistant s. aureus ; mssa methicillin - susceptible s. aureus ; rbmd revised broth microdilution ; s. aureus staphylococcus aureus ; van vancomycin among the 528 isolates with reduced susceptibility to vancomycin (mic 1 g / ml), telavancin mic50/90 values obtained using the rbmd method were 0.06/0.12 g / ml. among these samples, 166 were mssa and 362 were mrsa, with mic50/90 values of 0.06/0.06 g / ml for mssa and 0.06/0.12 g / ml for mrsa, respectively (table 1). these values were lower than the telavancin mic50/90 values for isolates with reduced vancomycin susceptibility generated by the original method, which were 0.25/0.5 g / ml and 0.5/0.5 g / ml for mssa and mrsa, respectively. the isolates with reduced susceptibility to vancomycin remained susceptible to telavancin (rbmd mic50/90) when using the revised fda - approved breakpoint of < 0.12 g / ml. a total of 183 isolates exhibited a 1 dilution change in the vancomycin mic 1 g / ml upon retesting. of those, 1 increased from 0.25 to 0.5 g / ml, 110 increased from 0.5 to 1 g / ml, 11 increased from 1 to 2 g / ml, 50 decreased from 1 to 0.5 g / ml, and 11 decreased from 2 to 1 g / ml. a total of 2 isolates exhibited a 2-dilution change in the vancomycin mic upon retesting with 1 increasing from 0.5 to 2.0 g / ml and 1 decreasing from 2.0 to 0.5 g / ml. these changes can be attributed to random testing differences between two laboratories (table 1). the clinical cure rate by revised mic was assessed in the microbiologically evaluable population of telavancin - treated patients (195 patients). the overall telavancin clinical cure rate for s. aureus was 78% (153/195 patients), with clinical cure rates of 83% (62/75 patients) and 76% (91/120 patients) for mssa- and mrsa - infected patients, respectively (table 2). the clinical cure rate did not decrease with increasing telavancin mic values for s. aureus obtained using the rbmd method, which ranged from 0.03 to 0.12 g / ml, as clinical cure rates remained 81% and 70% for mssa- and mrsa - infected patients, respectively, for all mic values (table 2). among the 166 microbiologically evaluable telavancin - treated patients with s. aureus isolates that demonstrated reduced susceptibility to vancomycin (mic 1 g / ml), the clinical cure rate was 79% (131/166 patients) for telavancin. the microbiological eradication rates for the overall s. aureus, individual mssa and mrsa isolates, and isolates with reduced vancomycin susceptibility (mics 1 g / ml) were comparable (table 2).table 2clinical cure and microbiological eradication rates in the microbiologically evaluable population of the attain studies using telavancin mic values obtained using the rbmd methodtotalmic (g / ml)0.0150.030.060.12clinical cure, n / n (% ; 95% ci) s. aureus (all)153/195 (78 ; 72, 84)36/46 (78 ; 64, 89)103/134 (77 ; 69, 84)14/15 (93 ; 68, 100) s. aureus (mssa)62/75 (83 ; 72, 90)22/26 (85, 65, 96)39/48 (81 ; 67, 91)1/1 (100 ; 3, 100) s. aureus (mrsa)91/120 (76 ; 67, 83)14/20 (70 ; 46, 88)64/86 (74 ; 64, 83)13/14 (93 ; 66, 100) s. aureus vancomycin mic 1 g / ml 131/166 (79 ; 72, 85)26/31 (84 ; 66, 95)91/120 (76 ; 67, 83)14/15 (93 ; 68, 100)microbiological eradication, n / n (% ; 95% ci) s. aureus (all)152/195 (78 ; 71, 84)34/46 (74 ; 59, 86)104/134 (78 ; 70, 84)14/15 (93 ; 68, 100) s. aureus (mssa)62/75 (83 ; 72, 90)22/26 (85 ; 65, 96)39/48 (81 ; 67, 91)1/1 (100 ; 3, 100) s. aureus (mrsa)90/120 (75 ; 66, 82)12/20 (60 ; 36, 81)65/86 (76 ; 65, 84)13/14 (93 ; 66, 100) s. aureus vancomycin mic 1 g / ml 130/166 (78 ; 71, 84)24/31 (77 ; 59, 90)92/120 (77 ; 68, 84)14/15 (93 ; 68, 100) attain assessment of telavancin for treatment of hospital - acquired pneumonia ; ci confidence interval ; mic minimum inhibitory concentration ; mrsa methicillin - resistant s. aureus ; mssa methicillin - susceptible s. aureus ; rbmd revised broth microdilution ; s. aureus staphylococcus aureus these values included 48/56 patients with mssa and 83/110 patients with mrsa these values included 48/56 patients with mssa and 82/110 patients with mrsa clopper pearson (exact) confidence interval clinical cure and microbiological eradication rates in the microbiologically evaluable population of the attain studies using telavancin mic values obtained using the rbmd method attain assessment of telavancin for treatment of hospital - acquired pneumonia ; ci confidence interval ; mic minimum inhibitory concentration ; mrsa methicillin - resistant s. aureus ; mssa methicillin - susceptible s. aureus ; rbmd revised broth microdilution ; s. aureus staphylococcus aureus these values included 48/56 patients with mssa and 83/110 patients with mrsa these values included 48/56 patients with mssa and 82/110 patients with mrsa clopper pearson (exact) confidence interval lipoglycopeptide drug loss due to its binding to plastics is known to affect the mic determination. the addition of p-80 to the mic assay has resolved this issue and the fda - approved rbmd method includes dmso and p-80 to improve drug solubility and reduce drug loss, respectively [8, 1417 ]. telavancin is a lipoglycopeptide active against a wide range of susceptible gram - positive pathogens, including s. aureus. the objective of this study was to re - evaluate the attain clinical isolates with the rbmd method that has previously demonstrated lower telavancin mic values [8, 16, 17 ]. telavancin mic values for mssa and mrsa obtained using the rbmd method were lower than those that have been published elsewhere using the original method [21, 22 ], indicating that telavancin is more active in vitro against s. aureus isolates than previously considered. in this study, the rbmd method produced an overall 4-fold decrease in the telavancin mic50/90 for s. aureus compared with the original method. all isolates were susceptible to telavancin, consistent with other studies reporting telavancin susceptibility of s. aureus using the rbmd method [18, 19 ]. telavancin also retained its in vitro potency against isolates with reduced susceptibility to vancomycin (mic 1 g / ml). furthermore, the reassessment of attain study clinical isolates using the rbmd method demonstrated robust clinical cure and microbiological eradication even at the highest telavancin mic (0.12 g / ml) observed in the study. the patients were not prospectively stratified by mic and the reduced sample size prevented extensive statistical analyses of the clinical cure rates. however, the clopper pearson (exact) confidence interval method was applied to demonstrate that the clinical cure rates were comparable across all s. aureus isolates and telavancin mics. reassessment of the attain isolates using the rbmd method demonstrated increased in vitro potency of telavancin against s. aureus. these data support lowering the telavancin susceptibility breakpoint to 0.12 g / ml for s. aureus. moreover, these results suggest that published studies using the previous bmd underestimated the in vitro potency of telavancin [23, 24 ]. barriere is a former employee of theravance biopharma us, inc., and received personal fees during the conduct of the study. corey reports personal fees from theravance during the conduct of the study. in addition, g.r. corey reports personal fees from nabriva, medtronic, pfizer, cempra, cerexa / forest / actavis, trius / cubist / merck, achaogen, glaxosmithkline, melinta, and motif ; and grants and personal fees from the medicines company and dr. in addition, dr. stryjewski reports personal fees from cerexa and achaogen ; and grants from duke clinical research institute, the medicines company, jmi laboratories, theravance, and cempra outside the submitted work. this article is based on previously conducted studies, and does not involve any new studies of human or animal subjects performed by any of the authors. this article is distributed under the terms of the creative commons attribution - noncommercial 4.0 international license (http://creativecommons.org/licenses/by-nc/4.0/), which permits any noncommercial use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. | introductionthe broth microdilution method (bmd) for testing telavancin minimum inhibitory concentrations (mics) was revised (rbmd) in 2014 to improve the accuracy, precision, and reproducibility of the testing method. the aim of this study was to determine the effect of the revised method on telavancin mic values for staphylococcus aureus (s. aureus) clinical isolates obtained from hospital - acquired pneumonia (hap) patients.methodsisolates from patients who participated in the phase 3 assessment of telavancin for treatment of hap studies were retested using the rbmd method.resultsretesting of 647 isolates produced a range of telavancin mic values from 0.015 g / ml to 0.12 g / ml with mic50/90 values of 0.06/0.06 g / ml for the total pool of samples. for methicillin - resistant s. aureus (mrsa), mic50/90 values were 0.06/0.12 g / ml. these values are up to 4-fold lower than mic50/90 values obtained using the original method. these results were used in part to justify lowering the telavancin breakpoints. all tested isolates remained susceptible to telavancin at the revised susceptibility breakpoint of 0.12 g / ml. overall, the clinical cure rate for microbiologically evaluable telavancin - treated patients was 78% for s. aureus, 76% for patients with mrsa, and 79% for patients with isolates with reduced susceptibility to vancomycin (mic 1 g / ml).conclusionresults from the rbmd method support the in vitro potency of telavancin against s. aureus.trial registrationattain (nct00107952 and nct00124020).fundingtheravance biopharma antibiotics, inc. |
health issues of migrant populations are an important public health concern. the term migrant covers both emigrants and immigrants. in some of the literature including this paper, it is used synonymously with immigrant defined as a person with two foreign born parents.. the healthcare needs of migrant women have been an area of concern both nationally and internationally, with particular focus on issues of sexual and maternal health including access to maternity care as well as maternal and neonatal outcomes [35 ]. state that although varying considerably, the reproductive health of immigrant women in europe is fairly unsatisfactory. research indicates that many migrant women find it difficult to access optimal maternity care and have worse maternal outcomes than native born women [79 ]. in sweden and norway the reproductive health of immigrant women is similar to that of the native born population, while it is inferior in uk and italy, which may be due to the fact that receiving countries have different attitudes to immigrants and that the health and welfare of immigrant communities vary.. found that migrant women in maternity care in europe were concerned with preserving their integrity in the new country. many struggled to find meaning, which was related to inadequate communication, lack of connection, striving to cope, struggling to ensure a safe pregnancy and childbirth, and maintaining bodily integrity. some felt insecure and not taken seriously during childbirth. organizational barriers to maternity care also existed, as they were not designed for migrants or adjusted to their needs. on the other hand, although migrant women face many challenges, they also encounter opportunities in their new country of residence. appropriate maternity care presupposes recognition of and feedback from immigrant women. according to mccourt and pearce, minority ethnic women share similar fundamental expectations of maternity care services to those of the wider population of which they are a part, but they experience a greater dissonance between expectations and experiences. small. found that immigrant women were less positive about maternity care than nonimmigrant women, although few differences were found in terms of their wishes. migrant women focused on the need for respectful care, assistance with communication, and better information about how care is provided in their new country. according to heaman., migrant women are more likely to have inadequate prenatal care and the factors that contribute to their prenatal care utilization are poorly understood. research on migrant women in maternity care has traditionally focused on the reduction of risk during pregnancy and adverse birth outcomes, as well as the difficulties involved in caring for these women. although there has been little interest in the coping strategies of migrant women, some research has been carried out in this area. for example, gagnon. demonstrated that migrant women in canada draw on a wide range of coping strategies and resources to respond to maternal - child health and psychosocial concerns. among the 16 participants in the study, social inclusion was identified as an overarching factor for enhancing resiliency, while margioula - siarkou. revealed that immigrant women had better obstetric and neonatal outcomes compared to native women in greece. the increasing diversity of the population in many countries presents a challenge for healthcare professionals. correa - velez and ryan discussed the development of the best practice model of refugee maternity care comprising continuity of care, quality interpreter services, educational strategies, and psychosocial support. in sweden, women from the middle east and sweden who were interviewed three months postpartum were found to focus on well - being and ability to care for the baby. however, beliefs about health and illness differ and affect risk awareness as well as self - care practice. the researchers concluded that professionals play a significant role in supporting women and their families during the transition to motherhood, particularly those who are migrants. a critical factor for women 's experiences at all stages of care is the nature of the women 's interactions with caregivers. the number of immigrants living in norway has more than doubled since 2004 to 600,000 persons in 2013, corresponding to 12% of the population. norwegian immigrants mainly come from pakistan, iraq, vietnam, somalia, bosnia and herzegovina, iran, turkey, serbia, sri lanka, and poland. as a result, norwegian society has become more multicultural than ever before in the sense that different ethnic groups are a natural part of the same society. the growth in the number of immigrants and the fact that most immigrant women are of child - bearing age explain why an increasing number of babies born in norway have an immigrant mother. in 2012, 23% of newborns, nearly one in four, had an immigrant mother. equal rights are enshrined in norwegian legislation enabling immigrants and asylum seekers to access health care and medical treatment. the patients ' rights act is intended to ensure that all citizens have equal access to good quality health care by granting patients ' rights in their relations with the health service. the provisions of this act are designed to promote a trusting relationship between the patient and the health service while safeguarding respect for life, integrity, and human dignity. in norway refugees and their relatives who joined them under the family reunification scheme have a right and duty to participate in the introductory programme, the purpose of which is to promote their participation in work and social life. adult immigrants with a residence permit who are eligible for a settlement permit also have a right and duty to attend 300 hours of norwegian language training and social knowledge. the norwegian national clinical guideline for antenatal care states that women with a normal pregnancy should be cared for by a midwife or a general medical practitioner (gp). the woman herself should choose whether to be attended by a gp, a midwife, or a gp and midwife working in cooperation. the midwife in the community works in the maternal and child health clinic together with public health nurses. the national professional guideline for postnatal care emphasizes equity in the postnatal health services, implying that healthcare personnel must respect diversity and recognize all individuals as equal members of society with the same right to health services. a study from norway on healthcare professionals ' perspectives revealed that equity in maternity care for migrant woman is related to managing and supporting educational, relational, and cultural diversity, about which public health nurses and midwives require more information and knowledge. the maternal health coping strategies of migrant women should be viewed in the context of where they live. psychologist urie bronfenbrenner stated that human abilities and their realization depend to a significant degree on the larger social and institutional context. he presented an ecological model, which provides the framework from which researchers study an individual 's relationship within communities and society in general. the model was originally used to explain how a child 's development is influenced by different environments, but is now considered a valuable perspective in health promotion [28, 29 ]. in the present paper a description of the systems in the ecological model thurston and vissandje argue that the health of immigrant women is best understood in a framework that takes gender and the migratory experience into consideration, caring for the individual by focusing on social factors at meso- and macrolevels. the salutogenic perspective means focusing on what creates health rather than taking a traditional medical approach that explores the roots of illness. the sociologist aron antonovsky posed the question : what are the origins of health ? he explored the idea of health as a continuum from ease to dis - ease. migration as well as pregnancy and childbirth can be considered stressful life events. in a salutogenic approach, stressors are open - ended, initiating an interaction between the individual and her / his immediate environment. the emphasis is on the person 's ability to use both internal and external general resistance resources (grrs) to manage stressful situations. grrs are found within people bound to their person and capacity, but also as material and nonmaterial resources in their immediate or distant environment. grrs constitute the prerequisites for the development of a person 's sense of coherence (soc). soc is a coping resource that enables a person to choose different strategies for solving different problems or managing life events. the theory of salutogenesis corresponds closely with the key principles of the who ottawa charter for health promotion and is useful in the making of healthy public policy. the focus of this study was migrant women 's movement on the health / dis - ease continuum and how to strengthen their ability to make the best choices for safeguarding health. the aim is to explore the maternal health coping strategies of migrant women in norway. the research questions were as follows : what are migrant women 's expectations and experiences of the maternal and child health services ? in this study a qualitative exploratory, descriptive design with a hermeneutic approach was employed [35, 36 ]. the study, in which 17 migrant women participated, was conducted at a maternal and child health centre by means of semistructured interviews. the participants had attended the centre during pregnancy and after childbirth during the previous year. they were purposively selected and invited to take part in the study by a midwife at the health centre. the midwife contacted all women with a foreign background who had given birth during the previous year. the inclusion criteria were as follows : women who (1) were born in another country with two foreign born parents, (2) attended the maternity and child health centre, and (3) had given birth in the previous 12 months up to six weeks before the interview. they comprised refugees, labour migrants, students, and women married to norwegian men. most of them spoke norwegian, but somali, amharic, and kurdish interpreters were employed in five of the interviews (table 1). the interviews were conducted between may and december 2012 at the participants ' usual maternal and child health centre. each interview lasted between 45 and 75 minutes, was audio - taped, and later transcribed verbatim. the interviewers (berit viken and anne lyberg) encouraged the participants to narrate their expectations and experiences of norwegian maternity services and their life situation in general in the new country and as mothers in particular. some examples of the questions posed are as follows : what are your experiences of maternity health care ? was the cooperation between you and the midwife, public health nurse, and doctor satisfactory ? how did the health providers show respect for you ? do you have any suggestions for changes in the health services ? who do you ask when you are uncertain about your own or your baby 's health ? the interviewers listened to the women 's narratives and posed probing questions to further illuminate their experiences. the study was approved by the manager of the maternal and child health centre. the midwives and public health nurses working at the centre were also informed about the study, which was carried out according to the ethical guidelines for nursing research in the nordic countries. the participants were provided with written and verbal information about the purpose and design of the study by the midwife who tried to ascertain the women 's ability to understand it. the other women who needed a translation spoke different languages and received a verbal translation of the information from an interpreter. the women were advised about the voluntary nature of participation that their name and identity would not be disclosed as well as their right to withdraw at any time without any consequences in terms of maternal and child health care. they were also informed that the audio - tapes and transcripts would be destroyed at the end of the project. as the researchers are responsible for the safety of the participants, the latter were invited to contact a named midwife at the maternity and child health centre if they in any way felt upset after the interview. it started with the authors reading the transcribed text to gain an overview of the data as a whole as well as an understanding of each interview. thereafter, the two research questions what are migrant women 's expectations and experiences of the maternal and child health services ? and what are the maternal health coping strategies of migrant women ? were focused on. the codes were compared, combined, reduced, and sorted into condensed meaning, subthemes, themes, and a main theme (table 2). to avoid misunderstanding the meaning of the text and ensure validity, the analysis moved back and forth between the emerging categories and the data. when interpreting the interview texts, the three authors discussed and shared the meanings arrived at, thus expanding and adjusting their individual understandings of the participants ' expectations and experiences. thereafter the results of the analysis were interpreted in the light of bronfenbrenner 's ecological model. bronfenbrenner identified four environmental systems with which an individual interacts : the micro-, meso-, exo-, and macrosystem. the microsystem refers to the institutions and groups that most directly influence the child 's development (or a person 's adaptation, author 's note), including family, school, religious institutions, neighbourhood, and peers. the exosystem comprises links between a social setting in which the individual does not play an active role and her / his immediate context. the two interviewers are both public health nurses. this could have affected the participants ' answers in that they might have wanted to be positive and not criticize the norwegian health services. on the other hand, all participants had a residence permit in norway and did not have a reason to fear that their answers could have negative consequences for them, as described by liamputtong. it became clear when the interviews started that some of the participants had not understood all the information provided beforehand. informed consent presupposes that the individual understands the information and can make a voluntary decision to enrol and participate in the research. this can be a challenging task in cross - cultural research because issues of culture come into play. when the participants afterwards were asked how it was to talk with the interviewer, they all had a positive attitude towards giving their opinions about subjects that were important to them. the interview data revealed the participants ' strong will to integrate into the new society while at the same time maintaining traditions from their country of origin. the analysis revealed one main theme : keeping original traditions and at the same time being willing to integrate into norwegian society, based on four themes, balancing their sense of belongingness, seeking information and support from healthcare professionals, being open to new opportunities, and focusing on feeling safe in the new country. the results will be presented and explained in the light of the four systems in the ecological model (table 3). the migrant women 's life in a family and connections to other relatives and friends are part of the microsystem. several mothers stated that having a child gave them a feeling of belonging and that they followed the child 's rhythm. for example, one of the women could not understand why she should pump breast milk and keep it in a bottle because as she expressed : passes so quickly, i breastfeed at least three times every night and sleep when the baby sleeps. i do not have much time to do other things than taking care of her. (p15) the day passes so quickly, i breastfeed at least three times every night and sleep when the baby sleeps. i do not have much time to do other things than taking care of her. (p15) faith in god was important for several of the migrant women and gave them confidence in daily life.i am comforted when i read the koran, as you do not get any negative thoughts, just positive ones and trust. (p6) i am comforted when i read the koran, as you do not get any negative thoughts, just positive ones and trust. (p6) other women stated that their belief in god is not strong, but that it is important to follow some religious traditions such as the celebration of eid al - fitr after ramadan. this is a very common activity among norwegian families with children and everybody has a pram, but for migrants it can be a new experience.at weekends we normally take a walk in the forest. many took cod liver oil to prevent lack of vitamin d, which is a tradition in norway and recommended for both mothers and children. at the same time many of the women said that they made use of advice from their country of origin, for example, herbal tea for the baby 's stomach ache. an asian woman struggled with contradictory advice from healthcare professionals in norway and from her mother and healthcare professionals in her native country. she says that her gp had advised her to continue exercising as there was no need to change her lifestyle. she believed that exercise was the reason for her miscarriage because in her native country it is usual for women to be very careful during the first three months of pregnancy. therefore it was confusing to receive different advice in norway both from her norwegian husband and the gp. when this woman became pregnant again, she travelled back to her country of origin and followed the advice not to exercise. she also brought some medicine back to norway to prevent a miscarriage and this made her feel safer. (p14) the participants appreciated contact with other migrants but also longed for friendships with norwegians. some women found it difficult to get to know people in their new surroundings.i feel a bit lonely in this town. my midwife introduced me to a woman from poland in the same situation as myself and she has become my friend now. it is important to be open and speak norwegian. in another town, where i used to live, the health centre organised a group for women who had given birth, in which i felt very welcome. my midwife introduced me to a woman from poland in the same situation as myself and she has become my friend now. it is important to be open and speak norwegian. in another town, where i used to live, the health centre organised a group for women who had given birth, in which i felt very welcome. (p12) it was of major importance for all the migrant women to keep in contact with family and relatives in their country of origin, which they achieved by means of telephone and the internet. they also travelled there on holidays or to a neighbouring country if they were unable to go back to their own country, especially in the winter, to experience sun and hot weather. (p11) many women highlighted family unity as the most important aspect of life.unity in the family is the most important thing in life. having gone through what we experienced, we never ever want to be separated again. (p5) unity in the family is the most important thing in life. having gone through what we experienced, we never ever want to be separated again. (p5) a woman who had no family from her native country in norway said that she received a great deal of help from her husband 's family. the women wanted to strengthen their sense of belonging to norwegian society while at the same time maintaining their sense of belonging to their native country. one woman who came from a country during a war twenty years ago commented that she could not imagine living anywhere other than norway now. however, her dream was to move back to her native country and be buried there. migrant women 's interactions with persons and institutions in the local community, such as the health services, make up the mesosystem. with the exception of one woman, all the participants had undergone several check - ups with the midwife during pregnancy and found the prenatal care very satisfactory. some women compared the health services in norway with the poor health services in their native country. a woman from an african country plagued by war said : i think norway is best when you are pregnant. the culture is very different to that of my home country and i miss my family, but i had very good discussions with the midwife, who made me relax and not think too much about the delivery. we have midwives in my country too, but we do not have a big health centre. sometimes the mother or the child can die. but here in norway it is the best you can get. the child check - ups, the hospital and the midwife are all very good. the culture is very different to that of my home country and i miss my family, but i had very good discussions with the midwife, who made me relax and not think too much about the delivery. we have midwives in my country too, but we do not have a big health centre. sometimes the mother or the child can die. but here in norway it is the best you can get. the child check - ups, the hospital and the midwife are all very good. (p4) the participants required knowledge about pregnancy to gain confidence and feel secure about their own and their baby 's health. they were well informed and prepared for the birth, while many had visited the maternity ward before delivery. some said they were lucky to have had the same midwife at the pregnancy check - ups and at childbirth. the women who had experienced pregnancies or deliveries that required extra care were very grateful to be in norway.i had long contractions and they helped me so that everything went well. i am thankful to the midwives who stood beside me the whole day and ensured the baby came out. if my child had been born in my home country he would not have survived. (p16) i had long contractions and they helped me so that everything went well. i am thankful to the midwives who stood beside me the whole day and ensured the baby came out. if my child had been born in my home country he would not have survived. (p16) some of the women were circumcised and needed surgery to open the vagina before delivery.it was my first child and i had just arrived in norway. she sent the information to the hospital and i got the help i needed to open the vagina before the baby came out. (p6) it was my first child and i had just arrived in norway. she sent the information to the hospital and i got the help i needed to open the vagina before the baby came out. (p6) there were both good and bad experiences of childbirth in norway, sometimes due to poor communication. when this woman was in labour she described being treated with indifference and lack of respect, which she thought was because she was new in norway and did not speak the language. (p10) the women considered it important to accept what was offered by the health services, also after childbirth.i am satisfied with the health centre. i got very good follow - up when i had difficulty with breast - feeding. i got very good follow - up when i had difficulty with breast - feeding. (p12) the women appreciated the information they received from the midwife during pregnancy and from the public health nurse in early motherhood. the information was provided during regular check - ups as well as open hours at the maternal and child health centre when there was no need to make an appointment. some women need more follow - up from the maternal and child health centre because certain aspects of knowledge are less familiar to them compared with many ethnic norwegian women. this can concern relatively minor issues such as different cultural traditions when the child is ready to eat solid food, for example, what sort of porridge they should start with. another more serious matter is lack of knowledge about norwegian legislation that prohibits hitting children. she explained that she was new in norway, did not speak the language, and did not know anything about norwegian culture and parenting. she did not understand what to do and the child welfare service took her children into care. she has become very preoccupied with obtaining new knowledge and wants new immigrants to be informed about the right thing to do. this system consists of decision makers (such as municipal officials and politicians) with whom the women have no direct contact but who still affect their lives. many saw life in norway as a new opportunity. living in a welfare state allowed them an opportunity to learn norwegian and study culture and society whilst receiving financial benefit. all the participants wanted to learn more norwegian and obtain a higher educational qualification and at a later stage paid employment. they wanted their children to attend kindergarten and were impressed by the opportunities it presented to their children and themselves. learning norwegian as a part of the introductory programme was very important to all the participants. they wished to improve their language skills and have an opportunity to practise the language.i have a positive opinion of the municipality. i also had the opportunity to learn norwegian and earn money by attending the introductory programme. i also had the opportunity to learn norwegian and earn money by attending the introductory programme. (p6) one woman appreciated not needing social security payment because her husband was employed and she was in receipt of introduction money. the migrant women wanted to work outside the home and were prepared to send their children to kindergarten. nevertheless, not all of them could find suitable work. a woman from africa had a job for several years in another european country, but she found it difficult to obtain a permanent position in norway. she had been in trial placements but when they finished she did not obtain any further work.if you try you can manage anything, i just need a chance. if we are given a chance we will communicate and learn the language. i will go to high school next year and then my child will attend kindergarten. then i will learn to drive and maybe start studying. (p8) if you try you can manage anything, i just need a chance. if we are given a chance we will communicate and learn the language. many came from countries in the throes of war and conflict with no public schools and social security. when they longed for their family and native country, the thought of their own and their children 's safe future in norway helped them to manage their feelings.sometimes when i think of moving back, i remind myself of how safe our future is in norway. i think it is a good environment for children, with fresh air and clean, safe food. (p14) sometimes when i think of moving back, i remind myself of how safe our future is in norway. i think it is a good environment for children, with fresh air and clean, safe food. (p14) one woman who had lived for many years in norway expressed her need to process memories from the war but also to move on. a woman from asia explained how her opinion changed about the use of medications for the common cold.when i first came i was very irritated because i could not buy medication without a prescription. after a while i understood that we do not need medications if we just have a cold. (p2) when i first came i was very irritated because i could not buy medication without a prescription. after a while i understood that we do not need medications if we just have a cold. (p2) other women wanted more examinations by specialists, for example, by a gynaecologist instead of a gp when pregnant and by a paediatrician to check their children in the maternal and child health centre. several women wished for more ultrasound scans during pregnancy.i would have liked more ultrasound scans. (p7) this woman paid for extra check - ups with a private doctor to obtain the ultrasound examinations she wanted and felt safer after those examinations. the aim of this study was to explore the maternal health coping strategies of migrant women in norway. the main theme that emerged was keeping original traditions while at the same time being willing to integrate into norwegian society. they had mostly positive experiences of the maternal health services, which were reflected in their willingness to integrate into society. in situations where they had negative experiences, the women used a wider range of strategies that involved keeping their original traditions and travelling to their native country to seek advice. the discussion will focus on migrant women 's coping strategies in accordance with a salutogenic approach, including their expectations and experiences, both within and outside the maternal health services. sense of coherence (soc) appears to influence the women 's actions and coping strategies when they become pregnant and after childbirth. balancing their sense of belongingness to family members and institutions in norway with that to their relatives in their country of origin affected the women 's sense of meaningfulness, which is the motivational component of the soc. the most meaningful task for the women is taking care of their own and the baby 's health during pregnancy and childbirth. this is consistent with other studies which underline childbirth as a positive life event [41, 42 ]. another important strategy is being open to new opportunities, which involves the willingness and efforts of migrant women to learn norwegian by taking part in the introductory programme. despite the fact that this programme is mandatory for all new refugees, the migrant women in our study were all very interested in it, especially learning norwegian. the programme is provided in the community and is thus a part of migrant women 's mesosystem. in the mesosystem can increase the women 's capabilities ; for example language skills can help them to comprehend health information and social structures in norwegian society. it also strengthens their social networks and makes their daily life more manageable by creating independency through not having to rely on translation by family members and interpreters. ability to communicate in norwegian also provides a stronger sense of belonging to society as it enables women to strengthen their social network and start new activities such as entering into paid employment. nussbaum contends that economic independence enhances women 's self - respect and capacity for choice. balancing their sense of belongingness can also be seen from a global perspective in accordance with haith - cooper and bradshaw and the model the pregnant woman in the global context, which emphasizes the environment of migrant women prior to, during, and after migration. another coping strategy is seeking information and support from family and healthcare professionals, where the women interact both within their microsystem and with the maternal and child health services, which are a part of their mesosystem. this strategy can be seen in connection with personal agency, the notion that the person has the capacity to make her (or his) own choices and exercise autonomy over personal life events and circumstances. support during childbirth was also significant for their capacity building. however, some of the women felt exposed when meeting healthcare professionals as they experienced a lack of communication and social interaction. the women also searched for information from the media both in norway and other countries as well as from healthcare professionals and family in their native country. similar findings are described in a study where somali women said they received health information from, among other sources, an english television channel. most of the women in the present study maintained close links with family members in their country of origin through the internet, telephone, and/or international travel. this can be considered part of the macrosystem and is supported by other studies that have a transnational perspective on migration. the identity and social practices of migrants transcend national borders [48, 49 ]. for example, a traditional medical perspective on pregnancy and childbirth was maintained by several of the participants, which contrasts with the concept of normal childbirth held by many norwegian midwives and described in the international literature [50, 51 ]. the maternal health coping strategies of migrant women are also influenced by their health literacy, which is a composite term used to describe the capacities of persons to meet the complex demands related to health information in modern society. it is a resource that equips people to navigate healthcare systems, critically assess information, and take greater control of their health. two different concepts reflect health literacy, a clinical risk and a personal asset. boerleider. found that suboptimal health literacy was one of the difficulties midwives encountered when caring for nonwestern women in netherlands, which can be said to represent the risk approach. in the present study we used the asset approach, in which health literacy applies to the development of skills and capacities that enable migrant women to exert greater control over their health and the factors that shape it. consequently, health literacy affects women 's maternal health coping strategies. the introductory programme, maternal and child health services, and social media interact and contribute to the women 's health literacy and thus their comprehensibility and manageability. aasen highlighted the importance of language skills for refugee women to cope with their daily life. some of the participants in this study had poor education, which affected their health literacy. the women who could not read and write were largely dependent on verbal information from healthcare professionals to increase their comprehensibility. consistent with these findings is the need for more innovative forms of communication that go beyond the written word [10, 47 ]. in their encounters with healthcare professionals the women who did not speak norwegian were highly dependent on interpreters to achieve understanding. an important coping strategy is focusing on feeling safe in the new country, which is connected to structures in norwegian society and thus part of the macrosystem. when the women longed for their home country, they focused on the safety of themselves and their children. while this is related to norway being a relatively peaceful country, it can also be seen as confidence in the welfare system. experiencing comfort and social security in a safe society constituted a major part of the women 's meaningfulness. norway 's welfare system, migrant, and refugee policy affect the social and economic conditions as well as the health services offered to migrant women. this is in accordance with the results of a group of researchers from germany, canada, and the united kingdom, who compared migration and maternity care in the three countries. they found that while the design and delivery of maternal health services play a crucial role in ensuring positive experiences and good maternity outcomes for migrant women, these services must be understood within a much wider framework. the main resources of migrant women in this study were their own capacity to readjust their lives, support from family and friends both in norway and abroad, and information and support from healthcare professionals as well as financial support and educational opportunities provided by the municipality. thus migrant women 's maternal health coping strategies are affected by participation in the micro- and mesosystems as well as being influenced by the exo- and macrosystems. reorienting health services is an important strategy in the ottawa charter for health promotion and implies that the health sector should move in a health promotion direction, beyond its responsibility for providing clinical and curative services. it is vital that this leads to a change of attitude towards the organization of health services, where the focus is on the total needs of the individual as a whole person. the results of this study reveal that migrant women 's maternal health coping strategies involve multisetting participation and can be understood by means of a system approach. we advocate a salutogenic approach to maternity care research in order to strengthen the capabilities of migrant women to take care of their own and their baby 's health. this will encourage healthcare professionals, policy makers, and maternity service users to consider maternity care from a health oriented and person centred perspective as opposed to one based on morbidity and mortality. maternal healthcare services should therefore be adjusted to the needs of migrant women. in order to provide culturally competent care, healthcare professionals must be culturally sensitive and aware of their own cultural background. cooperation with other sectors and multidisciplinary work can be part of an ecological approach to strengthen migrant women 's capabilities and wellbeing. this study had a salutogenic approach with focus on migrant women 's resources and capabilities. it was concluded that migrant women were concerned with keeping their own traditions in pregnancy and childbirth while at the same time showing a willingness to integrate into norwegian society. they used different coping strategies connected to family, healthcare professionals, and the education sector. in relation to ecological systems they were also affected by the guidelines and decisions taken in the exo- and macrosystems. to provide quality care, healthcare professionals should use a system approach and focus on the development of migrant women 's capabilities. adaptation of maternal health services for culturally diverse migrant women also requires a culturally sensitive approach on the part of healthcare professionals. | the aim of the study was to explore the maternal health coping strategies of migrant women in norway. the ethnic and cultural background of the norwegian population have become increasingly diverse. a challenge in practice is to adjust maternal health services to migrant women 's specific needs. previous studies have revealed that migrant women have difficulty achieving safe pregnancies and childbirths. data were obtained by means of 17 semistructured interviews with women from south america, europe, the middle east, asia, and africa. qualitative content analysis was employed. one overall theme is as follows : keeping original traditions while at the same time being willing to integrate into norwegian society, and four themes emerged as follows : balancing their sense of belongingness ; seeking information and support from healthcare professionals ; being open to new opportunities and focusing on feeling safe in the new country. the results were interpreted in the light of bronfenbrenner 's ecological model. to provide quality care, healthcare professionals should focus on the development of migrant women 's capabilities. adaptation of maternal health services for culturally diverse migrant women also requires a culturally sensitive approach on the part of healthcare professionals. |
placental transmogrification, first described in 1979, is a rare and benign disease of the lung, whose cause is still unknown (1, 2). radiologically, placental transmogrification of the lung mainly shows bullous changes and presents, very rarely, as cysts or nodules (2, 3). pathology of the disease characteristically shows papillary structures similar to placental villi surrounding the pulmonary epithelium (3). through this study, we a thoroughly review previous literatures on placental transmogrification, a rare disease entity, and report an unusual case of the disease with both a bullous change and consolidation. a 31-year - old female patient presented with cough and mild dyspnea to the outpatient department. she had a history of being diagnosed with pulmonary tuberculosis 7 years ago, which she had completely recovered after 6 months of antituberculosis drug regimen. complete blood count revealed no abnormal finding, and no acid - fast bacilli was discovered on the sputum culture test. plain chest radiography showed giant bulla in the left lung and subsegmental consolidation in the left lower lung field. chest computed tomography (ct) scan revealed the left upper lobe of the lung being replaced by bullae of various sizes, and subsegmental consolidation had broad contacted with bullae at the lingular segment of the left upper lobe (fig. wedge resection of the left lung using video - assisted thoracoscopy was performed under the preoperative diagnoses of giant bullae and pulmonary emphysema. during operation, gross examination of the wedge resected lung was 15 9 4 cm in size with dilated giant bullae (fig. spongiform emphysema was observed at the cut surface, and there were partial solid changes and fibrotic progression. examination of the solidified sites by light microscope indicated general emphysematous changes with destruction of the alveolar wall in addition to numerous tissues replaced by structures similar to the chorionic villi. the villous structure was adjacent to, or in some cases adhered to, the destroyed alveolar wall and interlobular septa. immunohistochemical staining of the epithelium lining the villous structure was partly positive for thyroid transcription factor-1, and the interstitial cells at the core was positive for cd-10, d2 - 40 and vimentin (fig. chest ct scan was taken to rule out the possibility of remnant bullae or transmogrified tissue. the giant bullae at the left upper lobe were removed, but some fibrotic tissue still remained. additional left upper lobectomy was planned to remove the remaining tissue in order to minimize recurrence. however, the patient did not complain of any symptoms and refused further operation. she was explained about the possibility of recurrence and scheduled visits to the outpatient clinic on a regular basis for follow - ups. placental transmogrification of the lung, or pulmonary placental transmogrification, is also referred to as placentoid bullous lesion. it is known to be a very rare disease (1, 2) with less than 30 adequately documented cases. clinically, placental transmogrification usually occurs in men between 20 to 50 years old ; however, our case was even a more rare case due to the occurrence in a female patient. it may be asymptomatic or perhaps associated with pneumothorax or chronic obstructive lung disease (4). it may be congenital, but no placental transmogrification has been reported in children (5). placental transmogrification is not only associated with cystic or emphysematous lung lesion, but also with pulmonary fibrochondromatous hamartomas and pulmonary lipomatosis. thus, the radiologic presentation can be bullous change or pulmonary lung nodules (6, 7). the papillary placentoid and villous structures resembling chorionic villi, which are characteristically seen in this patient, are known as placental transmogrification. a villous transformation is thought to arise from edema and fibrosis of pulmonary strands of severe emphysema or giant bullous emphysema. lipomatosis is also implicated as a cause due to the expression of fat tissues inside the villi as well as frank lipomatosis in the placental villi and in the interstitium of the lung (4). others report the proliferation of interstitial clear cell as primary occurrence and emphysema - like cystic changes as secondary occurrence (8). placental transmogrification of the lung is distinguished from typical emphysema by the tendency to occur in the younger age group along with the local progression of the disease (8). next, the disease is expressed on radiography with a mixed pattern of thin - walled cystic lesion and nodule (6, 7, 9). the disease itself is a rare disease entity, and our case had even more unusual findings of both giant bulla and subsegmental consolidation patterns. radiologically, differential diagnosis of the lesion includes cystic or bullous lung disease, such as bullous emphysema, particularly giant bullous emphysema (vanishing lung syndrome), and solitary pulmonary lung nodules, such as hamartoma (3, 9). however, bullous emphysema generally demonstrates a diffuse bilateral lung involvement (8, 9). in addition, although bullous emphysema can have rapid progression of lung parenchyma destruction in drug abuse, acquired immune deficiency syndrome or autoimmune disease, it commonly affects old age with a history of smoking and alpha-1 antitrypsin deficiency and develops over a long period of time (5). in particular, giant bullous emphysema is also referred to as primary bullous emphysema or vanishing lung syndrome, which is characterized by bulla occupying at least one third of the hemithorax. other radiologic findings of placental transmogrification are emphysema with pulmonary nodules or solitary pulmonary nodule patterns. in rare cases, however, the image finding alone has limited value to differentiate the lesion from placental transmogrification. the image finding of placental transmogrification presents unilateral large bullous emphysema with or without an associated nodule or consolidation. from a clinical standpoint, making an accurate preoperative diagnosis is difficult. although it is an extremely rare disease, it must be ruled out in patients with unilateral bullous lesion who do not have high risk of bullous emphysema. | placental transmogrification is a very rare lung disease, where the alveoli resemble the chorionic villi of placenta, and this change is a characteristic finding. a 31-year - old female patient presented with cough and dyspnea that had begun 2 weeks prior to admission. along with giant bulla found in the left upper lung field, subsegmental consolidation was also identified in the lingular segment on plain chest radiograph and ct scan. wedge resection was performed to remove the bulla. pathologic examination of the resected bulla revealed destruction of the normal structures and characteristic villous and papillary changes. these changes led to a diagnosis of placental transmogrification. we made an encounter of an unusual placental transmogrification which had different image findings from other reported transmogrification cases. thus, we report an atypical placental transmogrification case where both consolidation and giant bulla coexist. |
diabetes mellitus, which is today considered a society - wide disease of a chronic / degenerative nature, includes a group of metabolic disorders characterized by a hyperglycemic state that may derive from the altered secretion or the altered uptake and peripheral action of insulin. individuals with by diabetes face microvascular and macrovascular complications. among the secondary effects of diabetes, several studies have attested to a close link between diabetes and periodontal disease, due to alterations in microcirculation in the periodontium and in oral mucosa [24 ]. in the presence of hyperglycemia, the metabolic process of the endothelial cells encounter an excessive oxidative stress that in turn increases non - enzymatic glycation and the oxidation of proteins. therefore the excess of glucose in the blood favors the glycation of low density lipoproteins, the basic process behind atherosclerosis (macroangiopathy) and peripheral microangiopathic phenomena. at an oral mucous and periodontal level, it is clinically expressed by an increased tendency for tissue atrophy (above all on the back of the tongue) and by an increased tendency for the development of periodontal disease, which is also caused by the increase of glucose in sulcular fluid. videocapillaroscopy is an interesting way of studying microcirculation because of the possibility of examining small vessels in vivo by means of a microscope. from the study of periodontal microcirculation, the presence of characteristic capillaries can be observed the arterial and venous ends with their different diameters ; the presence and number of capillary crossing ; and the capillary density. videocapillaroscopy can have a predictive role in evaluating the presence of inflammatory phenomena. until now, it appears that no in vivo studies on oral and periodontal microcirculation have been performed that show a correspondence between variations in the periodontal capillary bed and the diabetic pathology. thanks to its particular anatomical and histological characteristics and easy accessibility, oral mucosa is indicated as being the preferential centre for capillaroscopic examination. the aim of this study was to underline and objectify microcirculatory variations at a periodontal mucous level in subjects diagnosed with type 2 diabetes. eighty subjects were enrolled and divided into 2 groups : 40 subjects with a diagnosis of diabetes mellitus type ii (18 males and 22 females, between 44 and 85 years of age) ; and 40 healthy subjects (17 males and 23 females, between 44 and 78 years of age) (table 1). all the subjects gave their consent for the capillaroscopic examination to be performed and for the processing and use of their personal medical data in scientific papers, in accordance with italian laws on privacy and the use of personal data (law 675/1996, art. the subjects were selected according to the following criteria : an established diagnosis of diabetes mellitus type ii, oral hypoglycemic therapy for at least 1 year, with routine checks on the level of glycemia, levels of glycated hemoglobin between 6.3% and 7.1% (indicative of a good glycemic control), absence of other pathological conditions that could cause alterations to the peripheral microcirculation of oral mucous such as hypertension, rheumatoid arthritis, sjogren s syndrome, oral lichen planus, pemphigus, pemphigoid, scleroderma, hashimoto s thyroiditis, absence of exposure to risk factors such as cigarette smoke and alcohol, absence of exposure to radiologic and chemotherapeutic agents. a complete objective examination of the soft tissues and hard tissues of the oral cavity were carried out for each subject. all the subjects (health subjects and diabetic subjects) were enrolled in the study if they had a healthy periodontal condition. the state of oral hygiene was meticulously rechecked before the capillaroscopic examination. for every subject the following indexes of periodontal health were considered : bleeding on probing (bop) ; plaque index (pli) ; and clinical attachment level (cal). at the time of the capillaroscopic examination, all the enrolled subjects had a plaque and bleeding index equal to 0 and cal was not significantly different between healthy subjects and diabetic subjects in a healthy periodontal condition. all the subjects, both diabetic and healthy, were submitted to a videocapillaroscopic examination of the mucous of the oral cavity. the examination was conducted through the videocap 200 videocapillaroscope produced by ds medica s.r.l., the videocapillaroscope used for this study was made up of : a central body that includes a light source consisting of a cold halogen light emitted by a 100 w lamp fitted with an automatic or manual control device to regulate luminosity and white balancing (figure 1), a probe with an optic terminal connected to the central body by a fiberoptic cable 2 meters in length ; the optic terminal is in turn constituted by a color high - definition micro - television camera (420.000 pixels), a support for holding the different lenses, a ferrule for fine focussing regulation, and a radial ferrule at the tip with annular illumination (for a uniform illumination without shadows). the lenses that can be used can be either contact or non - contact types with varying enlargements : 20, 50, 100, 200, 500, 1000 (figure 2), a personal computer with dedicated graphics card connected to the central body of the videocapillaroscope through an s - video cable. videocap software release 8.0 capillaroscopic imaging software is installed on the pc, a high resolution color cathode ray tube monitor. the capillaroscopic examination was conducted under standardized conditions of temperature and illumination : room temperature of 241c was maintained constant through air - conditioning systems, illumination was through neon light for medical use with a white point equal to 6500k. the investigated site was selected according to criteria of accessibility and legibility of the capillaroscopic data : vestibular masticatory / gingival mucous of the iii and v sextant. all the acquisitions were obtained in the morning, with the subjects in a seated position. the lens chosen was the one with a constant enlargement of 200 and a varying focal spot (from 0 to 2 mm). this lens was chosen from those available for the good quality of its images in terms of the details offered (good reading of the capillary bed) and the size of the examined area (1.818 mm). all the acquisitions and the subsequent elaboration of the capillaroscopic images were conducted by the same operator (gas) (figures 3, 4). the capillaroscopic parameter evaluated was the following : density of the capillary loops (dc), defined as the number of loops per surface unit ; this parameter, as has emerged in previous capillaroscopic studies on oral mucous, is the one most directly correlated to the inflammatory state of the tissues and to angiogenetic activity. over 800 capillaroscopic images were examined in total (2 sites of investigation for each of the 80 subjects and a minimum of 5 images taken for each site investigated), and for each picture or capillaroscopic image a minimum of 5 measurements were taken, for a total of over 4000 measurements. the measurements of the density (expressed in the number of loops / mm) of the capillary loops presented differences between the healthy subjects and the diabetic subjects. the data obtained was grouped together and submitted for statistical analysis to verify the significance of the differences between diabetic and healthy subjects. the test selected was mann - whitney, a test of statistical comparison for non - parametric ordinal data. the differences between the groups with a value of p below 0.05 were considered statistically significant. the differences with p for all the measurements and the statistical analyses, we were aided by the computer software p.a.s.t. ryan in 1995 and updated to the latest version, release 1.97, in january 2010. the statistical analysis of the data selected presented highly significant variations in capillary density at the periodontal mucous level. the average periodontal capillary density (dc - p) was clearly superior in diabetic subjects (35.6210.40 nloop / mm) compared to healthy subjects (17.553.88 nloop / mm). the value of p, well below the level of significance (p=0.000000986), demonstrates the high significance of the result obtained (table 2) (figure 5). diabetes mellitus has important effects on macrocirculation and on peripheral microcirculation, as demonstrated by the frequent cardiovascular pathologies and by diabetic microangiopathy, with its organ - specific and local effects [79 ]. the dysfunctions in microcirculation involve the arterioles and the capillaries of the retina and of the kidneys, the vasa nervorum and the vasa vasorum. they often lead to irreversible consequences such as blindness, renal dysfunction with the need for dialysis or organ transplant, peripheral neuropathy with alterations in sensitivity and, finally, peripheral vasculopathy with damage from altered trophism. periodontal disease has been recognized as the sixth - most important complication of diabetes mellitus. it has been demonstrated that the severity of periodontal disease increases with the duration of diabetes, and the chronic nature of periodontal infection may contribute to worsening of diabetes status leading to more severe diabetes complications. the periodontal vasculature is profoundly affected during the progression of diabetes mellitus, and there is evidence that inflamed tissues enhance the expression of the various cytokines and growth factors that may regulate angiogenesis. angiogenesis is defined as the process by which new blood vessels are produced by sprouting from established vessels. it occurs under physiological and pathological conditions, and can contribute to the degree of inflammation as a result of the ability of new blood vessels to transport proinflammatory cells to the lesion and to supply oxygen and nutrients to the inflamed tissues. vascular endothelial growth factor (vegf), an angiogenic growth factor, potently increases microvascular permeability, stimulates endothelial cell proliferation, and induces proteolytic enzyme expression and the migration of endothelial cells, monocytes and osteoblasts, all of which are essential for angiogenesis. a meta - analysis of data (keles.) has demonstrated that diabetes mellitus increases the risk for periodontal disease 2-fold, independent of the effect of age and local factors. although type 2 diabetes has a significant genetic component, pre - existing obesity, and concomitant elevation in free fatty acids and pro - inflammatory cytokines increase risk. glycation of proteins (eg, hemoglobin, collagen) and accumulation of sorbitol and fructose (eg, in nerves, lens) contribute to tissue toxicity and oxidative stress. the biological link between diabetes and periodontal disease has been established in a number of studies. several different mechanisms have been proposed to explain this link : increased oxidative stress, advanced glycation end - products, altered immune function, and changes in collagen. oxidative stress occurs through a number of pathways, including the effects of hyperglycemia and of increased circulating free fatty acids. hyperglycemia increases generation of superoxides in the mitochondria and increased levels of glucose increase the proton gradient across the inner mitochondrial membrane. when the gradient exceeds a threshold, electron transfer is blocked, leading to leakage of electrons from ubiquinone, with formation of superoxide. the formation of superoxide in turn mediates activation of the polyol pathway, the hexosamine pathway, protein kinase c, and the formation of advanced glycation end - products. increased free fatty acid delivery to peripheral tissues seen in diabetes can activate inflammatory processes through activation of toll - like receptors. although this mechanism of oxidative stress has been demonstrated in other tissue (eg, muscle), very little work has been done relating free fatty acids to increased inflammation in periodontal disease. on the basis of these recent studies, aggressive periodontitis is now recognized as the sixth - most common complication of diabetes ; according to loe, multiple epidemiologic studies have demonstrated that both insulin - dependent diabetes mellitus (iddm ; type i) and noninsulin - dependent diabetes mellitus (type ii) are predictors of periodontal disease when the systemic disease is poorly controlled. several mechanisms have been proposed that may explain how diabetes produces alterations in the organs and tissues, including the periodontium. early studies demonstrated that the advanced glycation end - products (age), synthesized due to hyperglycemia, can convert macrophages into cells with a destructive phenotype, producing high levels of interleukin-1, interleukin 6 (il-6) and tumour necrosis factor- (tnf-). moreover, age have the capacity to increase the endothelium permeability and express high levels of molecular adhesion receptors. these changes could explain the greater susceptibility to infections and the delayed wound healing in diabetic subjects. this depressed immune response could explain why it may not be possible to eradicate periodontal infection totally in diabetics after conventional periodontal therapy. this might be one of the reasons why antibiotics may be suggested with mechanical therapy for diabetic subjects, especially for uncontrolled cases. by contrast, in trying to determine the capacity of periodontal disease to adversely affect the control of diabetes by influencing glycemia levels, it has been hypothesized that chronic low - grade inflammations such as this might result in insulin resistance. at a microscopic level, diabetic damage occurs in the endothelial cells that constitute the lining of the small and large vessels. the principal function of vasal endothelium is homeostasis through the synthesis and the release of a great variety of molecules and through pro - coagulative, vasoconstrictive and vasodilatatory activity ; among these are found coagulation factors, prostacyclin, endothelin, prostaglandin and nitric oxide (no). together, these substances contribute to modulating vascular tone, permeability, coagulation and, in a wider sense, the reparative processes of the small vessels. an intact and perfectly functioning endothelium protects against the development of atheromatous plaques and constitutes the basis for the health of tissues. this protective barrier is strengthened by the maintenance of a smooth and homogeneous vasal surface that prevents thrombogenesis, the adhesion of monocytes, macrophages and platelets and the transportation of lipoproteins through the cell wall. in the presence of hyperglycemia, this barrier is destroyed and the reactive mechanisms become dysfunctional, resulting in micro- and macro - vascular complications. the dysfunction of vasal endothelium is also reflected at a capillaroscopic level, as seen in the results obtained. diabetes mellitus is a chronic pathology that causes progressive damage to peripheral microcirculation, rendered explicit by alterations in vascular patterns, as widely demonstrated by previous studies on conjunctival and periungual capillaroscopy and on retinal fundus examinations. with regards to the vascular bed of the superficial periodontium (masticatory / gingival mucous), at a capillaroscopic level there is an increase in the number of capillary loops per mm, which is synonymous of strong angiogenic activity. the density of the vessels in diabetic subjects is approximately double that of healthy subjects. another characteristic of the periodontal capillary pattern in diabetic subjects is that there appears a leopard spot morphology, with diffused microhemorrhages and capillaries with a cockade pattern. the capillaries, which are usually seen in capillaroscopic images of the site of masticatory mucous as pinheads, seem broader, almost forming little circles close to one another, (figures 69). the increase in capillary density could suggest the presence of active inflammatory phenomena or, more probably, a tendency to a greater susceptibility to inflammatory phenomena. several authors have stressed a greater propensity for periodontal disease in diabetic subjects, linking it to more or less visible subclinical signs and symptoms (eg, spontaneous or provoked bleeding, an increase in the flow of sulcular fluid, alteration of the composition of the sulcular fluid). until now, the periodontal microcirculatory pattern in diabetic subjects has not been focussed on and objectified in vivo in the absence of a diagnosis of periodontal disease. it is evident that videocapillaroscopy applied to the study of the superficial periodontium is important for early diagnosis and monitoring of periodontal disease. several studies in recent years have been carried out to appraise in vivo the morphological alterations of the capillary bed in diabetic subjects using videocapillaroscopy, a sign of the need of the medical community to be able to stage diabetic illness, monitor it, and make early diagnosis the damages correlated to it [1619 ]. in the past this method was used to investigate the conjunctival bed and the ungual bed, and in both cases it succeeded in directly revealing alterations correlated to diabetes. the results obtained by the present study appear to be in accordance with the literature and add a new point of view to it. the mouth has numerous advantages in terms of accessibility, non - invasivity and visibility in comparison to conjunctival mucous and the nailfold [2023 ]. ultimately, this study shows that there is some peripheral damage to microcirculation at the masticatory mucous level in diabetic subjects, and that such alterations are instrumentally objectifiable and quantifiable through the videocapillaroscopic method. | summarybackgrounddiabetes mellitus is today considered a society - wide disease of a chronic / degenerative nature. among the secondary effects of diabetes, the one that interests the dental surgeon most is diabetic parodontopathy. the aim of this study was to underline and objectify microcirculatory variations at a periodontal mucous level in type 2 diabetics.material/methodsthe study enrolled 80 subjects : 40 subjects with a diagnosis of diabetes mellitus type ii (18 males and 22 females, between 44 and 85 years of age) ; and 40 healthy subjects (17 males and 23 females, between 44 and 78 years of age). all the subjects, both diabetic and healthy, were submitted to a videocapillaroscopic examination of the mucosa of the oral cavity.resultsthe measurements concerning the density (expressed in the number of loops / mm2) of the capillary loops presented differences between the healthy subjects and the diabetic subjects. the average periodontal capillary density (dc - p) was clearly superior in diabetic subjects (35.6210.40 nloop / mm2) compared to healthy subjects (17.553.88 nloop / mm2). the statistical analysis was performed by means of the mann whitney test. the value of p (p=0.000000986), well below the level of significance, demonstrates the high significance of the results obtained.conclusionsthe increase in capillary density could suggest the presence of active inflammatory phenomena or, more probably, a tendency to a greater susceptibility to inflammatory phenomena. ultimately, this study shows that there is some peripheral damage to microcirculation at the masticatory mucous level in diabetic subjects and that such alterations can be instrumentally objectified and quantified through the videocapillaroscopic method. |
septic arthritis in neonates and infants has increased in prevalence and severity over time and has important functional and social handicap.12 it is caused by a variety of microorganisms, most common being gram - positive, followed by gram - negative bacteria and fungi.34 however, there is a report of an important etiologic shift, with gram - negative organisms constituting 33%, gram - positive 20%, and fungal organisms 7%.5 septic arthritis caused by candida albicans is not seen widely and the actual incidence is not known.2 however, there are reports of increasing prevalence, from a few reported cases to almost 11 fold increase in the last 15 years.6 in almost all studies c. albicans is the most common fungal pathogen (53%),478 though other agents such as candida tropicalis, candida parapsilosis, candida glabrata, candida guilliermondii, and candida krusei have also been implicated.127 this is mostly seen in premature infants with incidence of 10 - 25% in low birth weights infants,67 with neonatal septicemia having a long history of hospitalization. this predisposes the child to frequent colonization with microorganisms hence there is increased prevalence of neonatal septicemia. other associated risk factors are prolonged hyperalimentation, gastrointestinal defects, prolonged intravenous (iv) catheters (up to 67%, and increase survival rates if removed under 3 days), long term steroid and antibiotic therapy and immunosuppression. complications of disseminated neonatal candidiasis include arthritis, endocarditis, meningitis, endophthalmitis and respiratory disorders.1234567 nearly, 85% pediatric fungal arthritis occurs in infants.8 in general, two forms of arthritis are seen primary as a result of acute candidiasis seen in hospitalized, preterm, septicemic infants with indwelling catheters and secondary presenting later following disseminated candidiasis.9 it mostly occurs as mono or oligoarthritis with knee joint most commonly involved. it can also lead to osteomyelitis in 70 - 80% of cases, which can be a simultaneous occurrence or late manifestation of hematogenously disseminated candidiasis.8 though the incidence of osteomyelitis has reduced over time, this condition requires prompt diagnosis and aggressive treatment to prevent and overcome the potential crippling sequel associated with it.9 a 25-day - old preterm neonate with a birth weight of 2.7 kg admitted since birth for neonatal septicemia elsewhere presented with pain and swelling of the left hip with the loss of spontaneous movements of the left lower extremity from 2 weeks. on clinical examination, there was swelling, pain on passive movements and instability on examination of the left hip (barlow 's positive). x - ray pelvis with both hips anteroposterior (ap) view showed subluxation of the left hip while the right hip was lateralized. there were changes of early osteomyelitis in the upper end of the left femur [figure 1a ]. while the x - ray of right shoulder ap and axial views showed no abnormality ultrasound of both hips revealed left sided intra - capsular collection with synovitis. laboratory findings showed total leucocyte count (tlc) as 13,000/cu mm, c - reactive protein (crp) as 4.42 mg / dl, erythrocyte sedimentation rate (esr) as 90 mm/1 h. blood culture was positive for c. albicans. (case 1) - x - ray pelvis both hip joints anteroposterior view showing (a) left hip subluxation, osteomyelitis upper end femur left side (b) followup at 2 years and 8 months. there is appearance of capital femoral epiphysis on both sides and coxa magna left side the child was taken up for aspiration and arthrotomy of the left hip. purulent material and inflamed synovium obtained from the hip joint was sent for culture which was positive for candida, while the histopathological examination of the inflamed synovium revealed synovitis. repeat laboratory investigations after 4 weeks showed improvement with tlc-4600/cu mm, crp-2.2 mg / dl, esr-15 mm / h. pain and swelling in the left hip reduced progressively. the hip was stabilized in the pavlik harness within 1 week and he regained spontaneous movements 2 months following arthrotomy. hip was protected in a hip abduction brace for 3 months after discontinuing the pavlik harness. at followup, at the last followup at 2 years 8 months of age, the child was ambulatory without a limp. 20 days old infant presented with multiple joint swellings involving bilateral shoulders, elbows, knees, ankles and loss of spontaneous movements and painful movements of the left hip for 1 month. he was a preterm child with a birth weight of 2.4 kg and was diagnosed as a case of neonatal septicemia and kept in a neonatal intensive care unit for 28 days before being referred to our hospital. prior to being referred his left knee was aspirated which was positive for candida on culture. the child was irritable and had pain on passive movements of both hip joints and instability on the left side. the upper limbs had mild swelling around the elbows and shoulders but the child had spontaneous movements of both upper limbs. x - ray pelvis with both hips ap view revealed the hip was lateralized on both sides with early changes of osteomyelitis in the upper ends of femur bilaterally and osteomyelitis of the lower end of femur on the right side and upper end of the tibia on the left side [figure 2a ]. (case 2) - (a) preoperative radiograph of both lower limbs anteroposterior view showing osteomyelitis of lower end of femur on right side and upper end of tibia on the left side. hip was lateralized on both sides (b) standing orthogram at 2 years age showing coxa valga left femur, growth arrest proximal tibia left and distal femur right (c) x - ray left elbow (anteroposterior and later views) showing destruction of lower end of humerus laboratory investigations showed crp 15.6 mg / dl, white blood cells (wbc) 11920/cu mm. the child was taken up for aspiration and drainage of the left hip and repeat aspiration of left knee. purulent fluid was obtained on aspiration of the knee and on arthrotomy of the hip joint. pus culture and tissue culture from the hip was negative for any growth as was the aspirate from the knee. clinical examination and laboratory investigations of leucocytosis and raised crp were suggestive of some infection. the patient was started on antifungal therapy amphotericin b which was continued for 5 weeks. repeat laboratory investigations after 10 days showed improvement with crp 2.35 mg / dl, wbc 9450/cu mm. the hip joint was stabilized in the pavlik harness within a week and the pain and swelling reduced progressively with time. a hip abduction brace was used for another 3 months for protection of the hips. followup examination revealed that the child had started walking without support at 16 months of age. at the most current followup at 2 years of age, the patient was ambulatory without support and walked without a limp. x - ray showed well formed capital femoral epiphysis, with coxa valga on the left, growth arrest of the distal femur on the right and at the proximal tibia on the left [figure 2b ]. left elbow had a cubitus varus deformity, with deformation of the lower end of the humerus [figure 2c ]. a 25-day - old preterm neonate with a birth weight of 2.7 kg admitted since birth for neonatal septicemia elsewhere presented with pain and swelling of the left hip with the loss of spontaneous movements of the left lower extremity from 2 weeks. on clinical examination, there was swelling, pain on passive movements and instability on examination of the left hip (barlow 's positive). x - ray pelvis with both hips anteroposterior (ap) view showed subluxation of the left hip while the right hip was lateralized. there were changes of early osteomyelitis in the upper end of the left femur [figure 1a ]. while the x - ray of right shoulder ap and axial views showed no abnormality ultrasound of both hips revealed left sided intra - capsular collection with synovitis. laboratory findings showed total leucocyte count (tlc) as 13,000/cu mm, c - reactive protein (crp) as 4.42 mg / dl, erythrocyte sedimentation rate (esr) as 90 mm/1 h. blood culture was positive for c. albicans. (case 1) - x - ray pelvis both hip joints anteroposterior view showing (a) left hip subluxation, osteomyelitis upper end femur left side (b) followup at 2 years and 8 months. there is appearance of capital femoral epiphysis on both sides and coxa magna left side the child was taken up for aspiration and arthrotomy of the left hip. purulent material and inflamed synovium obtained from the hip joint was sent for culture which was positive for candida, while the histopathological examination of the inflamed synovium revealed synovitis. repeat laboratory investigations after 4 weeks showed improvement with tlc-4600/cu mm, crp-2.2 mg / dl, esr-15 mm / h. pain and swelling in the left hip reduced progressively. the hip was stabilized in the pavlik harness within 1 week and he regained spontaneous movements 2 months following arthrotomy. hip was protected in a hip abduction brace for 3 months after discontinuing the pavlik harness. at followup, at the last followup at 2 years 8 months of age, the child was ambulatory without a limp. a 1 month 20 days old infant presented with multiple joint swellings involving bilateral shoulders, elbows, knees, ankles and loss of spontaneous movements and painful movements of the left hip for 1 month. he was a preterm child with a birth weight of 2.4 kg and was diagnosed as a case of neonatal septicemia and kept in a neonatal intensive care unit for 28 days before being referred to our hospital. prior to being referred his left knee was aspirated which was positive for candida on culture. the child was irritable and had pain on passive movements of both hip joints and instability on the left side. the upper limbs had mild swelling around the elbows and shoulders but the child had spontaneous movements of both upper limbs. x - ray pelvis with both hips ap view revealed the hip was lateralized on both sides with early changes of osteomyelitis in the upper ends of femur bilaterally and osteomyelitis of the lower end of femur on the right side and upper end of the tibia on the left side [figure 2a ]. (case 2) - (a) preoperative radiograph of both lower limbs anteroposterior view showing osteomyelitis of lower end of femur on right side and upper end of tibia on the left side. hip was lateralized on both sides (b) standing orthogram at 2 years age showing coxa valga left femur, growth arrest proximal tibia left and distal femur right (c) x - ray left elbow (anteroposterior and later views) showing destruction of lower end of humerus laboratory investigations showed crp 15.6 mg / dl, white blood cells (wbc) 11920/cu mm. the child was taken up for aspiration and drainage of the left hip and repeat aspiration of left knee. purulent fluid was obtained on aspiration of the knee and on arthrotomy of the hip joint. pus culture and tissue culture from the hip was negative for any growth as was the aspirate from the knee. clinical examination and laboratory investigations of leucocytosis and raised crp were suggestive of some infection. the patient was started on antifungal therapy amphotericin b which was continued for 5 weeks. repeat laboratory investigations after 10 days showed improvement with crp 2.35 mg / dl, wbc 9450/cu mm. the hip joint was stabilized in the pavlik harness within a week and the pain and swelling reduced progressively with time. a hip abduction brace was used for another 3 months for protection of the hips. followup examination revealed that the child had started walking without support at 16 months of age. at the most current followup at 2 years of age, the patient was ambulatory without support and walked without a limp. x - ray showed well formed capital femoral epiphysis, with coxa valga on the left, growth arrest of the distal femur on the right and at the proximal tibia on the left [figure 2b ]. left elbow had a cubitus varus deformity, with deformation of the lower end of the humerus [figure 2c ]. the examination findings confirmed the multifocal involvement of candida arthritis, especially in premature infants.4 in lab investigations, crp is a better indicator then esr because it increases immediately following infection and decreases as the infection subsides. timely diagnosis and intervention by aspiration, arthrotomy and antifungal therapy goes a long way in salvaging a joint.5 in the above cases, the joint could be salvaged despite presenting after 2 weeks. iv amphotericin b remains the standard treatment for candida sepsis.10 iv or oral fluconazole is an alternative to amphotericin b.1112 while antibiotic resistance to fluconazole has been found to be from 18% to 24% no resistance was found against amphotericin b.1112 though candida if present can grow on routine bacteriological media,13 it is advisable to screen at - risk neonates with both bacterial and fungal cultures. neonatal septic arthritis has a potential for disastrous sequels with long term disability, especially with joint destruction, instability and growth disturbances.9 other conditions affecting the neonatal joints such as multifocal osteomyelitis, chronic granulomatous disease, neonatal onset multisystem inflammatory disease, leukemia, eosinophilic granuloma, juvenile rheumatoid arthritis, langerhans cell histiocytosis should also be kept in mind. progress of infection, nonspecificity of laboratory data of culture negative septic arthritis (up to 18 - 48%),14 and failure to recognize candida as a potential pathogen may also lead to delay in diagnosis and complications.15 fungal arthritis should be kept in mind while treating a neonatal septic arthritis case especially in at - risk neonates who are preterm, low birth weight with a long history of hospitalization having multifocal involvement and have blood and pus samples, which are negative for bacterial culture. from our experience in these cases proper timely diagnosis and aggressive line of treatment with arthrotomy and long term duration of amphotericin b and fluconazole as an alternative should be used with a prolonged followup to monitor and treat the sequels of arthritis and osteomyelitis. | fungal arthritis is an uncommon yet serious disorder in the newborn. delay in diagnosis and management can lead to significant morbidity. we report our experience with management of two such cases. two preterm neonates with multifocal arthritis caused by candida were studied. diagnosis was made by clinical examination, laboratory investigations, radiological investigations and culture. both were treated by aspiration, arthrotomy and antifungal therapy. one patient recovered fully from the infection while the other had growth disturbances resulting in limb length inequality at recent followup. prompt and expeditious evacuation of pus from joints and antifungal therapy is imperative for treatment. associated osteomyelitis leads to further difficulty in treatment. |
the problem of quantizing general relativity (gr) is a very hard one. to this day, and despite continuing efforts, there is not a completely satisfactory theory of quantum gravity. in order to acquire the necessary intuition to deal with the very many issues whose resolution is required, it is often useful to concentrate on simplified models that exhibit only some of the difficulties present in the full theory (and hopefully in a milder guise). a natural way to get simplified versions of a physical system is to introduce symmetries. they often allow us to get particular solutions in situations where a complete resolution is out of reach. usually this happens because the effective dimensionality of the problem is reduced by the symmetry requirements. in the case of classical (i.e., non - quantum) systems, symmetry is often used at the level of the equations of motion. actually, a good way to start exploring the concrete physics of a given model is to look for symmetric configurations. the effectiveness of this approach is reinforced by the fact that, in many instances, they are good approximations to real physical situations. for example, the waves produced on a water pond by a falling stone are very well described by rotationally - invariant functions satisfying the two - dimensional wave equation. this is so because the initial disturbance producing the wave is rotationally symmetric to a good approximation. this very same philosophy is used in many branches of physics. in gr, for instance, the most important and useful metrics solving the einstein field equations exhibit some type of symmetry just think of the schwarzschild, kerr, or friedman spacetimes. actually only a few closed form solutions to the einstein field equations with no killing fields are known. there is a vast literature devoted to the classical aspects of the symmetry reductions that covers topics ranging from purely mathematical issues to physical applications in the fields of cosmology and black hole physics. these simplified systems also provide interesting quantum theories that are easier to handle than full gravity. there are two main types of models that can loosely be defined as those with a finite number of degrees of freedom (minisuperspaces) and those that require the introduction of infinitely many of them (midisuperspaces). the purpose of this living review is to explore the quantization of the latter, hence, we will only discuss those classical aspects that are of direct relevance to their quantization (for example the hamiltonian description). after this introduction we will review the history of midisuperspaces in section 2. to this end a very important idea that plays a central role in this subject is the principle of symmetric criticality. it provides a very useful simplification especially when considering the hamiltonian framework because it allows us to derive everything from a symmetry - reduced variational principle obtained by restricting the einstein - hilbert action to the family of symmetric solutions of interest. though not every reduced system is of this type, this happens to be the case for all the models that we will consider in the paper. after a discussion on some aspects concerning the mathematical description of superspace we will comment on the differences between minisuperspaces and midisuperspaces. general issues concerning quantization will be addressed in section 3, where we will quickly review with the idea of dealing with the quantization of symmetry reductions the different approaches to the quantization of constrained systems, i.e., reduced phase - space quantization, dirac quantization, quantization of fully and partially gauge - fixed models and path - integral methods. we will end this section with a discussion of the differences between the quantizing first and then reducing and the reducing first and then quantizing points of view. we will consider, in particular, one - killing vector reductions, two - killing vector reductions and spherically - symmetric models and leave to section 5 the main subject of the paper : the quantization of midisuperspaces. there we will review first the one - killing vector case and then go to the more important and developed two - killing vector reductions for which we will separately consider the quantization of einstein - rosen waves, gowdy cosmologies and other related models. we will look at both metric and loop quantum gravity (lqg) inspired quantizations for the different models. this is so because we have chosen to highlight the main ideas and emphasize the connection between the different approaches and models. we provide a sizable bibliography at the end of the paper ; technical details can be found there. we feel that the proper way to master the subject is to read the original papers, so we believe that we will have reached our goal if the present work becomes a useful guide to understanding the literature on the subject. we have tried to give proper credit to all the researchers who have made significant contributions to the quantization of midisuperspaces but of course some omissions are unavoidable. classical systems are (usually) described in terms of field models whose dynamics is given by partial differential equations derived from a variational principle. a symmetry - reduced model associated with a given classical system is defined as one obtained by considering only those solutions to the equations of motion that satisfy a certain symmetry condition. in order to describe these reduced models one has to follow several steps (see for a careful and complete discussion of these issues) : defining a group action on the solution space of the full model.finding a suitable parametrization of the solutions invariant under the group action.obtaining the field equations describing the symmetric configurations. defining a group action on the solution space of the full model. finding a suitable parametrization of the solutions invariant under the group action. obtaining the field equations describing the symmetric configurations. when these steps can be successfully carried out, the final outcome of this process is a set of equations for the symmetry - reduced system. the direct one consists in particularizing the general field equations to the invariant solutions obtained in the second step (by using some of the parametrizations introduced there). a second more indirect way this may seem an unnecessary detour but, if we intend to quantize the reduced system, it becomes an unavoidable step as we need a hamiltonian formulation to define the dynamics of the quantized model. though one may naively expect that the reduced action can be obtained by just restricting the one describing the full (i.e., non - reduced) model to the parameterized symmetric configurations, there are subtleties that may actually prevent us from doing so. the transit to the quantum version of symmetry reductions of classical theories (involving either mechanical systems or fields) is quite non - trivial. this is a very important topic that plays a central role in the present paper so we discuss it here in some detail. there are several questions to be addressed in this respect : definition of the symmetric quantum states and/or quantum symmetry reductions.evolution of the symmetric states under full dynamics and the reduced dynamics.comparison between the two : can we derive one from the other ? comparison between the two : can we derive one from the other ? the first of these issues is usually discussed as the problem of understanding the commutativity of symmetry reduction and quantization, i.e., to figure out if the result of first quantizing and then reducing is the same as the one of first reducing and then quantizing. the other two items are also important, for example, to assess to what extent the results obtained in quantum cosmology (in its different incarnations including lqc) can be taken as hard physical predictions of quantum gravity and not only as suggestive hints about the physics of the complete theory. of course the usual problems encountered in the quantization of constrained systems will also be present here. the original formulation of the principle of symmetric criticality, telling us when symmetric extremals of a functional can be obtained as the ones corresponding to the symmetry reduction of it, was stated by palais in a variety of different settings. the adaptation of this principle to gr was discussed in detail by fels and torre though its importance was recognized since the early seventies (see for an excellent review). as mentioned above, the classical reduction process for a field theory is performed in several steps [186, 203 ]. one starts by defining a group action on the space of fields of the model, find then a parametrization of the most general configuration invariant under the group action and, finally, obtaining the form of the equations of motion restricted to these symmetric field configurations (the reduced field equations). general solutions to these equations correspond to symmetric solutions of the full theory. in the case of gr one can ask oneself if the reduced field equations can be obtained as the euler - lagrange equations derived from some reduced lagrangian and also if this lagrangian can be obtained by simply restricting the einstein - hilbert action to the class of metrics compatible with the chosen symmetries. obviously this would be the simplest (and more desirable) situation but one can not exclude, in principle, that the reduced field equations could come from an action that is not the symmetry - reduced one (or even that they can not be derived from a well - defined action principle). if a hamiltonian formulation can be obtained for a symmetry reduction of a physical system then it is possible to consider its quantization. this is the path followed in quantum cosmology and in the study of the midisuperspace models that are the subject of this review. the parametrization of the invariant field configurations usually involves the introduction of a set of arbitrary functions whose number is smaller than the number of original field components. furthermore, a judicious choice of coordinates adapted to the symmetry normally restricts the number of variables upon which these functions depend. in some instances it is possible to work with a single independent variable this happens, for example, in bianchi models where these unknown functions depend only on a time coordinate that labels compact homogenous spatial slices of spacetime. another instance of this behavior is provided by static, spherical, vacuum spacetimes where the arbitrary functions appearing in the metric depend on an area variable (usually proportional to r). in both cases this, in turn, shows that these particular symmetry reductions of gr describe systems with a finite number of degrees of freedom, i.e., purely mechanical models. necessary and sufficient conditions guaranteeing that the principle of symmetric criticality holds in gr are given in theorem 5.2 of. they are technical in nature but their role is to prevent the occurrence of the two conceivable scenarios in which the symmetric criticality principle may fail. the first has to do with the possibility that the surface terms coming from integration by parts after performing variations in the full action do not reduce to the ones corresponding to the reduced action (this is what happens for bianchi b models). the second is related to the fact that considering only symmetric variations may not give all the field equations but only a subset of them. an important comment to make at this point is that it is always possible to check if the symmetric criticality principle holds just by considering the group action because it is not tied to the form of a specific lagrangian. this remarkable fact allows us to check the validity of the principle for whole families of symmetric models irrespective of their dynamics. in fact, for the types of vacuum models that are the main subject of this living review, symmetric criticality can be shown to hold [86, 203 ] and, hence, we have a simple way to get a hamiltonian for the reduced systems. in the spherically - symmetric case the result holds as a consequence of the compactedness of the group of symmetries (in the case of the two - killing symmetry reductions the validity of the principle is justified in the papers [86, 203 ]). if scalar fields are coupled to gravity the principle still holds, however the introduction of other matter fields must be treated with care because their presence may influence the action of the symmetry group. wheeler s notion of superspace is inextricably linked to the problem of understanding quantum gr. in a nutshell superspace can be defined as the space of geometries for the three - dimensional manifolds that constitute space in the dynamical picture of gr that we have come to know as geometrodynamics. as the study of symmetry reductions requires us to restrict the possible configurations to a subset of the full superspace, it is convenient to discuss, at least briefly, some of its basic features. superspace plays the role of the configuration space for general relativity in the traditional metric representation. the associated cotangent bundle, when properly defined, is the phase space for the hamiltonian formulation of the theory. as a hamiltonian formulation is the starting point for the quantization of any mechanical or field system, the role of superspace and the need to understand its mathematical structure can not be overemphasized. however, it should be noted at this point that even in the quantization of the simplest field theories such as scalar fields it is necessary to suitably enlarge this configuration space and allow for distributional, non - smooth objects to arrive at a consistent model (see, for example,). how and if this can be done in the geometrodynamical setting is an interesting, if hard, question. this is directly related to the wheeler - dewitt approach to the quantization of gr. the precise definition of the geometry of a three manifold requires some discussion (see [87, 98 ] and references therein for a nice introduction to the subject). here it is important to remark at this point that the non - generic character of geometries with non - trivial isometry groups has a very clear reflection in superspace : they correspond to singularities. the geometry of a four - dimensional manifold in relativists parlance refers to equivalence classes of suitably smooth lorentzian metrics defined on it. though one might naively think that this is just a mathematically - sensible requirement, in fact, it is quite natural from a physical point of view. this, in turn, demands an operational definition of the (possibly idealized) physical processes allowing us to explore actually measure it. this is in the spirit of special and general relativity, where the definition of physical magnitudes such as lengths, distances, velocities and the like requires the introduction of concrete procedures to measure them by using basic tools such as clocks, rulers and light rays. every transformation of the manifold (and the objects defined on it) that does not affect the operational definition of the measuring processes will be physically unobservable. notice that this prevents us from identifying physical events with points in the spacetime manifold as a diffeomorphism can take a given event from one point of the manifold to another (see). the precise definition and description of the space of geometries requires the introduction of mathematical objects and structures at different levels : the three - dimensional smooth manifold with the required differential structure.a class of smooth riemannian metrics on this manifold that should be considered (smooth in the sense of being c()). we will denote this as riem().a suitable topology and differential structure on this space.an equivalence relation between different smooth metrics provided by the action of (a class of) smooth (c) diffeomorphisms diff() on riem(). the three - dimensional smooth manifold with the required differential structure. a class of smooth riemannian metrics on this manifold that should be considered (smooth in the sense of being c()). an equivalence relation between different smooth metrics provided by the action of (a class of) smooth (c) diffeomorphisms diff() on riem(). after doing naturally, superspace will inherit some background properties from those carried by the different elements needed to properly define it. the resulting space has a rich structure and interesting properties that we will very quickly comment on here (the interested reader is referred to and the extensive bibliography cited there). an important issue is related to the appearance of singularities in this quotient space associated with the fact that in many instances the spatial manifold s allows for the existence of invariant metrics under non - trivial symmetry groups (leading to a non - free action of the diffeomorphisms). this turns out to be a problem that can be dealt with in the sense that the singularities are minimally resolved (see). it is important to mention at this point that the symmetry reductions that we will be considering here consist precisely in restrictions to families of symmetric metrics that, consequently, sit at the singularities of the full superspace. this fact, however, does not necessarily imply that the reduced systems are pathological. in fact some of them are quite well behaved as we will show in section 4. finally, we point out that both the space of riemannian metrics riem() and the quotient space mentioned above are endowed with natural topologies that actually turn them into very well - behaved topological spaces (for instance, they are metrizable and hence paracompact, second countable and connected). the space of metrics riem() can be described as a principal bundle with basis riem()/diff() and structure group given by diff() (that is, the proper subgroup of those diffeomorphisms of that fix a preferred point and the tangent space at this point). some of these properties are inherited by the spaces of symmetric geometries that we consider here. other approaches to the quantization of gr, and in particular loop quantum gravity, rely on spaces of connections rather than in spaces of metrics. hence, in order to study symmetry reductions in these frameworks, one should discuss the properties of such connection superspaces and then consider the definition of symmetric connections and how they fit into these spaces. the technical treatment of the spaces of yang - mills connections modulo gauge transformations has been developed in the late seventies by singer and other authors [198, 166 ]. these results have been used by ashtekar, lewandowski and others to give a description of the spaces of connections modulo gauge (encompassing diffeomorphisms) and their extension to symmetry reductions have been explored by bojowald and collaborators as a first step towards the study of symmetry reductions in lqg. minisuperspaces appear when the symmetry requirements imposed upon spacetime metrics are such that the dimension of riem() (and, hence, of riem()/diff()) becomes finite. historically, these were the first symmetry reductions of gr that received serious consideration from the quantum point of view [79, 80, 81, 164 ]. their main advantage in the early stages of the study of quantum gravity was the fact that the resulting models were finite - dimensional and their quantization could be considered in a more - or - less straightforward way. important conceptual problems received attention within this setting ; in particular those related to the interpretation of the universe wave function and the resolution of cosmological singularities. they are receiving renewed attention these days as very useful test beds for lqg (called loop quantum cosmology or lqc in short). in particular the resolution of the initial singularity in lqc is a tantalizing hint of the kind of fundamental knowledge about the universe that a complete theory of quantum gravity could provide. these spacetimes are obtained (see [193, 224 ] for a pedagogical presentation) by requiring that the spacetime admits a foliation by smooth three - dimensional hypersurfaces t that are the orbits of a group of isometries g. when the action g is required to be simply transitive (i.e., for each pair of points p, q t there exist a unique element of g g such that g in addition to the bianchi models there are other spatially - homogeneous spacetimes for which the group action is not simply transitive (or does not have a subgroup with a simply transitive action). these are the kantowski - sachs models with g = r so(3) and such that the spatial homogeneous hypersurfaces are r s. metrics for the bianchi models are parameterized by functions of the time variable that labels the sheets of the spacetime foliation and can be conveniently written by using a basis of invariant one forms. the killing vector fields of the metric induced on each t are in one to one correspondence with the right invariant vector fields in the group g and satisfy the commutation rules of the lie algebra of g. the einstein field equations reduce in these cases to a system of ordinary differential equations. bianchi models are classified as type a and type b depending on some invariant properties encoded in the structure constants \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${c_{ab}}^c$\end{document } of the isometry group. if they satisfy the condition \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${c_{ab}}^b = 0$\end{document } the resulting model is type a, otherwise it is called type b. only the type a ones satisfy the principle of symmetric criticality and can be quantized in a straightforward way. two main approaches are possible to studying the classical dynamics of minisuperspace models and, in particular, the bianchi models : the covariant spacetime textbook approach (see, for example,) that directly looks for the spatially - homogeneous solutions to the einstein field equations, and the hamiltonian one that can be applied when the principle of symmetric criticality holds. of course, they are ultimately equivalent, but the descriptions that they provide for the classical dynamics of these systems are surprisingly different. a very good account of these issues can be found in. among the points that are worthwhile singling out, maybe the most striking one refers to the identification and counting of the number of degrees of freedom. as can be seen, these numbers generically disagree in the case of open spatial slices. this can be easily shown for the bianchi i model for r spatial slices. from the covariant point of view the family of solutions of bianchi type i is fully described by a single parameter ; on the other hand the hamiltonian analysis (this is a constrained hamiltonian system) tells us that the number of phase - space degrees of freedom is ten corresponding to five physical degrees of freedom. the resolution of this problem requires a careful understanding of several issues : the role of the spatial topology. it can be seen that this mismatch does not occur for compact spatial topologies (which, by the way, are impossible for the bianchi type b case). in this case the appearance of global degrees of freedom reconciles the covariant and hamiltonian points of view.the difference between gauge symmetries and non - gauge symmetries. the first ones are those generated by the constraints in the hamiltonian formulation such as the familiar u(1) gauge invariance of electromagnetism and connect physically - indistinguishable configurations. the non - gauge ones correspond to homogeneity preserving diffeomorphisms that connect physically distinct solutions. a standard example is the poincar invariance in standard quantum field theories.the need to understand the different roles played by diffeomorphisms in the spacetime and hamiltonian pictures. whereas from the spacetime point of view solutions to the einstein equations that can be connected by the action of a diffeomorphism are considered to be physically equivalent, they may not be so from the hamiltonian point of view in which a spacetime foliation must be introduced. it can be seen that this mismatch does not occur for compact spatial topologies (which, by the way, are impossible for the bianchi type b case). in this case the appearance of global degrees of freedom reconciles the covariant and hamiltonian points of view. the first ones are those generated by the constraints in the hamiltonian formulation such as the familiar u(1) gauge invariance of electromagnetism and connect physically - indistinguishable configurations. the non - gauge ones correspond to homogeneity preserving diffeomorphisms that connect physically distinct solutions. the need to understand the different roles played by diffeomorphisms in the spacetime and hamiltonian pictures. whereas from the spacetime point of view solutions to the einstein equations that can be connected by the action of a diffeomorphism are considered to be physically equivalent, they may not be so from the hamiltonian point of view in which a spacetime foliation must be introduced. the bottom line can be summarized by saying that the extra structure present in the hamiltonian framework provides us with sharper tools to separate gauge and symmetries than the purely geometric point of view of the standard covariant approach. if one is interested in the quantization of these minisuperspace reductions, the hamiltonian framework is the natural (and essentially unavoidable) starting point. it is obvious that essentially all the points discussed here will also be relevant in the case of midisuperspaces, though to our knowledge the current analyses of this issue are far from complete and definitely much harder because one must deal with infinite dimensional spaces. in this case, as we will see, the gauge symmetry remaining after the symmetry reduction will include a non - trivial class of restricted diffeomorphisms. this is, in fact, one of the main reasons to study these symmetry reductions as they may shed some light on the difficult issue of dealing with diffeomorphism invariance in full quantum gravity. a final interesting point that we want to mention is the problem of understanding how minisuperspace models sit inside the full superspace. the type of phenomena that can be described by a minisuperspace is rather limited because the metrics in these models effectively depend on a finite number of parameters. a less drastic simplification would consist in allowing some functional freedom but not the most general one. more specifically the idea is to impose again symmetry requirements to restrict the set riem() used in the superspace construction in such a way that the allowed metrics are parametrized by functions rather than by numerical parameters. by doing this the hope is to increase the number of degrees of freedom of the models and eventually have local degrees of freedom. notice that, as we will discuss below, the presence of fields at this stage does not preclude the possibility of having a finite - dimensional reduced phase space. this can be accomplished, in particular, by restricting ourselves to metrics having a low number of spatial killing vector fields. as we will see in the following, the case in which spacetime metrics are required to have two commuting killing vector fields is specially appealing because some of these models are solvable both at the classical and quantum levels while, on the other hand, it is possible to keep several interesting features of full gr, such as an infinite number of degrees of freedom and diffeomorphism invariance. the einstein - rosen (er) waves [84, 29 ] were the first symmetry reduction of this type that was considered from the hamiltonian point of view with the purpose of studying its quantization. as a matter of fact, kucha introduced the term midisuperspace precisely to refer to this system [141, 142 ]. other configurations of this type are the well - known gowdy spacetimes [102, 103 ] that have been used as toy models in quantum gravity due to their possible cosmological interpretation. a different type of systems that has been extensively studied and deserves close investigation is the spherically - symmetric ones (in vacuum or coupled to matter). these are, in a sense, midway between the bianchi models and the midisuperspaces with an infinite number of physical degrees of freedom such as er waves. general spherically - symmetric - spacetime metrics depend on functions of a radial coordinate and time, so these models are field theories. on the other hand, in vacuum, the space of physically - different spherical solutions to the einstein field equations is finite dimensional (as shown by birkhoff s theorem). this means that the process of finding the reduced phase space (or, alternatively, gauge fixing) is non - trivial. the situation usually changes when matter is coupled owing to the presence of an infinite number of matter degrees of freedom in the matter sector. the different approaches to the canonical quantization of these types of models is the central topic of this living review. the canonical treatment of the symmetry reductions of gr requires the understanding of constrained hamiltonian systems. in the cases that we are going to discuss (and leaving aside functional analytic issues relevant for field theories), the starting point consists of the classical unconstrained configuration space \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}$\end{document } of the model and the cotangent bundle over \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal c}$\end{document } endowed with a suitable symplectic form. a dynamical hamiltonian system is said to be constrained if the physical states are restricted to belonging to a submanifold \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\bar \gamma}$\end{document } of the phase space, and the dynamics are such that time evolution takes place within \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\bar \gamma}$\end{document }. in the examples relevant for us the space \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\bar \gamma}$\end{document } will be globally defined by the vanishing of certain sufficiently regular constraint functions, ci = 0. in the case of gr these constraint functions are the integrated version of the scalar and vector constraints and the subindex i refers to lapse and shift choices (see, for example,). notice, however, that there exist infinitely - many constraint equations that define the same submanifold \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\bar \gamma}$\end{document}. the choice of one representation or another is, in practice, dictated by the variables used to describe the physical system. we will assume that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\bar \gamma}$\end{document } is a first - class submanifold of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\gamma$\end{document}. this is geometrical property that can be expressed in terms of the concrete constraint equations describing \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\bar \gamma}$\end{document } as 1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\{{c_i},{c_j}\ } { \vert_{\bar \gamma } } = 0.$$\end{document } the pull - back of the symplectic structure of to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\bar \gamma}$\end{document } is degenerate and the integral submanifolds defined by the degenerate directions are the gauge orbits. the reduced phase space \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\tilde \gamma}$\end{document } is the quotient space whose points are the orbits of the gauge flows. it can be endowed with the natural symplectic structure \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\tilde \omega}$\end{document } inherited from. if a non - trivial dynamics describes the evolution of the system in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\tilde \gamma}$\end{document } this will be given by the reduced hamiltonian \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\tilde h}$\end{document } obtained by restricting the original one to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\tilde \gamma}$\end{document}. this restriction is well defined whenever h is gauge invariant and, hence, constant on the gauge orbits. the reduced phase - space quantization is simply the quantization of the reduced space \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$(\tilde \gamma, \tilde \omega, \tilde h)$\end{document } of the constrained hamiltonian system whenever this is possible. notice that the process of taking quotients is highly non - trivial and many desirable regularity properties need not be preserved. in the models that we consider in this paper we will suppose that no obstructions appear so that the reduced phase space is well defined. even in this case some difficulties may (and in practice do) arise, in particular : the characterization of the quotient space \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\tilde \gamma}$\end{document } is usually very difficult even when the quotient itself is well defined. in practice this reduced phase space is effectively described by using a gauge fixing procedure that picks a single field configuration from each gauge orbit in a smooth way (whenever this is possible).in general, there is no guarantee that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\tilde \gamma}$\end{document } will be the cotangent space of a reduced configuration manifold \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\tilde { \mathcal c}}$\end{document}. although there are techniques that may allow us to tackle this situation (i.e., geometric quantization) they are not always straightforward to apply.it may be difficult to extract physics from the reduced phase - space description. in practice, even when the reduction can be carried out in an explicit way, it is very difficult to reexpress the results in terms of the original variables in which the problem is naturally written. the characterization of the quotient space \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\tilde \gamma}$\end{document } is usually very difficult even when the quotient itself is well defined. in practice this reduced phase space is effectively described by using a gauge fixing procedure that picks a single field configuration from each gauge orbit in a smooth way (whenever this is possible). in general, there is no guarantee that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\tilde \gamma}$\end{document } will be the cotangent space of a reduced configuration manifold \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\tilde { \mathcal c}}$\end{document}. although there are techniques that may allow us to tackle this situation (i.e., geometric quantization) they are not always straightforward to apply. it may be difficult to extract physics from the reduced phase - space description. in practice, even when the reduction can be carried out in an explicit way, it is very difficult to reexpress the results in terms of the original variables in which the problem is naturally written. the reduced phase - space description amounts to the identification of the true physical degrees of freedom of the system. as a rule, for many physical models (and certainly in the case of gravitational theories) is forced to learn how to live with the redundant descriptions provided by gauge theories and how to handle the constraints both at the classical and quantum levels. finally, it is important to notice that whenever the reduced phase space can be characterized by means of a gauge fixing, the quantization ambiguities that may arise do not originate in the different gauge choices as long as they are acceptable but rather in the possibility of having different quantizations for a given classical model. this is so because from the classical point of view they are explicit representations of the same abstract object : the reduced phase space. there are several approaches to the quantization of gauge systems that we will briefly discuss next. in dirac s approach to quantization one starts from a kinematical vector space v adapted (i.e., with the right dimensionality among other requirements) to the description of a physical system defined on the phase space. the constraints ci = 0 are then represented as operators whose kernels define the physical states v of the quantum theory, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\hat c}_1}\psi = 0$\end{document}. finally, to define probabilities, the physical subspace vphys is endowed with a suitable inner product, such that (vphys,,) becomes a hilbert space \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal h}_{{\rm{phys}}}}$\end{document}. in order to make these ideas explicit, the following concrete points must be addressed : the identification of a set of elementary phase - space variables for the full (non - constrained) phase space of the classical system.the selection of a suitable poisson algebra on the full phase space generated by the elementary variables.the construction of a representation for this poisson algebra on the complex vector space v.the implementation of the first class constraints ci = 0 as operators acting on the representation space.the characterization of the physical states, i.e., the space vphys spanned by those vectors in the kernel of all the constraint operators.the identification of physical observables (the operators that leave vphys invariant).finally, if we want to answer physical questions such as probability amplitudes or expectation values we need to endow vphys with an hermitian inner product. the identification of a set of elementary phase - space variables for the full (non - constrained) phase space of the classical system. the selection of a suitable poisson algebra on the full phase space generated by the elementary variables. the construction of a representation for this poisson algebra on the complex vector space v. the implementation of the first class constraints ci = 0 as operators acting on the representation space. the characterization of the physical states, i.e., the space vphys spanned by those vectors in the kernel of all the constraint operators. the identification of physical observables (the operators that leave vphys invariant). finally, if we want to answer physical questions such as probability amplitudes or expectation values we need to endow vphys with an hermitian inner product. the wheeler - dewitt approach and lqg both follow the spirit of the dirac quantization of constrained systems mentioned here. in lqg, the kinematical vector space v is endowed with a hilbert space structure defined in terms of the ashtekar - lewandowski measure. however, the identification of the inner product in the space of physical states is not as simple as the restriction of the kinematical hilbert structure to the physical subspace because the spectrum of the constraint operators may have a complicated structure. in particular, it may happen that the kernel of these operators consists only of the zero vector of the kinematical hilbert space. the wheeler - dewitt approach is less developed from the mathematical point of view but many constructions and ideas considered during the mathematical development of lqg can be exported to that framework. it is important to mention that under mathematical restrictions similar to the ones imposed in lqg some crucial uniqueness results (specifically the lost and fleischack theorems on the uniqueness of the vacuum state) do not hold. though the approach can probably be developed with the level of mathematical rigor of lqg this result strongly suggests that lqg methods are better suited to reach a complete and fully consistent quantum gravity theory. in any case we believe that it could be interesting to explore if suitable changes in the mathematical formulation of the wheeler - dewitt formalism could lead to uniqueness results of the type already available for lqg. as mentioned above the reduced phase space is the space of gauge orbits endowed with a symplectic structure \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\tilde \omega}$\end{document } inherited from the original one in the full phase space. a strategy that is useful in the context of midisuperspaces is to partially fix the gauge. in practice this means that the dimensionality of the constraint hypersurface (and, as a consequence, of the gauge orbits) is reduced. this may be useful if one is interested in leaving some residual gauge symmetry in the model on purpose (such as radial diffeomorphisms in spherically - symmetric models) to check if one can deal with it in some quantization scheme. in other situations the natural gauge fixing conditions simply fail to fix the gauge completely ; this happens, for example, in the compact gowdy models. in such cases the residual gauge invariance is usually treated by employing dirac s procedure, though other approaches are, of course, possible. a very attractive feature of the resulting formulation is that the quantum dynamics of the model is given by a time dependent hamiltonian that can be studied in great detail due to its relatively simple structure. this is possible because its meaning can be understood by using results developed in the study of the time - dependent harmonic oscillator (see and references therein). an alternative quantization, that has been successfully employed in standard quantum field theories, consists in using a path integral to represent relevant physical amplitudes and then develop perturbative techniques to extract the physical information as some kind of expansion (usually asymptotic) in terms of coupling constants. the main idea is to represent transition amplitudes as integrals over a set of interpolating configurations (trajectories for particle systems or, more generally, field histories). for example, the expression 2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$({g_2},{\phi _ 2},{\sigma _ 2}\vert{g_1},{\phi _ 1},{\sigma _ 1 }) = \int { \exp \left({is(g,\phi) } \right)\mu ({ \rm{d}}g,{\rm{d}}\phi)}$$\end{document } would represent the probability amplitude to go from a state with metric g1 and matter fields 1 on a 3-surface 1 to a state with metric g2 and matter fields 2 on a 3-surface 2. here s is the classical action and is the measure on the space of field configurations determined by the phase - space measure. the integral has to be computed over the field configuration on the region of the spacetime manifold, which has 1 and 2 as boundaries. one of the advantages that is usually attributed to the path integral is that it provides a however, it is important to notice that only the phase - space path integral can be shown to be formally equivalent to canonical quantization. if the integral in the momenta can be performed in a closed algebraic form, one gets a configuration space path integral whose integrand can be seen to be, in some cases but not always, just the action expressed in terms of configuration variables and their derivatives. in the case of reduced phase - space quantization the correct writing of the path integral requires the introduction of the fadeev - popov terms that take into account the fact that the integration measure is the pullback of the formal liouville measure to the hypersurface defined by the first - class constraints and gauge fixing conditions (or alternatively to the space of gauge orbits). the path integral method can be rigorously defined in some instances, for example in the quantum mechanics for systems with a finite number of degrees of freedom and some field theories, such as topological models and lower dimensional scalar models. in other cases, the first proposals to use path integrals in quantum gravity go back to the fifties (see, for example, the paper by misner) and were championed by hawking, among many other authors, in the study of quantum cosmology, black hole physics and related problems. path integrals are also useful in other approaches to quantum gravity, in particular regge calculus, spin foams and causal dynamical triangulations (see the living review by loll and references therein). although the majority of the work on quantum midisuperspaces uses the canonical approach, there are nonetheless some papers that use standard perturbative methods based on path integrals to deal with some of these models, for example the einstein - rosen waves [183, 27 ]. the results obtained with these methods suggest that this model, in particular, is renormalizable in a generalized sense and compatible with the asymptotic safety scenario. for instance, in the case of a free particle moving in three dimensions all the relevant information about the quantum evolution can be encoded in the propagator (x1, t1|x2, t2) giving the probability amplitude to find the particle at x2 in the time instant t2 if it was at x1 in the instant t1. a nice but somewhat heuristic way to obtain this amplitude the main contribution to it comes from the value that the action takes on the classical path connecting (x1, t1) to (x2, t2). however, we also have to consider the contributions given by other paths, especially those close to the classical trajectory. it is clear now that the amplitude will depend both on the class of paths used in the definition of the integral and the specific form of the action that has to be evaluated on these. notice that it is possible to have different lagrangians leading to the same equations of motion. furthermore, these lagrangians do not necessarily differ from each other in total derivative or divergence terms (an example of this phenomenon in the context of gr is provided by the self - dual action [127, 194 ] and the holst action). one expects that a modification either in the class of allowed paths and/or in the action will generically change physical amplitudes. a natural way to think about a quantum symmetry reduction of a model (again in the heuristic setting provided by path integrals) would consist in first restricting ourselves to computing probability amplitudes between symmetric configurations and then considering only a restricted class of paths in the path integral : precisely those that are, themselves, symmetric. first, the value of the probability amplitude will generically differ from the one obtained by considering unrestricted trajectories connecting the two symmetric initial and final configurations. this is expected because we are ignoring paths that would be taken into account for the non - reduced system. second, it will be generally impossible to recover the amplitudes corresponding to the full theory from the symmetry - reduced ones because information is unavoidably lost in the process of rejecting the non - symmetric trajectories (which can be thought of as a projection, see and also for a more general point of view). this is even more so because, in principle, completely different mechanical or field systems may have the same reduced sectors under a given symmetry. though it can be argued that we can learn very important lessons from a quantum symmetry - reduced model, and even get significant qualitative information about the full quantized theory, it will be generally impossible to recover exact results referring to the latter. this would be so even if we restricted ourselves to computing transition amplitudes between symmetric classical configurations. there the authors compare in a quantitative way the physical predictions derived from two different symmetry reductions of gr such that one of higher symmetry (the taub model) is embedded in the other (the mixmaster model). they do this by constructing appropriate inner products and comparing the probabilistic interpretations of wave functions in both models. this result sends an important warning signal : one should not blindly extrapolate the results obtained from the study of symmetry reductions. on the other hand it does not exclude that in physically - relevant situations one can actually obtain interesting and meaningful predictions from the study of the quantization of symmetry reductions. finally it is also important to disentangle this last issue from the different one of understanding to what extent the processes of symmetry reduction and quantization commute. to see this, consider a certain classical field theory derived from an action principle and a symmetry reduction thereof obtained by restricting the action to symmetric configurations (this procedure will be consistent if the principle of symmetric criticality holds as we will discuss in the next section). one can consider at this point the quantization of the classical reduced model by using as the starting point the reduced action. supposing that one has a consistent quantization of the full theory, one can try to see if it is possible to recover the results obtained by first reducing and then quantizing by a suitable restriction requiring a correct and consistent implementation of the symmetry requirement of the fully quantized model. this has been done in detail for the specific example of a rotationally symmetric klein - gordon field in. the main result of this paper is that it is indeed possible to show that using a suitable quantum symmetry reduction both procedures give the same result, i.e., in a definite sense reduction and quantization commute. giving a general prescription guaranteeing the commutation of both procedures on general grounds would certainly be a remarkable result, especially if applicable to instances such as lqg. this is so because many details of the quantization of full gr in this framework are still missing. it would be very interesting indeed to know what lqg would say about concrete symmetry reductions of full quantum gravity that could conceivably be obtained by considering the comparatively simpler problem of loop - quantizing the corresponding reduced classical gravity model. even if this can be effectively done we would not learn the answer to the problem of computing the amplitudes predicted by lqg for transitions between symmetric configurations. this implies that the results derived in symmetry - reduced implementations of the full lqg program such as lqc, no matter how suggestive they are, can not be extrapolated to completely trustable predictions of full quantum gravity. a rough classification of the symmetry reductions of general relativity can be made by considering the dimension of the isometry group of the metric. in many cases this is equivalent to classifying the spacetime metrics for the midisuperspace model in question according to the number of killing vector fields that they have. though this is a sensible approach, especially when the killing fields commute, this is not always the most natural way to describe all the interesting symmetry reductions, in particular, when spherical symmetry is present. let us start by considering the simplest example of symmetry reduction corresponding to one - dimensional spatial isometry groups. in practice one considers r or u(1) and, hence, the spacetime metrics are required to have a single killing vector field. these models are interesting because they retain important features of full general relativity, in particular diffeomorphism invariance, an infinite number of physical degrees of freedom and a non - linear character. the local aspects of the one - killing vector reductions were first considered by geroch. in that paper 3 + 1 dimensional geometric objects to the 2 + 1 dimensional space of orbits of the killing field (required to have a non - vanishing norm). here the world refers to the fact that some topological aspects are sidestepped in a first look ; however, if the quotient itself is well behaved (for example, it is haussdorff) the projection is globally defined and has a clear geometrical meaning. the most important result of this work was to show that the reduced system can be interpreted as 2 + 1 gr coupled to certain matter fields with a concrete geometrical meaning : the norm and twist of the four dimensional killing vector field (a scalar and a one - form field respectively). this link between one - killing vector reductions and 2 + 1 dimensional gravity theories opened the door to quantum treatments relying on techniques specially tailored for lower dimensional models. for example it allows one to write the four - dimensional scalar curvature as a curvature on the 2 + 1 dimensional orbit manifold plus some extra terms involving the norm of the killing. this is very useful to write the 3 + 1 dimensional action as a 2 + 1 dimensional one. the global aspects and the hamiltonian formalism (for vacuum gr) have been discussed by moncrief in the case when the symmetry group is u(1) with compact cauchy surfaces. the spatial slices in this case can be taken to be u(1) bundles (or rather s) over the sphere (though the analysis can be extended to arbitrary surfaces). the discussion presented in is relevant to studying some of the compact gowdy models, in particular those with the s s and s spatial topologies, though it is possible to employ other approaches that rely on the geroch reduction as discussed in. the non - compact case with asymptotically - flat two - geometries (in the sense relevant in 2 + 1 gravity developed in) has been studied by varadarajan. the next natural step is to consider two - dimensional spacelike isometry groups. a local approach that parallels the one given for the one - killing vector reduction was developed by geroch in for the abelian case (corresponding to commuting killing fields). the global aspects for these models, in the case of considering commutative, connected and two dimensional isometry groups with effective and proper action, have been discussed in detail in [38, 66 ]. in the following we will simply refer to them as two - killing vector reductions despite the fact that they do not correspond to the most general situation. the classification of the smooth, effective, proper actions by isometries of a commutative, connected, two - dimensional lie group on a connected, smooth 3-manifold has been studied in the literature both for the compact [176, 177 ] and non - compact topologies. of all the possible choices of group action and spatial topology only two have been considered with sufficient detail from the quantum point of view : the einstein - rosen cylindrical waves, with isometry group r u(1) and spatial topology r.the gowdy models, whose isometry group is u(1) u(1) and the spatial topologies are t:= s s s, s s, s, and the lens spaces. the einstein - rosen cylindrical waves, with isometry group r u(1) and spatial topology r. the gowdy models, whose isometry group is u(1) u(1) and the spatial topologies are t:= s s s, s s, s, and the lens spaces. in the restricted case when the group orbits are hypersurface orthogonal we have the polarized models (also known as linearly polarized models). historically, two - killing vector reductions were introduced to explore some concrete problems ; in particular, the original motivation by einstein and rosen [84, 29 ] was to use cylindrical symmetry as a simplifying assumption to explore the existence of gravitational waves (see, however,). gowdy considered the u(1) u(1) model as a first step to study inhomogeneous cosmologies [102, 103 ]. as we are mainly interested in the quantization of midisuperspaces we discuss next only those classical issues of direct use in the quantum treatment of these models, in particular their hamiltonian description. the first hamiltonian analysis of the einstein - rosen waves was carried out by kucha in the early seventies. however, a work that was very influential in getting the final hamiltonian formulation was, in which ashtekar and varadarajan study the hamiltonian formalism of asymptotically - flat 2 + 1 dimensional gr coupled to matter fields satisfying positive energy conditions. they find that, due to the peculiarities associated with the definition of asymptotic flatness in the 2 + 1 setting, the hamiltonian of those systems is always bounded both from below and from above. this is a very important result for the class of midisuperspaces whose 2 + 1 description can be performed in terms of non - compact cauchy surfaces. as we pointed out before, this is the case for certain one - killing symmetry reductions and especially for einstein - rosen waves. before we proceed further we want to point out that the asymptotic analysis mentioned above has been extended to discuss the structure of the null infinity in the 2 + 1 description of one - killing symmetry reductions of 3 + 1 gr in and the behavior of einstein - rosen waves at null infinity has been studied in detail in. in the case of the einstein - rosen waves the two killing vector fields correspond to translations and rotations. the translational killing field has a non - vanishing norm whereas the rotational one vanishes at the symmetry axis. the main steps to arriving at the hamiltonian formulation of the einstein - rosen waves can be summarized as follows : start from the einstein - hilbert action with the appropriate surface terms depending on the extrinsic curvature at the boundary.use then the translational killing vector to perform a geroch reduction and obtain a 2 + 1 dimensional action written in terms of the 2 + 1 dimensional metric in the space of orbits and a scalar field (the norm of the killing).get the hamiltonian from this 2 + 1 dimensional action in the standard way by using an axially symmetric foliation adapted to the minkowskian observers with proper time t at infinity. in this process it is necessary to introduce and take into account the fall - off conditions for the fields corresponding to the relevant asymptotics and the fact that the action depends on a minkowskian fiducial asymptotic metric.finally use a gauge fixing condition to select a single point in each gauge orbit. start from the einstein - hilbert action with the appropriate surface terms depending on the extrinsic curvature at the boundary. use then the translational killing vector to perform a geroch reduction and obtain a 2 + 1 dimensional action written in terms of the 2 + 1 dimensional metric in the space of orbits and a scalar field (the norm of the killing). get the hamiltonian from this 2 + 1 dimensional action in the standard way by using an axially symmetric foliation adapted to the minkowskian observers with proper time t at infinity. in this process it is necessary to introduce and take into account the fall - off conditions for the fields corresponding to the relevant asymptotics and the fact that the action depends on a minkowskian fiducial asymptotic metric. finally use a gauge fixing condition to select a single point in each gauge orbit. after all these steps the hamiltonian is obtained in closed form as a function of the free hamiltonian h0 for an axially - symmetric massless scalar field evolving in an auxiliary, 2 + 1 dimensional minkowskian background 3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h = 2\left({1 - \exp (- { h_0}/2) } \right).$$\end{document } the fact that this hamiltonian is a function of a free one can be used to study the exact classical dynamics of this system. it is very important to point out that the minkowskian metric (induced by the metric in the asymptotic region) plays an auxiliary role. although the hamilton equations are non - linear, it is possible to achieve a remarkable simplification by introducing a redefined time variable of the form t:= exp(h0/2)t where t is the asymptotic inertial time. when this is done the scalar field that encodes the gravitational degrees of freedom of the model must simply satisfy the free field equation for an axisymmetric field. in particular the exact unitary evolution operator can be written in closed form and, hence, closed form expressions can be written for many interesting physical objects such as two - point functions. we want to mention that some generalizations of the einstein - rosen waves to a class of cylindrical spacetimes endowed with angular momentum have been considered by manojlovi and mena in, where the authors found boundary conditions that ensure that the resulting reduced system has consistent hamiltonian dynamics. this work lead mena to consider a definition of cylindrical spacetimes that is less restrictive than that usually employed in the literature. the key idea is to remove the symmetry axis from the spacetime and, as a consequence, relax the regularity conditions in the fields when they approach this spacetime boundary. little is presently known about the quantization of these systems so we will not consider them further. the hamiltonian formalism for the gowdy models has been developed by many authors both in vacuum [159, 189 ] and coupled to scalar fields for all the possible topologies. the hamiltonian analysis for the t topology can be seen in [159, 189 ]. an interesting technical point that is relevant here concerns gauge fixing. in this model the natural gauge fixing condition gives rise to a deparametrization because the fixing is not complete. one of them is linear in the momentum canonically conjugate to a variable that can be interpreted as time. as a consequence of this it is possible to reinterpret the system as one described by a time dependent hamiltonian. in particular, it discusses the constraints that must be included in the hamiltonian formulation to take into account the regularity conditions on the metric in the symmetry axis for the s s and s topologies. the main difficulty that arises now is the vanishing of some of the (rotational) killing vector fields at some spacetime points. in the case of the s s topology only one of the two killings vanish so the problem can be dealt with by using the non - vanishing killing to perform a first geroch reduction and carefully deal with the second by imposing suitable regularity conditions for the fields in the 2 + 1 dimensional formulation. nevertheless the problem can be successfully tackled by excising the axes from the spacetime manifold and imposing suitable regularity conditions on the fields when they approach the artificial boundary thus introduced. an interesting feature that shows up in the hamiltonian analysis for the ss and s topologies is the presence of the polar constraints induced by the regularity conditions. the final description for these topologies is somehow similar to the one corresponding to the t case, in particular the fact that the dynamics of the system can be described with a time dependent hamiltonian that clearly shows how the initial and final singularities appear. the main difference is the absence of the extra constraint present in the t model. other classical aspects related to gowdy models coupled with matter (concerning integrability or the obtention of exact solutions) have been covered in detail in [62, 61, 63, 57, 56 ] ; in particular, the identification of a complete set of dirac observables for the einstein - rosen and the gowdy t midisuperspace was obtained in [202, 205, 140 ] (and in by using ashtekar variables). the relation of two - killing reductions and sigma and chiral models have been considered by many authors [9, 118, 158 ]. we want to mention also an interesting paper by romano and torre where they investigate the possibility of developing an internal time formalism for this type of symmetry reduction. they also show there that the hamiltonian of these models corresponds to that of a parametrized field theory of axially symmetric harmonic maps from a 3-dimensional flat spacetime to a 2-dimensional manifold endowed with a constant negative curvature metric (though in the compact cases the presence of constraints must be taken into account). spherically - symmetric systems in gr are another type of midisuperspace models (in a sense the other type) that has received a lot of attention. they are enjoying a second youth these days as very useful test beds for lqg. a 3 + 1 spacetime (m, g) is called spherically symmetric if its isometry group contains a subgroup isomorphic to so(3) and the orbits of this subgroup are 2-spheres such that the metric g induces riemannian metrics on them that are proportional to the unit round metric on s. notice that in the standard definition of spherically symmetry the spacetime manifold is taken to be diffeomorphic to r, where the cauchy hypersurface is r s (notice, however, that this is not the only possibility [67, 197, 200 ]). in this case the so(3) symmetry group acts without fixed points (there is no center of symmetry). the spherically - symmetric metric on the cauchy slices r s is given by (t, r)dr + r(t, r)d (where d denotes the metric of the unit 2-sphere). this metric depends only on two functions (t, r) and r(t, r). the radial coordinate r r is such that the r limits correspond to the two different spatial infinities of the full schwarzschild extension and t denotes a time coordinate. the first attempt to study spherically - symmetric models in gr from the hamiltonian adm point of view goes back to the paper by berger, chitre, moncrief and nutku. here the authors considered vacuum gravity and also coupled to other fields such as massless scalars. the problem with this approach, as pointed out by unruh in, was that they did not reproduce the field equations. the cause for this was identified also by unruh : a boundary term needed to guarantee the differentiability of the hamiltonian was missing in the original derivation. it must be pointed out that the paper predates the classic one by regge and teitelboim where the role of surface terms in the correct definition of the hamiltonian framework for gr is discussed in detail. we also want to mention that was the starting point for an interesting series of articles by hjek on hawking radiation [106, 104, 105 ]. the spacetime slicings chosen in the first studies of spherically - symmetric models covered only the static regions of the extended schwarzschild spacetime (the kruskal extension). this means that, in practice, they only considered the schwarzschild geometry outside the event horizon. this problem was tackled by lund who used a different type of slicing that, however, did not cover the whole kruskal spacetime with a single foliation. an interesting issue that was explored in this paper had to do with the general problem of finding a canonical transformation leading to constraints that could give rise to a generalized schrdinger representation (as was done by kucha in the case of cylindrical symmetry). one of the conclusions of this analysis was that this was, in fact, impossible, i.e., there is no time variable such that the constraints are linear in its canonically conjugate momentum. this negative conclusion was, however, sidestepped by kucha in by cleverly using a less restrictive setting in which he considered foliations going from one of the asymptotic regions of the full kruskal extension to the other. this paper by kucha is in a sense the culmination of the continued effort to understand the quantization of schwarzschild black holes in the traditional geometrodynamical setting. it must be said, however, that it was predated by the analysis performed by thiemann and kastrup [130, 201 ] on the canonical treatment of schwarzschild black holes in the ashtekar formalism. in the authors found a pair of canonical variables that coordinatize the reduced phase space for spherically - symmetric black holes consisting of two phase - space variables m and t where m is the black hole mass and t is its conjugate variable that can be interpreted as time (more precisely the difference of two time variables associated with the two spatial asymptotic regions of an eternal black hole). this description of the reduced phase space precisely coincides with the ones given by kucha. we want to mention also that an interesting extension of kucha s work appears in. in this paper, varadarajan gave a non - singular transformation from the usual adm phase - space variables on the phase space of schwarzschild black holes to a new set of variables corresponding to kruskal coordinates. in this way it was possible to avoid the singularities appearing in the canonical transformations used by kucha. the hamiltonian formulations obtained by these methods provide a precise geometrical description of the reduced phase for vacuum spherically - symmetric gr. in particular an exact parametrization of the reduced phase space is achieved. at this point it is just fitting to quote kucha : primordial black holes, despite all the care needed for their proper canonical treatment, are dynamically trivial. primordial black holes, despite all the care needed for their proper canonical treatment, are dynamically trivial. a possible way to have spherically - symmetric gravitational models with local degrees of freedom and avoid this apparent triviality consists in coupling matter to gravity. it must be said, nevertheless, that for some types of matter couplings the reduced phase space of spherically - symmetric systems is finite dimensional. this is so, for example, in the case of adding infinitesimally spherical thin shells. the hamiltonian analysis of the massive and the null - dust shell cases has been extensively studied in the literature [90, 33, 151, 107, 109 ]. the presence of additional null shells has been also analyzed [110, 111 ]. it is perhaps more surprising to realize that this finite - dimensional character is also a property of spherically - symmetric einstein - maxwell spacetimes with a negative cosmological constant, for which the gauge symmetries exclude spherically - symmetric local degrees of freedom in the reduced phase space. in this case canonical transformations of the kucha type can be used to obtain the reduced phase - space hamiltonian formulation for the system. once matter in the form of massless scalar fields is coupled to gravity, the reduced system is a (1 + 1)-dimensional field theory and some of the techniques developed by kucha can not be applied. in particular, romano has shown that the coupled einstein - klein - gordon system does not have a suitable extrinsic time variable. as we mentioned above, the hamiltonian formulation for the gravity - scalar field model was clarified by unruh in. recently, some simplifications have been obtained by using flat slice painlev - gullstrand coordinates. other types of matter that can be coupled to gravity giving rise to infinite - dimensional reduced phase spaces are those including collapsing dust clouds [223, 220 ]. to end this section, we should mention that another interesting way to gain insights into the quantization of more realistic gravity models, such as the collapse of spherically - symmetric matter in 3 + 1 dimensions, is to consider two - dimensional dilaton gravity as in the callan, giddings, harvey and strominger (cghs) model and similar ones that admit a phase - space description close to the 3 + 1 spherically - symmetric spacetimes. these systems are usually exactly solvable (both classically and quantum mechanically) and hence can be used to study the consequences of quantizing gravity and matter. from a technical point of view these models are close to spherical symmetry because they can be treated by using the same type of canonical transformations introduced by kucha in. they lead to descriptions that are quite close to the ones obtained for the vacuum schwarzschild case [211, 49, 95 ]. the quantization of this class of midisuperspaces has been considered by some authors but it is fair to say at this point that only a very partial knowledge has been achieved. a preliminary analysis within the lqg framework (with complex variables) was carried out in. the fact that these models can be interpreted as 2 + 1 dimensional gravity coupled to some matter fields suggests that a quantization of this lower dimensional system essentially solves the problem for one - killing vector reductions. in fact, some claims about the perturbative renormalizability of 2 + 1 gravity coupled to scalar fields have appeared in the literature although, in our opinion, it is rather unclear how these results can be extrapolated to symmetric gravity in 3 + 1 dimensions. the reason for this scepticism is the non - trivial structure of the hamiltonian for these systems when the right asymptotic behavior is incorporated (taking into account that, as shown by ashtekar and varadarajan in, the hamiltonian that corresponds to the generator of time translations at spatial infinity is actually bounded from above). in any case, the physical consequences of these perturbative analyses have not been explored. the first historic attempt to canonically quantize (vacuum) einstein - rosen waves goes back to the pioneering paper by kucha. there he carefully studied cylindrical metrics for the polarized case and derived the hamiltonian formulation for the system. the author used the full four - dimensional picture in a very effective way in order to develop a suitable hamiltonian formalism, in particular, the canonical transformations leading to a convenient coordinatization of the reduced phase space. one of the key achievements of the paper was the identification of a phase - space function that could play the role of a time variable for er waves. this provides an extrinsic time representation similar to the one used in the spherically - symmetric case. by defining an appropriate canonical transformation it is possible to turn this time into a canonical variable and make it part of a new set of canonical coordinates. in terms of them the action functional takes the particularly simple form of the parameterized formalism for an axially - symmetric scalar field evolving in a (fictitious) minkowskian background. an interesting comment is that the canonical transformation mentioned above mixes configuration and momentum variables in such a way that the original configuration space is traded for a rather different one, which is not a subset of a space of metrics. the main problem with was that it did not take into account the necessary boundary terms needed to render the variational problem well defined. the results of the derivation given by kucha can be obtained in a more systematic and straightforward way by using the principle of symmetric criticality, substituting the form of the cylindrically - symmetric metrics corresponding to polarized einstein - rosen waves in the einstein - hilbert action and getting the hamiltonian formulation from there. the dirac quantization of the einstein - rosen waves that kucha gives is interesting from a pedagogical and intuitive point of view but arguably quite formal. the main consistency issues related, for example, to the path independence with respect to the foliations interpolating between two given ones, are formally taken into account as well as the definition of the scalar product of schrdinger picture functionals. however, no attention is payed to the subtle functional analytic and measure theoretic issues that come up. cho and varadarajan have studied the relationship between the schrdinger and fock representations and considered several issues related to the unitary implementability of the evolution of the free axisymmetric scalar field in a minkowskian background. in particular they have discussed the existence of unitary transformations on the fock space implementing the evolution between two axisymmetric, but otherwise arbitrary, cauchy slices of the auxiliary flat spacetime in such a way that their infinitesimal version gives the functional schrdinger equations obtained by kucha. the analysis is based on work by torre and varadarajan [208, 209 ] on the evolution of free scalar fields between arbitrary cauchy surfaces. in this respect it is interesting to remark on the different behaviors in the 1 + 1 dimensional case and the higher dimensional ones. it is also important to point out that polarized einstein - rosen waves are remarkably close to this type of model. the main result of is that in the half - parameterized case, when the radial coordinate is not changed, the dynamics can be unitarily implemented as a consequence of shale s theorem. however, if no condition is imposed on the radial coordinate thus allowing for the possibility of having radial diffeomorphisms the quantum counterpart of these transformations can not be unitarily implemented because it is not given by a hilbert - schmidt operator. the unitary equivalence of the schrdinger and fock quantizations for free scalar fields has been studied in a slightly more general setting by corichi. in. the authors of this paper take into account several functional issues that are relevant for a rigorous treatment of the rather subtle issues that crop up in the quantization of free field theories defined in arbitrary globally - hyperbolic spacetimes. a different approach to the quantization of einstein - rosen waves by standard quantum field theory methods consists in performing a complete gauge fixing and studying the reduced space of the model. the quantization of the system is performed after paying special attention to the asymptotic conditions relevant in the 2 + 1 dimensional case (and, consequently, to the necessary boundary terms in the gravitational action). specifically, some of the peculiarities of this system come from the fact that, due to the translational part of the isometry group, the (non - trivial) einstein - rosen solutions can not be asymptotically flat in four dimensions (or alternatively, the 2 + 1 dimensional metric does not approach a minkowski metric at spatial infinity because a deficit angle is allowed). the numerical value of the hamiltonian of this system, when the constraints are satisfied, is given by an expression originating in the surface term of the action for einstein - rosen waves. as mentioned before, this has the form given by equation (3) and is a function of h0, the free hamiltonian corresponding to an axisymmetric massless scalar field in 2 + 1 dimensions evolving in a minkowskian background metric. in fact, as the hamiltonian is just a function of a free one it is possible to obtain the exact quantum evolution operator from the one corresponding to the free auxiliary model and use it to obtain close form expressions for many objects of physical interest such as field commutators and n - point functions. it is important to point out that only after the gauge fixing is performed will the hilbert space of states take this form. this means that other quantizations or gauge fixings describing the gravitational degrees of freedom in a different way, could lead to a different form for the quantized model. it is important to notice that ashtekar and pierri do not perform the canonical transformation used by kucha to introduce the extrinsic time representation, but directly fix the gauge in the original canonical formulation. by using this scheme varadarajan has studied the mathematical properties of the regularized quantum counterpart of the energy of the scalar field in a spherical region of finite ratio of 2 + 1 flat spacetime. in particular, he has given a proof of the fact that this regularized operator is densely defined and discussed its possible self - adjoint extensions (this is only a symmetric operator). in the aftermath of, several papers have used the quantization presented there to derive physical consequences of the quantization of einstein - rosen waves. among the most influential is. in this paper ashtekar considers 2 + 1 gravity coupled to a maxwell field with the additional condition of axisymmetry as a solvable toy model to discuss quantum gravity issues. it is important to point out that in 2 + 1 dimensions the maxwell field can be interpreted as a free massless scalar, with the usual coupling to gravity, and dynamics given by the wave equation. this system is equivalent to the symmetry reduction of 3 + 1 dimensional gravity provided by einstein - rosen waves, so the results on the paper are relevant to studying the quantum physics of this midisuperspace model. the main result of is the somewhat surprising appearance of large quantum gravity effects in the system. the presence of these has some importance because, if we trust this quantization, it points to the spurious character of many classical solutions to the model, as they can not appear in the classical limit. one key element here is the form of the hamiltonian, given by equation (3) and the fact that it is a non - linear function of a free hamiltonian. the large quantum gravity effects manifest themselves in the expectation values of the metric components (that are functions of the scalar field that describes the degrees of freedom). specifically these do not correspond to the classical values in some limits, in particular for high frequencies. furthermore the metric fluctuations in this regime are very large even for states that are peaked around a classical configuration of the scalar field and grow with the number of quanta (photons) of the scalar field. a reflection of these large quantum gravitational effects is also manifest in the behavior of the field commutators, especially at the symmetry axis, as shown in. an extension of these results to four dimensions has been carried out in the paper by angulo and mena. they do this by expressing the four - dimensional metric of the einstein - rosen waves in terms of the maxwell scalar and the 2 + 1 dimensional metric. it is important to mention at this point that the scalar field enters the four - metric in a highly non - linear way. the most important result of is the actual verification of the possibility to extend the conclusions reached by ashtekar in the 2 + 1 dimensional setting to four dimensions (far from the symmetry axis). however, the authors argue that to reach an acceptable classical description in the asymptotic region in four dimensions, it is not mandatory to require as in three that the number of quanta of the maxwell scalar field be small. also, the behavior on the symmetry axis is interesting because the relative uncertainties of the metric become very large there. this casts some doubts on the appropriateness of the classical regularity conditions usually introduced at the axis. there are other papers in which the physical consequences of the fock quantization of einsteinrosen waves given in have been considered. in [24, 25, 28, 18, 19, 20 ] several physical issues have been discussed in some detail, in particular microcausality, n - point functions, 2-point functions, matter couplings and coherent states. microcausality in poincar - invariant models, formulated with the help of a background minkowskian metric, reflects itself in the vanishing of the commutator of the field operators at spatially separated spacetime points. the fact that einstein - rosen waves can be quantized offers the possibility of quantitatively testing some of the expected features of quantum gravity such as the smearing of light cones due to quantum fluctuations of the metric. the presence of this effect was suggested already in the original paper by ashtekar and pierri. in the explicit form of the commutator was obtained and a direct numerical analysis showed the expected smearing effect and clear indications about how the classical limit is reached when the effective gravitational constant of the model goes to zero. a quantitative understanding of this phenomenon has been given in [25, 28 ] by performing an asymptotic analysis of the integrals that define the field propagators. an interesting result coming from these analyses is the different way in which the classical limit is reached on and outside the symmetry axis. outside the symmetry axis the large scale regime is reached in a rather smooth way, but on the axis large quantum gravitational effects persist even at macroscopic scales. this is probably another manifestation of the kind of effects described by ashtekar in. a way to incorporate quantum test particles that could further help explore the quantized geometry of the model is to couple matter fields to gravity and use their quanta as probes. this is very difficult for generic matter fields but can be remarkably achieved for massless scalars keeping both the classical and quantum resolubility of the system. this was done in [18, 19 ] though the classical integrability was understood by several preceding authors, in particular lapedes, charach, malin, feinstein, carmeli and chandrasekhar [145, 62, 63, 60 ]. it is also fair to mention at this point that the effective decoupling of the gravitational and matter scalar modes (in the flat space picture), that is the key ingredient in the fock quantization of the einstein - rosen waves coupled to massless scalar fields presented in, was discussed in essentially the same form by lapedes in, though he treated the quantization in the heuristic way customary at the time. in newton - wigner localized states were used to build actual position space wave functions for the massless quanta in order to study how they evolve in full interaction with the quantized geometry. the resulting picture shows, in a convincing way, that the quantum particles in their motion define approximate trajectories that follow the light cones given by the microcausality analysis. also, the study of 2-point functions (extended to n - point functions in) gives a consistent picture when they are interpreted as approximate propagation amplitudes ; in particular, the persistence of large quantum gravity effects in the symmetry axis is confirmed. finally, the issue of obtaining coherent states for the einstein - rosen waves has been considered in. an interesting set of papers by korotkin, nicolai and samtleben [182, 137, 136, 138, 139 ] explores a family of systems consisting of two - dimensional gr coupled to non - linear sigma models. these generalize the symmetry reductions from 3 + 1 dimensions that we are considering in this section, in particular the einstein - rosen waves, and treat genuine midisuperspace models with non - linear interactions and an infinite number of degrees of freedom. a unified treatment of them appears in, where a number of issues related to their classical integrability and quantization are discussed. by assuming the presence of two commuting killing symmetries it is possible to make a first simplification of the functional form of the metrics for this system by restricting the number of coordinates on which the fields depend to just two. in this way they effectively correspond to 2-dimensional non - linear sigma models coupled to a dilaton and gravity. a key observation is that the resulting models are integrable and their solutions can be obtained from an auxiliary linear system of equations. in fact, the matter dynamics can be derived from the compatibility conditions for this linear system. the hamiltonian formulation can be written in terms of two constraints generating translations along the light cone that become partial differential equations when quantized. these quantum constraints are precisely the knizhnik - zamolodchikov equations that play a fundamental role in conformal field theory. their solutions, as discussed in, provide concrete physical states of the quantized theory. some of the specific models collectively considered in this paper are individually studied in a series of works by the authors [137, 136, 138 ]. the limits of this line of work are related to the impossibility of solving the coset constraints for the non - compact spaces [such as sl(2, r)/so(2) ] by using discrete unitary representations [of sl(2, r) in the previous example ]. we want to mention here that is of particular interest because korotkin and samtleben extend the fock quantization techniques used in to the much harder non - polarized case where the non - linearity of the model shows up in full strength. in this case, the einstein field equations can be written as the ernst equation for the unimodular metric on the killing orbits and an integral expression for the conformal factor of the metric in terms of the solutions to this ernst equation. the quantization is achieved by finding a complete set of quantum observables and a representation of them in a fock hilbert space. these generalize another set of variables that can be defined in the polarized case in terms of the positive and negative frequency modes that appear in the fourier decomposition of the axisymmetric scalar field that encodes the gravitational degrees of freedom. the poisson algebra of the new observables is quadratic (i.e., the poisson bracket of two basic observables can be written as a linear combination of products of two of them) ; this introduces some complications in the treatment due to the necessity to deal with operator ordering issues after quantization. however, the solution to this problem is known in the theory of integrable systems. the final step of the process consists in finding a representation of the quantum algebra in a fock hilbert space. the availability of this representation opens up the possibility of computing expectation values of important operators, in particular certain components of the metric. in principle this can be used to derive physics from the non - polarized er waves, although the non - local character of these new observables introduced by the authors and, in particular, their lack of an explicit spacetime dependence, makes it difficult to make contact with other results, especially those related to the study of microcausality in the polarized case. as a final comment on the approach by korotkin, nicolai and samtleben, we want to mention the possibility of mapping the non - polarized cylindrical models to free theories as discussed in. to end this section we want to comment that the quantization of einstein - rosen waves has also been considered from other points of view, such as perturbative methods and lqg inspired techniques. the use of perturbative techniques in particular is a very natural path to follow because it offers the possibility of comparing the results with those obtained by non - perturbative methods and ultimately trying to get an additional understanding of the causes leading to the perturbative non - renormalizability of gr. in a thorough study of the quantization of two - killing vector reductions the main goal of this paper is to find out if the perturbative quantization of einstein - rosen waves is consistent with the asymptotic safety scenario of weinberg. in fact, the main result of is to show that two - killing symmetry reductions of gr are asymptotically safe. however, this result is not straightforward because two - killing vector reductions are not renormalizable in the standard sense (beyond one loop). despite this, the model can be declared to be renormalizable if the space of lagrangians is expanded by allowing conformal factors that are functions of the radion field (the determinant of the pull - back of the metric to the integral manifolds of the killing vector fields). in fact, when this is done, the renormalization flow has a unique ultraviolet stable fixed point where the trace anomaly vanishes. a similar result has been obtained in the case of non - polarized einstein - rosen waves in by using a path integral approach and the algorithm developed by osborn in to deal with position dependent sigma models. these papers provide an alternative point of view from the esentially non - perturbative methods of korotkin, nicolai and samtleben several other papers can be found in the literature that consider different aspects of some two - killing vector reductions from the perturbative point of view ; in particular the loop quantization of the einstein - rosen midisuperspace is an interesting open problem that deserves some comments. in their seminal paper about fock quantization of cylindrically symmetric spacetimes, ashtekar and pierri computed the holonomies around those loops that are integral curves of the rotational killing vector and showed that their traces are functions of the energy (in a box of finite radius) of the scalar field encoding the reduced degrees of freedom. in particular, in the large radius limit, those traces reduce to a simple function of the total energy of the system. hence, as they point out in the paper, the question of whether those traces are well - defined operators in quantum theory, reduces to the question of whether the operator corresponding to the energy of a scalar field in a box can be satisfactorily regulated (see). in any case, the dynamic issues of the polymeric quantization of the scalar field (including the classical limit and the relation with standard quantizations) need to be analyzed in detail. as usual in lqg, one follows a two - step route to quantization by first constructing the kinematical hilbert space of the theory and then defining the hamiltonian constraint (and, for er waves, also the hamiltonian) of the model. in this last step, the geometric operators (in particular the volume operator) are thought to play a relevant role in the rigorous definition of the hamiltonian constrain operator. in his living review, bojowald discusses the kinematical hilbert space for er waves and also certain properties of the volume operator. he pays special attention to the differences of the cylindrically - symmetric sector and the homogeneous cosmologies. there are also other papers, much more qualitative in nature, by neville [181, 180 ], in which the construction of a kinematical hilbert space for the loop quantization of cylindrically - symmetric spacetimes and planar waves is sketched. gowdy cosmological models [102, 103 ] are described by time - oriented, globally - hyperbolic, vacuum spacetimes, which can be constructed from the evolution of u(1) u(1)-invariant cauchy data defined on a 3-dimensional closed (compact without boundary) hypersurface. the action of the u(1) u(1) group of spatial isometries is assumed to be effective and the topology of is restricted to be t, ss, s, or the lens spaces. these spacetimes describe inhomogeneous cosmologies with initial and/or final singularities and represent gravitational waves propagating in a closed universe. for the t topology, only one singularity is present, whereas in the case of the ss, s topologies there are both initial and final singularities. the quantization of these models has been considered only in the polarized case (for which both killing vector fields are hypersurface orthogonal). the gowdy t model describes an inhomogeneous cosmology with one singularity (that can be thought of as initial or final). misner was the first researcher to recognize its relevance as a test bed for quantum cosmology [164, 165 ]. pioneering work on its quantization was carried out in the 1970s and 1980s by him and berger [35, 34, 36 ]. actually it is fair to say that many of the ideas that have been used to this day in the attempts to achieve a rigorous quantization for this system actually originate in these works. in particular : the hamiltonian analysis for this type of model, including the identification of the extra constraint present for the t topology [165, 34].the introduction of the deparametrization customarily used to achieve a (partial) gauge fixing.the identification of a suitable real fourier expansion that decouples the modes appearing in the hamiltonian.the identification of the time - dependent field redefinitions that have been used in recent works to find a unitary implementation of the quantum dynamics (though these were used by berger in a different context to study the wbk regime).the role of the harmonic oscillator with time - dependent frequency in the dynamics of the gowdy models [165, 34 ]. the hamiltonian analysis for this type of model, including the identification of the extra constraint present for the t topology [165, 34 ]. the introduction of the deparametrization customarily used to achieve a (partial) gauge fixing. the identification of a suitable real fourier expansion that decouples the modes appearing in the hamiltonian. the identification of the time - dependent field redefinitions that have been used in recent works to find a unitary implementation of the quantum dynamics (though these were used by berger in a different context to study the wbk regime). the role of the harmonic oscillator with time - dependent frequency in the dynamics of the gowdy models [165, 34 ]. the approach to quantization followed in these papers consisted in a rather formal treatment in which the hilbert space was taken to be the infinite tensor product of the (countably) infinite hilbert spaces associated with each of the oscillator modes appearing in the fourier transformation of the fields. the use of an infinite tensor product of hilbert spaces is problematic, as emphasized by wald, because this hilbert space is non - separable and the representation of the canonical commutation relations is reducible. misner and berger have discussed a number of issues related to graviton pair creation in gowdy universes and the semiclassical limit. a nice and intuitive picture developed in these papers is the idea that their quantum dynamics can be interpreted as scattering in superspace. an interesting problem that received significant attention even at this early stage was the issue of the singularity resolution. berger approached it by working in a semiclassical approximation, where it was possible to describe the gravitational radiation by means of an effective energy momentum tensor depending on some of the metric components. by using this framework this very same question was considered by husain in, where he used the same kind of hilbert space as berger but followed a different approach consisting in quantizing the kretschmann invariant (the square of the riemann tensor) after finding an appropriate operator ordering. this was done by imposing the sensible requirement that the expectation values of the kretschmann operator in coherent states equal their classical values sufficiently far from the singularity. the main result of was that, at variance with the findings of berger, the classical singularity persisted in the quantized model. it is interesting to mention that this quantum version of the kretschmann invariant for gowdy t was also used by husain to explore a conjecture by penrose pointing to a relation between the weyl curvature tensor and gravitational entropy. in the mid-1990s, the work on the quantization of the einstein - rosen waves developed by ashtekar and pierri led naturally to the revision of the quantization of other two - killing midisuperspace models, and in particular the gowdy t cosmologies. this was done, among other reasons, to open up the possibility of using this type of symmetry reductions as toy models for lqg. since then the system has been considered not only within the traditional geometrodynamical approach but also in the ashtekar variables framework. the hamiltonian formalism of the gowdy t model, with a detailed analysis of the gauge fixing procedure, was studied in terms of (complex) ashtekar variables by mena in (see also). in that paper, the quantization of the reduced model was also sketched, however, the first attempt to study the fock quantization of the polarized t gowdy model (in the geometrodynamical setting) appears in. in this paper, pierri used one of the u(1) subgroups of the isometry group to perform a dimensional reduction and represent the model as 2 + 1 gravity coupled to a massless scalar field. she showed that the reduced phase space could be identified with the one corresponding to a u(1) symmetric, massless scalar field propagating in a 2 + 1 background geometry and satisfying a quadratic constraint. by using this description of the reduced phase space, she proposed a fock quantization that relied on a quantization of the modes of the free field propagating in the 2 + 1 background geometry. the main drawback of this approach, as later pointed out by corichi, cortez and quevedo in, was that the quantum dynamics of the free scalar field used in pierri s quantization does not admit a unitary implementation. it is important to realize that this type of behavior is not a specific pathology of the gowdy models but actually an expected (and somehow generic) feature of quantum field theories. the results of were confirmed and extended by torre who was able to show that, even after restricting the quantum dynamics to the physical hilbert space obtained by imposing the constraint present in the model la dirac, the quantum evolution is not given by a unitary operator. this important problem was tackled and solved in a satisfactory way in a series of papers by corichi, cortez, mena, and velhinho [69, 68, 70, 71, 74, 76, 77, 75 ]. these authors have shown that it is actually possible to have unitary dynamics if one redefines the basic scalar field in the description of the gowdy t model [69, 68 ] by introducing an appropriate time - dependent factor (inspired by a similar field redefinition used by berger in). an additional important uniqueness result appearing in these papers is that, up to unitary equivalence, this is the only way in which the dynamics can be unitarily implemented in this reduced phase - space quantization of the system. the quantum description of the s s and s gowdy models in terms of a fock quantization of their reduced phase spaces can be found in (the details of the hamiltonian formulation for these topologies were studied in). in those cases, the reduced phase spaces can be identified with the ones corresponding to u(1)-symmetric massless scalar fields. the problem of unitarily implementing the quantum dynamics is present also for these topologies but, as in the t case, the quantum dynamics can be implemented in a unitary way if the scalar fields are suitably redefined [23, 99, 75 ]. the description of the reduced phase space of gowdy models in terms of massless scalar fields has been used to explore the quantum schrdinger representation of the system in terms of square - integrable functions on a space of distributional fields with a gaussian probability measure [206, 71, 99, 100 ]. this representation is, in this case, unitarily equivalent to the fock one, but in some situations it is actually more convenient due to the availability of a spacetime interpretation. we want to end this section by commenting that gowdy models have been used as a test bed for other approaches to quantum gravity. in, the t model has been studied within the consistent discretizations approach (though the paper mostly deals with classical issues aimed at the problem of showing that the discretizations reasonably reproduce the expected classical results, in particular the preservation of constraints). the analysis presented in this paper is relevant because the difficulties to determine the lapse and shift suggest, according to the authors, that the quantization of the non - polarized case will have to rely on numerical methods. the loop quantization of the non - polarized gowdy t model in terms of complex ashtekar variables was considered for the first time by husain and smolin in. in this work, husain and smolin found a loop representation of the unconstrained algebra of observables and gave sense to the (regulated) constrains in this representation. they also constructed a large and non - trivial sector of the physical state space and identified the algebra of physical operators on the state space. the loop quantization of the polarized gowdy t model has been studied recently in terms of real ashtekar variables by barnerjee and date [16, 17 ], where the authors recast the model in terms of real su(2) connections as a first step towards quantization and also discuss the gauge fixing procedure. a preliminary description of the kinematical hilbert space for the polarized gowdy t model and some issues related to the volume operator are given in. the previous papers do not discuss the quantum resolution of the classical singularity, a natural question to consider after the success of lqc in the study of this problem in the simpler setting provided by homogeneous models. this very important issue has been recently considered by using a combination of loop and fock quantizations in [157, 51 ]. in the authors use the formulation of the theory as 2 + 1 gravity coupled to a massless scalar field (with a residual u(1) symmetry). by introducing the usual fourier mode decomposition of the solutions in terms of the relevant angle variable, the authors define a hybrid quantization consisting in a polymeric quantization for the homogeneous mode (angle - independent) and a fock quantization for the inhomogeneous (angle - dependent) ones. the follow - up study appearing in further considers the quantum dynamics of the polarized gowdy t model, in particular the description of the initial big bang singularity that appears to be replaced by a big bounce as in the popular lqc models. two - killing vector reductions of gr, in the case when the killing vectors are hypersurface orthogonal, can be classified according to some properties of the gradient of the determinant of the restriction of the metric to the group orbits (the area function). the familiar cases of the einstein - rosen waves and the gowdy cosmologies correspond to spacelike and timelike gradients respectively, whereas plane waves correspond to the null case. the geometrodynamical approach to the quantization of plane wave midisuperspaces was considered in, where the authors study both the polarized and non - polarized cases. they show that the reduced phase - space models have vanishing hamiltonians in the coordinates adopted for their description. in the case of polarized plane waves, the reduced phase space can be described by an infinite set of annihilationand creation - like variables (that are classical constants of motion) and therefore, it is possible to quantize the system by finding a fock representation for these variables. in this respect the model is quite similar to the gowdy cosmologies and einstein - rosen waves that can also be quantized by using fock space techniques. the results of this paper have been used in to study the appearance of large quantum effects in the system (similar to the ones described by ashtekar for einstein - rosen waves in). the plane wave case is rather interesting in this respect because of the focusing of light cones characteristic of this system. important quantum gravity effects are expected precisely at the places where this focusing takes place. by introducing suitable coherent states, the authors show that the expectation value of a regularized metric operator coincides with a classical plane wave solution whereas the fluctuations of the metric become large precisely in the vicinity of the regions when focusing of light cones occurs (and this happens for every coherent state). these papers nicely complement and expand in a rigorous language the preliminary analysis carried out in [179, 48 ] in terms of (complex) ashtekar variables. a complete discussion based in the modern approach to connection dynamics and symmetry reductions in this framework would be interesting indeed. finally, we want to mention another cosmological midisuperspace model, due to schmidt, that has some interesting features. the spacetime of this case has the topology rt, i.e., it is the product of a plane and a torus, and the isometry group is u(1)u(1) with orbits given by tori. schmidt cosmologies have initial singularities that are similar to gowdy t. beetle has studied its hamiltonian formulation (in the polarized case) by choosing appropriate asymptotic conditions for the fields in such a way that the resulting reduced phase space is very similar to the one corresponding to the gowdy t model. in fact, the system can be described as a u(1) symmetric massless scalar field evolving in a fixed time - dependent 2 + 1 background (topologically rs) and with no extra constraints (at variance with the gowdy t case). the same unitarity problems that show up in the quantum evolution of the gowdy models also appear here and can be solved again with a time dependent canonical transformation. spherically - symmetric reductions of gr have an obvious appeal as they can serve both as interesting toy models to test fundamental aspects of quantum gr and also describe very interesting physical situations involving schwarzschild black holes. spherical symmetry is peculiar in the sense that classically the space of spherically - symmetric solutions to vacuum gr is parameterized by a single quantity the black hole mass but general spherically - symmetric metrics require the introduction of functions for their most general description that can be taken to depend only in a radial coordinate and a time variable. as a consequence, and although the reduced phase space for vacuum spherical gr is actually finite dimensional (this is essentially the content of birkhoff s theorem), the hamiltonian analysis for this type of system must be performed in the infinite dimensional phase space corresponding to a field theory. this means that, in principle, there is an important difference between a reduced phase - space quantization (or one involving gauge fixing), where the field theory aspects and the issues related to diff - invariance are hidden, and the use of a dirac approach that will require the introduction of constraint operators to select the physical states from those in the linear space of quantum states of a proper field theory. to further complicate matters (or, actually, making them more interesting) some parts of the schwarzschild solution this is the case for the interior schwarzschild metric that can be isometrically mapped to a kantowski - sachs model. as we will mention later, this fact has been used to discuss issues related to the quantum resolution of black hole singularities by adapting lqc methods. though this living review is devoted to midisuperspace models, for the sake of completeness we deem it necessary to include a discussion of the relevant results obtained in this minisuperspace setting, especially those devoted to black hole singularity resolution, before dealing with the midisuperspace aspects of spherical symmetry. early suggestions of singularity resolution coming from the use of lqg inspired techniques go back to the works of husain, winkler and modesto. in an exotic quantization developed in later, in, the well - known fact that the interior of a schwarzschild black hole is isometric to the kantowski - sachs homogeneous cosmological model is mentioned. this idea, combined with the exotic quantizations considered in, leads in a natural way to the claim that black hole singularities are resolved in this scheme. though some attempts to derive this result within the lqg framework by quantizing the kantowski - sachs model appear in the literature [168, 169 ], the first complete and rigorous account of it is due to ashtekar and bojowald. in this paper the authors import some of the results obtained in the framework of lqc to the study of black hole interiors and the issue of singularity resolution. this is important because the degenerate triads play a significant role in the quantization process in particular the phase - space points corresponding to them do not lie on a boundary of the constraint hypersurface. these singular configurations can be naturally accommodated in the ashtekar variables framework but not in standard geometrodynamics. then the loop quantization of the kantowski - sachs model is carefully carried out to completion. some important results can be derived from it, in particular it is possible to prove that the physical spacetime corresponding to the interior of a black hole does not end at the classical singularity but can be extended beyond it. on the other hand the quantum fluctuations close to the singularity are such that a classical description breaks down. the issues of matter coupling and the extension of the quantization to the exterior region outside the horizon were not considered in. these problems and the work related to them will be mentioned and described in section 5.3.3, which is devoted to the loop quantization of spherically - symmetric spacetimes. within a semiclassical approach the study of what is the kind of physical object that replaces the classical singularity after quantization has been attempted by several researchers by employing different approximations. in the authors suggest that the original quantization used in may not have the correct semiclassical limit (as it happens when an analogue quantization is performed in lqc). the proposed way to remedy this problem is to allow the parameter (playing the role of the polymeric parameter of lqc) appearing in the construction of the hamiltonian constraint operator to be a function of the triads (in the same way as 0 is allowed to depend on the scale factor in lqc) and work with an effective hamiltonian constraint. this generalizes and improves the results of (see also for other different types of suggested improvements) where modesto uses exactly the approach of. though the results presented in these works are not rigorous derivations within lqg, they support the idea that the singularity is resolved and replaced by either a wormhole type of solution or a swollen singularity described by a spherical surface that is asymptotically approached but never reached by infalling particles. further refinements on the ideas and approximations presented in these papers can be found in (where a fractal type of spacetime is generated by the creation of a series of smaller and smaller black holes spawned by quantum collapses and bounces). more general examples have been considered in the minisuperspace framework and using lqg inspired methods. for example, the quantum collapse of a spherical dust cloud is described in both in the adm and ashtekar formulations and some issues concerning black hole evaporation are discussed in. additional references on the minisuperspace treatment of black hole singularities are [170, 187, 188 ]. in this section, we will review the literature relevant to study the geometrodynamical quantization of spherical midisuperspace models. the upcoming section 5.3.3 will describe the results that are being obtained these days by using lqg - inspired methods. as a general comment we would like to say that the level of mathematical rigor used in the standard quantization of these systems is sufficient in some cases but, as a rule, the attention payed to functional analytic issues and other mathematical fine points does not reach the level that is standard now in lqg. this should not be taken as a criticism but as a challenge to raise the mathematical standards. in our opinion a rigorous treatment within the geometrodynamical framework could be very useful in order to deepen our understanding of quantum gravity (see for some interesting results in this respect). a fundamental paper in the study of the classical and quantum behavior of spherically - symmetry reductions of gr is due to kucha. for example, it gives canonical coordinates leading to a very simple description of the reduced phase space for an eternal black hole and explains along the way how the transformation to this coordinate system can be obtained from the knowledge of the extended schwarzschild solution (in kruskal coordinates). this canonical transformation is widely used to analyze the hamiltonian dynamics of other spherically - symmetric gravitational systems coupled to matter as will be commented on later in this section. this work discusses the quantization of the system in several possible schemes : the direct reduced phase - space quantization and the dirac approach. this paper is a basic reference where subtle but important issues are taken into account, in particular those related to the asymptotic behavior of the fields and the introduction of the necessary boundary terms in the einstein - hilbert action. it must be said, nonetheless, that the identification of the canonical pair of variables that describe the reduced phase space for eternal black holes was first found by kastrup and thiemann in their study of the very same system within the ashtekar variables approach [201, 130 ]. the quantization of schwarzshild black holes was also considered around the same time in [58, 59 ]. these papers rely on the study of what the authors call the r - dynamics. the reason they have to use some non - standard dynamics for the system to develop a hamiltonian formalism is that the parametrization of the spherical metrics that they employ involves only functions of a single radial coordinate r (and do not involve an additional time this amounts to introducing from the start the ansatz that the metrics are not only spherical but also static. these papers also rely on the use of a rather formal wheeler - dewitt approach to quantization. it must be said that, at the end of the day, some of the results of [58, 59 ] are compatible with the ones discussed in and (in particular the unavoidable identification of the black hole mass as the configuration variable for the spherical - black - hole reduced phase space, but in our opinion this approach has been superseded by the treatment provided by kucha, kastrup and thiemann that considers the full kruskal spacetime for eternal black holes and discuses the hamiltonian framework and the identification of the reduced phase space in that setting. an interesting way to go beyond vacuum spherically - symmetric gravity is by coupling a single particle - like degree of freedom, which in this case corresponds to an infinitely thin spherical matter shell. after the system is quantized, this shell degree of freedom can be conceivably used as a quantum test particle allowing us to extract interesting information about the (spherical) quantized geometry. the paper by berezin, boyarsky and neronov studies this system both from the classical and quantum points of view by using the standard geometrodynamical approach. one of the key ingredients is the use of the canonical transformation introduced by kucha in his famous paper on spherically - symmetric gravity. although the quantization presented in is quite formal, some interesting features are worth comment, in particular the fact that the schrdinger equation becomes a difference equation (a feature reminiscent of results obtained with lqg methods). by using analyticity arguments the authors identify the quantum numbers characterizing the system (actually a pair of integer numbers that parameterize the mass spectrum). this result, however, does not correspond to the one proposed by bekenstein and mukhanov and derived by other authors in different frameworks. the collapse of a thin null dust shell has been extensively studied by hjek in the context of a midisuperspace quantization [107, 108, 109, 110, 111 ]. classically this system gives rise to black holes and, hence, its quantization may shed light on the issue of singularity resolution. in this case the unitary evolution of the wave packets representing the collapsing shell degree of freedom suggest that the singularity is resolved because they vanish at the place where the singularity is expected to be (see and references therein). a series of papers [219, 215, 221, 222, 223, 220, 217, 218, 216, 135, 134 ] dealing with the quantization of spherically - symmetric models coupled to matter is due to kiefer, louko, vaz, witten and collaborators. a very nice summary of some of these results appears in the book by kiefer. the authors use the kucha canonical quantization and the idea of introducing a preferred dust - time variable (the brown - kucha proposal) to quantize eternal black holes. by an appropriate selection of the lapse function it is possible to make the proper dust time to coincide with the proper time of asymptotic observers. this selects a particular time variable that can be used to describe the system (furthermore the total energy can be seen to be the adm mass of the black hole). an interesting result derived in this reference is the quantization law for the black hole mass of the type \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${m_n } = \sqrt n { m_p}$\end{document } (where mp denotes the planck mass and n a positive integer) for the normalized solutions of the wheeler - dewitt equation with definite parity (notice that this is precisely the mass spectrum proposed by mukhanov and bekenstein [31, 178 ]). it is interesting to point out that the authors do not use a purely reduced phase - space quantization, but a dirac approach. this means that the wave functions for the quantized model do not only depend on the mass (in the vacuum case) but also on some embedding variables that can be used to distinguish between the interior and the exterior of the black hole. the matching conditions for the wave function in these two regions give rise to the quantization of the mass. the fact that the wave function of the system vanishes outside and is different from zero inside can be interpreted by realizing that observers outside the horizon should see a static situation. the results obtained by kastrup, thieman and kucha have also been used by kastrup in to get similar results about the black hole mass quantization. the methods introduced in were used in the midisuperspace approach for the computation of black hole entropy discussed in. here the authors have shown that it is actually possible to reproduce the bekenstein - hawking law for the entropy as a function of the black hole area after introducing a suitable microcanonical ensemble. in order to do this, they first show that the wheeler - dewitt wave functional can be written as a direct product of a finite number of harmonic oscillator states that can themselves be thought of as coming from the quantization of a massless scalar field propagating in a flat, 1 + 1 dimensional background. the finite number of oscillators that originates in the discretization introduced on the spatial hypersurfaces is then estimated by maximizing the density of states. this is proportional to the black hole area and suggests that the degrees of freedom responsible for the entropy reside very close to the horizon. a curious feature of the derivation is the fact that, in order to get the right coefficient for the bekenstein - hawking area law, an undetermined constant must be fixed. this is reminiscent of a similar situation in the standard lqg derivation, where the value of the immirzi parameter must be fixed to get the correct coefficient for the entropy. in our opinion there are some arbitrary elements in the construction, such as the introduction of a discretization, that make the entropy computation quite indirect. partially successful extensions of the methods used in [219, 215 ] to the study of other types of black holes appear in [221, 222, 223 ]. in the first of these papers the authors consider charged black holes. by solving the functional schrdinger equation it is possible to see that the difference of the areas of the outer and inner horizons is quantized as integer multiples of a single area. this is similar to the bekenstein area quantization proposal but not exactly the same because the areas themselves are not quantized and the entropy is proportional to this difference of areas. the second paper studies gravitational collapse as described by the lematre - tolman - bondi models of spherical dust collapse and considers a dirac quantization of this midisuperspace model. the main technical tool is, again, a generalization of the methods developed by kucha, in particular the canonical transformations introduced in. in addition, the authors use the dust as a way to introduce a (natural) time variable following the brown - kucha proposal. the physical consequences of this quantization (for the marginally bound models) are explored in [220, 217, 135, 134 ] where the authors describe hawking radiation and show that the bekenstein area spectrum and the black hole entropy can be understood in a model of collapsing shells of matter. in particular, the mass quantization appears as a consequence of matching the wave function and its derivative at the horizon. some subtle issues, involving regularization of the wheeler - dewitt equation are sidestepped by using the de witt regularization (0) = 0, but the whole framework is quite attractive and provides a nice perspective on quantum aspects of gravitational collapse. in dust the entropy of the black holes formed after the collapse of these n shells depends, naturally, on n. for black holes in equilibrium the authors estimate this number by maximizing the entropy with respect to n (this is similar to the result mentioned before for the schwarzschild black hole). as far as hawking radiation is concerned the authors model this system by taking the dust collapse model as a classical background and quantizing a massless scalar field by using standard techniques of quantum field theory in curved spacetimes. they separately consider the formation of a black hole or a naked singularity. in the first case they find that hawking radiation is emitted whereas in the second one the breaking of the semiclassical approximation precludes the authors from deriving meaningful results. the treatment provided in this paper is only approximate (scalar products are not exactly conserved) and semiclassical (the wbk approximation is used), but the resulting picture is again quite compelling. black holes in ads backgrounds have been considered in [218, 216 ] (see also). the most striking result coming from the analysis provided in these papers is the fact that different statistics (boltzmann and bose - einstein) must be used in order to recover the correct behavior of the entropy from the quantization of area in the schwarzschild and large cosmological constant limits respectively. finally, we want to mention that the collapse of null dust clouds has been partially discussed in. a number of papers by louko and collaborators [150, 152, 149 ] study the hamiltonian thermodynamics of several types of black holes, in particular of the schwarzschild, reissner - nordstrom - anti - de sitter types, and black holes in lovelock gravity. the idea proposed in is to consider a black hole inside a box and use appropriate boundary conditions to fix the temperature. the black hole thermodynamics can now be described by a canonical ensemble and standard statistical physics methods can be used to compute the entropy. in particular the authors provide a lorentzian quantum theory and obtain from it a thermodynamical partition function as the trace of the time evolution operator analytically continued to imaginary time. from this partition function it is possible to see that the heat capacity is positive and the canonical ensemble thermodynamically stable. one of the remarkable results presented in is that the partition function thus obtained is the same as the one previously found by standard euclidean path integral methods [227, 50 ]. canonical transformations inspired in the one introduced by kucha in play a central role in all these analyses. also the methods developed in have been extended to study the thermodynamics of spherically - symmetric einstein - maxwell spacetimes with a negative cosmological constant and spherically - symmetric spacetimes contained in a one parameter family of five - dimensional lovelock models. in both cases the most important conclusion of these works is that the bekenstein - hawking entropy is recovered whenever the partition function is dominated by a euclidean black hole solution. in the lovelock case the results suggest that the thermodynamics of five - dimensional einstein gravity is rather robust with regard to the the introduction of lovelock terms. another paper where the kucha canonical transformation is used is, where the authors consider extremal black holes and how their quantization can be obtained as a limit of non - extremal ones. the obtention of the bekenstein area quantization in this setting (for schwarzschild and reissner - nordstrm black holes) is described in [148, 155 ]. we end with a comment about the quantization of spherically - symmetric gr coupled to massless scalar fields. the authors study this problem in the geometrodynamical setting by using painlev - gullstrand coordinates (that are especially suitable for this system). they use the non - standard quantization described in that displays some of the features of lqg and suggest that the black hole singularity is resolved. the key idea is to use operator analogues of the classical null expansions and trapping conditions. as the authors emphasize, their proposal can be used in dynamical situations (at variance with isolated horizons). the construction of semiclassical states in this context and their use is further analyzed in. historically the first author to consider the treatment of spherically - symmetric gravity within the ashtekar formalism was bengtsson in. there he started to develop the formalism needed to describe spherically - symmetric complex su(2) connections and densitized triads and used it to discuss some classical aspects related to the role of degenerate metrics in the ashtekar formulation and its connection with yang - mills theories. the quantization of spherically - symmetric midisuperspace models written in terms of ashtekar variables was undertaken rather early in the development of lqg, by kastrup and thiemann. at the time the mathematical setting of lqg was at an early stage of development and, hence, the quantization that the authors carried out was not based in the proper type of hilbert space and made use of the old complex formulation. a key simplification is due to the fact that the constraints can be written as expressions at most linear in momenta. an interesting point that kastrup and thiemann discuss in is related to the physically - acceptable range of black hole masses (that somehow define the configuration space of the model, at least in the reduced phase - space formulation) and how this should be taken into account when representing the algebra of basic quantum operators. the (unitary) equivalence of the reduced and dirac quantizations for this system also found and discussed by kucha can be proven once the right ordering is found for the operators representing the constraints of the system. the modern approach to spherical - symmetry reductions in lqg starts with, where bojowald carefully introduces the necessary mathematical background to consider the quantization of spherically - symmetric models. in this paper bojowald constructs the kinematical framework for spherically - symmetric quantum gravity by using the full lqg formalism ; in particular he shows how the states and basic operators (holonomies and fluxes) can be derived from those in lqg. an important result of is the realization of the fact that significant simplifications take place that make these symmetry reductions tractable. as expected they are midway between the full theory and the homogeneous models that have been considered in lqc. a very useful feature of the spherically - symmetric case is the commutativity of the flux variables (thus allowing for a flux representation in addition to the connection representation that can be used as in the case of homogeneous models). this work particularizes the general framework developed in to study the quantum symmetry reduction of diff - invariant theories of connections based on the isolation of suitable symmetric states in the full 3 + 1 dimensional theory and the subsequent restriction to this subspace (defining quantum symmetry reductions). the study of the volume operator for spherically - symmetric reductions was carried out in, where its basic properties were derived. an important property of the eigenfunctions is that they are not eigenstates of the flux operator (and, in fact, have a complicated dependence on the connection). the fact that on the volume eigenstates the holonomy operators have a complicated dependence makes it quite difficult to study the hamiltonian constraint because it contains commutators of the volume with holonomies. these are difficult to compute because the volume eigenstates are not eigenstates of the triads (upon which the holonomies act in a simple way). nonetheless, an explicit construction of the hamiltonian constraint in the spherically - symmetric case that makes use of non - standard variables that mix the connection and the extrinsic curvature has been given in. these new variables can not be generalized, but are especially tailored for the spherically - symmetric case. the main consequence of using them is the simplification of the volume operator that they provide. notice, however, that the hard problem of finding the kernel of the hamiltonian constraint still has to be solved. an obvious application of the formalism developed in these papers is the study of singularity resolution for schwarzschild black holes in lqg. as shown in the structure of the quantized hamiltonian constraint for spherical - symmetry reductions may allow us to understand the disappearance of spacelike singularities. this issue has also been considered for other spherically - symmetric models, such as the lematre - tolman - bondi collapse of an inhomogeneous dust cloud. the (approximate) numerical analysis carried out in this paper shows a slow - down of the collapse and suggests that the curvature of naked singularities formed in gravitational collapse can be weakened by effects. this is in agreement with the behavior expected in lqg, where effective repulsive forces of a quantum origin usually make the singularities tamer. the main problem of this spherically - symmetric approach followed by bojowald and collaborators as emphasized by campiglia, gambini and pullin in is related to the difficulties in finding a particularization of the construction proposed by thiemann for the hamiltonian constraint with the appropriate constraint algebra in the diff - invariant space of states. this has led the authors of to explore a different approach to the quantization of spherically - symmetric models in lqg [54, 55, 92, 93, 94 ] based on a partial gauge fixing of the diffeomorphism invariance. the quantization of the exterior schwarzschild geometry has been carried out in where the asymptotic behavior of the fields relevant in this case was carefully considered. this corrected a problem in related to the fall - off of some connection components. the gauge fixing is performed in such a way that the gauss law is kept so that the reduced system has two sets of constraints the gauss law and the hamiltonian constraint with a non - trivial gauge algebra. two approaches are then explored, in the first of them the standard dirac method is used after abelianizing the constraints. the second is inspired by the fact that generically one does not expect this abelianization to be possible. this leads the authors to use uniform discretizations [82, 83 ] to deal with the general, non - abelianized constraints. although the study of the exterior region of a black hole gives no information about singularity resolution, according to the authors there are hints of singularity removal because the discrete equations of the model are similar to those appearing in lqc. the result of the lqg quantization of the exterior of a spherically - symmetric black hole is in agreement with the one obtained by kucha in terms of the usual geometrodynamical variables ; in particular, the number and interpretation of the quantum degrees of freedom (the mass of the spacetime) are the same in both approaches. this means that the quantum dynamics are trivial : wave functions depend only on the mass and do not evolve. after studying the exterior problem, the interior problem for a schwarzschild black hole is considered by these authors in (in a minisuperspace model similar to the one by ashtekar and bojowald). here a suitable gauge fixing leads to a description in terms of a kantowski - sachs metric. in this case it is possible to describe the exact quantum evolution as a semi - classical one with quantum corrections. the model is quantized in the connection representation and behaves as a lqc model, where a certain type of bounce replaces the cosmological singularity. when the quantum solution evolves past the singularity it approaches another regime that behaves again in an approximate classical way. a key ingredient here is the choice of a gauge fixing such that the radial component of the triad is a function selected in such a way that in the limit when the polymerization parameter goes to zero one recovers the schwarzschild metric in kruskal coordinates. the authors give a classical metric that represents some of the features of the semiclassical limit for this spherical black - hole system, in which the singularity is effectively avoided. the suggested picture consists of an eternal black hole continued to another spacetime region with a cauchy horizon. far away from the singularity, the issue of the residual diff - invariance in spherically - symmetric models quantized with lqg techniques is discussed in. it is shown there that it is possible to reconstruct spacetime diffeomorphisms in terms of evolving constants of motion (dirac observables on the physical hilbert space), but some memory of the polymerization introduced by loop variables remains because only a subset of spacetime diffeomorphisms are effectively implemented. according to gambini and pullin this is a reflection of the fact that sub - planck scales should behave differently from the macroscopic ones. finally, it is worth pointing out that the system consisting of spherical gravity coupled to a massless scalar field has been discussed in. the same system was also considered by husain and winkler in in the context of geometrodynamical variables. in the authors use the uniform discretization technique to deal with the thorny problem of working with a lie algebra of constraints with structure functions. specifically they consider a discrete version of the master constraint and it is important to understand that one should find the kernel of this master constraint. the fact that this is not achieved in the present model is interpreted by the authors as a hint that there is no quantum continuum limit. nevertheless the theory provides a good approximation for gr for small values of the lattice separation introduced in the discretization. the lowest eigenvalue of the master constraint corresponds to a state with a natural physical interpretation, i.e., the tensor product of the fock vacuum for the scalar field and a gaussian state centered around a flat spacetime for the gravitational part. the authors argue that it is impossible that lqg regulates the short distance behavior of this model in the gauge that they use. this leads them to conclude that one should face the challenging problem of quantization without gauge fixing (keeping the diffeomorphism and hamiltonian constraints). to end this section we want to mention the paper by husain and winkler that considers the quantization of the spherically - symmetric gravity plus massless scalar fields after a gauge fixing that reduces the theory to a model with a single constraint generating radial diffeomorphisms. though they do not work in the framework of lqg proper, they employ a type of polymeric quantization somehow inspired in lqg. in this quantization there are operators corresponding to the curvature that can be used to discuss issues related to singularity resolution (at the dynamical level). from a technical point of view an interesting detail in this work is the use of painlev - gullstrand coordinates that avoid the necessity to consider the interior and exterior problems separately. as we have seen, the quantization of midisuperspace models has been and still is a very useful test bed to understand many different methods and techniques proposed for the quantization of gr. in this respect the models described in this living review will certainly be of use in the near future, in particular to explore new avenues to the quantization of gravity both in the geometro - dynamical approach and in lqg. these models have also been used with some success to extract qualitative physical results that are expected to be present in full quantum gravity (black hole mass quantization, hawking radiation, unitarity of quantum evolution, microcausality, singularity resolution, semiclassical limit,). certainly a complete full quantization of the symmetry reductions described here will shed light on many methods and approaches and may be useful to arrive at a fully functioning theory of quantum gravity. among the open problems that we think should be considered we provide the following list : from a purely mathematical point of view it would be interesting to study how midisuperspace models sit inside full superspace (both in metric and connection variables) in the spirit of analysis carried out by jantzen for minisuperspaces.advance in the study of one - killing reductions (or equivalently 2 + 1 gravity coupled to matter fields). it is important to find out if these systems can be treated perturbatively after carefully introducing the necessary boundary terms in the action. from the non - perturbative point of view it is necessary to obtain the concrete form of the bounded hamiltonians for open spatial topologies and understand their physical implications.give a complete and unified hamiltonian treatment of all the midisuperspaces that admit a general two - dimensional spatial isometry group, generalizing in this way the two - killing vector reductions of gr considered so far. special attention should be paid to the analysis of non - compact spatial topologies and general polarization.obtain physical predictions from these models.it is important to attempt the quantization of two - killing vector reductions coupled to different types of matter fields beyond massless scalars. though there are some papers dealing with scalar and electromagnetic fields, there is still a lot of work to do. it must be said, however, that it may actually be very difficult to get exact solutions to them.using the non - perturbative quantizations provided by korotkin, nicolai and samtleben to obtain physical predictions and check if expected quantum gravitational phenomena do actually occur. among these another interesting issue may be the coupling of matter fields in particular massless scalars. if they can be described by using fock spaces, their particle - like quanta might be used as quantum test particles to explore quantum geometry and the emergence of a classical spacetime.it would be interesting to provide consistent and complete quantizations inspired in lqg for einstein - rosen waves and gowdy models for which the available results are rather incomplete. in particular, it is important go beyond the current hybrid formulations to purely polymeric ones.complete the quantization of gravitational plane waves.advance in the geometrodynamical quantization of spherically - symmetric gravitational systems coupled to matter. in particular, in those situations where the reduced phase spaces are infinite dimensional. it would be desirable to reach a level of mathematical rigor on par with the one customary within lqg.complete the program started by gambini, pullin and collaborators to understand spherically - symmetric gravitational systems (with and without matter) in lqg.answer the following questions in the midisuperspace setting : does lqg predict the mass quantization of black holes ? is it possible to describe hawking radiation in this framework?understand the fate of classical singularities both cosmological and in black holes. in the case of black holes. one should understand the possible relationship between both approaches. from a purely mathematical point of view it would be interesting to study how midisuperspace models sit inside full superspace (both in metric and connection variables) in the spirit of analysis carried out by jantzen for minisuperspaces. advance in the study of one - killing reductions (or equivalently 2 + 1 gravity coupled to matter fields). it is important to find out if these systems can be treated perturbatively after carefully introducing the necessary boundary terms in the action. from the non - perturbative point of view it is necessary to obtain the concrete form of the bounded hamiltonians for open spatial topologies and understand their physical implications. give a complete and unified hamiltonian treatment of all the midisuperspaces that admit a general two - dimensional spatial isometry group, generalizing in this way the two - killing vector reductions of gr considered so far. special attention should be paid to the analysis of non - compact spatial topologies and general polarization. it is important to attempt the quantization of two - killing vector reductions coupled to different types of matter fields beyond massless scalars. though there are some papers dealing with scalar and electromagnetic fields, there is still a lot of work to do. it must be said, however, that it may actually be very difficult to get exact solutions to them. using the non - perturbative quantizations provided by korotkin, nicolai and samtleben to obtain physical predictions and check another interesting issue may be the coupling of matter fields in particular massless scalars. if they can be described by using fock spaces, their particle - like quanta might be used as quantum test particles to explore quantum geometry and the emergence of a classical spacetime. it would be interesting to provide consistent and complete quantizations inspired in lqg for einstein - rosen waves and gowdy models for which the available results are rather incomplete. in particular, it is important go beyond the current hybrid formulations to purely polymeric ones. in particular, in those situations where the reduced phase spaces are infinite dimensional. it would be desirable to reach a level of mathematical rigor on par with the one customary within lqg. complete the program started by gambini, pullin and collaborators to understand spherically - symmetric gravitational systems (with and without matter) in lqg. answer the following questions in the midisuperspace setting : does lqg predict the mass quantization of black holes ? understand the fate of classical singularities both cosmological and in black holes. in the case of black holes we expect that a lot of progress on these issues will happen in the near future | we give a comprehensive review of the quantization of midisuperspace models. though the main focus of the paper is on quantum aspects, we also provide an introduction to several classical points related to the definition of these models. we cover some important issues, in particular, the use of the principle of symmetric criticality as a very useful tool to obtain the required hamiltonian formulations. two main types of reductions are discussed : those involving metrics with two killing vector fields and spherically - symmetric models. we also review the more general models obtained by coupling matter fields to these systems. throughout the paper we give separate discussions for standard quantizations using geometrodynamical variables and those relying on loop - quantum - gravity - inspired methods. |
sensory processing is the ability to receive, organize, and interpret sensory stimuli, including, but not limited to, oral, visual, vestibular, and auditory experiences.1 one of the best tools to evaluate the sensory symptoms in young children is the infant / toddler sensory profile (itsp).1 it is a judgment - based caregiver questionnaire that characterizes a child s behaviors and performance in relation to sensory processing. it provides a standardized method to measure a child s sensory processing abilities and to profile the effect of sensory processing on functional performance in the daily life of a child aged 036 months.1 the itsp was created to extend the age range of the original instrument, the sensory profile.2 the itsp standardization process took place between 2000 and 2001 and evaluated > 1,100 infants and toddlers with and without disabilities between birth and 36 months of age. the theoretical and conceptual features of the itsp are based on dunn s model of sensory processing. neurological threshold refers to the amount of stimuli required for a neuron or neuron system to respond. it is a continuum, with very high thresholds on one end and very low thresholds on the other end. the balance between these two extreme states is adjusted through modulation, which refers to the brain s regulation of neural messages by facilitating or inhibiting responses. when modulation is intact, the nervous system responds to some stimuli while ignoring other stimuli, and the child generates an appropriate adaptive response to the situation.1 poor sensory processing can take the form of over - responsivity or lack of responsivity. behavioral response / self - regulation refers to the way children act relative to their thresholds. passive responses to threshold indicate that the child behaves consistently with neurological threshold ; however, active responses to thresholds indicate that the child behaves more actively and works against thresholds.1 although difficulties with sensory processing have been associated with autism since it was first defined as a diagnosis,3 there has been a long - lasting debate whether sensory symptoms are a component of core autistic deficits or a co - morbid phenomenon.4,5 two theories of sensory dysfunction in autism have been suggested : (1) over - arousal and (2) under - arousal theory. according to a review by rogers and ozonoff,6 there is more evidence that children with autism are hypo - responsive to sensory stimuli ; however, there is very little support for hyper - arousal and failure of habituation in autism. sensory symptoms are often evaluated in the diagnosis of autism and are scored in gold - standard autism diagnostic measures. sensory symptoms are more frequent and prominent in children with autism than in typically developing children. leekam showed that > 90% of children with autism had sensory abnormalities and had sensory symptoms in multiple sensory domains. sensory abnormalities were pervasive, multimodal, and persistent across age and ability in children and adults with autism. certain groups, including children with fragile x syndrome or those who are deaf blind, seem to demonstrate higher rates of sensory symptoms than children with autism,6 even though fragile x syndrome might be one of the many etiologies behind autism. the development of parent - caregiver - administered, standardized norm - referenced sensory questionnaires has allowed quantification of these behaviors relative to age norms. findings from standardized questionnaires show that 45%95% of individuals with autism spectrum disorders (asd) present with high frequencies of sensory behaviors that are > 1 standard deviation (sd) away from norms.810 a meta - analysis of 14 studies by ben - sasson indicated a significant difference between asd and typical groups relative to the presence and frequency of sensory symptoms, with the greatest difference in 1) under - responsivity, followed by 2) over - responsivity, and 3) sensation seeking. sensory differences were greatest in studies of children aged 69 years, samples with an autism diagnosis in > 80% of cases, and compared to a chronological age - matched group versus mental age / developmental delay - matched group. asd prevalence in the general pediatric population is currently believed to be in the range of 1%1.5%.11,12 however, there are populations with increased risk for asd. recent studies on the prevalence of asd in prematurely born children have indicated high prevalences of asd, ranging between 3.65% and 12.9%.1317 there is a challenging question, whether different performance on the sensory profile could be used to indicate which prematurely born infants are at greatest risk of developing asd in the future. as for sensory symptoms and prematurity, dunn1 described some significant differences in the data for children aged 06 months born prior to 38 weeks of gestation. these babies tended to be hyper - responsive to stimuli during the early months of development. however, dunn found no significant differences in the data for children aged 736 months based on the time they were born in relation to their gestation. dunn hypothesized that the effects of prematurity fade as the child gains more experience with sensation. on the other hand, several studies have published data about atypical sensory profile scores in the population of preterm infants. atypical sensation seeking18 and atypical oral and auditory sensory processing19 have been reported in late preterm infants (gestational age between 34 and 35 weeks). wickremasinghe reported that ~40% of sensory profiles have been found to be atypical among children born very prematurely (32 weeks of gestation). eeles compared the sensory profiles of very preterm children born before 30 weeks of gestation with the profiles of term - born controls at 2 years of age. they found that very preterm children had scores consistent with stronger patterns on sensory profiles compared with term - born children ; an association with male sex, higher social risk, longer hospital stays, and moderate to severe white matter abnormalities were also described. recently, rahkonen described that atypical sensory processing in very preterm children was common, and children with neonatal neuroanatomical lesions tended to present specific behavioral responses to sensory stimuli. however, none of the studies was focused on the simultaneous presence of sensory symptoms and autism in a population of prematurely born children. it is striking that preterm children may have both a risk of atypical sensory behaviors as well as increased rates of asd. it is possible that atypical sensory behaviors in very low and extremely low birth weight children may result in false - positive asd diagnoses.22 on the other hand, atypical sensory behaviors might be important components of the asd phenotype, at least in some preterm infants. our team was the first to use a standardized norm - referenced sensory parent questionnaire as an autism - specific screening tool in prematurely born children. the other screening instruments used in this study were broadband screens, the modified checklist for autism in toddlers (m - chat), and the communication and symbolic behavior scales developmental profile infant - toddler checklist (csbs - dp - itc). the first article was based on preliminary data and involved first 101 examined preterm children.16 the second article comprised the final sample of 157 preterm children and was focused on asd prevalence in preterm children, as well as on specific psychometric values of the three tests.17 our assessment of the screening potential of the itsp led to skepticism. the itsp was significantly inferior to the csbs - dp - itc (14.3% versus 27.1% of positive screens, p=0.022) and nonsignificantly inferior to the m - chat (14.3% versus 17.8%, p=0.522) in our study.17 although the present study is based on the same final data set, its aim and methods are completely different. the objective of the present study was to determine whether there is enough evidence to recommend the use of itsp or some of its subscales as an additional tool for the detection of autism for use in combination with other screening instruments. although our previous papers16,17 dealt with the traditional comparison of the screening tests by means of standard statistical methods, the present study is mainly based on innovative mathematical methods to achieve a more complex model of autism screening. preterm children with birth weights + 1 sd represent less frequent atypical responses than others because itsp never gives the highest points. the itsp is not generally used for screening ; therefore, we established a new criterion for a positive screening. participants were considered to have screened positive if results were outside 2 sd of population norms (definite difference) on at least two scores involving the section and/or quadrant scores. the parents of prematurely born children with very low birth weights (vlbw + 1 sd represent less frequent atypical responses than others because itsp never gives the highest points. the itsp is not generally used for screening ; therefore, we established a new criterion for a positive screening. participants were considered to have screened positive if results were outside 2 sd of population norms (definite difference) on at least two scores involving the section and/or quadrant scores. the parents of prematurely born children with very low birth weights (vlbw < 1,500 g) and extremely low birth weights (elbw < 1,000 g) (2 years of age, corrected for prematurity) completed the screening battery questionnaires and sent them to the department of child psychiatry at motol university hospital. all the children who were screened positive on any of the screening tools were subsequently invited for a detailed follow - up assessment. the assessment involved testing using the autism diagnostic observation schedule (ados)23 and a clinical examination by two experienced child psychiatrists with expertise in autism. the concept of the best estimate clinical diagnosis (bed) was used as the gold standard.24 in cases of disagreement between the ados diagnosis and best estimate clinical diagnosis, the latter was preferred. the international classification of diseases, tenth edition (icd-10) provided the criteria for the clinical diagnoses.25 statistical analysis was performed using the statistical package for the social sciences (spss, version 22.0 ; ibm corporation, armonk, ny, usa) and r (r core team, 2015).26 descriptive statistics for the samples were used. the cochran s q test was used for analyzing differences in positive screening results among tests. more detailed pair comparisons between tests were performed using the paired wilcoxon sign test false discovery rate adjusted for multiple testing. classification trees2729 were used as a basic analysis tool to identify promising scales or subscales of the three diagnostic instruments (m - chat, csbs - dp - itc, and itsp). classification trees are a mathematical tool used to partition a set of objects into subsets (classes) based on the values of a nominal or ordinal discrete variable (eg, correct diagnoses). the classification is performed based on the values of one or more features of the objects (or values of specific scales / diagnostic tools). from the diagnostic point of view, the variable defining the classes can be binary (ie, a positive or negative diagnosis) and the characteristic features can be the values / outputs / scores of various diagnostics or screening tools (methods) this is the case of binary classification trees. binary classification - tree models employed in the analysis presented in this paper hierarchically select features that best distinguish between two classes (ie, positive diagnosis [p ] and negative diagnosis [n ]) and specify the threshold values of these features (the classification tree is then split at the given node into sub - branches based on the value of these thresholds or cutoffs and appropriate classes are assigned to the sub - branches). this is the established system behind many diagnostic methods used in psychology and medicine to identify those features that provide the best discrimination power (eg, between people who should be assigned a diagnosis and people who should not) and to specify the cutoff value(s) of the identifying feature ; that is, the values that are characteristic for the given class (diagnosis). in this sense, classification trees can be seen as a tool for the selection of appropriate cutoffs of given scales (features) and as a tool that is not dependent on specific standardizations of the measurement instruments. for those diagnostic (or screening) tools that deal with raw scores or their linear transformation, the classification tree can itself be considered a standardization procedure (if the sample is representative of the given population). classification trees also allow for reflection on the severity of false negative (fn) and false positive (fp) errors. the selection of features for classification is done step by step based on the minimization of the cost function, reflecting the relative severity of fn - type and fp - type errors sometimes called the impurity, which is a weighted sum of fn and fp. in the first step, the feature that provides the largest reduction of impurity is identified as the root node of the tree structure representing the classification process ; at that node, the set of data to be classified is split into two disjointed subsets with respect to the threshold value for which the impurity of classification, based solely on the root node feature, is minimal. two branches of the classification tree are thus defined each representing a different class and the features representing their end nodes (leaves) are identified analogically. the process of splitting nodes (creating branches) stops when zero impurity is reached (ie, all the data instances in the given branch are correctly classified) or no reduction of impurity is possible. a classification tree obtained this way is a representation of the classification process. as such it is a description of how to assign a class to each data instance based on the values of the selected features (figure 1 shows our proposed classification tree). to avoid overfitting, that is, to make the resulting classification tree more robust, we prune the resulting classification trees so that relatively few levels or decision nodes remain (during the actual analysis of the data, we identified two levels or a maximum of three decision nodes as a reasonable level of pruning). the resulting classifier is then examined by the leave - one - out cross - validation procedure to assess its robustness in more detail.27,30 the objective of this study was to determine whether itsp (or some of its subscales) can be combined with other screening tools (eg, the m - chat, csbs - dp - itc, or its subscales) into an effective asd screening tool that could better discriminate between autistic and nonautistic cases. in order to address this, we applied classification trees to the sets of available data (ie, variables / criteria) and overall results or subscales of the itsp, m - chat, and csbs - dp - itc, which consisted of : the overall scores for the m - chat and csbs - dp - itc (raw - scores) two featurestwo separate raw scores from the m - chat (score for critical questions and score for overall questions) two featuresthe raw scores of the subscales of the csbs - dp - itc (social composite, speech composite, and symbolic composite) three featuresthe scores from the itsp subscales (auditory processing, visual processing, tactile processing, vestibular processing, oral sensory processing, low registration, sensation seeking, sensory sensitivity, and sensation avoiding) nine features. the overall scores for the m - chat and csbs - dp - itc (raw - scores) two features two separate raw scores from the m - chat (score for critical questions and score for overall questions) two features the raw scores of the subscales of the csbs - dp - itc (social composite, speech composite, and symbolic composite) three features the scores from the itsp subscales (auditory processing, visual processing, tactile processing, vestibular processing, oral sensory processing, low registration, sensation seeking, sensory sensitivity, and sensation avoiding) nine features. in this case, in accordance with the diagnostic manual, the raw scores of the scales were transformed into z - scores. the screening results obtained from each tool separately three features.the aggregated results of pairs of screening tools (a screening was considered positive if at least one of the tools from the pair was positive) three features.the overall result of the screening : a combination of all the three screening tools ; the m - chat, csbs - dp - itc, and itsp used together (screening was considered positive if any of the three methods was positive) one feature. the aggregated results of pairs of screening tools (a screening was considered positive if at least one of the tools from the pair was positive) three features. the overall result of the screening : a combination of all the three screening tools ; the m - chat, csbs - dp - itc, and itsp used together (screening was considered positive if any of the three methods was positive) one feature. for the itsp, we converted raw scores to z - scores (since the diagnostic manual1 suggests the use of z - scores). however, we did not use the absolute values of the distance from the mean, in terms of standard deviation multiples, that is, the absolute values of z - scores as applied in the screening and suggested in the itsp diagnostic manual, since these values assume symmetry in the diagnostic power of positive and negative deviations. therefore, we worked with z - scores of the scales (ie, both positive and negative values) to allow for possible asymmetry. this is a more general approach and does not violate the reasoning used in the construction of the method and the cutoff scores. in total, 23 features were considered. based on the purpose of the screening, we considered misclassification costs of fn - type errors to be higher than the misclassification costs of fp - type errors. the optimal resulting screening tool (classifier) was one that had an fn as close to 0 as possible, while at the same time having a reasonably low fp count. having selected these 23 features, we obtained a data set of 131 cases (for description of excluded cases, see methods, subsection sample). best estimate clinical diagnosis made by two experienced child psychiatrists with expertise in autism (the concept is discussed in the methods section). the classification trees were pruned to two levels (with a maximum of three decision nodes), and subsequently the leave - one - out cross - validation procedure was applied to the pruned tree (leaving only the selected features corresponding to the decision nodes in the pruned tree). the following characteristics of the classifier were computed both for the classifier and for its cross - validation : fn count, fp count, tn count, tp count, accuracy, sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv), false - positive rate (frp), false - negative rate (fnr), f measure, area under the roc curve (auroc), and cohen s kappa coefficient all under their standard definitions.31 applying the classification tree method to the 23 features, we identified the classification tree presented in figure 1 as the best classifier that can be constructed and remain sufficiently robust. note, that it is possible to achieve fn = 0 (npv = 1 and sensitivity = 1) while the count of fp = 18 is reasonably low. using the csbs - dp - itc overall score and the sensation seeking subscale of the itsp, we obtain a screening tool that could identify all the children with asd in our sample. since the tree is pruned to just two decision nodes, the risk of overfitting is low. the robustness of the proposed classifier (screening tool) is demonstrated by the results of the leave - one - out cross - validation. this validation technique was applied to assess how the results would generalize to an independent data set. the main purpose of using the leave - one - out cross - validation was to see how accurately the classification model would perform in practice (also for independent samples outside the current data set). the basic idea was to separate data into a training data set (used to establish our model) and a testing data set (which is then used to validate the model) the classifiers built into the cross - validation never use the whole data set for fitting. the cross - validation in this case resulted in same identical characteristics as the original classifier. this can be interpreted to mean that no single data instance carries crucial information for its classification. the proposed screening tool (see figure 1) suggests the following reasoning for screening : if the overall value of the csbs - dp - itc is < 45.5, then the screening is positive.if the overall value of the csbs - dp - itc is 45.5 and the z - score for the sensation seeking subscale of the itsp is 1.54, then the screening is positive.otherwise, the screening is negative. if the overall value of the csbs - dp - itc is < 45.5, then the screening is positive. if the overall value of the csbs - dp - itc is 45.5 and the z - score for the sensation seeking subscale of the itsp is 1.54, then the screening is positive. note that csbs - dp - itc is positive when its overall raw score (denoted as itc sum in this paper) is 42. the classification tree depicted in figure 1 suggests a threshold for a positive outcome at 45.5. since this value is higher than the value in the diagnostics manual for the csbs - dp - itc, every child screened positive with respect to the csbs - dp - itc would also be classified as positive according to the proposed classifier (ie, a screening based on the proposed classification tree would be positive). (since fn errors are a more significant problem, our screening might identify more children as positive, but it can never identify fewer tp than the csbs - dp - itc with a threshold of 42). according to the itsp diagnostics manual for the given age of children, the raw scores outside the interval (mean 2 sd, mean + 2 sd) are considered to be definitely different from the norm (the corresponding interval in z - scores is [2, 2 ]). our classifier identifies the z - score cutoff at 1.54, values above this threshold indicate positive screening results for asd. above the mean value, this threshold is again more conservative in the same sense as in the csbs - dp - itc discussed above. note that negative deviations from the mean (over - responsiveness according to the itsp) are not considered to be significant for screening purposes. figure 2 provides a clear illustration that negative sample values of z - scores have low classification power. this is a clear argument for the use of z - scores over their absolute values in connection with the itsp in this case. the effect of the screening rules 1, 2, and 3 are summarized in figure 2, where the values of overall csbs - dp - itc score are plotted against itsp sensation seeking (sd) values. our work has allowed us to identify two features that can be used in the screening for asd in prematurely born children. figure 2 clearly illustrates that the itsp sensation seeking (sd) values provide valuable screening information. the data confirm (figure 2) that the decision not to deal with the absolute values of z - scores regarding itsp was reasonable (ie, values of itsp sensation seeking [sd ] greater than the mean value provide the necessary additional information for classification). additionally, if z - score values < 1.54 were considered to be representative of asd, then the fp count would increase without any improvement in the performance of the suggested screening tool. we should also note that from the clinical perspective, four measures are being considered in the proposed classifier the itsp sensation seeking scale, and the three subscales of csbs - dp - itc : social composite, speech composite, and symbolic composite (since the sum of their scores constitutes the overall csbs - dp - itc score). the objective of this study was to determine whether itsp (or some of its subscales) can be combined with other screening tools (eg, the m - chat, csbs - dp - itc, or its subscales) into an effective asd screening tool that could better discriminate between autistic and nonautistic cases. in order to address this, we applied classification trees to the sets of available data (ie, variables / criteria) and overall results or subscales of the itsp, m - chat, and csbs - dp - itc, which consisted of : the overall scores for the m - chat and csbs - dp - itc (raw - scores) two featurestwo separate raw scores from the m - chat (score for critical questions and score for overall questions) two featuresthe raw scores of the subscales of the csbs - dp - itc (social composite, speech composite, and symbolic composite) three featuresthe scores from the itsp subscales (auditory processing, visual processing, tactile processing, vestibular processing, oral sensory processing, low registration, sensation seeking, sensory sensitivity, and sensation avoiding) nine features. the overall scores for the m - chat and csbs - dp - itc (raw - scores) two features two separate raw scores from the m - chat (score for critical questions and score for overall questions) two features the raw scores of the subscales of the csbs - dp - itc (social composite, speech composite, and symbolic composite) three features the scores from the itsp subscales (auditory processing, visual processing, tactile processing, vestibular processing, oral sensory processing, low registration, sensation seeking, sensory sensitivity, and sensation avoiding) nine features. in this case, in accordance with the diagnostic manual, the raw scores of the scales were transformed into z - scores. the screening results obtained from each tool separately three features.the aggregated results of pairs of screening tools (a screening was considered positive if at least one of the tools from the pair was positive) three features.the overall result of the screening : a combination of all the three screening tools ; the m - chat, csbs - dp - itc, and itsp used together (screening was considered positive if any of the three methods was positive) one feature. the aggregated results of pairs of screening tools (a screening was considered positive if at least one of the tools from the pair was positive) three features. the overall result of the screening : a combination of all the three screening tools ; the m - chat, csbs - dp - itc, and itsp used together (screening was considered positive if any of the three methods was positive) one feature. for the itsp, we converted raw scores to z - scores (since the diagnostic manual1 suggests the use of z - scores). however, we did not use the absolute values of the distance from the mean, in terms of standard deviation multiples, that is, the absolute values of z - scores as applied in the screening and suggested in the itsp diagnostic manual, since these values assume symmetry in the diagnostic power of positive and negative deviations. therefore, we worked with z - scores of the scales (ie, both positive and negative values) to allow for possible asymmetry. this is a more general approach and does not violate the reasoning used in the construction of the method and the cutoff scores. in total, 23 features were considered. based on the purpose of the screening, we considered misclassification costs of fn - type errors to be higher than the misclassification costs of fp - type errors. the optimal resulting screening tool (classifier) was one that had an fn as close to 0 as possible, while at the same time having a reasonably low fp count. having selected these 23 features, we obtained a data set of 131 cases (for description of excluded cases, see methods, subsection sample). best estimate clinical diagnosis made by two experienced child psychiatrists with expertise in autism (the concept is discussed in the methods section). the classification trees were pruned to two levels (with a maximum of three decision nodes), and subsequently the leave - one - out cross - validation procedure was applied to the pruned tree (leaving only the selected features corresponding to the decision nodes in the pruned tree). the following characteristics of the classifier were computed both for the classifier and for its cross - validation : fn count, fp count, tn count, tp count, accuracy, sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv), false - positive rate (frp), false - negative rate (fnr), f measure, area under the roc curve (auroc), and cohen s kappa coefficient all under their standard definitions.31 applying the classification tree method to the 23 features, we identified the classification tree presented in figure 1 as the best classifier that can be constructed and remain sufficiently robust. note, that it is possible to achieve fn = 0 (npv = 1 and sensitivity = 1) while the count of fp = 18 is reasonably low. using the csbs - dp - itc overall score and the sensation seeking subscale of the itsp, we obtain a screening tool that could identify all the children with asd in our sample. since the tree is pruned to just two decision nodes, the risk of overfitting is low. the robustness of the proposed classifier (screening tool) is demonstrated by the results of the leave - one - out cross - validation. this validation technique was applied to assess how the results would generalize to an independent data set. the main purpose of using the leave - one - out cross - validation was to see how accurately the classification model would perform in practice (also for independent samples outside the current data set). the basic idea was to separate data into a training data set (used to establish our model) and a testing data set (which is then used to validate the model) the classifiers built into the cross - validation never use the whole data set for fitting. the cross - validation in this case resulted in same identical characteristics as the original classifier. this can be interpreted to mean that no single data instance carries crucial information for its classification. the proposed screening tool (see figure 1) suggests the following reasoning for screening : if the overall value of the csbs - dp - itc is < 45.5, then the screening is positive.if the overall value of the csbs - dp - itc is 45.5 and the z - score for the sensation seeking subscale of the itsp is 1.54, then the screening is positive.otherwise, the screening is negative. if the overall value of the csbs - dp - itc is < 45.5, then the screening is positive. if the overall value of the csbs - dp - itc is 45.5 and the z - score for the sensation seeking subscale of the itsp is 1.54, then the screening is positive. note that csbs - dp - itc is positive when its overall raw score (denoted as itc sum in this paper) is 42. the classification tree depicted in figure 1 suggests a threshold for a positive outcome at 45.5. since this value is higher than the value in the diagnostics manual for the csbs - dp - itc, every child screened positive with respect to the csbs - dp - itc would also be classified as positive according to the proposed classifier (ie, a screening based on the proposed classification tree would be positive). therefore, the screening tool proposed in this paper is more conservative (since fn errors are a more significant problem, our screening might identify more children as positive, but it can never identify fewer tp than the csbs - dp - itc with a threshold of 42). according to the itsp diagnostics manual for the given age of children, the raw scores outside the interval (mean 2 sd, mean + 2 sd) are considered to be definitely different from the norm (the corresponding interval in z - scores is [2, 2 ]). our classifier identifies the z - score cutoff at 1.54, values above this threshold indicate positive screening results for asd. above the mean value, this threshold is again more conservative in the same sense as in the csbs - dp - itc discussed above. note that negative deviations from the mean (over - responsiveness according to the itsp) are not considered to be significant for screening purposes. figure 2 provides a clear illustration that negative sample values of z - scores have low classification power. this is a clear argument for the use of z - scores over their absolute values in connection with the itsp in this case. the effect of the screening rules 1, 2, and 3 are summarized in figure 2, where the values of overall csbs - dp - itc score are plotted against itsp sensation seeking (sd) values. our work has allowed us to identify two features that can be used in the screening for asd in prematurely born children. figure 2 clearly illustrates that the itsp sensation seeking (sd) values provide valuable screening information. the data confirm (figure 2) that the decision not to deal with the absolute values of z - scores regarding itsp was reasonable (ie, values of itsp sensation seeking [sd ] greater than the mean value provide the necessary additional information for classification). additionally, if z - score values < 1.54 were considered to be representative of asd, then the fp count would increase without any improvement in the performance of the suggested screening tool. we should also note that from the clinical perspective, four measures are being considered in the proposed classifier the itsp sensation seeking scale, and the three subscales of csbs - dp - itc : social composite, speech composite, and symbolic composite (since the sum of their scores constitutes the overall csbs - dp - itc score). preterm children are at increased risk of both atypical sensory behaviors1922 and asd.1317 the role of sensory abnormalities in the higher prevalence rate of asd in the preterm population is not thoroughly understood. it has been stated that for autism, in general, most evidence supports the hypo- responsiveness to sensory stimuli, while theories about hyper - arousal and failure of habituation lack significant support.6 on top of that, children with asd differ significantly from typical groups regarding the presence and frequency of mainly under - responsivity, followed by over - responsivity, and sensation seeking.4 on the other hand, atypical sensory profile scores have been described in preterm infants,1922 without agreement on the typical sensory profile pattern for this group. some studies of preterm infants included information about special neurological abnormalities, which might be the root of abnormal sensory symptoms. we infer that abnormal sensory processing might be an important component of the diagnosis of asd in at least some preterm children ; however, in other children, these symptoms might lead to false - positive screening results for asd, which has already been suggested by wickremasinghe and rahkonen.22 the strength of our screening study is that all toddlers with positive screening results on any of the screening tools were invited for the detailed diagnostic assessment, during which an asd diagnoses was confirmed or excluded. our assessment of the screening potential of the itsp, performed also on preterm population, led to skepticism.17 however, we still foresee a possible role for the itsp, or some of its subscales, when used as an adjunct with other screening instruments, to identify some cases of asd in the preterm population. this assumption is based on previous research that found abnormalities in sensory behaviors in 45%95% of individuals with asd;710 however, in general, evaluation of sensory symptoms is underrepresented in autism screening tools. in our data analysis, we used classification trees to examine all possible combinations of overall results as well as results from subscales from three screening tools (23 features in total) to find the optimal screening result of our sample. based on clinical implications, an optimal screening result was one with an fnr close to 0, and with the false positive rate also being reasonably low. on the basis of this approach, we identified that the optimal positive screening result is achieved when the overall csbs - dp - itc value was < 45.5, or when the overall csbs - dp - itc value was 45.5 and the sensation seeking subscale of the itsp was 1.54. this finding was in line with our theoretical assumption that abnormal sensory processing might help identify some preterm children with asd. the positive overall csbs - dp - itc score used in our study (45.5) was greater than the positive overall score recommended by the authors less than or equal to 42,34 therefore our screening might identify more children with asd. the importance of the sensation seeking subscale, however, is surprising, given that the subscale value (1.54) means that the child engages in sensation seeking behavior less frequently than others. ermer and dunn35 found that a lower incidence of sensation seeking behavior was one of the main characteristics that differentiated children with asd from both typically developing children and children with adhd and suggested that since the sensory profile was able to discriminate between the studied groups, the measure might be useful during the screening process for autism, pdd, or adhd. in contrast, dunn1 did not find a difference in sensation seeking scores for toddlers with pdd and typically developing children, when using the itsp. on top of that, the higher incidence of sensation seeking behaviors in children with asd compared with typically developing children was described by dunn and watling.37 ben - sasson suggested that this inconsistency may relate to differences in the age range, which ranged from 3 to 15 years. in the study of ben - sasson,4 toddlers with asd were most distinct from typically developing groups in their low frequency of sensation seeking, high frequency of under - responsiveness, and avoidance behaviors. in a meta - analysis by ben - sasson,4 a lower incidence of sensation seeking in studies of 0- to 3-year olds was the only finding of a lower frequency of any sensory seeking behavior in asd cases relative to typical groups. chronological age was found to be the only factor that contributed to the variability in sensation seeking in persons with asd. these behaviors in toddlers with asd may not differ from typically developing toddlers in frequency (as described by the itsp) but rather in quality.38 the low incidence of sensation seeking behavior might be that fortuitous feature that can discriminate between preterm children who will develop asd and those who will not, relative to those with negative screening results on the csbs - dp - itc. the results of our study may encourage future development of screening tests for autism that include questions related to sensation seeking behavior. before making any recommendations regarding clinical practice based on our results, our results on this particular population should be replicated ; although, we think that using screening instruments in preterm children with more than a 10-fold increase in prevalence does not significantly handicap the data, since a lower - order number of probands is needed in order to obtain reliable results.16 the limitation of our study was that a large number of children with positive screens did not undergo clinical evaluation because of their parent s lack of interest (23 of 56 [41.1%]).17 however, drop - outs are a well - known problem in the follow - up studies. in the only follow - up study performed in preterms (besides ours),39 the drop - out rate was 14% even when the authors used telephone follow - ups, which are much more convenient for parents than the clinical evaluation that was used in our study. the utility of itsp alone as a screening tool for autism, initially, did not seem to be very promising ; however, when the sensation seeking subscale of the itsp was used in combination with the csbs - dp - itc, the screening accuracy for autism in preterm children was substantially improved. | objectivethe objective of this study was to explore the potential of the infant / toddler sensory profile (itsp) as a screening tool for autism spectrum disorders (asd) in prematurely born children.methodsparents of 157 children with birth weights < 1,500 g (aged 2 years, corrected for prematurity ; 88 boys, 69 girls) completed a screening battery that included the itsp, modified checklist for autism in toddlers (m - chat), and the communication and symbolic behavior scales developmental profile infant - toddler checklist (csbs - dp - itc). children with known disabilities were excluded. all the children who were screened positive on any of the screening tools subsequently underwent clinical examination including the autism diagnostic observation schedule.resultswe used classification trees to answer the question whether itsp (or some of its subscales) could be combined with the m - chat and/or the csbs - dp - itc or its subscales into an effective asd screening tool. using the csbs - dp - itc, overall score, and the sensation seeking subscale of the itsp, we obtained a screening tool that was able to identify all of the asd children in our sample (confirmed by cross - validation). the proposed screening tool is scored as follows : 1) if the overall csbs - dp - itc value is < 45.5, then the screening is positive ; 2) if the overall csbs - dp - itc value is 45.5 and the z - score of the sensation seeking subscale of itsp is 1.54, then the screening is positive ; 3) otherwise, the screening is negative.conclusionthe use of csbs - dp - itc in combination with the sensation seeking subscale of the itsp improved the accuracy of autism screening in preterm children. |
self - expanding metal stent (sems) have a longer patency than plastic stents and offer adequate palliation in patients with an unresectable malignant distal bile duct obstruction.1 however, semss and plastic stents may cause complications such as stent migration, tumoral or nontumoral reocclusion, stent - induced ulceration, duodenal obstruction, perforation, and upper gastrointestinal bleeding.2 - 5 in particular, covered sems (csems) tend to migrate more frequently than uncovered sems (usems). to resolve these complications, several techniques for sems removal have been described in the literature. sems were successfully removed by using a polypectomy snare, large - mouthed rat tooth forceps, biopsy forceps or an endoscopic suture - cutting device.6 - 9 although removal of these complicated csems is usually successful and easier than removing a usems in most cases, some cases of csems may be difficult to remove using conventional methods.6 - 11 several studies have reported that difficult cases of distally migrated sems can be treated by argon plasma coagulation (apc) trimming. however, there has been no report about endoscopic removal of proximally migrated, trimmed sems during balloon sweeping after apc trimming, which may be difficult to handle using conventional methods. we report an unusual complicated case of proximal migration of a remnant metal stent during balloon sweeping following complete stent trimming by apc, the case which was successfully removed by using a basket and revised endoscopically. a 74-year - old woman was admitted due to jaundice and epigastric pain which lasted for 7 days. laboratory tests revealed the following : white blood cell count, 5.8410/l (reference range, 4.0 to 10.810/l) ; aspartate aminotransferase, 123 iu / l ; alanine aminotransferase, 144 iu / l ; total bilirubin, 5.2 mg / dl ; alkaline phosphatase, 488 iu / l ; amylase, 315 u / l ; lipase, 689 u / l ; carbohydrate antigen 19 - 9, 35.86 u / ml ; and carcinoembryonic antigen, 1.65 ng / ml. an abdominal computed tomography scan showed a ~2.1-cm low - attenuated mass lesion abutting on the superior mesenteric vein with dilatation of the intrahepatic bile duct, common bile duct (cbd) and pancreatic duct (pd), which suggested pancreas neoplasm. subsequent endoscopic retrograde cholangiopancreatography (ercp) showed short segmental distal cbd narrowing and diffuse irregular narrowing of the pd with marked upstream ductal dilatation (fig. 1). after a 5-fr single pigtail 7-cm - long stent was inserted into the pd following brushing cytology and an 8.5-fr tannenbaum, 7-cm - long (cook endoscopy, winston - salem, nc, usa) stent was inserted into the cbd, her symptoms improved and the obstructive jaundice resolved. finally, we diagnosed her as a pancreas adenocarcinoma with cbd obstruction. two months later, she underwent repeat ercp for stent exchange using a 60-mm - long, 10-mm diameter csems (bonastent ; standard sci tech inc., however, she was readmitted in 2 months due to epigastric pain, fever, and jaundice. a subsequent endoscopy showed a partial distally migrated csems that was impossible to access and revise through the migrated stent. thus, stent trimming was performed with an apc system (erbe elektromedizin gmbh, tbingen, germany). the power setting was 60 w, and the argon gas flow was set to 2 l / min. stent amputation progressed in a circumferential manner until it was completely cut after 10 minutes (fig. then, a retrieval balloon was used to remove the clogging sludge following insertion of a guidewire through the remnant fragmented stent. however, while moving the balloon back and forth, the remnant stent migrated proximally very abruptly and floated to the dilated cbd. immediate retrieval of the stent was not attempted because of the sharp cutting plane edge. two 7-fr double pigtail stents (9- and 10-cm long, respectively ; cook endoscopy) were inserted in both intrahepatic bile ducts through the proximally migrated metal stent (fig. however, the patient was readmitted twice because of cholangitis and obstructive jaundice after 1 month, so removal of the proximally migrated metal stent was planned. following removal of the double pigtail stent with rat tooth forceps, the stricture was dilated to 8 mm with a balloon catheter (hurricane biliary balloon dilatation catheter ; boston scientific, natick, ma, usa). the proximal end of the floating metal stent was captured with a basket, rotated 180 in the cbd, and then removed successfully (figs. 4, 5). no procedure related complications occurred during the removal of the migrated stent, and then a usems was inserted successfully. since then sems placement has the advantage of greater long - term patency and cost - effectiveness compared to placement of a plastic stent for the palliative treatment of malignant distal biliary strictures.1,2 nevertheless, some complications, such as occlusions and migration of sems, can be problematic. proximal migration is a less common mode of migration and less frequently reported by other investigators. there was limited number of cases about removal of proximally migrated sems and also these migrated stents had a normal configuration. bakhru.12 reported one patient who experienced proximal migration 58 days following csems insertion but did not describe retrieval technique. ho.13 reported that proximal migration of partially covered sems occurred in seven cases (1.7%), and migrated stents was extracted by balloon dilation combined with rat tooth extraction. unlike plastic stents, semss are relatively hard to extract after being inserted and fixed. endoscopic removal has been successfully achieved in selected cases, particularly when a csems was the issue.6 - 8 several techniques for sems removal have been described in the literature. kahaleh.9 successfully removed a sems using a polypectomy snare and large - mouthed, rat tooth forceps. usemss have been removed using ' hot ' biopsy forceps to extract individual wire filaments from the distal end. levy and wiersema14 removed a distally migrated biliary wallstent using an endoscopic suture - cutting device. even with csemss, only 4.5% to 22.2% of cases are limited by removal of the stent.6,9 apc has been used successfully to trim sems in selected patients, particularly in cases where the prolapsing part of the stent is extremely long and can not be held with a snare or holding forceps. the safety and usefulness of apc trimming have been confirmed in experimental and clinical studies.6,10,11,15,16 similarly, we used an apc trimming method in our case, because the sems was partially distally migrated, and we failed to remove the stent with a snare and forceps. however, in our case, the remnant stent migrated proximally and was difficult to remove because of sharp cutting plane edge. first, excessive balloon sweeping inside the remnant stent to remove sludge might have facilitated stent migration. second, as a predisposing factor, the decreased frictional force between the metal stent and the stented cbd wall due to thermal injury by apc trimming might facilitate migration. in an animal study, which reported gross and histological findings after biliary stent trimming, apc stent trimming had the potential to cause superficial tissue damage by heat transmission through the treated metal stent.15 in these circumstances, excessive balloon sweeping might have facilitated stent migration. third, relatively short length of malignant stricture due to pancreas malignancy might be an additive factor for proximal migration. accordingly, after trimming a metal stent, excessive balloon sweeping inside the stent should be performed with caution, particularly with csems. retrieval techniques for removal of proximally migrated plastic and metal stents include forceps, a dormia basket, snare, balloon, and a soehendra stent retriever.12,17,18 a migrated stent may cause frequent cholangitis, as in our case, even if endoscopic revision is successful. although two plastic stents were inserted through the migrated metal stent, cholangitis developed twice within 2 months. a remnant stent may affect bile flow and more frequently clog the stent. in our case, the basket - capture technique was used to retrieve the migrated metal stent. after capturing the proximal end of the migrated stent with a basket, the stent was rotated 180 in the markedly dilated proximal cbd and then finally removed, as the distal end of the stent was sharp and may have caused bleeding or perforation during passage through the strictured distal cbd. proximal migration of a remnant stent with sharp cutting plane edge after stent trimming by apc has not been reported in the english literature, making it difficult to establish ideal methods for stent removal. our method might have also caused injury to the bile duct during rotation if cbd dilatation was not enough. folding or crushing with basket or snare can be used for the same purpose. however, considering the sharp edges of cutting plane, our method may be helpful to minimize injury during removal. no complications were observed during the removal of the fragmented stent, and then an usems was placed successfully. as another option, secondary reinsertion of a metal stent through the migrated remnant stent should be considered, if guidewire initially passed through the migrated metal stent. in conclusion, although endoscopic apc trimming of a distally migrated csems facilitated the removal of the migrated stents that were difficult to remove by conventional methods, a rare complication, such as proximal migration, can develop in association with excessive balloon sweeping in short segmental stricture. when trimming the sems to resolve a distal migration, adequate remnant stent material should be ensured to prevent proximal migration during additional endoscopic procedures such as ballooning, particularly with csems. furthermore, since retrieval of a stent using a basket can be attempted safely, early removal of the migrated stent is necessary to prevent other complications. | placement of a self - expanding metal stent (sems) is an effective method for palliation of a malignant biliary obstruction. however, metal stents can cause various complications, including stent migration. distally migrated metal stents, particularly covered sems, can be removed successfully in most cases. stent trimming using argon plasma coagulation may be helpful in difficult cases despite conventional methods. however, no serious complications related to the trimming or remnant stent removal method have been reported due to the limited number of cases. in particular, proximal migration of a remnant fragmented metal stent after stent trimming followed by balloon sweeping has not been reported. we report an unusual case of proximal migration of a remnant metal stent during balloon sweeping following stent trimming by argon plasma coagulation. the remnant metal stent was successfully removed with rotation technique using a basket and revised endoscopically. |
mucous membrane pemphigoid, a new denomination of cicatricial pemphigoid, encompasses a group of chronic subepithelial autoimmune blistering diseases that predominantly affect the oral cavity and the eyes (conjunctivitis and symblepharon). a rare case of mucous membrane pemphigoid (mmp) in a human immunodeficiency virus (hiv) positive patient is discussed with clinicohistopathological presentation. since our patient was hiv - positive and had lesions restricted to the oral mucosa with ocular involvement, only topical and intralesional steroids were preferred as the first line of treatment. mmp is autoimmune in nature and probably includes an autoantibody - induced, complement mediated sequestration of leukocytes with resultant cytokine and leukocyte enzyme release and detachment of the basal cells from the basement membrane zone, but there may also be complement - mediated cell lysis (1). mmp is characterized by junctional separation at the level of the basement membrane, causing a sub - basilar split. we report a case of mmp in a male patient who was also diagnosed as hiv positive during investigations for mmp and did not show any other oral manifestations. we would also like to emphasize the need to investigate the immune status along with histopathology, immunofluorescence microscopy and antibody confirmation tests for hiv, since hiv might have varying interactions with the immune system, which may lead to the development of autoimmune processes. a forty - eight years old male patient reported with a chief complaint of painful ulcerations in his mouth and lip along with eye discomfort that had started before 10 days. he did not complain of the presence of any other systemic diseases or ongoing medical therapy, and did not report any changes in the skin or genital areas and related family history at the reporting date. clinical assessment : oral examination revealed large ulcerations formed after the rupture of bulla involving upper left buccal vestibule and mucosa surrounded by epithelial sloughing (figure 1). superficial, hemorrhagic crusted ulcerations on upper and lower labial mucosa and irregular ulceration involving the floor of the mouth (figure 2) were well appreciable. a mild conjunctivitis with yellowish fibrinous slough and severe conjunctivitis were easily observed in the right and left eyes respectively (figures 3). further evaluation did not disclose any other lesions of buccal mucosa, perioral skin and genital areas. a mucosal biopsy was performed and the patient was recalled after five days for follow - up observation. however, on third day, the patient came with more extensive lesions on the left buccal mucosa, vestibule, upper and lower labial mucosa (figure 4. it might be due to pressure on the normal mucosa applied during biopsy procedure (positive nikolsky 's sign). the possibility of a drug - induced bullous eruption was effectively excluded in this patient because he did not use any drugs. clinical picture showing the lesion showing lesion on floor of the mouth and lips showing eye involvement extensive lesions after three days of biopsy the patient was treated with topical and intralesional corticosteroids (0.1% triamcinolone acetonide and dexamethasone 4mg / ml respectively). he was given instructions for oral hygiene care with chlorhexidine mouthwash (0.2%) for five days and was referred to an ophthalmologist for conjunctival lesions. a significant improvement was noticed in the lesion size and symptoms after ten days, and thereafter, the patient did not return for follow - up. initially enzyme - linked immunosorbent assay (elisa) and later western blot were positive for hiv antibody confirmation tests with 1180 cd4 cells /microlitre. immunofluorescence examination : immunofluorescence testing could not be done because of unavailability of preservative transport media during biopsy. histopathological findings : histopathology revealed the presence of stratified squamous epithelium supported by fibrocellular connective tissue stroma and presence of subepithelial prominent areas of separation between the epithelium and connective tissue (subepithelial cleft) with edematous connective tissue. also dense infiltration of chronic inflammatory cells, predominantly the lymphocytes was seen (figure 5). there was also a presence of muscle tissue and adepocytes in the deeper portion of the tissue which was also infiltrated with inflammatory cells. photomicrograph of the lesion showing sub - epithelial cleft differential diagnosis : the following differential diagnosis of blistering diseases was considered before final diagnosis : erythema multiformepemphigus vulgarisbullous pemphigoiderosive lichen planusbehcet 's syndrome erosive lichen planus mucous membrane pemphigoid has been known by many different names in the medical literature including benign mucous membrane pemphigoid, cicatricial (scarring) pemphigoid, and ocular cicatricialpemphigoid. a group of researchers in 2002(1) determined that mucous membrane pemphigoid was the best designation for this group of disorders. the term benign mucous membrane pemphigoid was deemed inappropriate because of the potential for serious complications in cases where the lesions were very severe. site - specific terms such as ocular cicatricialpemphigoid excluded individuals with multiple site involvement (1). since in this patient characteristic lesions are restricted only to the mucosa and the eye, it was diagnosed as mmp (2, 3, 4). pathogenesis of mmp probably includes an autoantibody - induced, complement mediated sequestration of leukocytes with resultant cytokine and leukocyte enzyme release and detachment of the basal cells from the basement membrane zone, but there may also be complement - mediated cell lysis. mmp is characterized by junctional separation at the level of the basement membrane, which gives rise to a sub - basilar split (5), which was observed in the present histopathological section. the association of immunodeficiency and autoimmunity in one patient is two sided clinical immune response. however, the reports of high frequency of auto antibodies in hiv - infected patients are increasing in the literature. autoimmunity may develop during acute viral infection with normal to low cd4 counts (6), which may be a part of the nonspecific polyclonal b - cell stimulation secondary to interleukin (il)-1 and il-2 released by hiv - infected macrophages (6,7). the frank loss of specific immunomodulatory cd4 t cells may allow the expansion of b - cell clone, which is responsible for the autoantibody formation (8). the actual pathogenesis and mechanism in development of mucous membrane pemphigoid secondary to hiv are still not confirmed. the possible mechanisms for autoimmune manifestations of hiv infection include the direct effect of hiv on endothelial, synovial, and other hematopoietic cells resulting in destruction of cd4 cells, increased cytotoxic cell activity and increased expression of auto - antigens (6). the exogenous infectious agent may have molecular similarity with a self - antigen and may therefore induce an autoimmune response (7, 8, 9). pemphigus is similar in its clinical presentation with mmp, but it produces acantholysis with cleavage of the spinous cell layer and the vesicle is intraepithelial, whereas in mmp as a result of the separation of connective tissue from the epithelium the vesicle is subepithelial (10). keratinocyte necrosis, intracellular edema in prickle layer, acanthosis and a perivascular inflammatory cell infiltrate with a thin basement membrane adhered to the epithelium are characteristic histopathological features of erythema multiforme (10). in contrast to lichen planus, the inflammatory infiltrate is non - specific in nature comprising of lymphocytes, plasma cells neutrophils with few eosinophils. patients with mmp rarely have circulating autoantibodies to the basement membrane zone components ; thus, indirect immunofluorescence is usually not indicated as a diagnostic procedure. treatment of mmp is based on the severity of symptoms and the site involved (1, 2). patients with mild localized lesions may often benefit from topical steroids such as beclomethasonedipropionate, betamethasone, clobetasol propionate, fluocinomide. patients with more extensive lesions can be prescribed systemic steroids like prednisolone (1). other treatment regimens, which are effective in certain resistant cases, are immunosuppressive agents such as azathioprine, cyclophosphamide, cyclosporine and dapsone. sulphononamides and tetracyclines can also be implemented (11, 12). in hiv - infected individuals our patient was hiv - positive and had lesions restricted to the oral mucosa with ocular involvement, only topical and intralesional steroids were preferred as first - line treatment (1, 12) accompanied by referral of the patient to an ophthalmologist. | backgroundmucous membrane pemphigoid, a new denomination of cicatricial pemphigoid, encompasses a group of chronic subepithelial autoimmune blistering diseases that predominantly affect the oral cavity and the eyes (conjunctivitis and symblepharon).case detailsa rare case of mucous membrane pemphigoid (mmp) in a human immunodeficiency virus (hiv) positive patient is discussed with clinicohistopathological presentation.conclusionsince our patient was hiv - positive and had lesions restricted to the oral mucosa with ocular involvement, only topical and intralesional steroids were preferred as the first line of treatment. systemic corticosteroid therapy raises a concern regarding immunosuppression. |
meningococcal disease is an acute bacterial disease caused by neisseria meningitides which is a gram negative capsulated coffee - bean - shaped bacteria seen in pairs. thirteen serogroups of n. meningitidis have been identified based on capsular polysaccharide antigen and of these ; four sera groups namely a, b, c, and w135 are known to cause epidemics. multiple outbreaks have occurred in 1966, 198586, 2005, 2007, and 2009. an outbreak of meningococcal disease was reported in three districts of andhra pradesh namely, vishakhapatnam, vijayanagram, and srikakulam in 20052006. the etiological agent was again found to be of the sera group a. there are no other cases reported from other parts of south india. we are reporting two cases of meningococcal meningitis from an urban health centre from vellore, tamil nadu. two male children, 12 years and 7 years, presented to the casualty of the urban health centre within 2-week interval. they came from the same community and lived half a kilometre of each other. the first child presented with history of fever, headache, neck pain, and vomiting for 4 days. the second child had these symptoms for one day and approached the urban health centre within 1 day. the past history, immunization history, and developmental history were normal for both children. on examination both children were drowsy, febrile with a glasgow coma scale (gcs) of 13/15 for first child and 15/15 for the second child. they were both admitted and underwent lumbar puncture for cerebrospinal fluid (csf) analysis. at lumbar puncture both the csf was turbid. the investigation results [table 1 ] were as follows : results of cerebrospinal fluid analysis for both children both children had predominant leucocytosis and increased neutrophil predominance in the blood investigation. the children were referred to the tertiary centre for further treatment and management due to deterioration in the sensorium. the polymerase chain reaction (pcr) test revealed the causative strain to be neisseria meningitis serotype c. both children were administered injection cefotaxime by parenteral route for two weeks and were well at discharge. once the diagnosis was established, we initiated preventive measures to the health - care providers, immediate family, and the community. health education about the signs and symptoms of this disease was given to the community and the health volunteers after the admission of the first child. the second child was brought to the health centre within a day of the symptoms because of the intervention of local health volunteers. after the diagnosis of the second child, further health education was planned and done to the surrounding urban community. as schools were closed for the summer vacation, chemoprophylaxis was not given to the school children. volunteers and health workers did intensive monitoring for 3 weeks consecutively after the diagnosis of the first case. limited data available on the epidemiology of meningococcal infection in india reveal a low background incidence of disease. the disease is characterized by fever, headache, and vomiting, and neck stiffness. if not detected early it can run a fulminating course leading to septicaemia and death. the age group mostly affected are children and young adults as observed in our case report. human beings are reservoirs for this bacterium and 1015% of healthy subjects harbour it in the upper respiratory tract. the transmission of disease is by droplet infection from nose and throat of patients and carriers. the incubation period is 210 days and outbreaks are more common in spring and winter months. the second child was brought to the centre early due to increased awareness initiated by the volunteers and health care workers after diagnosis of the first child. the management of suspected meningococcal disease includes admission to a health centre for lumbar puncture and csf examination. primary care physicians are also responsible for the health of the community, the management of cases also involves prevention of secondary cases. it should be initiated within 48 h of diagnosis of cases to all identified persons who had close contact with the case within 7 days of onset of the disease. drugs with dosage recommended for chemoprophylaxis in meningococcal disease two different types of meningococcal conjugate vaccine (mcv) are available in india. one is a quadrivalent (a, c, w135, y) mcv and the other is a monovalent serogroup a vaccine. the indian academy of paediatrics (iap) state that routine use of mcvs in india is not justified because of low incidence of disease. mass vaccination is recommended during outbreaks (defined as more than 10 cases per 100,000 populations). | meningococcal meningitis has rarely been reported in tamil nadu. we report here two children diagnosed with meningococcal meningitis in vellore, tamil nadu, on may 2014. the causative strain was neisseria meningitidis serotype c. the role of the primary care physician in early diagnosis, appropriate referral, and preventive measures of this disease to the immediate family and community is stressed. |
corrosive esophagitis is characterized by caustic injury following the ingestion of chemical agents, mainly alkaline substances such as detergents, cleaning compounds, and bleaches. the ingestion of acidic substances can also cause caustic injury, but these cases are less frequently reported than those caused by alkaline agents in the united states.1 early endoscopic intervention is very useful in suspected cases of corrosive esophagitis, not only for determining the degree of mucosal injury and severity, but also for predicting prognosis.2 high - degree corrosive esophagitis may result in esophageal bleeding, perforation, and even death in the worst cases. in particular, esophageal stricture, one of the more serious complications of corrosive esophagitis, may require recurrent ballooning procedures, which are highly expensive. moreover, the rates of esophageal carcinoma are 1,000 to 3,000 times higher in patients with esophageal complaints than those in healthy individual.3 picosulfate (picolight powder ; pharmbio, seoul, korea), a widely used laxative, is used to prepare the intestine for colonoscopy or colon surgery. this laxative may induce side effects including abdominal pain, persistent diarrhea, or electrolyte imbalance. however, picosulfate is still considered to be relatively safe, as these complications only occur in rare cases. generally, it is recommended that picosulfate powder should be completely dissolved in water and cooled before drinking because an exothermic reaction may occur immediately after the substance comes into contact with water, which can result in chemical burning of the esophagus and stomach. here, we report the first case of corrosive esophagitis due to the ingestion of picosulfate powder. he had ingested picosulfate that was not completely dissolved in water as preparation for colonoscopy. he had lost a cup of blood, but was hemodynamically stable and alert (blood pressure, 130/70 mm hg ; heart rate, 72 beats per minute). his initial hemoglobin level was 14.9 g / dl, and other laboratory tests results, including coagulation and electrolytes, were normal. fifteen hours after admission, the patient underwent upper gastrointestinal flexible endoscopy. during the procedure, we detected diffuse linear mucosal breaks with spontaneous blood oozing from the mid - esophagus to the esophagogastric junction caused by caustic esophageal injury. in addition, linear erosion at the gastric antrum and ulceration at the greater curvature of the gastric body were detected (fig. the patient was treated with conservative methods, including total parenteral nutrition and an intravenous proton pump inhibitor (esomeprazole, 40 mg). we regarded the fever as transient, but administered antibiotics (cefotaxime, 2 g) because we could not rule out the possibility of infection. linear ulcerations and signs of healing were detected in the esophagus, but there were no more visible oozing lesions or stricture (fig. 2). after this endoscopic procedure, he started eating a soft diet and stopped taking antibiotics and intravenous esomeprazol. he was stable and had no further symptoms, and was discharged with no complications. after 5 months, there were no mucosal abnormalities in the esophagus and stomach, except for a linear whitish scar in the stomach (fig. generally, this laxative is dissolved with water before drinking, and activated by hydrolysis of arylsulfate, an enzyme derived from colonic bacillus plexus.4 this mechanism facilitates peristalsis, blocks the absorption of water in the intestine, and finally induces diarrhea.4 picosulfate is widely used, and many physicians mix it with magnesium and citric acid for bowel preparation before colonoscopy or bowel surgery.5 however, side effects such as abdominal pain, thirst, vomiting, or electrolyte imbalance can follow the use of picosulfate.6 electrolyte disturbances, mainly hyponatremia, are present in 7.5% of patients, and can lead to seizures and rhabdomyolysis in severe cases.6 corrosive esophagitis is often caused by ingestion of alkaline agents, but rarely by the ingestion of acidic agents. although cases of corrosive esophagitis caused by acidic agents are very rare compared to those caused by alkaline agents, higher rates of side effects and complications such as perforation and stricture have been reported for acidic agent - driven corrosive esophagitis.1,2 additionally, a case of corrosive esophagitis due to the ingestion of liquid glue, which includes toluene,6 and diluted pendimethalin, a herbicide, have also been reported.7 as previously stated, both alkaline and acidic substances can induce corrosive esophagitis, but the effects of these two types of agents are explained by different pathophysiologic theories. alkaline agents cause liquefaction necrosis, which leads to the destruction of mucosa within a few seconds. one or 2 days after the ingestion of alkaline substances, destruction of the mucosa is exacerbated by thrombosis in small vessels and the production of heat. in contrast, acidic agents cause coagulation necrosis with eschar formation, which may limit tissue penetration.1 once picosulfate is dissolved in water, the substance is acidic and releases hydrogen ions by hydrolysis. to reduce the possibility of corrosive esophagitis occurring, dissolving picosulfate in a high volume of water is strongly recommended because this renders the dissolved picosulfate less acidic. on the other hand, dissolving picosulfate in a low volume of water produces a more strongly acidic liquid, which more frequently leads to corrosive esophagitis.1 furthermore, picosulfate powder itself may cause chemical burning in the esophagus and stomach by an exothermic reaction after it has been dissolved in water. presumably, the present case was caused by strong acidity due to incomplete dilution, and chemical burning from an exothermic reaction. although each study has some different criteria, grade of corrosive esophagitis is in general classified according to endoscopic findings according to zagar 's grade.8 the present case was regarded as grade 2a corrosive esophagitis (hemorrhage, exudates, and superficial [not deep ] ulceration).9 esophageal stricture can be complicated in grades 2 to 3.10,11 however, in the present case, esophageal stricture did not occur after the event. it can be inferred that crucial esophageal muscular damage did not occur after corrosive injury. all things considered, we report here the first case of corrosive esophagitis due to the ingestion of picosulfate powder incompletely dissolved in water due to the patient not taking sufficient care, diagnosed through endoscopy in the early stage, and completely cured without complications by conservative treatment. | corrosive esophagitis is characterized by caustic injury due to the ingestion of chemical agents, mainly alkaline substances such as detergents. esophageal bleeding, perforation, or stricture can be worsened by high - degree corrosive esophagitis. picosulfate is a commonly used laxative frequently administered for bowel preparation before colonoscopy or colon surgery. picosulfate powder should be completely dissolved in water before ingestion because the powder itself may cause chemical burning of the esophagus and stomach. here, we report a case of corrosive esophagitis due to the ingestion of picosulfate powder that was not completely dissolved in water. |
hepatic resection is considered first - line therapy for many primary and metastatic liver tumors. thanks to a more careful perioperative management, the use of highly accurate pre - operative imaging and the refinement of surgical techniques, hepatic resection has been applied more extensively and successfully. post - operative mortality has currently been reduced to less than 5% in almost all groups, whereas 5-year survival after hepatic resection for colorectal metastases, primary tumors or other types of non - colorectal metastases has increased from 30 to 50%. in the past, patients with large tumors involving the inferior vena cava (ivc) were not considered candidates for surgical resection. however, these untreated patients had a poor survival rate, less than 12 months, even when using palliative chemotherapy. in recent years, the improvement of surgical techniques with a detailed increase in knowledge of the segmental anatomical structure of the liver has permitted liver resections that were until recently deemed high - risk surgery. innovative and aggressive surgical techniques, principally derived from transplant surgery, such as hepatic vascular exclusion (hve), veno - venous bypass and ex vivo hepatic resection, have been reported in dealing with hepatic tumors involving the ivc. graft prosthetics and autologous or synthetic patches are methods extensively mentioned in the literature for repair of the ivc. in a woman of 67 years in follow - up at our clinic after right hepatectomy performed 3 years earlier, for colorectal hepatic metastasis, a new lesion was detected in the liver. two years before the hepatic resection, the patient had been operated on laparoscopically for left colectomy for adenocarcinoma g2, t3, n1, m0 (according to tnm, sixth edition). quadriphase contrast - enhancement abdomen and chest computed tomography (ct) showed a hypodense lesion in liver segment iv, 4 cm in diameter, infiltrating the intrahepatic portion of the inferior vena cava. prior to surgery the patient underwent magnetic resonance imaging (mri) with vascular reconstruction in order to evaluate the relationship between the tumor, the vena cava and the hepatic veins in detail. in addition, the patient underwent a stress echocardiogram so as to evaluate the heart function in view of a total hepatic vascular exclusion. the access laparotomy consisted of a bilateral subcostal incision on the previous surgical scar. after extensive and laborious adhesiolysis, since part of the colon and small intestine completely occupied the right upper quadrant, it was possible to expose the liver. an intraoperative ultrasound scan (ious) confirmed the preoperative findings and excluded other metastases in liver segments ii iii. the operation began by exposing the inferior vena cava below the tumor and above the hepatic vein ; both parts of the ivc were surrounded by vessel loops. the liver parenchyma was divided along the umbilical fissure (on the left side), by using bipolar forceps. central venous pressure was constantly maintained at or below 5 cm of h2o during the parenchymal transection to minimize the risk of bleeding. during the transection, non - selective we then proceeded with the placement of a vascular clamp on the infrahepatic vena cava and the other clamp on the suprahepatic vena cava (above the hepatic vein), completing the resection of segment iv associated with the excision of retrohepatic vena cava (fig. the caval reconstruction was via the interposition of a 20 mm ringed - ptfe tube graft, in total vascular exclusion for about 25 min. in the icu, heparin iv anticoagulation was started, maintaining an activated partial thromboplastin time ratio (aptt) of 2.53. on the fifth post - operative day, the infusion of iv heparin was stopped and changed to subcutaneous injection of low molecular weight heparin (enoxaparin) ; at the same time, we started the administration of antioral platelet drugs. the postoperative period was characterized by no major surgical complications, although the patient developed right pleural effusion. six months post - operatively, the patient is in good health and tumor - free. hepatic resection for liver mts has been shown to result in better prognosis than other treatments. hepatic resection of tumors located in the middle of the liver and invading the ivc was considered until recently an absolute contraindication to surgery. however, thanks to innovative techniques, these types of liver resections offer a chance of cure to patients who until recently were not considered candidates for any surgical treatment. the benefit of hepatic resection combined with resection and reconstruction of the vena cava is obvious, since it is an attempt to achieve oncological radicality, otherwise impossible. recent papers have clarified the safety and advantages of combining excision of the ivc in continuity with hepatic resection for liver malignancy invading the ivc or hepatic vein. several surgical techniques are reported in the literature in dealing with tumors involving the ivc (table 1). the type of surgical strategies that can be used depends on the extension of the tumor along the inferior vena cava. when the tumor invades a small portion of the ivc, it is possible to apply a clamp tangentially ; in this case, the ivc can be repaired primarily with a venorraphy or with a patch in enlargement, provided that there is no excessive narrowing of the vascular lumen. caval stenosis, in fact, causes persistent edema of the lower limbs and, in severe cases, renal dysfunction. in cases where tumor invasion is quite extensive, resection of the inferior vena cava requires the interruption of the caval flow. in this case, two different strategies can be used : when the involvement of the ivc is below the hepatic veins, then a clamp is placed above the tumor but below the outlet of the hepatic veins into the ivc (clamping of the intrahepatic cava) with the advantage of being able to maintain the blood flow from the liver to the heart while following resection and reconstruction of the ivc. when the clamp is placed on the cava above the hepatic veins, then total vascular exclusion (hve) the latter can be performed with or without veno - venous bypass, depending on the hemodynamic stability of the patient. in this case, it is advisable to perform the vascular exclusion test in order to check the stability of the circulation and heart function ; however, bypass is recommended in those patients with heart disease or kidney failure. it has been suggested, in cases of total vascular exclusion, to begin to minimize warm ischemia of the liver before the caval resection. in our case, a total vascular exclusion was achieved as not enough room was available to place the clamp below the outlet of the hepatic vein, since the tumor was close to the hepato - caval junction. however, it is preferable to reduce this time to 60 min in cases of diseased liver. in fact, despite undergoing ischemia for 25 min (hve) plus 12 during the pringle maneuver, our patient did not show any signs of postoperative liver failure. in situ and ex situ (bench surgery) hepatic resection is a particularly demanding surgical alternative, applicable in those cases in which the resection of the ivc is also associated with the reconstruction of the hepatic veins. in the literature, many varied surgical options are described in the reconstruction of the ivc, when this can not be repaired primarily. autologous veins have been used with success ; however, prosthetic materials are feasible and produce a long patency. an 1820 mm goretx or dacron graft appears superior in terms of patency of that resistance, and this prosthesis is, therefore, the one most frequently used in different surgical settings to reconstruct the cava. the most serious problems that may arise with the use of the prosthesis are infection and thrombosis. the risk of infection after liver resection is a well - documented problem, which varies between 8 and 28%, thus some authors have recommended the use of an omental wrap to protect the caval prosthesis. patency of the caval graft is 70% at 5 years, and systemic heparinization started postoperatively, with subsequent conversion to oral warfarin sodium, is therefore highly recommended. little has been published on the outcome of primary and metastatic liver tumors involving the inferior vena cava, as this surgery is still considered too complex and high risk for mortality and morbidity. in the most important cohorts that appear in the literature, the mortality rate is between 9 and 30%. despite the high risk faced by patients with this tumor, this high - risk surgery offers the only chance of curing tumors that would otherwise be considered non - resectable. however, it is worth noting that 5-year overall survival varies between 22 and 30%, an outcome entirely comparable to the survival of patients undergoing hepatic resection for colon - rectal metastasis. at present, patients with large tumors involving both liver and vena cava may be candidates for liver resection and replacement of the vena cava, achieving long - term survival in selected cases. the surgical techniques used in the resection of these tumors require specialized centers with surgeons experienced in complex processes of hepato - biliary surgery.key learning pointshepatic resection of tumors invading the ivc was considered until recently an absolute contraindication to surgery.innovative and aggressive surgical techniques, such as hepatic vascular exclusion (hve), veno - venous bypass and ex vivo hepatic resection, have been reported in dealing with hepatic tumors involving ivc.patients with large tumors involving both liver and vena cava may be candidates for liver resection and replacement of the vena cava achieving long - term survival in selected cases. key learning pointshepatic resection of tumors invading the ivc was considered until recently an absolute contraindication to surgery.innovative and aggressive surgical techniques, such as hepatic vascular exclusion (hve), veno - venous bypass and ex vivo hepatic resection, have been reported in dealing with hepatic tumors involving ivc.patients with large tumors involving both liver and vena cava may be candidates for liver resection and replacement of the vena cava achieving long - term survival in selected cases. hepatic resection of tumors invading the ivc was considered until recently an absolute contraindication to surgery. innovative and aggressive surgical techniques, such as hepatic vascular exclusion (hve), veno - venous bypass and ex vivo hepatic resection, have been reported in dealing with hepatic tumors involving ivc. patients with large tumors involving both liver and vena cava may be candidates for liver resection and replacement of the vena cava achieving long - term survival in selected cases. written informed consent was obtained from the patient for the publication of this case report and accompanying images. gian piero guerrini ph.d. contributed in data collection, analysis and writing the manuscript. paolo soliani md was the 1st surgeon who performed the hepatic resection and contributed in writing the article. | introductionadvanced tumors of the liver involving the inferior vena cava (ivc) have always been considered a contraindication to surgery.presentation of casewe report a case of a patient, who previously underwent right hepatectomy, with recurrence of colorectal liver metastasis invading the ivc. the patient had a liver resection together with replacement of the vena cava using a ringed polytetrafluoroethylene (ptfe) graft tube. the operation was carried out in hepatic vascular exclusion (hve) without the use of veno - venous bypass. the patient was healthy and tumor - free at 6 months post-surgery.discussionin patients with hepatic malignancy involving the ivc, extended hepatic resection and reconstruction of the ivc is often the prerequisite to obtaining a resection margin.conclusionextended hepatic resection with ivc reconstruction for hepatic malignancy may offer a chance of cure to selected patients who otherwise have poor survival rates. |
the prevalence of type 2 diabetes mellitus (t2 dm) continues to rise in adults worldwide. previous magnetic resonance imaging (mri) studies have found brain abnormalities, such as cortical and subcortical atrophy and white - matter hyperintensities (wmh) in t2 dm patients. however, the pathophysiological mechanism of t2dm - induced cognitive impairment is still largely unknown. previous studies have investigated the role of subcortical deep gray matter (sdgm) volume in t2dm - induced cognitive impairment, but conflicting results have been reported. some studies found abnormalities in the subcortical structures of t2 dm patients associated with impaired cognitive domains related to the speed of mental processes and memory. we recently reported gm volume reductions in the hippocampus and amygdala of patients with t2 dm with mild cognitive impairment. nevertheless, to date, the majority of morphometric studies of t2 dm have been conducted using voxel - based morphometry analysis methods or manual segmentation methods, and a relatively small sample sizes have led to conflicting results about subcortical pathology. for example, some research groups found no structural changes in the hippocampus in t2 dm patients. in contrast to the problem of tissue segmentation (gm, wm and cerebrospinal fluid [csf ]) on brain mri for which acceptable solutions have been found, the issue of subcortical structure segmentation has yet to be satisfactorily addressed. thus, using accurate and robust segmentation methods that are fully automated, quantitative evaluations of sdgm abnormalities in t2 dm patients could provide new insights into the pathogenesis of cognitive dysfunction in this disorder. here, we applied a novel image analysis technique (freesurfer), which is a voxel - based, automated software for brain reconstruction, to investigate the extent of sdgm structures directly. moreover, this technique reveals the anatomical features of the hippocampal formation at an unprecedented level of detail, providing the basis for hippocampal subfield measurement. in previous neuroimaging investigations, freesurfer was used successfully to study sdgm and hippocampal subfield structures in conditions such as alzheimer 's disease (ad). the aim of this study was to investigate the characteristics of sdgm structural changes and regional changes in the hippocampal subregions of t2 dm patients compared to control subjects. we also examined whether volumetric changes in hippocampal subfields in t2 dm were related to cognitive impairment. this study protocol was approved by the ethics committee of southwest hospital in chongqing, china. written informed consent and complete medical records were obtained from each participant prior to the study. the patients volunteered to participate in this study and were able to express their psychological states. two age-, gender- and education - matched groups of participants were studied, including 80 patients diagnosed with t2 dm and 80 healthy controls (hcs) subjects. patients with t2 dm were diagnosed using the criteria recommended by the american diabetes association in 2010. all of the patients were recruited from southwest hospital and were being treated with oral hypoglycemic agents, insulin secretagogue agents and metformin at various dosages. in addition, these patients used other nonpharmacologic strategies, including diet control and physical exercise. a group of hc with no history or symptoms of diabetes or psychiatric or neurologic disease was recruited. the exclusion criteria for all of the participants included central nervous system diseases, including stroke, drug or alcohol dependence ; a history of head trauma, major depression (excluded by the hamilton depression rating scale) or other neurological or psychiatric illnesses (excluded by clinical assessment and case history) ; contraindications to mri ; and indications of dementia (defined as a mini - mental state exam [mmse ] score of 0.05). in terms of cognitive assessment, the t2 dm patients had significantly lower moca scores than the control subjects, suggesting that their general cognition was impaired (p 0.05). in terms of cognitive assessment, the t2 dm patients had significantly lower moca scores than the control subjects, suggesting that their general cognition was impaired (p < 0.001). partial correlation analysis found that the hba1c was negatively correlated with the total moca score (r = 0.24, p = 0.032), mainly in terms of delayed recall (r = 0.309, p = 0.006) in the t2 dm patients. demographic, clinical and cognitive characteristics of the study patients and control subjects data are reported as mean (sd) ; p<0.05. sd : standard deviation ; hba1c : glycosylated hemoglobin (%) ; bp : blood pressure ; bmi : body mass index ; mmse : mini mental state exam ; moca : montral cognitive assessment ; tiv : total intracranial volume ; gm : gray matter ; wm : white matter ; na : not applicable ; wmhr : total white matter hyperintensity volume ; expressed as a percentage of the intracranial volume ; t2 dm : type 2 diabetes mellitus ; sbp : systolic blood pressure ; dbp : diastolic blood pressure. no significant differences were observed in gm (f = 2.877 ; p = 0.092), wm (f = 0.002 ; p = 0.969) volume, and wmhr (t = 0.925 ; p = 0.357) or tiv (f = 0.616 ; p = 0.434) between the patients and control subjects [table 1 ]. the total volumes of the left (f = 4.543 ; p = 0.035) and right (f = 6.081 ; p = 0.015) hippocampi of the t2 dm group were significantly smaller than those of the control group [table 2 ]. however, there were no significant volume differences for the nucleus accumbens, amygdala, caudate, pallidum, putamen or thalamus subfields between the t2 dm group and control group. mean subcortical nuclei volume in cubic centimeters per diagnosis : mancova analysis applied to estimate group differences adjusted for age, sex, education years and tiv, threshold for statistical significance was set at p<0.05. sd : standard deviation ; t2 dm : type 2 diabetes mellitus ; tiv : total intracranial volume. partial correlation analysis found that the hba1c was negatively correlated with the hippocampal volume (r = 0.267, p = 0.02). however, there were no significant association between the hba1c and nucleus accumbens (r = 0.191, p = 0.099), amygdala (r = 0.099, p = 0.397), caudate (r = 0.099, p = 0.394), pallidum (r = 0.099, p = 0.396), putamen (r = 0.122, p = 0.295) or thalamus (r = 0.109, p = 0.35) volumes. in the hippocampal subfield [table 3 ], the ca1 (f = 8.126 ; p = 0.005) and subiculum (f = 5.686 ; p = 0.018) volumes were significantly smaller in the t2 dm group compared with the control group. however, there were no significant volume differences between the t2 dm group and control group in the ca23 (f = 0.061 ; p = 0.805), ca4-dg (f = 0.079 ; p = 0.779), presubiculum (f = 0.042 ; p = 0.838), fimbria (f = 0.027 ; p = 0.870) or hippocampal fissure (f = 0.210 ; p = 0.647) subfields. results from the mancova analysis and effect sizes of differences between groups in hippocampal structures adjusted for age, sex, education years and tiv, indicates threshold for statistical significance was set at p<0.05. dg : dentate gyrus ; t2 dm : type 2 diabetes mellitus ; tiv : total intracranial volume. partial correlation analysis revealed that the total volumes of the ca1 and subiculum subfields were positively correlated with the total moca score (r = 0.516, p < 0.001, [figure 1a ] ; r = 0.307, p = 0.007, [figure 1b ]), mainly in terms of delayed recall (r = 0.430, p < 0.001, [figure 1c ] ; r = 0.385, p = 0.001 ; [figure 1d ]) in the t2 dm patients. the correlation of the bilateral ca1 volume (a) and the bilateral subiculum volume (b) with the total montreal cognitive assessment (moca) score in t2 dm patients. the correlation of the bilateral ca1 volume (c) and the bilateral subiculum volume (d) with the delayed recall score in t2 dm patients. the present study examined the volumetric changes in sdgm structures and hippocampal subfields in t2 dm patients. compared to the hc group, we found reduced bilateral hippocampal volume in t2 dm patients, mainly in the ca1 and subiculum subfields. additionally, the moca scores, particularly those regarding delayed memory, were significantly positively correlated with the ca1 and subiculum subfields in t2 dm patients. notably, hba1c levels were significantly negatively correlated with poor memory performance and hippocampal atrophy in t2 dm patients. the hippocampus has been shown to be specifically affected as a result of t2 dm, both structurally and functionally. in agreement with these previous studies, the current study found that compared to the hc group, significant bilateral hippocampal atrophy occurs in t2 dm patients. in addition, damaged hippocampal structures are known to impair hippocampus - mediated learning and aspects of memory function, such as recognition memory. our study also demonstrated that t2 dm patients presented significantly reduced moca scores compared to matched controls, and higher hba1c levels were significantly associated with poor memory performance and hippocampal atrophy among t2 dm patients. therefore, hippocampal damage and memory impairments are potentially serious complications in t2 dm patients. notably, in the present study, other sdgm structures were relatively preserved in t2 dm (the nucleus accumbens, amygdala, caudate, hippocampus, pallidum, putamen, and thalamus). in addition, no significant association between the hba1c and these sdgm structures was found in t2 dm patients. these results were likely observed because insulin resistance may be leading to neuronal loss in the hippocampus due to the high concentration of insulin receptors in that region. thus, degenerative pathology affects hippocampus structures to a greater degree in t2 dm than other sdgm areas, which suggests that the hippocampus may serve as the initial mediator of the association between t2 dm and cognitive decline. a previous study in streptozotocin - induced diabetic rats demonstrated widespread damage to the ca1 region of the hippocampus, indicated that despite t2 dm possibly having an overall effect on hippocampal volume, there also appeared to be region - specific effects. however, it remains unclear which hippocampus subregions are involved in cognitive dysfunction in t2 dm in vivo. in most studies on t2 dm, the hippocampus has been assessed as a single structure, without evaluating its various subregions, such as the ca1, ca23, ca4-dg and subiculum. thus, hippocampal subfield analysis could provide a more sensitive marker for t2 dm than whole - hippocampal volumetry. to the best of our knowledge, this was the first study to propose automated segmentation of the hippocampus in t2 dm. among the hippocampal subfields examined, we observed that degeneration within the ca1 and subiculum regions appeared to be more significant in t2 dm compared with other components of the hippocampus. the hippocampus is mainly organized as a unidirectional circuit starting at the dg, and this circuit is completed as the ca1 sends projections to the subiculum, which is the major output region of the hippocampus. therefore, our findings revealed selective involvement of specific hippocampal subregions in t2 dm, mainly in the output regions within the hippocampal circuit. interestingly, pathologic findings in patients with ad have suggested that severe degeneration of the perforant path, which provides input from layer iii of the entorhinal cortex to the ca1 and the subiculum, is a characteristic feature of ad. accumulating studies have shown that t2 dm and associated cognitive impairment are each associated with common pathophysiology of the central nervous system, whilst each is associated with increased mortality, an effect which is cumulative when both features coexist. compared with the results of the current study, the pattern of neurocognitive and hippocampal volumetric deficits in t2 dm populations show considerable similarity to those reported in populations of individuals with ad. in the correlation analysis, structural abnormalities in the ca1 and subiculum subfields were found to be related to impaired cognitive performance on the moca and delayed recall scores in the t2 dm group. a functional distinction has previously been reported between subfields, with ca1 pyramidal neurons of the hippocampus serving as an area crucial to the formation and encoding of memories, whereas the ca23 and dg regions are engaged by learning or the creation of new memories, and the subiculum is engaged specifically in the recall of these memories. our findings emphasized the role of the ca1 and subiculum regions within the hippocampal circuit in learning and memory deficits in these patients. first, the t2 dm subjects received various medications that may have produced confounding effects on neurocognitive. second, our study design was cross - sectional, and longitudinal neuroimaging studies of t2 dm patients with dementia should be conducted in the future to determine dynamic effects on hippocampal subfields. third, there are no diagnostic criteria for diabetes - related cognitive dysfunction, and this lack of objective and specific neurocognitive assessment limited our interpretation of the results. should such criteria become available, they would help to identify patients who are at risk for the development of dementia and would allow researchers to explore their brain patterns. finally, patients may complain about their poor memory to their health care providers, and although subjective memory complaints were not a reliable indicator of cognitive impairment in our study, these complaints may be a warning sign that patients ability to cope creatively with diabetes problems is reduced. in conclusion, among the sdgm structures, hippocampal might be the main affected region in t2 dm. these structural changes in the hippocampal subiculum and ca1 regions may serve as the main underlying neurobiological mechanisms of hippocampal dysfunction and may, therefore, be highly relevant to memory impairment in t2 dm. furthermore, these characteristics of hippocampal subfields atrophy in t2 dm might be very similar to those in ad. | background : little attention has been paid to the role of subcortical deep gray matter (sdgm) structures in type 2 diabetes mellitus (t2dm)-induced cognitive impairment, especially hippocampal subfields. our aims were to assess the in vivo volumes of sdgm structures and hippocampal subfields using magnetic resonance imaging (mri) and to test their associations with cognitive performance in t2dm.methods:a total of 80 t2 dm patients and 80 neurologically unimpaired healthy controls matched by age, sex and education level was enrolled in this study. we assessed the volumes of the sdgm structures and seven hippocampal subfields on mri using a novel technique that enabled automated volumetry. we used mini - mental state examination and montreal cognitive assessment (moca) scores as measures of cognitive performance. the association of glycosylated hemoglobin (hba1c) with sdgm structures and neuropsychological tests and correlations between hippocampal subfields and neuropsychological tests were assessed by partial correlation analysis in t2dm.results:bilaterally, the hippocampal volumes were smaller in t2 dm patients, mainly in the ca1 and subiculum subfields. partial correlation analysis showed that the moca scores, particularly those regarding delayed memory, were significantly positively correlated with reduced hippocampal ca1 and subiculum volumes in t2 dm patients. additionally, higher hba1c levels were significantly associated with poor memory performance and hippocampal atrophy among t2 dm patients.conclusions:these data indicate that the hippocampus might be the main affected region among the sdgm structures in t2 dm. these structural changes in the hippocampal ca1 and subiculum areas might be at the core of underlying neurobiological mechanisms of hippocampal dysfunction, suggesting that degeneration in these regions could be responsible for memory impairments in t2 dm patients. |
age estimation is an important part in personalization, especially when information regarding the deceased is unavailable. estimating the age narrows down the search among the missing profiles and enables a more efficient and time saving approach. teeth are the important aids in estimation of age, as they are least affected by variation in nutritional and endocrine status, therefore teeth can be used as accurate indicators of chronological age. dental radiography is, a non - destructive and simple technique, routinely employed in methods of age estimation. maturation stages of developing third molars can be used in estimation of chronological age of sub - adult individuals. the mandibular body and ramus show marked remodeling changes during growth and are sexually dimorphic. the main aim of the study is to identify the most reliable method for age estimation among height of condyle (hoc), length of mandibular body (lmb) and third molar maturation (mtm) by demirjian 's method using simple linear regression analysis. the study sample consisted of 60 subjects (30 males and 30 females) who visited the department of orthodontics in our college. out of 30 subjects in each gender 15 were below 18 years and 15 were above 18 years. lateral cephalograms were used to calculate the height of mandibular condyle and the length of the mandibular body. the tracings obtained were compared with modified demirjians comparison chart and maturity score was obtained following this the age was estimated after introducing the values in the formula derived using linear regression analysis [figure 1 ]. third molar traced on orthopantomograph placed on an illuminator lateral cephalograms of 60 subjects were traced using the same view box, hoc was obtained by marking reference points at the maximum hoc and minimum point at the tubercle, a line was drawn from maximum to minimum reference points and distance between two points was measured using a ruler [figure 2 ]. lateral cephalogram placed on a view box lmb was obtained by marking reference points on most antero - inferior part of mandible, that is, gnathion (gn) which is the constructed point between menton and pogonion, another point is marked at the gonion (go) that is the constructed point at the junction of ramus plane and the mandibular plane. line drawn from go to gn was measured with ruler and values were noted in millimeters. age estimation formula was derived for each parameter by simple linear regression analysis [table 1 ]. estimated age was calculated using the derived equation and compared with chronological age which was obtained by decoding the radiographs. all the data was subjected to statistical analysis using spss 14.0 software (software package used for statistical analysis). estimation or prediction of actual age by different independent variables (i.e. length of condylar height, length of mandibular body, maturity score of third molar) by simple linear regression analysis method - total, male and female samples the chronological age of the each subject was compared with the estimated age by three parameters (hoc, lmb and third molar calcification) of the respective subjects using a students t test. table 2 shows the comparison of estimated age with the chronological age of the patients. comparison of chronological age with estimated age by length of condylar height, length of mandibular body and third molar calcification in total samples when the chronological age was compared with the estimated age using hoc, lmb and third molar calcifications, the p values were 0.978, 0.9515, and 0.8611, respectively. all the values were more than 0.05, which indicates that there was no significant difference between the chronological age and the estimated age of the patients. table 3 shows the comparison of estimated age by condylar height, lmb and third molar calcification between males and females by student there was no significant difference observed in condylar height and third molar calcification with p values of 0.0548 and 0.5721 but little difference was observed with lmb with a p value of 0.0492. comparison of estimated age by height of condyle, length of mandibular body and third molar calcification between male and females by t test tables 46 show comparison of age groups and gender with estimated age calculated by length of condylar height, lmb and maturity score of third molar respectively by two - way anova test. comparison of age groups with gender with estimated age calculated by length of condylar height by two - way anova comparison of age groups with gender with estimated age calculated by length of mandibular body by two - way anova comparison of age groups with gender with estimated age calculated by maturity score of third molar calcification by two - way anova table 7 shows comparison of age assessed on combination of length of condylar height and lmb with chronological age, where there was no statistical significant difference. comparison of actual and estimated age calculated by length of condylar height and length of mandibular body in total, male and female sample by t test table 8 and graph 1 show the comparison of chronological age with the estimated age along with the standard deviation and standard error test to identify the most reliable method for age estimation. the standard deviation for the age estimated using lmb, was 1.45, followed by age estimated using condylar height was 1.51 while the third molar calcification was 3.6. standard error test for the age estimated using lmb was 0.188, condylar height was 0.194 while the third molar calcification was 0.466. standard error test to identify the most reliable method among height of condyle, length of mandibular body, third molar maturation comparison of chronological age with estimated age by all three parameters these values indicate that lmb is a more reliable method parameter for estimation of age followed by condylar height and third molar calcification. age estimation is a sub - discipline of the forensic sciences and should be an important part of every identification process. estimation of age is important for differentiating the juvenile from the adults in criminal law cases, social benefits, employment and marriage. determination of chronological age in persons within the range of 15 - 23 years remains a problem. skeletal indicators such as diaphysis - epiphysis fusion, hand - wrist examination, cervical vertebrae maturation, amino acid racemization, changes in pubic symphysis, fusion of cranial bones, fusion of cranial sutures or changes in the secondary sexual characters are most commonly used for age estimation in this age group. dental radiography is a non - destructive and simple technique used in dental practice for age estimation. comparison of ante - mortem and post - mortem radiographs is one of the cornerstones of positive identification of human remains. morphological changes of the mandible are thought to be influenced by the occlusal status and age of the subject. various studies have shown a decrease in ramus height, mandibular body height and mandibular body length with increase in age. hence, measurement of these parameters in the lateral cephalogram can be successfully used in estimation of age. teeth are the most useful material for age estimation and they remain unchanged for longer time because they are the most indestructible part of the body. compared to all teeth third molar is more useful in forensic dentistry because it continues to develop over a prolonged period and until a later age. in the present study, a comparison of the age estimation was done by measuring the lmb, hoc and by identifying the stage of third molar calcification. there was no statistically significant difference between the age estimated using height of the condyle, lmb and third molar calcification (p > 0.05) [table 2 ]. estimated the age and determined the sex using three mandibular parameters, namely gonial angle, ramus height and bigonial width. they observed that the bigonial width increases with age, ramus height increases in 2 and 3 decade and then decreases with age. in the present study but in a study conducted by annamalai., on various measurements of mandibular ramus which aid in sex determination, they observed a significant difference between males and female subjects. found mandibular ramus height to be the best parameter in their study with 75.8% accuracy and in the present study hoc can also be used for age estimation but has less reliability than lmb and more reliability than third molar calcification. demirjian. gave new method of age estimation by observing the radiological appearances of seven teeth on left side of the mandible and maturity score was given according to tanner. study conducted by krailassiri. in thai individuals of age group 7 - 19 years showed that tooth calcification stages from opg can be useful as maturity indicator of pubertal growth period. using this demirjians system willems. studied the age estimation in belgian children which showed over estimation of chronological age, which helps to state that there will be different rates of development in dental population. in 2004, conducted a study on the third molar development in relation to chronological age in turkish children and young adults and concluded that development of third molar in turkish people is rapid compared to other populations. rai. studied the role of development of mandibular third molar in age determination and sex identification in north indian children and young adults, and concluded that third molar development occurs at an advanced age relative to other populations and development staging of third molar has a linear relation to age in both genders and statistical analysis shows stronger correlation for males than females. in the present study using the demirjians indian formula given by acharya estimated age was calculated and no significant difference observed between estimated age and chronological age. the present study has underestimated the chronological age by a mean of 0.02 years with hoc, 0.03 years by lmb and overestimated with mean of 0.12 years with third molar calcification. age estimated using calcification of third molar, condylar height, mandibular body length showed no significant difference with the chronological age of the patient. | aim : to identify the most reliable method for age estimation among three variables, that is, condylar height, length of mandibular body and third molar calcification by demirjian 's method.materials and methods : orthopantomograms and lateral cephalograms of 60 patients with equal gender ratio were included in the study, among each gender 15 subjects were below 18 years and 15 subjects were above 18 years. lateral cephalograms were traced, height of condyle and mandibular body are measured manually on the tracing paper, opg 's were observed on radiographic illuminator and maturity score of third molar calcification was noted according to demirjian 's method. all the measurements were subjected to statistical analysis.results:the results obtained are of no significant difference between estimated age and actual age with all three parameters (p > 0.9780 condylar height, p > 0.9515 length of mandibular body, p > 0.8611 third molar calcification). among these three, length of mandibular body shows least standard error test (i.e. 0.188).conclusion : although all three parameters can be used for age estimation, length of mandibular body is more reliable followed by height of condyle and third molar calcification. |
the developmental defects of the enamel (dde) may be defined as the alteration of enamel that may affect an area of one surface or may be wide spread affecting all the surfaces throughout its full thickness. they may be quantitative in nature that is manifested as a deficiency in adequate thickness of enamel or qualitative in nature as enamel opacities. dde are associated with a wide spectrum of etiologic factors including systemic, genetic, local, and environmental conditions. enamel defects have significant impact on oral health and esthetics and act as a predisposing factor for caries. most epidemiological studies have shown that the frequencies of dde are on rise in all population streaming their clinical significance and evidence for public health initiatives. the prevalence of dde ranged from 6.7% to 67.1% in the developed countries and from 27% to 66.2% in the developing countries. assessed the prevalence and presentation of the dde of healthy school children residing in hills of himachal pradesh, india, and reported to be 66.2%. (2010) found a significant association between systemic illness during the first 5 years of life and the prevalence of dde. 2011) reported a significant association between intake of drugs or chemicals during the first 5 years of life and prevalence of dde. the knowledge of the epidemiology of enamel defects is important in order to provide basic information within a community or country and between countries ; and help in educating population. it is also important since it may contribute to the assessment and monitoring of environmental or systemic factors and for detecting possible etiological factors responsible for the occurrence of the enamel defects. the number of studies being done in this part of the country is scanty hence the present study was undertaken. the present study was done in 10 urban and 10 rural schools of kollam district with a study duration of 6 months with sample size of 2,500 students. a multistage stratified random sampling method was used to select the schools. from the selected schools, per school these children aged 1215 years from kollam district who had not migrated from any other school and had complete permanent dentition without any systemic illness were included in the study. those children who had migrated from other districts after 8 years of age and with mixed dentition were excluded. the subjects were examined seated on a straight back chair and daylight was the source of light. the teeth were examined in a wet state and the buccal surfaces of 10 fully erupted index permanent teeth namely maxillary first premolars, canines and incisors, and mandibular first molars were examined using the modified dde index based on the recommendations made in 1992 by the federation dentaire internationale (fdi) working group on the dde index. the sequence of examination was from maxillary right first premolar to maxillary left first premolar and from mandibular left molar to mandibular right first molar. missing, crowded, unerupted, severely fractured, or grossly carious teeth involving the buccal / labial surface of the teeth were recorded and excluded from the analysis. a sterilized mouth mirror was used to retract the cheeks or lips for better visualization. instruments were disinfected by immersing the mouth mirror in 2% glutaraldehyde for 10 min and then autoclaving it for 20 min by using a portable autoclave. a questionnaire was completed by the parents of the children diagnosed with any of the enamel defects. the modified dde index [table 1 ] the data were subsequently processed and analyzed using the statistical package for the social sciences (spss) statistical software programs. of the 1,255 males examined, 447 (p = 35.6%) were having dde, and of the 1,245 females examined, 352 (p = 28.3%) were having dde. the prevalence of the dde in children aged 12 - 15 years from kollam district was found to be 32%. in the present study where 2,500 students were examined, the prevalence of dde from urban schools was found to be 34.3% and the prevalence of dde from rural schools was found to be 29.6%. the most common tooth affected by the defect was maxillary right lateral incisor (p = 28.6%) and the tooth least affected was maxillary right first premolar (p = 3%) [table 2 ]. the most affected site was the incisal third and the least affected regions were cervical and middle third [table 3 ]. most ddes seen were extending less than one - third of the tooth and the least extended were more than two - third of the tooth [table 4 ]. the most common deformity was demarcated opacities (p = 28.76%) and the least common deformity was combination of diffuse opacities and hypoplasia and combination of demarcated, diffuse opacities, and hypoplasia (p = 0%) [table 5 and graph 2 ]. the prevalence of chronological dde (73.2%) was more than localized dde (p = 19%) followed by generalized dde (p = 6.5%) [table 6 ]. the prevalence of dde due to environmental etiologic factors (p = 95%) is more than due to genetic factors (p = 5%). number of children with the dde tooth wise prevalence of the dde site affected by the dde prevalence of type of deformity in patients with dde prevalence of the type of deformity in patients with dde prevalence of generalized, localized, and chronological dde the most prevalent prenatal factor associated with dde was found to be for mothers with systemic illness during their pregnancy (17.4%) followed by mothers who consumed medication during their pregnancy (10.1%) followed by mothers pregnancy age between 15 years and 20 years (1.5%). there was a very high significant association between mothers with systemic illness during their pregnancy, mothers pregnancy age between 15 years and 20 years, and medication taken during pregnancy by mother and dde. the most prevalent prenatal factor associated with dde was found to be for children with a history of birth weight between 1 kg and 2 kg (10%), followed by premature birth (8.3%), and intubation during prematurity (2%). there was a very high significant association between birth weight, premature birth, intubation during prematurity, and dde. the most prevalent postnatal factor associated with dde was nutritional status (31.7%) followed by systemic illness during the first 5 years of life (23.8%) followed by intake of drugs or chemicals during the first 5 years of life (11.8%) followed by trauma or infection to deciduous teeth (1.8%). there was a very high significant association between nutritional status, systemic illness during first 5 years of life, trauma or infection to deciduous teeth, and dde [table 7 ]. the table shows association between etiological factors and dde the prevalence of molar incisor hypomineralization was found to be 1.6%. among the children with dde, 42.8% of them had decay, missing, or filling [graph 3 ] the developmental of enamel can be defined as any alteration resulting from diverse disturbances during the process of odontogenesis. according to their clinical appearances, dde have been classified as demarcated opacity, diffuse opacity, or hypoplasia. in the present study, the prevalence of dde in children aged 1215 years from 10 urban and 10 rural areas of kollam district was found to be 32%. on international literature search, the least prevalence of dde reported was 6.7% (pasareanu.) among 600 schoolchildren aged between 8 years and 11 years from lasi and the highest prevalence of dde reported was 67.1% (sujak.) among 1,024 school children aged 16 years from the island of penang, malaysia. on national literature search, the least prevalence of dde of 27% was reported (ekanayake. in a study on the prevalence of dental caries and dde conducted in sri lanka. the highest prevalence of dde reported was 66.2% (chauhan. among healthy school children residing in the hills of himachal pradesh, india. epidemiological studies on the prevalence of dde exhibit a wide range of variability in the prevalence rate that may be explained by the specific characteristics and method adopted in the study such as indices used and the criteria used in the examination. the prevalence of dde was found to be higher in schools in urban area (34.32%) than the schools in rural area (29.6%), which was comparable to that of the study by gopalakrishnan. the prevalence was found to be higher in males (35.6%) compared to that in females (28.3%), which was similar to the findings of hussein. in the study by ramesh. (2011), the prevalence of defects was slightly higher in females (90.7%) as compared to males (87.9%), which was contradictory to the present study. the most common tooth affected by the defect was maxillary lateral incisor (p = 28.6%) and the tooth least affected was maxillary right first premolar (p = 3%), which was comparable to that reported by montero. the most affected site was the incisal third (15.4%) and the least affected site was cervical and middle third (0.9%), whereas ruiz. (2013) stated that the defects were located at the middle (40%) and incisal (33%) thirds. (2006) stated that the majority of opacities were involving less than one - third of the tooth surface that was comparable with the present study. (2013) found that the most common type of dde to be demarcated opacity similar to the present study while ramesh. (2013) found that diffuse opacity (25.3%) was found to be the most common defect followed by demarcated opacity (23.1%) and enamel hypoplasia was the least prevalent defect with prevalence of 2.9%. the prevalence of molar incisor hypomineralization was found to be 1.6% in the present study. gomez.) (2012) reported that 17.85% had molar incisor hypomineralization (mih). (2013) reported that the prevalence of mih in their study was found to be 7.7%. (2013) reported significant association between the prevalence of the dde and young maternal age. (2010) found significant association between illness during pregnancy for mother and the prevalence of the dde which they reported as 9.8% and children of mother 's with intake of drug during pregnancy reported to be 13.8% which was comparable to the present study. jacobsen. (2013) reported a prevalence of 34% in children exposed to antiepileptic drugs. in the present study, since the questionnaire - based approach was adopted the type of drug taken could not be assessed. (2010) and arrow in 2010 reported significant association between prematurity of birth and prevalence of dde. the prevalence of the dde of children with low birth weight in the present study was found to be 10%. while funakoshi. (2010) reported a prevalence of 3.7%, the wide variation in the prevalence is attributed to the difference in the study population. in contrary to the present study, cruvinel. (2012) did not find any significant association between the dde and systemic illness during the first 5 years of life. the percentage of children affected by dde with intake of drugs or chemicals during their first 5 years of life was found to be 11.8%. there was a very high significant association between the intake of drugs or chemicals during the first 5 years of life and the prevalence of dde. the percentage of children affected by the dde with low nutritional status was found to be 31.7%. there was a very high significant association between nutritional status and the prevalence of dde comparable to the results of pasareanu. the percentage of children with trauma or infection on deciduous teeth was found to be 8.6%. there was a very high significant association between trauma or infection on deciduous teeth and prevalence of dde, which was same as that of the result of taji. (2012) did not find any significant association between the dde and the systemic illness during the first 5 years of life, low nutritional status and trauma, or infection to deciduous teeth. the prevalence of decayed missing filled teeth (dmft) affected by dde was found to be 42.8%. there was a very high significant association between the dmft and the prevalence of the dde, which was comparable to the findings of idiculla. it was inferred that the poor nutritional factors and systemic illness were the most predominant risk factors for the dde and educating the public to bring down the risk factors help to reduce the prevalence. the dde present an important clinical problem since they may present esthetic problem, sensitivity, and act as predisposing factor for caries. | aim and objectives : to determine the prevalence of developmental defects of enamel in children aged 1215 years in kollam district and to examine the etiological factors associated with the developmental defects of the enamel (dde).materials and methods : a total of 2,500 children from 10 urban and 10 rural schools were examined using modified dde criteria for recording enamel defects. ten index permanent teeth were screened for the dde.results:the overall prevalence of the dde was found to be 32% and the prevalence is higher in urban schools (34.3%) compared to rural schools (29.6%). the most common tooth affected by the defect was maxillary right lateral incisor (p = 28.6%) and the tooth least affected was maxillary right first premolar (p = 3%). the most common deformity was demarcated opacities (p = 28.76%) and the least common deformity was combination of diffuse opacities and hypoplasia and combination of demarcated, diffuse opacities, and hypoplasia (p = 0%). there was a very high significant association between dde and the mothers pregnancy age, illness during pregnancy for mother, medication taken during pregnancy by mother, prematurity of birth, intubation done during prematurity, birth weight, systemic illness during the first 5 years of life, intake of drugs or chemicals during the first 5 years of life, nutritional status, and trauma or infection on deciduous teeth and dental caries.conclusions:the study population showed a prevalence of 32% and very high significant association between perinatal, natal, and postnatal etiological factors. it indicates the need for educating the population about the risk factors for the dde. |
ovarian cancer is the eighth most common type of cancer among women in the world. furthermore, it is the seventh leading cause of cancer - related mortality with a severe impact on socioeconomic and community health, globally.14 compared to other types of gynecologic malignancies, ovarian cancer has the highest mortality rate in developed countries worldwide.57 the american cancer society estimated that 22,240 women were going to be diagnosed with ovarian cancer in the us in 2013, while the number of ovarian cancer - related deaths was estimated to be 14,230.8 moreover, ovarian cancer was estimated to account for 3% of all cancers among women. currently, the lifetime risk of developing ovarian cancer is approximately 1 in 72, while the lifetime risk of dying from ovarian cancer is approximately 1 in 100.8 the international agency for research on cancer estimated that the age - standardized incidence rate (asir) and the age - standardized mortality rate for ovarian cancer in saudi arabia in 2012 was 3.4 per 100,000 women and 2.5 per 100,000 women, respectively.9 furthermore, the registry of the king faisal specialist hospital and research centre (2011) recorded 1510 cases of ovarian cancer with admission to the hospital from 19752011.10 in 2008, data from the saudi cancer registry (scr) suggested that ovarian cancer ranked seventh in cancer incidence among saudi women.11 the asir of ovarian cancer in saudi arabia is low in comparison with other countries in the arabian gulf. for example, in 2012, the reported asir of ovarian cancer for oman, united arab emirates, kuwait, qatar, and bahrain was 10.2, 6.4, 4.7, 4.6, and 4.4 per 100,000 women, respectively.9 despite a lack of data on the geographic distribution of ovarian cancer among women in saudi arabia, we aimed to investigate and describe the crude incidence rate (cir) and asir of ovarian cancer cases by providing an observational descriptive epidemiological analysis of cancer of the ovary while considering spatial / temporal distribution of recorded cases in the scr from 20012008.12 a retrospective descriptive epidemiological study of the ovarian cancer cases and diagnosed in saudi arabia from january 2001december 2008 was performed. the data of reports regarding cancer incidence in saudi arabia are publicly available and easily accessible to be downloaded from the website of scr. the main mission of scr is to gather, register, and provide high standard data for cancer incidence stratified by region, age group, and year of diagnosis. furthermore, the published reports by scr would be available to policy makers, cancer researchers, treating physician, cancer control, and prevention programs. the data source for this study was the scr, a population - based registry established in 1992 by the ministry of health of saudi arabia. the data can not be obtained directly from the scr, but it is published in reports. however, no data was available from 19942000 and the latest published report of the scr was available in 2008. since 2001, the scr has been providing reports on the patterns of cancers in saudi arabia with the primary objective of defining the population - based incidence of the disease. currently, there are comprehensive reports for each of the 13 administrative regions of saudi arabia. each report discusses the number and percentage of cases diagnosed, the cir, and the asir (adjusted by region and year of diagnosis) of cancer from 20012008. the current study utilized the scr reports to gather all the information on ovarian cancer with the aim of presenting the descriptive epidemiology of ovarian cancer in saudi arabia. data were analyzed using the statistical package for the social sciences version 20.0 (ibm corporation, armonk, ny, usa). the descriptive analysis of the epidemiological data of ovarian cancer was performed by calculating the overall percentage of ovarian cancer cases, cir, and the asir adjusted by the age group, region, or year of diagnosis. the analysis of variance (anova) test was performed to determine the effect of the geographical area and the year of diagnosis on cir and asir.13 simple linear regression was used separately with an independent variable (year of diagnosis) to predict the annual cir and the asir of ovarian cancer among saudi women.14 in addition, the poisson regression was conducted to calculate the incidence rate ratio (irr) of ovarian cancer cases for each region of saudi arabia. a total of 991 ovarian cancer cases were recorded in the scr from january 2001december 2008. figure 1a and b shows that there were 101 new ovarian cancer cases in 2001 (3.7% of all cancer cases diagnosed in women ; 95% confidence interval [ci ] : 1.7%5.7%) and 96 in 2002. this indicates a 0.4% decline in cases. by 2005, 136 ovarian cancer cases were reported, corresponding to a 0.2% increase. the number of ovarian cancer cases diagnosed annually remained slightly high, but the percentages were stable in 2005 and 2008 at 3.5%. the average number and percentage of ovarian cancer cases diagnosed from 20012008, adjusted by age group, was calculated using the data from the scr (table 1 and figure 1c). women aged 4559 years were most frequently diagnosed with ovarian cancer followed by those aged 6074 years, representing 31% (38 cases) and 26.0% (33 cases) of the total number of ovarian cancer cases. in contrast, the younger (044 years) and older (75 years and over) groups recorded the lowest overall number and percentage of cases. the cirs per 100,000 women, adjusted for the year of diagnosis, indicated a steady increase in ovarian cancer cases in saudi arabia from 20012008. as shown in table 2 and figure 2a, a cir of 1.3 (95% ci : 0.62.0) ovarian cancer cases per 100,000 women was estimated in 2001 ; and a cir of 1.8 (95% ci : 0.92.7) cases per 100,000 women in 2008. the latter cir was the highest rate observed, but it was not statistically significantly different compared to the other years (f[7, 96]=0.106 ; p>0.05). linear regression analysis suggested that the annual increase of cir for ovarian cancer in saudi arabia can be predicted by the equation 0.9 + (0.07 years), which indicates that the cir of ovarian cancer increased on average by 0.9 cases per 100,000 saudi women per year. table 3 and figure 2b show the overall cir of ovarian cancer per 100,000 women, adjusted for the region of saudi arabia from 20012008. the region of riyadh had the highest overall cir for ovarian cancer at 1.9 (95% ci : 1.62.2) cases per 100,000 women, followed by the regions of baha and makkah at 1.8 (95% ci : 1.02.6) and 1.7 (95% ci : 1.43.0) cases per 100,000 women, respectively. the anova test revealed that the incidence rates of ovarian cancer were significantly higher for these three regions compared to the other regions of saudi arabia (f[12, 91]=4.988 ; p 44 years of age were the most affected by ovarian cancer in the different provinces of saudi arabia. furthermore, the poisson regression showed that the irr of ovarian cancer cases in the regions of riyadh and makkah was more than six times higher than the reference region of jazan. this study also reported the changes in the percentage of ovarian cancer cases diagnosed, cir, and asir from 20012008. the baha region had the largest differences in cir and asir, which were both higher than their predicted values for the years 20012008. these differences in rates suggest that, from 20012008, more women in the region of baha were affected by ovarian cancer than predicted. in contrast, the rates of ovarian cancer decreased in the regions of najran and hail. these regions reported the lowest changes in the respective study outcomes, with a downtrend observed in the cir and asir from 20012008. however, with the exception of the scr reports, only limited information was available on the incidence of ovarian cancer in saudi arabia. this study intended to explore the real pattern of ovarian cancer in different regions of saudi arabia from 20012008. the findings of this study may be useful for researchers and decision makers in the field of medicine and health care administration in saudi arabia as they can provide opportunities for generating hypotheses about the potential risk factors of ovarian cancer in the highest affected regions. furthermore, the findings can be utilized to conduct analytical epidemiological studies that aim to identify the relationship between exposure and disease.13 the availability of data from the scr reports can also encourage further descriptive epidemiological studies of other types of cancers among the saudi population. the epidemiological analysis of the reports by the scr, from 20012008, revealed that the cir and asir of ovarian cancer were increasing slightly. the regions of riyadh, jouf, and asir had the highest overall asir in saudi arabia, while jazan and hail recorded the lowest rates. the irr of ovarian cancer cases indicated a concern in the number of ovarian cancer cases diagnosed in certain regions, such as makkah, riyadh, and the eastern region of saudi arabia. moreover, the region of baha documented the greatest changes in cir and asir from 20012008, while the regions of najran and hail reported the lowest. nonetheless, further analytical studies are required to determine and to explore the potential risk factors of ovarian cancer in saudi arabia. | purposethis study provides descriptive epidemiological data, such as the percentage of cases diagnosed, crude incidence rate (cir), and age - standardized incidence rate (asir) of ovarian cancer in saudi arabia from 20012008.patients and methodsa retrospective descriptive epidemiological analysis of all ovarian cancer cases recorded in the saudi cancer registry (scr) from january 2001december 2008 was performed. the data were analyzed using descriptive statistics, analysis of variance tests, poisson regression, and simple linear modeling.resultsa total of 991 ovarian cancer cases were recorded in the scr from january 2001december 2008. the region of riyadh had the highest overall asir at 3.3 cases per 100,000 women, followed by the jouf and asir regions at 3.13 and 2.96 cases per 100,000 women. however, hail and jazan had the lowest rates at 1.4 and 0.6 cases per 100,000 women, respectively. compared to jazan, the incidence rate ratio for the number of ovarian cancer cases was significantly higher (p<0.001) in the makkah region at 6.4 (95% confidence interval [ci ] : 4.139.83), followed by riyadh at 6.3 (95% ci : 4.109.82), and the eastern region of saudi arabia at 4.52 (95% ci : 2.936.98). the predicted annual cir and asir for ovarian cancer in saudi arabia could be defined by the equations 0.9 + (0.07 years) and 1.71 + (0.09 years), respectively.conclusionwe observed a slight increase in the cirs and asirs for ovarian cancer in saudi arabia from 20012008. riyadh, jouf, and asir had the highest overall asir, while jazan and hail had the lowest rates. makkah, riyadh, and the eastern region of saudi arabia had the highest incidence rate ratio for the number of ovarian cancer cases. further analytical studies are required to determine the potential risk factors of ovarian cancer among saudi women. |
it is a common belief that non - vegetarian diets are superior to the vegetarian diets and people who take them are healthier and stronger. however, there are no scientific data to prove this. the people who are vegetarians take such foods due to ethical, moral, cultural, religious, or political reasons. it is well - established that vegetarian diet fulfils protein requirements and provides all the essential amino acids. similar studies have also been conducted in athletes, long distance runners, and bodybuilders. in spite of that, in our part of country it is customary to give meat and eggs in maximum tolerable quantity to patients recovering from any type of injury and whenever diet rich in calories with high protein is advised. the magnitude of nutritional demand in burn patients is virtually unsurpassed by any other disease process. even those who are otherwise vegetarians are forced to take meat and eggs for expected better recovery. the question arises, is it really necessary for vegetarian burn patients to become non vegetarian ; or remain vegetarian, but constantly live under the threat of doubts regarding complete recovery. to find an answer, we decided to conduct a study comparing outcome in burned patients taking vegetarian and non - vegetarian diets. in india, need for such a study was very important because of strong religious beliefs regarding vegetarian and non - vegetarian food. the study was conducted in the department of plastic surgery of a tertiary referral teaching hospital having an 18 bedded burn unit. the patients suffering thermal burns involving 10 - 50% of total body surface area (tbsa) and between 15 and 50 years of age ; who reported to our centre within 24 hours of sustaining the injury were included in the study over a period of 1 year. among these, patients having pre - existing co morbidities such as respiratory disease, cardiac disease, renal disease, and diabetes mellitus were excluded from the study. a total of 42 patients were included in the study over period of 1 year. the patients were assigned to either of the two groups (group a on vegetarian diet and group b on non - vegetarian diet) depending on their preinjury food habits. thus, 23 patients were included in group a and 19 patients in group b. after initial fluid resuscitation, patients were put on enteral diets within 24 - 48 hours of suffering burn injury. in patients who were unable to meet the requirement we started one - fourth of the requirement at the time of admission and then progressed to full diet using the formula by 4 -5 post burn day. vitamins and minerals were added to both groups according to the weight of the individual (in similar dose). the vegetarian and non - vegetarian constituents of the diet were modified according to the group of the patient using food exchange list. nutritional status assessment was performed by measuring following parameters : serum albumin on day 1, 15, and 30.weight on day 0, 7, 15, 30, and 60. initial weight was taken either before resuscitation or pre burn weight of the patient was recorded.nitrogen balance on day 1, 15, and 30.serum ferritin on day 15 and 30.microbiological investigations on weekly basis (wound swab culture and sensitivity, blood culture and sensitivity, urine culture, and sensitivity). serum albumin on day 1, 15, and 30. weight on day 0, 7, 15, 30, and 60. initial weight was taken either before resuscitation or pre burn weight of the patient was recorded. nitrogen balance on day 1, 15, and 30. microbiological investigations on weekly basis (wound swab culture and sensitivity, blood culture and sensitivity, urine culture, and sensitivity). antibiotics were given only if indicated due to presence of infection based on culture and sensitivity. in group a, age of patients ranged from 15 to 42 years (mean, 25.04 years) with burns ranging from 15% to 50% tbsa (mean, 33.39%). in group b, age ranged from 15 to 50 years (mean, 25.21 years) with burns ranging from 15% to 45% tbsa (mean, 28.94%) as depicted in figure 1. haemoglobin and serum ferritin levels in both groups were comparable [figures 2 and 3 ]. results of serum albumin and total proteins in both the groups were equivocal [figures 4 and 5 ]. nitrogen balance in both groups were negative on 1 day, but was positive on 15 and 30 days [figure 6 ]. 31.8% in group a and 36.2% in group b showed weight loss, which was ranged from 1 to 4 kg at the end of 2 months follow - up. hospital stay ranged from 10 to 60 days (mean, 27.79 days) in group a and ranged from 11 to 60 days (mean, 27.26 days) in group b patients [figure 8 ]. the number of units given were decided by the anaesthetist according to the blood loss. the difference between two groups for wound swab culture (p value - 0.790), blood culture (p value - 0.703), and urine culture (p value - 0.358) were statistically not significant [table 1 ]. fifteen (65.2%) patients in group a and 11 (58%) patients in group b required systemic antibiotics during the stay. however, the difference between the two groups was not statistically significant (p value - 0.627). age and percentage burns in the study group hemoglobin levels during hospital stay serum ferritin levels nitrogen balance during stay weight variation during stay average hospital stay among two groups microbiological profile among both groups during hospital stay 11 patients of group a and 8 patients of group b underwent split skin grafting for their burn wounds. three patients of group a and two patients of group b could not complete the study since they developed sepsis and were put on parenteral nutrition and later on required ventilatory support. one patient of group a had major graft loss and had to be grafted again. effective cost of nutrition for each of the group was also analysed. as both groups primarily differed in the constituent for fulfilling the protein requirement ; the cost of this constituent in rupees per gram protein was calculated and is depicted in figure 9. none of the studies ever conducted have proved that non vegetarian diet is superior to vegetarian diet. in burned patients curreri formula remains the gold standard and was used in our study to calculate the energy requirements of the burned patients. we provided 50 - 60% of total calories from carbohydrates in patients of both groups. it was in accordance with various studies, which showed that only 50 - 60% of total energy should be from carbohydrates. overfeeding has to be avoided because excessive carbohydrate feeding can increase oxygen production, cause hyperglycaemia and fatty liver. the ability of burn patient to handle glucose is limited to 5 mg / kg / day, i.e., for an adult 500 g or 2000 cal / day. we provided 20% calories from proteins in patients of both groups. a high protein feed named as burn feed, prepared in our institute by the department of dietetics b, meat, chicken and eggs were included as replacement for vegetarian sources of proteins. high protein delivery of 1.5 - 3 g / kg body weight / day or 20 - 25% of total energy is the maximum required for burn patients. non protein calorie to nitrogen ratio should be maintained between 150:1 - 100:1 as done in our study. fat should constitute no more than 25 - 30% as energy, but in fact 15 - 20% of non - protein energy as fat is optimal. specific requirements of vitamins and minerals for burn patients have not been established however, provision of zinc and vitamins a and c has been suggested. target calories were achieved by 5 post burn day in all the patients. only those five patients who developed sepsis and complications were supported by parenteral nutrition. gastric bloating was taken care of by advising small quantities of food at more frequent intervals and early mobility. anorexia and aversion to food were major hindering factors which prevented the intake of total calorie and protein intake. patients were offered variations of diet to get maximum co - operation, but basic burn diet regimens as mentioned were followed. all patients on non - vegetarian diet showed aversion to meat, chicken and fish, but with a firm belief of this being a better choice took it with determination. nutritional status of patients was assessed by serial measurements of serum albumin levels, nitrogen balance, serum ferritin levels and weight measurements in our study which are the simplest and easily available indicators of protein anabolism and nutritional status. in addition to that, weekly progress of wound healing, presence of slough and conversion from superficial to deep thickness was recorded. in wounds which were grafted, graft take and graft loss were recorded. despite their limitations, many of these markers of nutritional status when trended or used collectively can help the clinician in monitoring day to day efficacy of diet in burn management. in our study, we found the difference of serum protein and serum albumin levels in both groups was not statistically significant. the nitrogen balance was also comparable in two groups and the difference was not statistically significant. this observation is consistent with various studies, which shows that the protein intake in both groups are found to be adequate. it has been found in various studies that protein intake in vegetarian and vegan diets is only slightly lower than in meat diets and can meet daily requirements for any person including athletes and bodybuilders. studies conducted in the united states, great britain, canada, australia, new zealand and various european countries, confirmed that vegetarian diets provide more than sufficient protein intake as long as a variety of plant sources are available and consumed. in our study, the difference of haemoglobin levels between two groups was found to be clinically insignificant. iron stores are lower in vegetarians because the iron from plant foods is poorly absorbed. the clinical importance of this, if any, is unclear because iron deficiency anaemia rates are found similar in vegetarians and non - vegetarians. major graft loss occurred in this patient of group a and had to be grafted again. meat and chicken need cumbersome cooking whereas cheese can be taken raw and milk and egg by merely boiling. the adequate amount of calorie and protein intake is important rather than the source, whether vegetarian or non - vegetarian. vegetarian food was however cost - effective and more palatable. in our study, we have included patient < 50% burns. further studies can be conducted including patients of larger tbsa and larger number of patients. however, all parameters in burned patients are subjected to so many variations, which can not be rigidly controlled or equalised such as, depth and distribution of burns, blood loss during surgeries, dressing changes, and mobility of the patients. however, vegetarian diet was found to be more palatable and cost - effective in our study. thus, vegetarian diet remains a safe and viable option for the patients suffering from burn injury. | background : the importance of adequate nutritional support in burned patients can not be overemphasised. for adequate long - term compliance by the patients, diet should be formulated in accordance with their pre - burn dietary habits, religious beliefs, and tastes.patients and methods : a study was conducted in 42 consecutive patients suffering from 10% to 50% of 2nd and 3rd degree thermal burns with the aim to compare nutritional status, clinical outcome, and cost - effectiveness of vegetarian and non - vegetarian diets. the patients were divided into two groups depending upon their pre - injury food habits. total calories were calculated by curreri formula. both groups were compared by various biochemical parameters, microbiological investigations, weight, status of wound healing, graft take, and hospital stay and they were followed for at least 60 days postburn.results:the results were comparable in both groups. vegetarian diet was found to be more palatable and cost-effective.conclusion:vegetarian diet is a safe and viable option for the patients suffering from burn injury. the common belief that non - vegetarian diet is superior to vegetarian diet is a myth. |
alzheimer 's disease (ad) is the most common neurodegenerative disease, accounting for more than 50% of all dementia types. its twice more common in females which could be due to increased longevity and sex difference in brain size. women with alzheimer have lower endogenous estrogen level which lead to the hypothesis that estrogen could be neuroprotective. hormone replacement therapy (hrt) has been extensively studied in the past, but results were inconclusive. recent studies suggest that 17 -estradiol based therapies may provide the most beneficial neuroprotective effect. early introduction and prolonged therapy preferably for < 5 years with 17 -estradiol prevents ad. observational studies have examined both hrt and estrogen replacement therapy (ert), in relation to ad. an inverse relationship was seen for the duration of ert and risk for alzheimer. increased risk of alzheimer is seen with younger age at oophorectomy (bilateral or unilateral). these findings suggest that earlier age of surgical menopause increases the risk of cognitive impairment. in women 's health initiative memory study (whims) a total of 4532 women with natural menopause were randomized into a trial comparing conjugated equine estrogen (cee) with medroxyprogesterone (mpa) versus placebo. study revealed that women who received cee with mpa demonstrated greater cognitive decline compared with the placebo group. additional analysis revealed that risk for dementia was doubled for women who received cee with mpa compared with the placebo group. taken together, data from the whims demonstrated a higher incidence of dementia and greater cognitive decline among hormone user. predominant estrogen in premenopausal women is estradiol and its decline is more than estrone in post - menopausal age. cee contains predominantly estrone rather than estradiol ; it also contains other hormones which are not desired. in contrast 17 -estradiol, administration achieved a hormone state close to that observed prior to menopause. studies suggest that 17 -estradiol significantly improved the verbal memory by enhancing verbal information processing and decreased forgetfulness. 17 -estradiol increased the metabolism in receptive language and auditory association area. for getting the desired result, a minimum of 3 months of therapy with no upper cut off is needed.-amyloid and tau proteins are involved in the structural changes that lead to ad pathology, particularly in the hippocampus, medial temporal, parietal and frontal cortical regions. evidence has shown that estrogen particularly 17 -estradiol provides protection against -amyloid induced damage and tau - related changes. neuroimaging outcomes have also been supportive of the benefits of 17 -estradiol, particularly in the brain regions that show preclinical abnormalities in individuals who are at risk for ad. 17 -estradiol, increases the blood flow to the hippocampus and superior temporal gyrus and it also activates left middle, superior frontal cortex and inferior parietal cortex during verbal memory encoding task on functional magnetic resonant imaging.17 -estradiol also reduces the level of amyloid precursor protein (app) through enhanced alpha secretase processing resulting in marked reduction of app - c terminal fragment, amyloid beta and plaque burden. it also enhances the level of transthyretin in the brain, which inhibits the aggregation of amyloid eta into plaque.17 -estradiol also promotes the growth and survival of cholinergic neurons, increases the density of hippocampal neurons and increases the synaptic plasticity in the hippocampus which enhance the short and long term memory. it selectively benefits healthy neurons (healthy cell bias) or when neuronal stress has just started. in degenerating neurons particularly in the presence of apolipoprotein e4, critical period for post - menopausal women during which 17 -estradiol selectively provides a beneficial effect. younger post - menopausal women have higher density of muscarinic receptor than an older one. 17 -estradiol in early post - menopausal women significantly decreased the anticholinergic induced verbal memory task. in cache county study investigators enrolled 1768 women between 1995 and 2006 at the age of menopause who were given hrt particularly17 -estradiol without progesterone. when started within a critical period of 5 years post - menopausal ideally 17 -estradiol should be started within a critical period of 5 years post - menopause in a dose of 50 g / day transdermally. transdermal route byepasses the hepatic metabolism and hence results in steady state plasma concentration of estradiol. it also maintains 1:1 estrone to estradiol ratio which is normally observed in selected phases of the menstrual cycle. favorable results are noted from both short and long term therapy. food and drug administration recommends hrt at lowest possible dose for the shortest duration but in 2013 british and international menopause society put no arbitrary limit on duration of hrt. if symptoms persist, the benefits of hormone therapy outweigh the risk. the major concern with estrogen therapy is increased risk of venous thrombosis, coronary artery disease, breast and endometrial carcinoma, dysmenorrhea, abnormal vaginal bleeding and hypersensitivity. screening of patients who are at risk to develop above complications and mammography should be done in every case. regular annual follow - up for early detection of complication is advisable just like any other hrt. recent studies suggest that estrogen particularly 17 -estradiol has a positive effect in increasing blood flow, stimulating dendrites, protecting against oxidative stress and modulating neurotransmitters. there appears to be a critical window (window of opportunity) between 50 and 60 years of age, ideally within first 5 years of menopause during which estrogens have this positive effect but thereafter the cells deteriorate and estrogen may accelerate cell damage, the so called healthy cell bias. so it is advised to start 17 -estradiol in early menopause for longer duration as an inverse relation exists between hrt and risk for ad. additional research is needed to optimize hormone therapy to delay, prevent and treat ad and to prevent estrogen related complications such as carcinoma and venous thrombosis. | alzheimer disease (ad) is a crippling neurodegenerative disorder. it is more common in females after menopause. estrogen probably has a protective role in cognitive decline. large amount of research has been carried out to see the benefits of hormone replacement therapy with regards to alzheimer still its neuroprotective effect is not established. recent studies suggest a reduced risk of ad and improved cognitive functioning of post - menopausal women who used 17 -estradiol in the critical period. use of 17 -estradiol in young and healthy post - menopausal women yields the maximum benefit when the neurons are intact or neuronal stress has just started. hence intervention in the critical period is key in the prevention or delay of ad in post - menopausal women. |
inflammatory myofibroblastic tumor (imt), the most common pulmonary neoplasm in children, is an uncommon neoplasm of the respiratory tract in adults. it can occur at any site of the body, lung being one of the common sites of origin. although a benign neoplasm on pathology, the clinical presentation of imt is variable. in a majority of the patients, imt presents as a benign, slow - growing tumor without local invasion or distant metastases. however, in upto 5% of the patients it can present as an aggressive locally invasive tumor or a metastatic disease. the symptoms at clinical presentation and radiologic features are non - specific and diagnosis of these tumors requires a high index of suspicion. an accurate diagnosis of these lesions can only be made after histopathology and immunohistochemical analysis. it is important to differentiate imt from its closest histopathologic differential, the igg4-related inflammatory pseudo tumor (ipt) as both share common histopathologic features of prominent fibroblastic / myofibroblastic proliferation in a background of inflammatory cells (plasma cells and lymphocytes). complete surgical resection is considered to be the treatment of choice for imt of the thorax. other modalities which have been tried include bronchoscopic resection (for endotracheal lesions), steroids, alk inhibitor crizotinib and non - steroidal anti - inflammatory drugs (nsaids). in this report, we describe a 28-year - old lady who was diagnosed with an imt during the first trimester of her pregnancy and was managed with surgical resection. a 28-year - old lady presented with dry cough for 2 years and hemoptysis for 1 week. a non - contrast computed tomography scan revealed a left hilar mass lesion causing abrupt cut off of the left lower lobe bronchus [figure 1a ]. the patient was subjected to flexible bronchoscopy, which showed a vascular growth occluding the left lower lobe bronchus. in view of the clinical presentation with massive hemoptysis and the presence of a vascular growth occluding the bronchus, a clinical suspicion of carcinoid tumor was considered. an endobronchial biopsy was obtained using an electrocoagulation - enabled biopsy forceps with the application of electrocoagulation current (40 w for 10 seconds in a monopolar mode). the histopathologic examination of the biopsy specimen showed a tumor composed of a mixture of spindle cells arranged in short and long fascicles [figure 1b ]. these cells were positive for smooth muscle actin (sma) confirming their myofibroblastic nature. immunostaining for anaplastic lymphoma kinase (alk) antigen was positive [figure 1c ] in the tumor cells and the igg4 stain did not highlight excess of igg4-positive plasma cells, thus confirming the diagnosis of an inflammatory myofibroblastic tumor. (a) computed tomography image (axial view) showing a well - defined mass lesion (4.5 3.5 4.4 cm) occluding the left lower lobe bronchus and causing partial obstruction of the left upper lobe bronchus (b) photomicrograph showing spindle - shaped tumor cells arranged in short and long fascicles admixed with few lymphoplasmacytic cells. (h and e, x200) (c) photomicrograph showing granular cytoplasmic positivity for alk (alk immunostaining, x400) (d) gross photograph of left lower lobectomy specimen, showing circumscribed greyish white tumor measuring 4 4 cm and occluding the main bronchus she had ongoing hemoptysis despite being treated with cough suppressants and antifibrinolytic agents (tranexamic acid). the treatment options were discussed with the patient and she opted for surgical resection following a medical termination of pregnancy (mtp). the resected left lower lobectomy specimen showed a circumscribed greyish white tumor (4 4 3 cm) occluding the left lower lobe main bronchus [figure 1d ]. to the best of our knowledge, this is the second case report of imt diagnosed during pregnancy. described the first case of a 21-year - old lady in her first trimester with an imt of the trachea who was managed with endoscopic resection of the tumor. the management of imt of the lung during pregnancy is difficult as the therapeutic options are limited. however, there have been reports of successful use of crizotinib for lung cancer in pregnancy. celecoxib is also considered a category c drug (category d if used beyond 30 weeks of gestation) for use during pregnancy. though steroids can be used safely in pregnancy, use in the first trimester has been associated with an increased risk of cleft lip. other options include endoscopic therapy (for endotracheal lesions) and close monitoring with definitive procedure performed after delivery (for asymptomatic or minimally symptomatic patients). as the index patient had ongoing life - threatening hemoptysis and a large mass with significant extraluminal component, it was decided to proceed for surgery at the earliest. the management of imt during pregnancy depends on the patient 's symptoms, the duration of pregnancy and the location of tumor. | inflammatory myofibroblastic tumors (imt) are uncommon neoplasms of the lung in adults. they constitute less than 1% of all lung neoplasms and usually present as parenchymal masses. diagnosis requires a high index of suspicion. they are characterized by spindle - shaped tumor cells (fibroblasts / myofibroblasts) in a background of lymphoplasmacytic infiltrate. about 50% of the tumors harbor an alk gene rearrangement. they have to be differentiated from inflammatory pseudotumors (ipt), which show increased number of igg4 plasma cells on immunostaining and are negative for anaplastic lymphoma kinase (alk) protein. herein, we present a case of a 28-year old female who presented with hemoptysis and was diagnosed with an imt of lung in the first trimester of pregnancy. we have not only reviewed the occurrence of imt during pregnancy but also discuss the management options for imt during pregnancy. |
surgical site infections (ssis), a significant postoperative complication, can lead to considerable patient morbidity and mortality. preventing postoperative infection is an essential factor in improving the results of surgical procedures. the benefits of preoperative antibiotics, which reduce bacterial contamination during clean - contaminated operations such as cholecystectomy and contaminated operations, are well known. at present, the incidence of infectious complications after lc is significantly lower compared with infections with open cholecystectomy. the use of prophylactic antibiotics as a means of preventing ssis is still controversial in elective lc, which has a low risk for infectious complications. many authors believe that antibiotic prophylaxis may not be necessary in low - risk patients undergoing elective lc. on the contrary, many other surgeons still use and recommend the administration of prophylactic antibiotics. mcguckin documented in their chart review that 79% of patients who had undergone lc were given prophylactic antibiotics preoperatively. because of its broad - spectrum antimicrobial effect, low toxicity, and low cost, the single - dose use of cefazolin has been recommended for patients undergoing open cholecystectomy and other biliary surgery, and it was recommended by the united states centers for disease control and prevention as the general principle for prevention of postoperative surgical site infection (ssi) in open cholecystectomy. because controversy still surrounds the routine use of prophylactic antibiotics in elective lc, this prospective study was conducted according to the united states centers for disease control and prevention guidelines with the aim of investigating the necessity and testing the efficacy of single - dose cefazolin as a prophylactic antibiotic to prevent postoperative infection complications in low - risk patients undergoing lc. a double - blind, prospective, randomized, controlled study comparing the prophylactic use of cefazolin (group 1) vs placebo (group 2) was performed between january 1, 2005 and december 31, 2007 in our clinic. in all, 343 lcs were performed in this period. following institutional research ethics committee approval of the protocol, 150 patients undergoing elective laparoscopic cholecystectomy were selected as suitable for the study protocol. exclusion criteria were patients older than 60 years ; antibiotic intake in the 7 days prior to surgery ; acute cholecystitis in the 6 months prior to the procedure ; evidence of cholangitis and/or obstructive jaundice and biliary pancreatitis ; regular corticosteroid therapy ; pregnancy or lactation ; previous biliary tract surgery or previous endoscopic retrograde cholangiopancreatography ; presence of american society of anesthesiologists classification (asa) higher than score ii ; evidence of diabetes mellitus ; body mass index higher than 30 ; and conversion to open cholecystectomy. cefazolin (1 g) or normal saline was administered intravenously by the anesthesiologist during induction of anesthesia. one of 2 packages containing either cefazolin or placebo was chosen randomly by the anesthesiologist for each patient. the package was opened by the anesthesiologist, and the type of solution administered (cefazolin or placebo) was recorded. the medical staff and after induction of anesthesia, the skin was disinfected with a 10% solution of povidone - iodine. a 10-mm trocar was placed in the epigastrium, a 5-mm trocar on the midclavicular line, and a 5-mm trocar in the right flank in line with the gallbladder fundus. if gallbladder rupture and spill of bile or stones was encountered, spilled stones were retrieved whenever possible, and local peritoneal lavage with 1000cc saline was performed. the gallbladder was removed through the umbilical port, always with the use of an endobag that was prepared using a sterile glove without talc. a sample of bile was removed by suction with a sterile syringe from the gallbladder immediately after its removal and sent to the microbiology laboratory for bacterial detection. a nasogastric tube was positioned during the induction of anesthesia and removed at the end of surgery. the postoperative course was monitored, and any incidents, such as fever, infection of the trocar site, or intraabdominal collection of pus, were recorded. after discharge from the hospital, the patients underwent weekly clinical and laboratory postoperative monitoring for ssi for a 30-day period. the following data were collected for each patient : age, sex, body weight, asa classification, blood biochemical data, times of antibiotic administration, time of skin incision, gallbladder rupture, bile and/or spillage, positive bile culture, type of fascia and skin closure, duration of surgery, conversion to open cholecystectomy, bile and wound culture results, length of hospital stay, and number of septic complications. postoperative superficial or deep incisional soft tissue ssi and intraabdominal abscess (organ / space ssi) were defined according to published criteria. if local signs of inflammation or a pus collection were present, bacteriological swabs were taken from the wound site. statistical analyses were performed using chi - square test, fisher 's exact test, and mann - whitney u - test. chicago, il, usa) with a p - value 0.05). the positive culture rates of bile in patients in groups 1 and 2 were 13.2% and 17.1%, respectively, and the difference was not significant. ssi occurred in 1 of 22 patients who had a positive bile culture and in 4 of 122 patients who had a negative bile culture (p>0.05). wound culture was negative in 2 of 4 patients who had negative bile culture and in the 1 patient who had a positive bile culture. in the other 2 of 4 patients who had a negative bile culture, coagulase - negative staphylococcus spp. and the average rate of ssis for lc has been reported in the literature to be between 0.4% and 6.3%, which is lower than rates reported for open cholecystectomy. our data show that the incidence of ssi in patients was 3.47% for the total study group, 4.41% for group 1, and 2.63% for group 2, and in agreement with previous studies, there was no significant difference in infection rate between the groups. although antibiotic prophylaxis is considered standard protocol in open cholecystectomy as a means of reducing the incidence of infectious complications, its use is still debated in lc. lippert and gastinger performed a prospective population - based multicenter study to evaluate antimicrobial prophylaxis in laparoscopic and conventional cholecystectomy. they recommend that both laparoscopic and conventional open cholecystectomy be performed with adequate perioperative antimicrobial prophylaxis because patients receiving prophylaxis fared significantly better than those with no prophylaxis in terms of the rate of postoperative infections, other complications, reoperation, and mortality. similarly, several other studies conclude that the use of prophylactic antibiotics in lc leads to a significant decrease in infectious complications. conversely, many prospective studies have suggested that antibiotic prophylaxis is probably not required in elective lc, because the infection rate of lc is already low and the use of prophylactic antibiotics does not decrease the incidence of ssis and other postoperative infection complications. goldfaden and birkmeyer reviewed the perioperative treatment of patients with laparoscopic interventions in 98 randomized studies on antibiotic prophylaxis since 1990. they stated that routine antibiotic use in lc is likely unnecessary for low - risk patients. catarci recommended in their review that a multicenter prospective randomized controlled clinical trial be designed to find a definitive answer to the question of routine antibiotic prophylaxis in lc, because there are no randomized trials with a sufficient number of cases to avoid a type ii error. the theoretical number of patients necessary for such a trial was estimated to include more than 3500 patients. the positive bile culture rate in patients with gallbladder stones has ranged between 10% and 42.5% in previous studies. perforation during gallbladder surgery is attributed to traction, grasping, dissection, and removal of the gallbladder and occurs in 11% to 35% of lcs. the effect of positive bacteria culture and bile or stone spilling due to perioperative gallbladder perforation on the occurrence of ssi infections is still controversial. shindholimath found that bactibilia was the most important predictor of wound infection in low - risk patients undergoing elective lc. they recommended prophylactic antibiotics to reduce the incidence of wound infection because it might not be possible to determine which patients have bactibilia by routine investigation. uchiyama reported that preoperative prophylaxis with 0.5 g sulbactam and 0.5 g cefoperazone significantly reduces the positive bile culture, resulting in a significant reduction in complications induced by postoperative infections. however, dervisoglou stated that both positive bile culture and intraoperative gallbladder rupture were strongly associated with development of ssi. additionally, they determined that ssi was caused by exactly the same pathogens found in intraoperative cultures. in our study, we detected that the overall rate of ssi did not correlate with the presence of bacteria in the bile or gallbladder rupture. many other studies have also indicated that ssis are not related to bile culture, rupture of the gallbladder, or spillage of gallbladder stones or bile. on the other hand, there was no relationship between the perioperative organisms isolated from the bile and the subsequent ssi in our study or in other studies. tocchi in their study identified different microorganisms in pus culture in 10/11 bile culture positive patients which had septic complications. however, harling detected that all organisms isolated from the wound sites of patients who developed ssis were skin commensals. they did not find a significant difference in rate of ssi between prophylactic antibiotic usage and mechanical isolation of the gallbladder with endobag during extraction from the abdomen. hamzaoglu also reported that the umbilical flora and the bile were not the source of the ssis after laparoscopic surgery. also in our study, possibly commensal coagulase - negative staphylococcus spp were isolated from 1 of 2 patients with negative bile culture, and staphylococcus aureus was isolated from the other patient in the pus culture. the wound culture was negative in the other 2 patients with negative and 1 patient with positive bile cultures. we believe that mechanical isolation of the gallbladder from the umbilical wound with routine endobag use in all patients while extracting the gallbladder from the abdomen and local peritoneal irrigation in the presence of gallbladder perforation is effective in preventing contamination with possibly infected bile. the preparation of an endobag from gloves without talc is an easily prepared, effective, and cost - effective method that we routinely use. in our study, despite prevention of contamination with the endobag of the umbilical port area, the fact that all ssi infections occurred at the umbilical port area leads us to believe that entering with the first port open procedure and extracting the gallbladder from the umbilical port leads to more tissue trauma than that sustained in the other wounds at the port entrances. based on our data, perioperative gallbladder perforation and possible positive bile culture do not increase ssi rates. local irrigation in the case of perforation and the mechanical isolation of the gallbladder while extracting it from the abdomen with an endobag in the case of bactobilia seem to be adequate to prevent contamination. antibiotic prophylaxis does not seem to affect the incidence of ssis and is not necessary for elective lc in low - risk patients. | background and objectives : elective laparoscopic cholecystectomy has a low risk for infectious complications, but many surgeons still use prophylactic antibiotics. the aim of this prospective study was to investigate the necessity and test the efficacy of prophylactic antibiotics on postoperative infection complications in low - risk patients undergoing laparoscopic cholecystectomy.methods:low-risk patients were randomly placed into 2 groups : 68 patients (group 1) received cefazolin 1 g intravenously after induction of anesthesia, and 76 patients (group 2) were not given prophylactic antibiotics. in both groups, septic complications were recorded and compared.results:positive bile culture and gallbladder rupture did not significantly increase the rate of surgical site infections. in group 1, there were 3 (4.41%) cases of wound infection, 3 (4.41%) cases of pulmonary infections, and 1 (1.47%) case of urinary tract infection. in group 2, there were 2 (2.63%) cases of wound infection, 2 (2.63%) case of pulmonary infections, and 3 (3.95%) cases of urinary tract infection. no significant difference existed in the complication rates.conclusions:based on our data, the use of prophylactic antibiotics does not decrease the rate of postoperative infection complications and surgical - site infections and is not necessary in low - risk patients undergoing laparoscopic cholecystectomy. |
cardiovascular disease is the leading cause of mortality in the united states and more than 80% of these deaths occur in those 65 years or older., advanced age is the most important predictor of poor outcome in patients with acute coronary syndromes and with the projected increase in the elderly population worldwide, it is important to understand the risk factors associated with aging in order to identify appropriate treatments. the aged heart has an increased sensitivity and decreased tolerance to ischemia / reperfusion (i / r) injury., aging also results in a loss of the heart 's ability to respond to cardioprotective stimuli such as pharmacological and ischemic preconditioning., it is believed that the aging cardiomyocyte develops a reduced tolerance to stress because of decreased mitochondrial function, increased oxidative stress, changes in gene expression, and aberrant cell signaling. lipid rafts are subcellular microdomains of the plasma membrane that consist of lipid clusters enriched in cholesterol and sphingolipids, in which particular proteins are concentrated. caveolae, a subset of lipid rafts, have a unique flask - like structure that is generated by caveolin and cavin proteins. caveolae and caveolins act to coordinate cellular signaling events in many cells, including those of the cardiovascular system. age - associated alterations in the composition of lipid rafts and caveolae could affect a variety of cellular functions and have been linked to diseases, such as alzheimer 's, atherosclerosis, and diabetes, which are more prevalent in the elderly population. the focus of this review will be on the role of lipids rafts and caveolae in the progression of age related cardiovascular diseases, such as heart failure, myocardial infarction, and pulmonary hypertension. caveolae, or little caves, are cholesterol and sphingo- lipid - enriched invaginations of the plasma membrane. caveolin-1 (cav-1) is required for caveolae formation in many non - muscle cell types and for the membrane localization of cav-2, while cav-3 is required for caveolae formation in striated (skeletal and cardiac) and smooth muscle cells. caveolins function as chaperones and scaffolds for signaling molecules in caveolae by providing temporal and spatial regulation of signal transduction. through their caveolin scaffolding domain (csd), caveolins not only anchor other proteins in caveolae, but also inhibit or enhance that protein 's signaling capacity. caveolins have a variety of other functions including vesicular transport, cholesterol and calcium homeostasis, and t - tubule formation., the generation of mice lacking the caveolin genes has made it possible to better understand the significance of each caveolin isoform and its contribution to whole animal physiology and human disease. while all caveolin null mice [cav-1, cav-2, cav-3 single knockouts and cav-1/3 double knockout (ko) ] are viable and fertile, each displays a unique phenotype. cav-1 ko mice have a complete loss of caveolae in non - muscle cells and a 90% loss of cav-2 expression due to protein degradation., thus, cav-1/3 double ko mice lack expression of all three caveolin proteins and do not form visible caveolae in any cell type. caveolin protein expression and association with caveolae is decreased with age and studies of the caveolin ko mice support the hypothesis that the loss of caveolin protein causes an aged phenotype., caveolins are, therefore, potential therapeutic targets in the treatment of age related disorders, such as cardiovascular disease. despite advances in diagnosis and treatment, heart failure remains one of the most common, costly, and deadly diseases. the prevalence of the disease is significantly increased in people over 65 years of age and arises as a consequence of abnormal cardiac structure, function, rhythm, or conduction. ventricular dysfunction resulting from myocardial infarction and/or hypertension is frequently the cause of heart failure and each will be discussed in more detail in the sections below. although cav-3 is the predominant caveolin in cardiac myocytes, cav-1 ko mice develop a severe cardiomyopathy, which appears to contribute to their significantly shortened life span. at 24 months, cav-1 ko mice show a 50% reduction in viability, with a major decline between 27 and 65 weeks of age. the hearts of cav-1 ko mice are structurally and functionally abnormal at 24 months of age. imaging and functional studies revealed that cav-1 ko hearts have significantly enlarged ventricular chambers, abnormal ventricular wall thickness, hypertrophy, and decreased contractility., these defects are worse by 12 months of age, which shows that loss of cav-1 causes a progressive cardiomyopathy. increased fibrosis in the heart has also been reported in cav-1 ko mice. since cav-1 is expressed in both cardiac myocytes and fibroblasts, it is not clear if this decline with aging is due to altered function of a specific cardiac cell (i.e., myocyte death and replacement by fibroblasts vs. increased activation of fibroblasts). as signaling scaffolds, caveolins have been shown to interact with receptor tyrosine kinases, src family tyrosine kinases, endothelial nitric oxide synthase (enos), and members of the p42/44 map kinase cascade (mek1/2 and erk1/2). at the molecular level, loss of cav-1 is thought to cause cardiac hypertrophy through disruption of signaling pathways. cav-1 is thought to negatively regulate the p42/44 map kinase cascade in cardiac fibroblasts and activation of the p42/44 mapk pathway in cardiac myocytes can drive cardiac hypertrophy. in cav-1 ko mice the p42/44 map kinase cascade is hyperactivated, which contributes to the cardiac defects seen in these mice. this finding suggests that the major effect of cav-1 loss may come from its function in the supporting cells of the heart (fibroblasts and endothelium). however, cav-1 is also found in the cardiac myocyte and its role in these cells can not be dismissed. in heart failure, -adrenergic signaling is reduced and this may lead to further deterioration of heart function since, with decreased contraction, the heart is unable to meet its needs. in cav-1 ko hearts, the levels of cyclic amp (camp), an important second messenger of -adrenergic signaling, and atp are decreased. cav-3 ko mice develop a progressive cardiomyopathy marked by significant hypertrophy, dilation, and reduced fractional shortening. similar to cav-1 ko mice, the p42/44 map kinase cascade is hyperactivated in the hearts of cav-3 ko mice and may partly account for these cardiac defects. similarly, a mutation in cav-3 has been found in familial hypertrophic cardiomyopathy. in cardiac myocytes, there is an extensive remodeling of the t - tubule network and this change may contribute to abnormal calcium handling. the disorganization of t - tubules in cav-3 ko skeletal muscle suggests that cardiac myocytes may display a similar phenotype that contributes to the progression of heart failure. the activity of dozens of ion channel proteins and membrane transporters generate the flux of ions across the sarcolemmal membrane, which is responsible for activation and contraction. many of these ion channels reside in caveolae and abnormalities in the function or regulation of these channels result in arrhythmias that can lead to heart failure. therefore, the loss of cav-3 and caveolae in the cardiac myocyte could contribute to arrhythmias as another potential source of heart failure. in fact, mutations in cav-3 have been found in the inherited arrhythmogenic syndrome, long - qt congenital syndrome. mice with a constitutive overexpression of 1-adenosine receptor demonstrate cardiac dilation and decreased left ventricular function. in this model of heart failure, cav-3 expression is decreased (cav-1 and cav-2 are unaffected) and there is a direct relationship between cav-3 expression and ventricular dysfunction. consistent with the findings that loss of cav-3 contributes to heart failure, overexpression of cav-3 has been shown to attenuate heart failure. caveolae contain natriuretic peptide receptors such as atrial natriuretic peptide (anp), which is closely associated with cav-3. overexpression of cav-3 attenuates cardiac hypertrophy, at least partially, by increasing natriuretic peptide expression and signaling. these results suggest cav-3 may not only be a marker for heart failure, but also a therapeutic target. this combined loss of caveolin protein does not produce any new phenotypes and only cardiac defects were exacerbated compared to single knockout animals. cav-1/3 double ko mice display a severe cardiomyopathy by two months of age in which left ventricular wall thickness, hypertrophy, ventricular dilation, and decreased fractional shortening that is more pronounced. the cardiac tissue also shows signs of interstitial inflammation, fibrosis, and myocyte necrosis. these results clearly establish a role for both cav-1 and cav-3 in maintaining normal cardiac function and further suggest that a loss of caveolin proteins with age could be a major contributing factor to the development of cardiovascular disease. ischemic heart disease and myocardial infarction result in loss of blood flow and oxygen to part of the heart, which causes damage and reduced cardiac function. changes in caveolin protein expression have been identified in renal, cerebral, hindlimb, and myocardial ischemia. cerebral artery occlusion causes a marked increase in endothelial cav-1 and cav-1 ko mice display a significant increase in the volume of cerebral infarcts, as compared with wild - type mice. in this model, cav-1 ko mice displayed decreased proliferation of endothelial cells and increased apoptosis. in a model of myocardial infarction, cav-1 ko mice subjected to left anterior descending coronary artery ligation display reduced survival and despite similar infarct sizes, cav-1 ko mice showed reduced cardiac function compared to wild - type mice. mechanistically, it appears that caveolins provide protection from ischemic injury through scaffolding signaling molecules and promoting cell survival., specifically, reduced cav-1 expression alters -adrenergic signaling through decreased camp production and protein kinase a phosphorylation, which alters cardiac contractility. ischemic preconditioning is an innate protective mechanism by which brief episodes of ischemia protect the heart from the damaging effects of prolonged i / r injury. in addition to sublethal ischemia, several pharmaceutical agents, such as opioids and volatile anesthetics, produce a similar preconditioning protective effect. preconditioning activates a complex signaling cascade known as the reperfusion injury salvage kinase pathway and many of these signaling molecules associate with caveolins in caveolae. caveolae play a pivotal role in the generation of survival signals in cardioprotection and ischemic preconditioning increases the number of caveolae present in the sarcolemmal membrane. preconditioning the heart in the presence of methyl--cyclodextrin abolishes the cardioprotective effects leading to decreased ventricular performance, increased myocardial infract size and cardiomyocyte apoptosis., additionally, aging is associated with a decrease in the protective effects of preconditioning in animal models and human patients.,, the loss of caveolae and caveolins with age may, therefore, contribute to the loss of signal regulation and protection.,, caveolins also function in preconditioning with isoflurane, a volatile anesthetic, and opioids. cardiac myocytes are protected from simulated i / r injury when incubated with -opioid receptor agonists. this effect is lost in the presence of methyl--cyclodextrin, indicating a critical role for caveolae. isoflurane - induced cardioprotection is dependent on the presence of caveolae, and is lost in both cav-1 and cav-3 ko mice. perhaps the most interesting evidence for the role of caveolins in cardioprotection comes from analysis of a transgenic mouse with myocyte specific overexpression of cav-3 (cav-3 oe). cav-3 oe mice subjected to i / r injury have significantly improved functional recovery, reduced infarct size and apoptosis relative to wild - type mice. this innate cardioprotection in cav-3 oe mice is similar to that seen in wild - type mice undergoing ischemic preconditioning. the precise molecular mechanism by which caveolins protect the heart from i / r injury remains to be determined. however, a generalized pathway has emerged in which preconditioning stimuli enhance release of agonists of one or more g - protein coupled receptor families (opioid, adenosine, bradykinin) to enhance activity of pro - survival kinase pathways (including pi3-k / akt and mapks) and inhibit activity of pro - death pathways (gsk3). the ability of caveolin to preserve such signaling as the heart ages may be a key feature of caveolin mediated stress adaptation. severe pulmonary hypertension (ph) is characterized by a progressive increase in pulmonary vascular resistance and vascular remodeling leading to right heart failure and early death. the ability of the right ventricle to respond to the increased vascular resistance is influenced by several factors, including age. disruption of the function of cav-1 in the vascular system can have profound effects on cardiac function and may serve as a marker for pulmonary hypertension, which would allow earlier detection and improved treatment strategies. cav-1 ko mice show lung abnormalities, with reduced alveolar spaces, increased wall thickening, fibrosis, and hypercellularity., direct measurement of pulmonary artery pressure and histological analysis revealed that cav-1 ko mice exhibit pulmonary hypertension, which may contribute to the right ventricle hypertrophy seen in these mice. additionally, lungs from cav-1 ko mice exhibit increased pulmonary vascular resistance associated with pulmonary vascular remodeling including increased medial thickness and muscularization of distal pulmonary vessels, which is an underlying feature of pulmonary vascular remodeling in ph. reduced expression of cav-1 in the lungs after myocardial infarction has been suggested to initiate the development of ph and lung structural remodeling. patients with severe pulmonary hypertension show reduced levels of cav-1 in total lung tissue and pulmonary vascular endothelial cells. these findings suggest an antihypertensive function of cav-1 with respect to the pulmonary vascular architecture. cav-1 ko mice display hyperactivation of enos and altered vasoconstriction and vasorelaxation responses of isolated aortic rings.,,, disruption of enos activity in cav-1 ko mice is likely involved in the development of the observed cardiopulmonary pathologies. enos is a critical mediator of cardiovascular homeostasis regulating multiple physiologic and pathophysiologic processes including vascular tone, vascular remodeling, platelet aggregation, and angiogenesis. through interactions with cav-1, direct interaction of enos with cav-1 significantly inhibits its enzyme activity in vitro and infusion of a membrane permeable cav-1 csd peptide performs similarly in vivo. the hyperactivation of enos in cav-1 ko mice results in increased no production, which is believed to cause the majority of the cardiovascular defects. double ko of cav-1 and enos (nos3) completely blocks the increase in no production in lung tissue. these double ko mice which do not develop pulmonary hypertension, have normal pulmonary vasculature and lung morphology, and no right ventricle hypertrophy. the adverse effects of cav-1 deficiency on lung architecture and pulmonary hypertension can also be reversed by inhibition of enos by l - name. interestingly, endothelial specific expression of cav-1 in cav-1 ko mice rescues the development of pulmonary and vascular defects. these data indicate that loss of cav-1 in the vasculature can have profound effects on cardiac function and the progression of pulmonary hypertension. the effects of cav-1 may function primarily through enos, but regulation of other pathways, including p42/44 and angiotensin - converting enzyme, can also influence hypercellularity and endothelial dysfunction contributing to pulmonary hypertension. cav-1 expression is ubiquitous and evidence suggests that its role in pulmonary hypertension may be cell specific. pulmonary hypertension may be characterized by a reduction of cav-1 in lung tissue, but cav-1 levels in vascular smooth muscle are elevated., human patients with pulmonary hypertension exhibit increased cav-1 levels and also display altered ca regulation. enhanced influx of ca in vascular smooth muscle causes hyper - proliferation of vascular smooth muscle, which leads to hypertrophy of the pulmonary vascular wall and increased pulmonary vascular resistance., additionally, cav-1 functions within caveolae to inhibit cell proliferation, but during increased mechanical stress, cav-1 translocates out of caveolae to other sites within the plasma membrane of smooth muscle cells. endothelial damage sustained during early stages of pulmonary hypertension may result in exposure of smooth muscle cells to high cyclic pressure, thus resulting in enhanced expression of cav-1 and cell proliferation. this data suggests that cav-1 may have a cell specific dual role in pulmonary hypertension, similar to how caveolin can both promote and inhibit tumor progression in cancer., two stages of cav-1 expression change promote pulmonary hypertension ; an initial loss of cav-1 in endothelial cells which is followed by subsequent increased expression of cav-1 in smooth muscle cells. conversely, there is evidence suggesting an increase in cav-1 in endothelial cells can have detrimental effects. increased cav-1 levels are associated with pathologic angiogenesis, breakdown of the blood - brain barrier, cardiac hypertrophy, and diabetic angiopathy. this must be considered when selecting therapeutic strategies. given the evidence presented here, it can be hypothesized that the aged population is vulnerable to cardiovascular disease as a consequence of lost caveolin expression. regulation or overexpression of caveolins promote signaling via enhanced receptor - effector coupling or enhanced receptor affinity. this improved signaling could prevent aging and the development of disease, or rescue impaired cardiac function. for example, in the failing heart, a left ventricular assist device initiates structural and functional changes through mechanical unloading. implantation of a left ventricular assist device in human patients increases expression of cav-1 and causes redistribution of cav-3. these data suggest that enhanced caveolin expression in the failing heart in response to mechanical unloading leads to reverse remodeling. mild exercise causes an increase in cav-3 expression, which may partially account for these results. similarly, exercise training of spontaneously hypertensive rats, which undergo pathologic hypertrophy as a consequence of hypertension, produces a reversal of this phenotype with increased expression of cav-3. infusion of a caveolin scaffolding domain (csd) peptide has cardioprotective effects against i / r injury. administration of the cav-1 csd has also been shown to effectively prevent the development of pulmonary hypertension and right ventricular hypertrophy. hyperactivation of enos caused by loss of cav-1 leads to an imbalance with vascular tetrahydrobiopterin (bh4), which acts as an essential enos cofactor. donation of bh4 to cav-1 ko mice causes improvement of both systolic and diastolic heart function and marked improvement of the impaired lung phenotype. statins were originally developed as lipid lowering drugs, but have additionally been shown to have vasoprotective properties through no dependent pathways., studies also suggest that statins may be useful in the treatment of pulmonary hypertension. fluvastatin causes dissociation of enos and cav-1, which has been shown to increase enos activity and improve endothelial function. this suggests that decreasing cav-1 expression in some cases could potentially be therapeutic as well. in contrast, the importance of caveolin / caveolae in cardiac systems suggest that though there may be beneficial effects of statins on multiple cellular systems that impact proper cardiovascular function, muscle specific myopathies associated with statin use may be of concern possibly through disruption of cardiac myocyte caveolae. from the evidence presented above, caveolins are central players in a number of cardiovascular diseases, and caveolin based therapeutics may be a powerful approach for addressing age - associated cardiovascular pathologies. certain limitations need to be addressed as future therapy is considered : as caveolins are ubiquitously expressed, can they be targeted to specific cell types ; current therapies are limited to delivery of gene, is it possible to develop pharmacologic agents that enhance the expression of caveolin ; what is the role of caveolin as a protein vs. its property as a structural protein for caveolae in modulating disease phenotypes ; is the regulation of caveolin expression transcriptional or translational with age, and can secondary processes, such as microrna or transcription factors, be potential targets ? | it is estimated that the elderly (> 65 years of age) will increase from 13%14% to 25% by 2035. if this trend continues, > 50% of the united states population and more than two billion people worldwide will be aged in the next 50 years. aged individuals face formidable challenges to their health, as aging is associated with a myriad of diseases. cardiovascular disease is the leading cause of morbidity and mortality in the united states with > 50% of mortality attributed to coronary artery disease and > 80% of these deaths occurring in those age 65 and older. therefore, age is an important predictor of cardiovascular disease. the efficiency of youth is built upon cellular signaling scaffolds that provide tight and coordinated signaling. lipid rafts are one such scaffold of which caveolae are a subset. in this review, we consider the importance of caveolae in common cardiovascular diseases of the aged and as potential therapeutic targets. we specifically address the role of caveolin in heart failure, myocardial ischemia, and pulmonary hypertension. |
myocardial hypertrophy is a typical response of the heart to changes in the conditions of cardiac functioning including the development of chronic pathologies. disturbances in the energy metabolism at the cellular level represent the important stage of myocardial dysfunction development in hypertrophic cardiomyopathies. the atp formation and utilization processes coordinated work of these ensembles directly depends on a state and structural changes of the cell itself. these ideas agree well with data suggesting that morphological changes in the cardiomyocytes seen in ischemic and diabetic cardiomyopathies accompany disturbances in the energy metabolism [4, 5 ]. normally, energy metabolism in the cardiomyocytes is supplied via the fatty acid and, partially, glucose oxidation. development of the heart failure of ischemic genesis predisposes to oxidation of glucose which is more efficient in regard to oxygen consumption. in diabetes, fatty acids are the main substrate due to, first of all, the abnormal insulin - sensitive transport of glucose. in this, massive permeation of fatty acids into mitochondria leads to excessive accumulation of acetyl coenzyme a in them. however, the experimental and clinical data about the predominance of one or another type of energy production in chronic cardiac pathologies are controversial. one of the adaptation myocardial reactions to the stimulated functioning in the adverse conditions is an upregulation of the apoptosis. development of the microvessels can bridge the gap between oxygen demand and oxygen supply in the cardiac myocytes. the aim of the experimental study was to perform a comparative analysis of chronic changes of the angiogenesis and energy metabolism in case of the postinfarction and diabetic heart damage. the animals were assigned to four groups with 5 animals in each group : intact animals (control group or group 1) ; animals with myocardial infarction modeling (group 2) ; animals with induced diabetes (group 3) ; and animals with combined pathology such as myocardial infarction plus diabetes (group 4). to model myocardial infarction, the etherized animals received ligature in the upper third of the left descending coronary artery. diabetes mellitus was induced by a single intraperitoneal infusion of streptozotocin (sigma, usa) in a dose of 60 mg / kg dissolved in 0.01 mol / l citrate buffer (ph 4.5). manifestation of diabetes mellitus was verified if there were 3-fold to 4.5-fold increase in the concentration of blood glucose, body weight reduction, polyuria, and polydipsia. when the combined pathology was modeled, the animals received a single intraperitoneal infusion of streptozotocin in a dose of 60 mg / kg 4 weeks after coronary occlusion. follow - up studies were performed 2 weeks after the streptozotocin infusion (total 6 weeks of the pathology duration)., the fragments of the preserved left ventricular myocardium adjacent to scar tissue were dissected from the animals of group 2 and group 4. anatomically similar samples were dissected from the hearts of the animals from group 1 and group 3. the histological processing in alcohols and paraffin embedding were performed according to the standard methods. to evaluate the status of myocardial vascularization, the histological sections (4 to 5 m), the number of capillaries was counted in 10 random visual fields with 100-fold magnification and then the arithmetic mean was calculated. microscopic examinations of the sections at the light - optical level were performed by using the binocular microscope axiolab a1 (carl zeiss, germany). for the histoenzymatic study, the sections were prepared from rat frozen left ventricular myocardium by using the cryostat tp - om-5 - 01 (russia). histochemical reactions were estimated to elucidate activity of succinate dehydrogenase, ec 1.3.99.1, (sdh) and l - lactate dehydrogenase, ec 1.1.1.27, (ldh). quantitative assessment of enzymatic activity was done by using the luminescent microscope lyumam - iz (russia) in transmitted light with the wave length of 546 nm in the areas of 0.5 m. myocardial homogenate and mitochondria from cardiomyocytes of rats were obtained by the standard procedure of differential centrifugation in sucrose media containing : 250 mm sucrose, 10 mm edta, and 10 mm hepes at ph 7.4. mitochondria were suspended and stored in the solution containing 250 mm sucrose and 10 mm hepes at ph 7.4. contents of free fatty acids (ffa) in blood plasma, myocardial homogenate, and mitochondrial suspension were determined enzymatically by the photoelectric colorimeter analyzer. commercial kits of diasys diagnostic systems (germany) were used to determine ffa contents. the studied parameters were not normally distributed (shapiro - wilk test, p > 0.05). due to small group sizes and their independence of each other, statistical significance of values between groups was evaluated by using the nonparametric mann - whitney rank test. within groups, statistical significance of differences was estimated via the wilcoxon test for dependent variables. single - factor analysis of variance was used for statistical processing of the morphological data. you can see that an increase in ffa amount in blood plasma was found for all studied pathologies. however, no statistically significant differences were found between groups of animals with modeled pathologies. this result obviously suggests that cardiomyocytes preserve effective control over ffa uptake in conditions of the modeled pathologies. essentially different result was obtained upon an estimation of ffa content in the mitochondrial suspension. the highest fatty acid content was found in group 3 (diabetes) : the fatty acid level exceeded control values by more than 5 times. in group 2 (myocardial infarction), there was 3-fold increase in fatty acid content. however, in group of combined pathologies (group 4) this value was close to control (table 1). it is quite possible that the found differences are characteristic of the differences in the mitochondrial capabilities to oxidize fatty acids in the studied pathologies. the results of the histological enzymatic study showed that cardiomyocyte lactate dehydrogenase (ldh) activity was significantly lower in rats with monopathologies (group 2 and group 3) than in control animals (table 2). however, ldh activity in the combination of pathologies was not statistically different relative to control group. analysis of data showed a decrease in succinate dehydrogenase (sdh) activity by 1.5- and 2.6-times in group 2 and group 3, respectively, compared to group 1 (control) (table 2). on the contrary, in group 4 (combined pathology), sdh activity was significantly higher by 2.5 to 3.5 times compared with monopathologies and control, respectively. the results of our study suggested that the efficiency of both aerobic and anaerobic energy production pathways decreased in diseased myocardium when the pathological conditions developed as monopathology, separately from each other. however, when combined pathologies developed, the level of aerobic processes remained preserved whereas the aerobic pathway of atp production was even elevated. obtained data can be explained by the adaptive remodeling of energy metabolism. in particular, the process of energy production in cardiac mitochondria can switch to succinate dehydrogenase pathway of oxidation. according to the existing knowledge, the very succinate is subjected to active oxidation in tissues when there is lack of oxygen [911 ]. augmentation of aerobic processes can be related to an activation of neoangiogenesis and, correspondingly, to increase in myocardial blood flow. in our study, its value statistically significantly exceeded the corresponding values in all other groups. in animals with combined pathologies, capillary density was significantly higher than in control animals and animals with myocardial infarction (figure 1). therefore, the development of both pathologies and their combinations triggers the statistically significant increase in myocardial vascularization. however, angiogenesis occurs via the activation of different endogenous mechanisms in group 2 and group 3. the less pronounced myocardial vascularization in myocardial infarction in case of monopathology is probably caused by the local vascular bed damage and, correspondently, by formation of collaterals bypassing damaged part. multiorgan microangiopathies are characteristic of type 1 diabetes mellitus ; this probably triggers the central mechanisms of angiogenesis and provides full - scale reaction. indirect confirmation of this hypothesis is the fact that upregulation of neoangiogenesis is not seen in combined pathologies compared to diabetes. moreover, capillary density is higher in case of combined pathology than in myocardial infarction alone (figure 1). obtained data suggest that increase in myocardial capillary network plays an important role in upregulation of anaerobic energy production in combined pathology. however, we did not obtain evidence for a direct association between sdh activity and capillary density. despite high density of capillaries, ldh activity was low in both groups of monopathologies, but high in case of combined pathology. there is no reason to claim that changes in intensity of aerobic and anaerobic processes of energy production in cardiac mitochondria are represented only by improvement of myocardial blood flow. the performed analysis suggests about normal activity of the aerobic and anaerobic processes of energy production in cardiomyocytes in case of the given combined pathologies. this aspect perhaps allows to simultaneously use glucose and fatty acids in a sufficiently enough way to provide the best heart functioning [4, 12 ]. less pronounced abnormality of the energy metabolism is probably related to adaptive activation of sdh. this hypothesis does not contradict data suggesting that the changes in sdh activity as well as the changes in succinate formation and oxidation provide adaptation and preservation of the cellular energy production under stress [9, 10 ]. | comparative study of changes in myocardial activity of lactate dehydrogenase (ldh), succinate dehydrogenase (sdh), and capillary density distribution in the experimental models of diabetic and postinfarction damage of rat heart was performed. data showed that decrease in ldh and sdh activities was observed in both pathologies which can suggest abnormal processes of glycolysis and oxidative phosphorylation in cardiac mitochondria. activity of ldh and sdh in combined pathologies was comparative with the corresponding values of these parameters in control group. the authors hypothesize that these differences can be caused by specifics of myocardial vascularization. the results of the study showed that an increase in capillary density was found in all groups of rats with pathologies compared with control group. however, no significant differences in the intensity of angiogenesis processes were found between groups with pathologies. |
flame retardants (frs) are common additives applied to consumer products and construction materials. as additives they are not chemically bound to these components and over time they migrate out. due to high octanol - air partitioning coefficients (log koa), many frs are ubiquitous and abundant in house dust. several studies have now found that house dust ingestion is one of the most important exposure pathways for frs, especially for infants and toddlers. in current risk assessments, 100% bioaccessibility is often assumed when evaluating human exposure to frs in house dust. however, previous studies have shown that hydrophobic organic compounds sorbed to organic matter (e.g., soil and sediment), can not be completely released from these matrices and subsequently absorbed into the gastrointestinal tract. therefore, understanding the bioaccessibility of frs in dust is of great significance for adequate risk evaluations. though some studies have examined the bioaccessibility (i.e., the fraction which can desorb from the ingested matrix) of polybrominated diphenyl ethers (pbdes) in house dust, no information is available for several new alternate frs such as organophosphate frs (opfrs) and firemaster550 (fm550), which are the major replacements for the pentabde commercial formulations following their phase - out. frs may be sorbed to organic material in the dust following partitioning from air, or be associated with debris in dust that results from product weathering (e.g., foam or plastic weathering). using microscopic forensic methods, webster. found that a strong bromine signal in a dust sample was associated with particles / debris suggestive of weathered commercial products. in another study using dust collected from a gymnasium, an abundance of polyurethane foam (puf) debris was observed using scanning electron microscopy, again suggesting the fr signatures in dust may be associated with weathered materials / polymers. infants or toddlers also tend to mouth toys or furniture made of puf impregnated with frs and phthalate additives. therefore, it is important to evaluate the bioaccessibility of frs from both dust particles and puf material. in vitro physiologically based extraction methods are predominately used in bioaccessibility studies due to the advantages of reduced cost / time and animal use. various models have been proposed and most of them use simulated digestive fluid to sequentially, or continuously extract contaminants within a matrix during relevant physiological residence times. however, absorption in the gastro - intestinal tract is a dynamic process and traditional in vitro methods might underestimate the bioaccessibility due to a failure to maintain and consider the concentration gradient, especially for very hydrophobic compounds. recent studies found that sorption - assisted bioaccessible extractions using a silicon rod, an activated carbon impregnated silicon rod, or a c18 membrane as an infinite sink could increase the bioaccessibility of polycyclic aromatic hydrocarbons (pahs) in sediment, and were more comparable with in vivo studies. however, activated carbon impregnated silicon rods may may not be amenable to back - extraction, and silicon rods need large surface areas (2 m) to ensure a high sorption capacity. tenax beads (ta), a porous polymer with desirable adsorption / desorption characteristics, have been validated as an effective material to evaluate bioaccessibilty due to their strong sorption capacity, easy back extraction and ability to be recycled. a 6 h ta extraction was widely used to predict the bioavailability of pahs and pesticides in soils and sediments. therefore, it seems feasible to predict that ta may also predict bioaccessibility of frs in dust ; however, to the authors knowledge, no studies have investigated this potential application. another knowledge gap is the stability of frs in digestive fluids. due to stricter environmental health regulations and intense public awareness, the fr market has moved from persistent frs like pbdes to less persistent frs such as opfrs and fm550. these latter chemicals have more labile functional groups, such as phosphate and carboxylic esters, which could be vulnerable to nucleophilic reactions. the half - times of these esters in water (ph 7) vary significantly between chemicals, which could range from several minutes to years. furthermore, hydrolysis reactions might also be different across the dynamic ph conditions along the human digestive system. thus, it is important to examine the stability of these less stable frs in digestive fluids. given these issues, the primary objectives of this study were to (1) develop an effective ta - sorption assisted in vitro physiologically based bioaccessible extraction method ; (2) examine the bioaccessibility of opfrs, fm550, and pbdes in house dust samples ; (3) test the bioaccessibility of several frs in puf and its dependence on particle size ; and (4) investigate the stability of several less persistent frs, for example, opfrs and components of fm550, in simulated digestive fluids. in our preliminary experiment, we found that the bile salts could precipitate the ta, which was probably due to the decreased tension force caused by the biodetergent. therefore, ta could not be used in the bioaccessible experiment until good separation from dust after incubation was achieved. in this study, ta beads (6080 mesh, supelco) were first cleaned by sonication using acetone : hexane (1:1, v / v) and sieved through 100 mesh (152 m, usa standard testing sieve) to minimize the lost of small beads during the experiment. an insert was designed for use in this study (see supporting information (si) figure s1) that would contain the ta. a 100 mesh stainless steel material (small parts, logansport, in) was cut into 11 7 cm (length width) dimensions. the mesh was rolled and fixed at the ends with 0.4 mm copper wire. a half - cut 4 ml glass vial after loading ta (0.5 g), another precleaned 4 ml vial was used as a cap on the other side. after incubation, the ta insert was rinsed thoroughly with deionized water to remove any dust residue attached to the ta, which were then collected in an aluminum weight boat. most of the dust remained in the colon fluid due to its smaller size (5). measured bioaccessibility of opfrs, eh - tbb, beh - tebp, and pbdes in srm2585 sieved to 5. one resistance is the organic barrier (e.g, ta) with lipid like properties while the second is an aqueous barrier. at low log kow values the lipid barrier provides the dominant resistance for absorption, which is independent of log kow. however, as log kow increases, diffusive transport through the aqueous barrier becomes increasingly limited and the absorption decreases. the relationship between kow and bioaccessibility observed in this study was very similar to a previous in vivo study in cows assessing the pbde absorption potential. reported that the bioaccessibility of individual pbde congeners did not appear to be correlated with degree of bromination for tri- to hepta - bdes using an in vitro method. and ruby. did not find any correlation between bioaccessibility of polychlorinated dibenzodioxins / furan (pcdd / fs) in soil and the degree of chlorination using a different in vitro method. in this study, the bioaccessibility of pbdes in srm2585 using the traditional incubation method (without ta) did not show a decreasing trend for tri- to hepta - bdes either. relationship between bioaccessibility and hydrophobicity (log kow) of the frs in 17 house dust samples. dust samples from different sampling years were also analyzed to examine the potential effect of aging on the bioaccessibility of frs, as previous studies have shown that aging could reduce the bioaccessibility of hydrophobic compounds in soils and sediments. in these previous studies, the mobility of hydrophobic compounds sorbed to organic matter was reduced with aging of the soils / sediments. as shown in si figure s6, the bioaccessibility of dust samples collected in 2006 (n = 7) was significantly different from those collected in 2010 (n = 10 ; p 5. in vivo absorption might not occur in the intestine if no active transportation was involved. overall, the hydrolysis experiment in this study revealed that more labile frs can undergo transformation in the digestive fluid prior to absorption and should be considered in further exposure / risk assessment. however, it should be noted that this in vitro method only included a limited set of enzymes and also did not include other factors such as microfloral activity, which could also affect absorption and fate in the gastrointestinal tract. in this study, an in vitro bioaccessibility test using ta as an adsorption sink was developed and our results are very comparable to an in vivo study, confirming several recent findings that the use of an infinite sink is necessary in evaluating in vitro bioaccessibility. the bioaccessibility of frs varied greatly between compounds / matrices and it should be considered in future exposure and risk assessments, particularly for highly hydrophobic compounds (log kow > 5). the results of this study also showed that less hydrophobic frs such as opfrs are quite bioaccessible in both dust and in puf, suggesting a higher risk of exposure for those compounds, despite the fact that they are generally less bioaccumulative. to date, the stability of the ingested organic contaminants in the gastro - intestinal tract was not well studied, although metabolism after absorption has been a focus of several studies. in this study, eh - tbb was readily transformed into tbba in the presence of intestinal enzymes. due to the abundance and variety of enzymes present in the digestive fluid, more labile organic contaminants with low ingestion rates this may be an important consideration as chemical industries shift from producing persistent / bioaccumulative chemicals to less persistent forms. however, it should be noted that the in vitro artificial digestive fluid in the present study does not completely resemble human or rodent digestive fluids. furthermore, the role of microflora on the metabolism and absorption should also be considered in future studies. | exposure to house dust is a significant source of exposure to flame retardant chemicals (frs), particularly in the us. given the high exposure there is a need to understand the bioaccessibility of frs from dust. in this study, tenax beads (ta) encapsulated within a stainless steel insert were used as an adsorption sink to estimate the dynamic absorption of a suite of frs commonly detected in indoor dust samples (n = 17), and from a few polyurethane foam samples for comparison. organophosphate flame retardants (opfrs) had the highest estimated bioaccessibility (80%) compared to brominated compounds (e.g., pbdes), and values generally decreased with increasing log kow, with 6) in foam was much less than that in house dust, and increasing bioaccessibility was observed with decreasing particle size. in addition, we examined the stability of more labile frs containing ester groups (e.g., opfrs and 2-ethylhexyl - tetrabromo - benzoate (eh - tbb)) in a mock - digestive fluid matrix. no significant changes in the opfr concentrations were observed in this fluid ; however, eh - tbb was found to readily hydrolyze to tetrabromobenzoic acid (tbba) in the intestinal fluid in the presence of lipases. in conclusion, our study demonstrates that the bioaccessibility and stability of frs following ingestion varies by chemical and sample matrix and thus should be considered in exposure assessments. |
aquaculture is the farming of aquatic organisms by intervention in the rearing process to enhance production and private ownership of the stock being cultivated. compared to fishing, this activity allows a selective increase in the production of species used for human consumption, industry or sport fishing. due to overfishing of wild populations, aquaculture 's contribution to world food production, raw materials for industrial and pharmaceutical use, and aquatic organisms for stocking or ornamental trade has increased dramatically in recent decades. the report world aquaculture 2012 found that global production of fish from aquaculture grew more than 30 percent between 2006 and 2011, from 47.3 million tons to 63.6 million tons. it also forecasts that by 2012 more than 50 percent of the world 's food fish consumption will come from aquaculture, so it is expected to overtaking capture fisheries as a source of edible fish. this growth rate is due to several factors : (1) many fisheries have reached their maximum sustainable exploitation, (2) consumer concerns about security and safety of their food, (3) the market demand for high - quality, healthy, low - calorie, and high - protein aquatic products, and (4) aquatic breeding makes only a minimum contribution to carbon dioxide emission [1, 2 ]. aquaculture has a long history, originating at least in the year 475 b.c. in china, but became important in the late nineteen - forties, since the methods of aquaculture could be used to restock the waters as a complement to natural spawning. nowadays, aquaculture is a lucrative industry [2, 4 ]. however, the intensification of aquaculture practices requires cultivation at high densities, which has caused significant damage to the environment due to discharges of concentrated organic wastes, that deplete dissolved oxygen in ponds, giving rise to toxic metabolites (such as hydrogen sulfide, methane, ammonia, and nitrites), that often are responsible for mortality. additionally, aquaculture has appropriated of water bodies used for recreational purpose, and sometimes makes a water 's waste because this natural resource is not reused in extensive aquaculture systems [5, 6 ]. moreover, under these conditions of intensive production, aquatic species are subjected to high - stress conditions, increasing the incidence of diseases and causing a decrease in productivity. outbreaks of viral, bacterial, and fungal infections have caused devastating economic losses worldwide, that is, china reported disease - associated losses of $ 750 million in 1993, while india reported $ 210 million losses from 1995 to 1996. added to this, significant stock mortality has been reported due to poor environmental conditions on farms, unbalanced nutrition, generation of toxins, and genetic factors. in recent decades, prevention and control of animal diseases has focused on the use of chemical additives and veterinary medicines, especially antibiotics, which generate significant risks to public health by promoting the selection, propagation, and persistence of bacterial - resistant strains [911 ]. resistance to antibiotics is acquired in two ways : chromosomal mutation or acquisition of plasmids. chromosomal mutations are not transferred laterally to other bacteria but resistance plasmids can be transferred very rapidly, producing a high percentage of pathogenic bacteria that develop mediated plasmid - resistance in a short period of time. for example, transfer of multidrug resistance occurred in ecuador during the cholera epizootic happened between 1991 and 1994, which originated from shrimp farm workers. although the original epidemic strain vibrio cholerae 01 was susceptible to 12 antimicrobial agents tested, on the coast of ecuador it acquired multidrug resistance due to transfer of resistance genes from other vibrio species that were pathogenic to shrimp. probiotic is a relatively new term which is used to name microorganisms that are associated with the beneficial effects for the host. kozasa made the first empirical application of probiotics in aquaculture, considering the benefits exerted by the use of probiotics on humans and poultry. he used spores of bacillus toyoi as feed additive to increase the growth rate of yellow tail, seriola quinqueradiata. in 1991, porubcan [15, 16 ] documented the use of bacillus spp, to test its ability to increase productivity of penaeus monodon farming and to improve water quality by decreasing the concentrations of ammonia and nitrite. in order to avoid or reduce the use of certain antimicrobials, biological control was tested, described as the use of natural enemies to reduce the damage caused by harmful organisms. strictly speaking, a probiotic should not be classified as a biological control agent because it is not necessarily a natural enemy of the pathogen. moriarty determined the ability of bacillus spp. to decrease the proportion of vibrio spp. in shrimp ponds, especially in sediments. further studies have stressed probiotics ability to stimulate appetite, improve absorption of nutrients, and strengthen the host immune system [19, 20 ]. the aim of this paper is to present an updated concept of probiotics, current uses in aquaculture, commercial presentations, and a final approach to prospect applications in this area. comes from greek pro and bios meaning prolife, having different meanings over the years. in 1905, elie metchnikoff was the first to describe the positive role played by some bacteria among farmers who consumed pathogen - containing milk and that reliance on gut microbes for food makes it possible to take steps to change the flora of our bodies and to replace harmful microbes by beneficial microbes. however, the term probiotic was introduced until 1965 by lilly and stillwell as a modification of the original word probiotika. it was used to describe substances produced by a microorganism that prolong the logarithmic growth phase in other species. later, sperti modified the concept of tissue extracts that stimulate microbial growth. the first use of the term to describe a microbial feed / food supplement was by parker in 1974. he defined it as organisms and substances that contribute to intestinal microbial balance. fuller expanded the definition to live microbial food supplement that benefits the host (human or animal) by improving the microbial balance of the body and said that it would be effective in a range of extreme temperatures and salinity variations. afterwards, it was suggested that probiotics were monocultures or mixed cultures of microorganisms applied to animals or humans, that benefit the host by improving properties of indigenous microflora. in 1998, guarner and schaafsma assumed that probiotics are live microorganisms which, when consumed in adequate amounts, confer health benefits to the host. gatesoupe in 1999, defined them as microbial cells administered in a certain way, which reaches the gastrointestinal tract and remain alive with the aim of improving health. in the same year, studies were carried out on the inhibition of pathogens using probiotics, this expanded the definition to live microbial supplement which benefits the host by improving its microbial balance. knowledge of probiotics has increased, currently it is known that these microorganisms have an antimicrobial effect through modifying the intestinal microbiota, secreting antibacterial substances (bacteriocins and organic acids), competing with pathogens to prevent their adhesion to the intestine, competing for nutrients necessary for pathogen survival, and producing an antitoxin effect. probiotics are also capable of modulating the immune system, regulating allergic response of the body, and reducing proliferation of cancer in mammals. because of this, in fact, terms such as friendly bacteria, friendly, or healthy are commonly used to describe probiotics. for many years, studies focused on microorganisms characteristic from intestinal microbiota, and the term probiotic was mainly restricted to gram - positive lactic acid bacteria, particularly representative of the genera bifidobacterium, lactobacillus, and streptococcus. in contrast to terrestrial animals, gastrointestinal microbiota of aquatic species is particularly dependent on the external environment due to the flow of water passing through the digestive tract. thus, the majority of bacteria are transient in the intestine, due to constant intake of water and food, together with microorganisms present in them. although in the gastrointestinal tract (git) of aquatic animals have been reported potentially pathogenic bacteria such as salmonella, listeria, and escherichia coli, probiotic bacteria and other microorganisms have also been identified. these include gram - positive bacteria such as bacillus, carnobacterium, enterococcus, and several species of lactobacillus ; gram - negative, facultative anaerobic such as vibrio and pseudomonas, as well as certain fungi, yeasts, and algae of the genera debaryomyces, saccharomyces, and tetraselmis, respectively [20, 67, 68 ]. due to the increasing interest of probiotics in aquaculture, moriarty proposed extending the definition of these to living microbial additives that benefit the health of hydrobionts and therefore increase productivity. a more general and common concept of probiotic is one or more microorganisms with beneficial effects for the host, able to persist in the digestive tract because of its tolerance to acid and bile salts. although the use of probiotics in aquaculture is relatively recent, interest in them has increased due to their potential in disease control ; however, gradually other applications have been proposed (summarized in table 1), that will be addressed in detail later. the interest in probiotics as an environmentally friendly alternative is increasing and its application is both empirical and scientific. according to soccol., the global market for probiotic ingredients, supplements and foods, reached us $ 15,900 million in 2008 and is projected to increase to us $ 19,600 million in 2013, representing an annual growth rate of 4.3%. at present, there are several commercial preparations of probiotics that contain one or more live microorganisms, which have been introduced to improve the cultivation of aquatic organisms. probiotics can be used as a food additive added directly to the culture tank or mixed with food. apart from laboratory preparation of bacteria, some commercially available products are now available. one of the first evaluations of commercial products focussed on a bacterial preparation called biostart that is derived from bacillus isolates. it was used during the production of cultured catfish studying the effect of inoculum concentration. in 1998, moriarty reported that the use of commercial probiotic strains of bacillus spp. increased the quality and viability of pond - raised shrimp. meanwhile, chang and liu evaluated the effect of enterococcus faecium sf68 and bacillus toyoi isolates present in cernivet lbc and toyocerin, respectively, to decrease the mortality of the european eel because of the edwardsielosis, ensuring greater efficiency with e. faecium sf68. it is relevant to note that e. faecium has long been known as a probiotic for humans, whereas b. toyoi has been used with terrestrial animals. moreover, a b. subtilis strain combined with hydrolytic enzymes to produce biogen, was used to supplement the feed of oreochromis niloticus, obtaining significant increases in productivity. the human probiotic, lactobacillus rhamnosus atcc (american type culture collection, rockville, md, usa), was used in rainbow trout for 51 days to reduce mortality by aeromonas salmonicida, the causative agent of the fish disease furunculosis mortality was reduced from 52.6 to 18.9% when 10 cells g were administered with feed, when probiotic dose was increased to 10 cells g of feed the mortality reached 46.3%. apparently, abasali and mohamad increased the gonadosomatic index and the production of fingerlings in females of reproductive age, using mixed cultures consisting of l. acidophilus, l. casei, e. faecium, and b. thermophilum (primalac). studies with penaeus vannamei showed that using mixed cultures of probiotics increases survival, feed conversion, and the final production of farmed shrimp. meanwhile, taoka. used alchem poseidon and alchem korea co and wonju korea co, which have mixed cultures of bacteria (bacillus subtilis, lactobacillus acidophilus, and clostridium butyricum) and yeast (saccharomyces cerevisiae), enhanced nonspecific immune parameters of tilapia oreochromis niloticus such as lysozyme activity, migration onf neutrophils, and plasma bactericidal activity, resulting in improvement of resistance to edwardsiella tarda infection. previous studies in humans and land animals using prebiotics (nondigestible ingredients of the diet that stimulates the growth of microorganisms) showed their ability to stimulate the activity of probiotic bacteria in the colon [73, 74 ]. some commercial aquaculture products included prebiotics in their formulation, such as mannans, glucans, and yucca extract that further increase the beneficial effects of the product [75, 76 ]. currently, commercial products are available in liquid or powder presentations, and various technologies have been developed for improvement. on the case of fermentation processes, the interest has been focused on optimizing the fermentation conditions to increase the viability and functionality of probiotics, improving performance. generally, the production is carried out in batch cultures due to the difficulty of industrial scale operation of continuous systems. more recently, systems have been developed for immobilization of probiotics, especially using microencapsulation. microbial cells at high density are encapsulated in a colloidal matrix using alginate, chitosan, carboxymethylcellulose, or pectin to physically and chemically protect the microorganisms. the methods commonly used for microencapsulation of probiotics are the emulsion, extrusion, spray drying, and adhesion to starch. focused on the application to aquaculture, rosas. have effectively encapsulated cells of shewanella putrefaciens in calcium alginate, demonstrating the survival of encapsulated probiotic cells through the gastrointestinal tract of sole (solea senegalensis). encapsulation in alginate matrices protects bacteria from low ph and digestive enzymes ; this protection helps to release the probiotic into the intestine without any significant damage. however, conditions for reconstitution of these preparations such as temperature, degree of hydration, and osmolarity of the solution are vital to ensure the viability of bacteria. it is important to emphasize that these products must provide a health benefit to the host ; for this, it is necessary that contained microorganisms have the ability to survive storage conditions, and after that in the digestive tract of aquatic species, remaining viable and stable, and finally improving production. according to the opinion of the producers, these preparations are safe to use and effective in preserving the health of aquatic animals. the need for sustainable aquaculture has promoted research into the use of probiotics on aquatic organisms. the initial interest was focused on their use as growth promoters and to improve the health of animals ; however, new areas have been found, such as their effect on reproduction or stress tolerance, although this requires a more scientific development. probiotics have been used in aquaculture to increase the growth of cultivated species, in reality it is not known whether these products increase the appetite, or if, by their nature, improve digestibility. some people are inclined to think that it could be both factors ; furthermore, it would be important to determine whether probiotics actually taste good for aquaculture species. according to balczar., probiotic microorganisms are able to colonize gastrointestinal tract when administered over a long period of time because they have a higher multiplication rate than the rate of expulsion, so as probiotics constantly added to fish cultures, they adhere to the intestinal mucosa of them, developing and exercising their multiple benefits. this also depends on factors such as hydrobionts species, body temperature, enzyme levels, genetic resistance, and water quality. the effect of probiotics has been tested on phytoplankton (microalgae), which forms the basis of aquatic food chains, due to its nutrient - producing photosynthetic machinery that in most cases, higher organisms are unable to synthesize such is the case of polyunsaturated fatty acids and vitamins. within groups of microalgae used in aquaculture, which have proven to be a good live food ; however, production has limitations due to the complexity of their nutritional requirements. assessed the growth of vibrio alginolyticus c7b probiotic in the presence of the microalgae chaetoceros muelleri, proving that these organisms can be grown together to achieve high density and fed to shrimp. rotifers are indispensable as the first live feed for larvae of most cultured aquatic species, due to their small size they are more accessible to larvae, for example, the nauplii of brine shrimp, which is a very common live feed used lactic acid bacteria to increase the growth of the rotifer brachionus plicatilis and obtained best results with the addition of lactococcus casei ssp. diet of nile tilapia (oreochromis niloticus) was amended with a probiotic streptococcus strain, increasing significantly the content of crude protein and crude lipid in the fish, also weight has increased from 0.154 g to 6.164 g in 9 weeks of culture. due to the commercial importance of this species, the effect of supplementing diet with probiotics produced an increase of 115.3% when commercial formulation was used at a concentration of 2%. examples of growth improvement of ornamental fishes include swordtail (xiphophorus helleri, x. maculatus) and guppy, (poecilia reticulate, p. sphenops), their feed was supplemented with bacillus subtilis and streptomyces, finding significative increases in growth and survival of xiphophorus and poecilia after 90 and 50 days of administration, respectively [27, 86 ]. probiotics also have been tested successfully in shellfish culture. macey and coyne isolated two yeasts and one bacterial strain (designated ss1, ay1, and sy9, resp.) from the digestive tract of abalone (haliotis midae). a diet was formulated with a mixture of the three putative probiotics. each probiont was added to the feed to achieve a final concentration of approximately 10 cells g of dry feed. the growth rate of small (20 mm) and large (67 mm) abalone was improved by 8% and 34%, respectively, in eight - month cultures. furthermore, abalones supplemented with probiotics had a survival rate of 62% to the pathogenic bacterium vibrio anguillarum compared to 25% survival of untreated animals. antibiotics were used for a long time in aquaculture to prevent diseases in the crop. however, this caused various problems such as the presence of antibiotic residues in animal tissues, the generation of bacterial resistance mechanisms, as well as an imbalance in the gastrointestinal microbiota of aquatic species, which affected their health. in fact, the european union has regulated the use of antibiotics in organisms for human consumption. today, consumers demand natural products, free of additives such as antibiotics ; moreover, there is a tendency for preventing diseases rather than treating them. thus, the use of probiotics is a viable alternative for the inhibition of pathogens and disease control in aquaculture species. probiotic microorganisms have the ability to release chemical substances with bactericidal or bacteriostatic effect on pathogenic bacteria that are in the intestine of the host, thus constituting a barrier against the proliferation of opportunistic pathogens. in general, the antibacterial effect is due to one or more of the following factors : production of antibiotics, bacteriocins, siderophores, enzymes (lysozymes, proteases) and/or hydrogen peroxide, as well as alteration of the intestinal ph due to the generation of organic acids. taoka. showed that viable probiotics administered to tilapia oreochromis niloticus, increased non - specific immune response, determined by parameters such as lysozyme activity, neutrophile migration, and bactericidal activity, which improved the resistance of fish to infection by edwardsiella tarda. in turn, robertson. administered alive to rainbow trout and atlantic salmon, demonstrating in vitro antagonism against known fish pathogens : aeromonas hydrophila, a. salmonicida, flavobacterium psychrophilum, photobacterium damselae, and vibrio species. there is also evidence on the effect of dead probiotic cultures consisting on a mixture of vibrio fluvialis a3 - 47s, aeromonas hydrophila a3 - 51, and carnobacterium ba211, in the control of furunculosis in rainbow trout. for this specific case, the number of leukocytes was greater than with live cells, in fact, the data suggest that cellular immunity more than humoral factors was involved in the benefits of these preparations of inactivated bacterial cells. in the case of shrimp, studies have focused on the evaluation of probiotics such as bacillus cereus, paenibacillus polymyxa, and pseudomonas sp. ps-102 as biocontrol agents against pathogens of various vibrio species [92, 93 ]. probiotic strains isolated from the gastrointestinal tract of clownfish (amphiprion percula) have been used to inactivate several pathogens such as aeromonas hydrophila and vibrio alginolyticus among others. it has been observed that probiotics in vivo generate a density such that allow the production of antimicrobial metabolites therefore, the bacteria isolated from adult clownfish have the potential to colonize the intestinal mucus and therefore can be used as prophylactic agent and/or therapeutic [94, 95 ]. furthermore, it has been found that concentrations of 10 to 10 cells g of probiotic promote the development of healthy microbiota in the gastrointestinal tract of ornamental fishes from the genera poecilia and xiphophorus, decreasing the amount of heterotrophic microorganisms. gmez. reported the use of vibrio alginolyticus strains as probiotics to increase survival and growth of white shrimp (litopenaeus vannamei), also by using probiotics in ecuadorian shrimp hatcheries, production increased by 35%, while with the use of antimicrobials it decreased by 94%. a study has suggested that probiotics have a beneficial effect on the digestive processes of aquatic animals because probiotic strains synthesize extracellular enzymes such as proteases, amylases, and lipases as well as provide growth factors such as vitamins, fatty acids, and aminoacids. therefore, nutrients are absorbed more efficiently when the feed is supplemented with probiotics. in this sense, probiotics have been used in edible fishes, as in the case of larvae of european bass (dicentrarchus labrax). it has been reported that the probiotic yeast debaryomyces hansenii hf1 has the ability to produce spermine and spermidine, two polyamines involved in the differentiation and maturation of the gastrointestinal tract in mammals. in addition this yeast secretes amylase and trypsin, enzymes that aid digestion in sea bass larvae. studies in juvenile common dentex dentex dentex l. showed that when diet is supplemented with 0.5 g of bacillus cereus strain e kg of food, increased fish growth due to more efficient use of the food. in the case of rainbow trout, similar results were obtained using b. subtilis, b. licheniformis, and enterococcus faecium, when these probiotics were provided for 10 weeks along with the diet of fish. in some trials, diet of european sea bass larvae (dicentrarchus labrax) has supplemented with probiotic yeast (saccharomyces cerevisiae strain x2180), assessing fish growth and activity and expression of antioxidative key enzymes (catalase, glutathione peroxidase, and superoxide dismutase), finding differences in enzyme activity and gene expression patterns between probiotic supplemented and nonsupplemented treatments, which was attributed to the presence of the yeast. in white shrimp litopenaeus vannamei boone and fenneropenaeus indicus, various strains of bacillus have been used as probiotics to increase apparent digestibility of dry matter, crude protein, and phosphorus. results showed higher sizes when the diet is supplemented with 50 g of probiotic kg of food. other research has suggested the importance of managing the probiotic in all ontogenetic stages of the shrimp to generate a constant effect on the production of digestive enzymes. in guppies (poecilia reticulata, p. sphenops), and swordtail (xiphophorus helleri, x. maculatus), the effect of incorporating bacillus subtilis, isolated from the intestine of cirrhinus mrigala into their diet has been evaluated. the results show an increase in the length and weight of the ornamental fishes as well as the specific activity of proteases and amylases in the digestive tract. according to moriarty, bacillus secretes a wide range of exoenzymes that complement the activities of the fish and increases enzymatic digestion. in fact, the bacteria isolated from the digestive tract of aquatic animals have shown chitinases, proteases, cellulases, lipases, and trypsin. in several studies, water quality was recorded during the addition of probiotic strains especially of the gram - positive genus bacillus. probably since this bacterial group is more efficient than gram - negative in transforming organic matter to co2. it is suggested that maintaining high levels of probiotics in production ponds, fish farmers can minimize the accumulation of dissolved and particulate organic carbon during the growing season. however, this hypothesis could not be confirmed on tests carried out during cultivation of shrimps or channel catfish, using one or more species of bacillus, nitrobacter, pseudomonas, enterobacter, cellulomonas, and rhodopseudomonas. thus published evidence for improving water quality is limited, except for the nitrification. in the case of edible fish, trout production farms generate high concentrations of nitrogen ranging from 0.053.3 mg l of total kjeldahl nitrogen and up to 6.4 mg concentrations of total ammonia (nh4 + nh3) increased from 4.73 to 14.87 mg l in a 21-day - experiment, while the nitrite concentration increased from 3.75 to 9.77 mg l. due to the high concentrations of produced nitrogen compounds, especially the highly toxic total ammonia ; haroun. supplemented the food of nile tilapia oreochromis niloticus l. with a commercial probiotic made from bacillus licheniformis and b. subtilis in 17 weeks of culture. assessment of water quality parameters showed an acceptable range for fish cultivation : 5.76.3 mg l for dissolved oxygen concentration, 0.360.42 mg isolated several strains of bacillus from cyprinus carpio and carried out tests to improve water quality in ornamental fish culture and to inhibit the growth of aeromonas hydrophila. three out of nine isolates showed high capacity to inhibit the pathogen in 78 of relative incidence rate ; moreover, concentrations of ammonia, nitrate, and phosphate were lowered in rates of 74%, 76% and 72%, respectively. contradictorily, queiroz and boyd tested a commercial probiotic in catfish (ictalurus punctatus), noting a survival and net fish production significantly higher when the probiotic was applied. however, very little differences were significant for the determined water quality variables (ammonia, chemical oxygen demand, nitrate, soluble reactive phosphorus, and dissolved oxygen) between the treated and control ponds. studied effects of commercial probiotics formulated from mixed cultures of bacteria and yeast on survival of japanese flounder paralichthys olivaceus, and water quality in a closed recirculating system. the probiotics - treated groups showed significantly greater survival rate as compared to the control group at the end of rearing experiment (50 days of culture), and water quality parameters were significantly lower in probiotics diet groups (from 0.24 0.22 to 0.12 0.10 mg l of no2, and from 13.0 3.9 to 10.2 3.0 mg, saccharomyces cerevisiae, nitrosomonas sp., and nitrobacter sp. had the ability to increase the beneficial bacterial microbiota of penaeus vannamei shrimp, further reducing the concentrations of inorganic nitrogen from 3.74 to 1.79 mg for example, it has been reported that chronic stress in zebra fish, danio rerio, induces a general depression on the synthesis of muscle protein. as a result, it was sought to increase stress tolerance by using probiotics. one of the firsts formal reports on this field studied the supplementation of lactobacillus delbrueckii ssp. delbrueckii in the diet of european sea bass (dicentrarchus labrax), at time intervals of 25 to 59 days. in addition to evaluating the growth improvement, hormone cortisol was quantified in fish tissue as stress marker, since it is directly involved in the animal 's response to stress. cortisol levels obtained in the treated fishes were significantly lower than those in the control (3.6 0.36 ng g and 5.1 0.47 ng g, resp.). another way to assess stress in fish involve subjecting them to heat shock, as in the case of japanese flounder (paralichthys olivaceus) grown in a recirculating system. the stress tests were carried out until half the population died, thus calculated the mean lethal time (lt50) in the absence and with addition of a commercial probiotic containing bacillus subtilis, lactobacillus acidophilus, clostridium butyricum, and saccharomyces cerevisiae. the group treated with probiotics showed greater tolerance in the stress test than the control group, the lt50 was 40 and 25 min, respectively. lactate and plasma glucose levels are considered appropriate indicators of stress as they increase as a secondary response during periods of stress to cover high energy requirements induced by this situation. therefore, varela. have conducted studies on the gilt - head bream (sparus auratus) ; the glycogen and triglycerides reserves in the livers of the control group were significantly decreased in relation to concentrations obtained when the fish feed was supplemented with the probiotic alteromonas sp. strain pdp 11. castex. evaluated the effect of pediococcus acidilactici ma 18/5 in the antioxidative response of the shrimp litopenaeus stylirostris to oxidative stress. the results showed high activities of antioxidant enzymes ; superoxide dismutase (283.7 and 153.7 mol min in control and treated groups, resp.) and catalase (3.08 and 1.34 mol min for the control and probiotic - treated, resp.). the results obtained so far raise the possibility of preparing the fish in advance with probiotic treatment, for conventional aquacultures practices that create stress in animals, such as transport, change in water temperature, and periodic manipulations. according to izquierdo., breeding aquaculture species have high nutritional requirements, thus reproductive capacity depends on appropriate concentrations of lipids, proteins, fatty acids, vitamins c and e, and carotenoids. furthermore, the relationship of these components influences reproduction in various processes such as fertility, fertilization, birth and development of larvae. at present, for most cultured fish species, there are commercially available broodstock diets that just are larger - sized diets. in practice, many fish hatcheries improve the nutrition of their broodstock by feeding them solely on fresh fish byproducts or in combination with commercial diets. the most common fresh organisms used to feed the use of these unprocessed fish products often do not provide adequate levels of nutrients needed by broodstock fishes, increasing the risk of pathogens transmission to the parents and offspring, including parasites, bacteria, and viruses. therefore, probiotics added to food or water were used in order to prevent infections and to explore their effect on reproduction. the pioneer study on the effect of probiotic supplementation on reproductive performance of fish was carried out by ghosh., using a strain of b. subtilis isolated from intestine of cirrhinus mrigala, incorporated at different concentrations to four species of ornamental fishes : poecilia reticulata, p. sphenops, xiphophorus helleri, and x. maculates, in a one - year experiment. the results showed that using b. subtilis concentrations of 1010 cells g of food, produced increases in the gonadosomatic index, fecundity, viability, and production of fry from the females of all four species. furthermore, these authors proposed that complex b vitamins synthesized by the probiotic, especially thiamine (vitamin b1) and vitamin b12, contribute to reduce the number of dead or deformed alevins. abasali and mohamad carried out similar studies with x. helleri, using a commercial probiotic containing lactobacillus acidophilus, l. casei, enterococcus faecium, and bifidobacterium thermophilum. their results showed significant differences between the control and probiotic - treated groups ; in the first parameter 105 and 150 alevins were found on average, respectively. while in the second, 28 females were fecundated in the control and 41 for the probiotic treatment. traditionally, probiotics used in food industry have been deemed safe, in fact, no human risks have been determined, remaining as the best proof of its safety. theoretically, probiotics may be responsible for four types of side effects in susceptible individuals : systemic infections, deleterious metabolic activities, excessive immune stimulation, and gene transfer. however, no hard evidence has been found. in practice, there are few reports of bacteremia in humans, where isolation of probiotic bacteria from infections seems to be the result of an opportunistic infection caused by skin lesions, cancer, chronic illness, or a drug - induced abnormality. these conditions lead to a decreased intestinal barrier that promotes the passage of the bacteria through the mucosal epithelium. subsequently, these microorganisms are transported to the mesenteric lymph nodes and other organs, leading to bacteremia that may progress to septicemia. all reported cases of bacteremia occurred in patients with chronic illness or a weakened immune system [69, 112 ]. in the case of some lactic acid bacteria that are regarded as probiotics, the resistance to antibiotics can be linked to genes on chromosomes, plasmids, or transposons. however, there is insufficient information about the circumstances in which these genetic elements could be mobilized. moreover, it is recognized that some enterococci may possess virulence characteristics and have the ability to transfer antibiotic resistance elements, so it is recommended that no reference should be made to these organisms as probiotics for human consumption, unless the producer can demonstrate that this strain can not acquire or transfer antibiotic resistance or induce infection. regarding safety of aquaculture products, in asia and more recently in latin america cultures of penaeus monodon have been reported with bacterial white spot syndrome (bwss), in farms with recurrent use of probiotics based on bacillus subtilis. the spots are similar to those generated in the white spot viral syndrome (wss), which is a deadly disease that spreads rapidly and causes mass mortalities in shrimp cultures [114, 115 ].. demonstrated in 2000 that bwss is a nonsystemic infection in p. monodon and lesions usually disappear after molting, under this condition cultures are still active and grow normally without significant mortality. however, it is of great concern because most farmers can not distinguish bwss from wss. farmers are advised in case of suspicion to send samples to the laboratory for confirmatory diagnosis. furthermore, since some aquaculture products are consumed raw or half cooked, has raised the question of whether residual probiotics may cause any infection in the final consumer. assessed the potential risk to humans caused by the use of probiotic shewanella algae in shrimp farms. studies were performed in mice that were given up to 10 cfu to reach the ld50 value for s. algae, proving the safety of using probiotics in mice. based on their results, these authors state that the use of s. algae is safe for the consumer of shrimps, such as for workers in the processing plants and farms. because there was no international consensus to ensure efficiency and safety of probiotics, fao and who recognized the need to create guidelines for a systematic approach for the evaluation of probiotics in food, in order to substantiate their health claims. a working group with experts in the field was formed in order to recommend criteria and methodology for the evaluation of probiotics, based on scientific evidence. as a result the guide for the evaluation of probiotics in food was presented, providing guidelines on the evaluation of health and nutrition properties of probiotics in food. the working group stated that no pathogenic or virulent properties were found in lactobacilli, bifidobacteria, or lactococci, although they acknowledged that under certain conditions, some strains of lactobacilli have been associated with rare cases of bacteremia. however, its incidence does not increase with raising the use of lactobacillus in probiotics. it was also mentioned that enterococci may possess virulence characteristics ; therefore, it is not recommended as a probiotic for human consumption. although the guide is not focused on aquaculture products, it creates a precedent for conducting studies to evaluate the safety of probiotics in this area. to date, the use of animal models including mice, rats, and fish has not revealed specific determinants of virulence or pathogenicity of the studied probiotic microorganisms, suggesting the overall safety of them. however, it is important to continue research using three approaches : (i) analyzing the intrinsic properties of probiotic strains, (ii) studying its pharmacokinetics (survival, activity in the intestine, dose response, and recovery from mucosa), and (iii) understanding the interactions between the microorganism and the host. the current global food crisis and increasing production costs has put pressure on governments and the international community to ensure sufficient food supply for a growing population. thus, aquaculture is presented as a way to meet the growing demand for fresh water food or seafood, and to meet current challenges relating to the ongoing globalization of trade, intensification and diversification of aquaculture, progress in technological innovations for food production, changes in ecological systems and human behavior, including a greater awareness to protect biodiversity, public health, and the environment. these challenges will lead to increased attention for improving aquaculture practices, and will become an important alternative to overexploitation and modification of aquatic ecosystems caused by capture fisheries. the use of probiotics can potentiate the benefits of this activity because, as presented in this paper, it offers viable alternatives for the generation of a higher - quality livestock product in terms of size, production time, and health. in the near future, it is necessary to conduct studies relating to probiotics resistance to antibiotics, and the chances of transmission of genetic elements to other microorganisms in the fish git, and thus to humans when consuming the aquaculture product. on the other hand, there is a need to strengthen studies of microbial ecology in aquaculture systems, correlating microbial communities (microorganisms on water and in the git of aquatic species) with animal growth and its relationship to the water quality. nowadays, a variety of probiotic strains present in the git of aquatic animals and nitrifying bacteria from biofilters have been isolated and characterized using biochemical, morphological, and molecular techniques [68, 119121 ]. the development of molecular techniques such as pcr, fish (fluorescent in situ hybridization), dgge (denaturing gradient gel electrophoresis), and generation of genomic libraries have started to unveil the diversity present in aquaculture systems. currently, next - generation sequencing methodologies offer great potential for phylogenetic identification of probiotic microorganisms without using conventional cultivation techniques. | the growth of aquaculture as an industry has accelerated over the past decades ; this has resulted in environmental damages and low productivity of various crops. the need for increased disease resistance, growth of aquatic organisms, and feed efficiency has brought about the use of probiotics in aquaculture practices. the first application of probiotics occurred in 1986, to test their ability to increase growth of hydrobionts (organisms that live in water). later, probiotics were used to improve water quality and control of bacterial infections. nowadays, there is documented evidence that probiotics can improve the digestibility of nutrients, increase tolerance to stress, and encourage reproduction. currently, there are commercial probiotic products prepared from various bacterial species such as bacillus sp., lactobacillus sp., enterococcus sp., carnobacterium sp., and the yeast saccharomyces cerevisiae among others, and their use is regulated by careful management recommendations. the present paper shows the current knowledge of the use of probiotics in aquaculture, its antecedents, and safety measures to be carried out and discusses the prospects for study in this field. |
from april 4 through may 2, 1947, 6.35 million new yorkers were vaccinated with the nyc board of health vaccinia strain (5). we used newspaper accounts and nyc department of health records to estimate the number of adults vaccinated on each of the 29 days (5). since all of the 2003 cardiac events occurred from 4 to 17 days after vaccination, the 1947 vaccination numbers were divided equally across the same 14-day period to calculate the person - time at risk for potential cardiac death. on the basis of these estimates, we identified the 2- and 4-week peak risk periods in 1947. we obtained all death certificates issued in nyc for the 4-month period between march and june, 19461948, from the nyc municipal archive. cause of death was coded according to the international classification of diseases, 5th revision (icd-5) (6). we abstracted the date of death, age of decedent, and icd-5coded primary and other cause of death into an electronic database. we defined cause of death as cardiac if the icd-5 codes for either cause included pericarditis (090), acute endocarditis (091), chronic endocarditis (092), myocardial disease (093), coronary artery diseases (094), and other disease of the heart (095). we compared daily death rates during the postvaccination risk periods with rates at other times during the study period. we used poisson regression, a generalized linear model appropriate for analysis of discrete data, to model counts of cardiac deaths (7). counts were used instead of rates, as nyc s population remained relatively constant during the study s 3-year timeframe. we also adjusted for temporal trends in the data : a long - term trend from 1946 to 1948 (defined by weeks since january 1, 1946) and a seasonal trend between march and june (defined by days since march 1 for any given year). the main model included all cardiac deaths as the outcome variable and a dichotomous exposure variable indicating whether the death occurred during the 2-week risk period. additional models examined subsets of cardiac disease and all - cause death as outcomes, as well as adjusting for noncardiac death volume. an a priori power analysis found that the model had > 90% power to detect a 5% increase in cardiac fatalities in the at - risk period. while this power would be more than sufficient to detect an excess of 2 deaths in 29,584 civilians (approximately 400 deaths in the 1947 nyc population of 6,000,000), it would not be able to detect very small elevations in risk. at the height of the 1947 vaccination campaign, from april 17 to april 21, the 2-week at - risk period in 1947 was estimated to be april 22 to may 5, which encompassed 84% of the projected at - risk person - time for adverse cardiac complications. the 4-week period was identified as april 16 to may 13 and included 99% of the at - risk person - time. adult vaccination doses administered and estimated person - time at risk for fatal cardiac adverse effects, new york city, 1947. during the months under review in 19461948, 81,529 death certificates were recorded, including 519 (0.6%) records with an illegible cause of death. of the remaining 81,010 records, 48% had heart disease listed as a cause of death. a total of 9,112 (11%) specifically referred to coronary artery or atherosclerotic disease. the number of daily deaths from heart disease in the months of march to june of 1946, 1947, and 1948 ranged from 72 to 149, with an increasing long - term trend and decreasing seasonal trend (figure 2). in the 2-week estimated risk period in 1947, 1,545 cardiac deaths occurred of 3,156 total deaths (average 110 deaths per day, range 91119 deaths) (table 1). daily deaths from cardiac causes, new york city, march to june, 19461948. in the main regression model (table 2), no independent association was found between cardiac deaths and the 2-week estimated risk period. the findings remained nonsignificant when the model was restricted to those 50 to 64 years of age and when adjustments for noncardiac deaths were made (rate ratio 1.01 ; 95% confidence interval 0.95 to 1.06). additional analyses examining different outcomes (all deaths, atherosclerotic deaths, or deaths due to myopericarditis) did not show any significant increase in deaths, nor did expanding the estimated risk period to 4 weeks. our analysis found no significant increase in reported cardiac deaths after the 1947 mass smallpox vaccination campaign in nyc. the campaign was unique in terms of the number of people vaccinated in one area in a short period. the high intensity and coverage of the vaccination campaign permitted a focused cardiac death assessment. recent reports of cardiac deaths after smallpox vaccination have raised concerns regarding the safety of the current vaccination initiative. the nyc board of health vaccinia strain used today is the same as was used in 1947 (5). as described in our prior publication (4), vaccinia is a dna virus with limited antigenic variability (8), and antigenic shifts are unlikely. regarding the vaccinated population, major risk factors, such as smoking and hypertension, were more widespread in 1947 than they are at present (911), and the death rate due to heart disease was nearly three times higher (11). if, as the 2003 cardiac fatalities suggest, cardiac risk factors increase vaccine - associated death rates, we should have seen an even - greater cardiac mortality risk in 1947. first, this analysis was ecologic, and we had no information on the vaccination status of decedents. more than 80% of the nyc population was vaccinated within the 4-week period, however, which minimizes the risk of faulty ecologic inference. the campaign urged all new yorkers to get vaccinated, irrespective of age, health, or pregnancy (5), and the likelihood of systematic bias that would mask an association is small. we extracted icd codes for > 99% of hardcopy death certificates, and missed codes were unlikely to affect the findings of the study. we also have no reason to believe that cardiac deaths were systematically misclassified in the peak risk exposure period as compared with other times. icd-5 heart disease codes and later icd revisions have been assessed to have a high comparability ratio, from 0.98 to 1.01 (6). third, assumptions pertaining to a poisson distribution may not be appropriate for these cardiac death data (11). however, no biologically plausible concern existed for underdispersion, and goodness - of - fit statistics suggested adequate fit. null findings of the study reduce concern for overdispersion, which could have otherwise potentially caused us to report an association that was not causal. finally, although the survey had substantial statistical power to detect small increases in cardiac deaths in the estimated at - risk period, extremely small increases may not have been detectable. in a large population, even small elevations in risk will produce a sizable absolute number of deaths. in light of this limitation, common to all observational studies any one study will not likely be able to definitively rule out a causal relationship between cardiac deaths among recent vaccinees and the vaccine itself, but findings from our study provide some reassurance that the current smallpox vaccination program is unlikely to increase risk for death from coronary disease. in 1947, the commissioner of health of nyc reminded his peers, whenever a large - scale vaccination program is undertaken, there is always the possibility that there may be some unfortunate complications. in new york city, there are thousands of people who become ill, and about two hundred of them die every day. since practically every person in new york city had a recent vaccination, it was inevitable that some of them would become ill and would die. vaccination does not stop the normal course of events. neither should vaccination be blamed for a death from cerebral hemorrhage, nephritis, or coronary occlusion (5). | in april 1947, during a smallpox outbreak in new york city (nyc), > 6,000,000 people were vaccinated. to determine whether vaccination increased cardiac death, we reviewed nyc death certificates for comparable periods in 1946 and 1948 (n = 81,529) and calculated adjusted relative death rates for the postvaccination period. no increases in cardiac deaths were observed. |
we describe a patient with a renal arteriovenous malformation with extension into the inferior vena cava (ivc) that was diagnosed as a renal cell carcinoma with ivc thrombus. to the best of our knowledge, this is the first report of a renal arteriovenous malformation presenting as a palpable renal mass with ivc extension. a 22-year - old female presented with fever off and on for 4 years and dull, aching, non - radiating pain in the epigastric and right loin region for 2 months. she complained of significant weight loss, mild loss of appetite and generalized weakness over the last 1 year. examination revealed a thinly built young lady with normal blood pressure and no pallor, icterus, pedal edema, palpable lymph nodes or clubbing. abdominal examination revealed a 15 12 12 cm ballotable lump in the right lumbar region that moved with respiration. blood investigations revealed a hematocrit of 34%, esr of 20 mm in the first hour, platelet count of 126,000 with giant platelets on the smear and normal renal and liver function tests. on contrast - enhanced computed tomography (cect) of the abdomen and pelvis, an 8.5 8.4 cm heterogeneously enhancing renal mass with solid and cystic components was seen extending into the right renal vein and ivc below the hepatic segment [figure 1 ]. on delayed images posteriorly, fat planes were preserved between the tumor and the abdominal wall ; medially, it was in relation to the uncinate process of pancreas (with infiltration considered unlikely) and laterally it was abutting segments 5 and 6 of the liver (where infiltration could not be excluded). contrast - enhanced computed tomography (arterial phase and venous phase) : heterogeneously enhancing right renal mass with thrombus extension into the right renal vein and inferior vena cava she underwent open right radical nephrectomy with ivc thrombectomy using a transperitoneal approach (chevron incision). gross examination revealed a tumor measuring 9 8 7 cm with a firm white surface on cut section, displacing the entire kidney and abutting the renal pelvis. a 2 cm fleshy mass with cut surface similar to the tumor was seen to extend out of the renal vein without infiltrating it (ivc thrombus). the stromal cells were negative for hmb45 [figure 2b ], sma, cd34, melan a, s-100 [figure 3a ], ema [figure 3b ] and desmin on immunohistochemistry. slides were reviewed by a second expert pathologist and the diagnosis of arteriovenous malformation of right kidney was confirmed. (a) hematoxylin and eosin stain (x50) : renal tissue with variably angulated and dilated blood vessels, distributed in a copious hyaline fibrous matrix. (b) human melanoma black 45 immunostaining (x200) : negative, rules out angiomyolipoma (a) s-100 immunostaining (x200) : negative, rules out neural lesions. (b) epithelial membrane antigen immunostaining under x200 : negative, rules out epithelial lesions, e.g. carcinomas she remained asymptomatic on follow - up for 2 years and there was no local recurrence on abdominal sonography. gross examination revealed a tumor measuring 9 8 7 cm with a firm white surface on cut section, displacing the entire kidney and abutting the renal pelvis. a 2 cm fleshy mass with cut surface similar to the tumor was seen to extend out of the renal vein without infiltrating it (ivc thrombus). the stromal cells were negative for hmb45 [figure 2b ], sma, cd34, melan a, s-100 [figure 3a ], ema [figure 3b ] and desmin on immunohistochemistry. slides were reviewed by a second expert pathologist and the diagnosis of arteriovenous malformation of right kidney was confirmed. (a) hematoxylin and eosin stain (x50) : renal tissue with variably angulated and dilated blood vessels, distributed in a copious hyaline fibrous matrix. (b) human melanoma black 45 immunostaining (x200) : negative, rules out angiomyolipoma (a) s-100 immunostaining (x200) : negative, rules out neural lesions. (b) epithelial membrane antigen immunostaining under x200 : negative, rules out epithelial lesions, e.g. carcinomas she remained asymptomatic on follow - up for 2 years and there was no local recurrence on abdominal sonography. gross examination revealed a tumor measuring 9 8 7 cm with a firm white surface on cut section, displacing the entire kidney and abutting the renal pelvis. a 2 cm fleshy mass with cut surface similar to the tumor was seen to extend out of the renal vein without infiltrating it (ivc thrombus). the stromal cells were negative for hmb45 [figure 2b ], sma, cd34, melan a, s-100 [figure 3a ], ema [figure 3b ] and desmin on immunohistochemistry. slides were reviewed by a second expert pathologist and the diagnosis of arteriovenous malformation of right kidney was confirmed. (a) hematoxylin and eosin stain (x50) : renal tissue with variably angulated and dilated blood vessels, distributed in a copious hyaline fibrous matrix. (b) human melanoma black 45 immunostaining (x200) : negative, rules out angiomyolipoma (a) s-100 immunostaining (x200) : negative, rules out neural lesions. (b) epithelial membrane antigen immunostaining under x200 : negative, rules out epithelial lesions, e.g. carcinomas she remained asymptomatic on follow - up for 2 years and there was no local recurrence on abdominal sonography. benign vascular lesions are classified as hemangiomas, lymphangiomas, vascular ectasias, reactive vascular malformations and glomus tumors. anatomically, they may arise from the arteriolar end, venous end or from capillaries, and have a variable amount of abnormal vessel density and mesenchymal tissue initial presentation may include painful or painless hematuria, hypertension or symptoms due to massive arterio - venous shunting of blood such as cardiomegaly, congestive heart failure or bruit. on cross - sectional imaging, they are seen as lesions of vascular density in the renal sinus and peripelvic areas with or without the presence of prominent renal vein. in cases where there is a large volume of blood shunting taking place in the vascular malformation this is due to the fact that renal cell carcinomas themselves have abnormal vasculature resulting in shunting of blood. the variable amount of stromal tissue in arteriovenous malformations also has a bearing on how they are seen on cross - sectional imaging. the typical homogenous appearance of vascular density may not be seen if there is a larger component of fibrous stroma. we hypothesize that the absence of bruit and pulsatility intra - operatively in this case could be attributed to this fact and the probable connection of abnormal vasculature with a low - pressure system (capillaries and venules). palpable flank lesion with flank pain is a very unusual presentation for a renal vascular malformation. there was no history of hematuria, she was normotensive and there was no bruit. cect revealed a mass that on arterial phase showed heterogeneous enhancement and had extension into the renal vein and infrahepatic ivc. intraoperatively, we could not recognize it as an arteriovenous malformation as it was non - pulsatile and firm in consistency. on reviewing the cect after the histopathology report was available, expert radiologists still felt that it was not possible to label the image as a vascular malformation of the kidney, although there were a few features not typical of renal cell carcinoma : the lesion was well encapsulated and showed heterogeneous enhancement in the region of 30 - 100 hounsfield units (somewhat more than what would be expected for a typical rcc). it is a matter of conjecture whether this lesion would have responded to angio - embolization if identified pre - operatively, and this renal unit could have been saved. | a young lady presented with complaints of right flank pain and a palpable mass. on contrast - enhanced tomography (cect), a renal mass with extension into the inferior vena cava (ivc) - suggestive of renal cell carcinoma - was diagnosed and she underwent radical nephrectomy with en bloc excision of the ivc thrombus. histopathology revealed a benign arteriovenous malformation that had extended into the ivc. arteriovenous malformation should therefore be kept in the list of differentials for a renal mass with ivc extension. |
chronic non - communicable diseases are assuming increasing importance among adult population in both developed and developing countries. the behavioral risk factors of smoking, nutrition, alcohol, and lack of physical activity (snap) are responsible for a substantial portion of chronic disease. public health interventions aimed at improving health often involve promoting healthy lifestyle by addressing these behavioral risk factors in general population. giving advice and educating the patient are viewed as a professional responsibility by all general practitioners and are expected by the patients too. however, researchers in many countries showed that medical students and graduates of medical colleges, as well as physicians, did not practice what they preach. high prevalence of smoking, drinking (alcohol), low level of physical activity, unsafe sex practices, and obesity among physicians were observed in studies conducted in japan, hungary, and israel. it is reported that 39.1% of physicians smoked in the presence of their patients, 34.7% did not think that they harmed themselves, and 45.7% did not consider that they harmed other people (nih, 2003). given the significant potential for negative outcomes to physicians own health as well as the health and safety of their patients, examination of the acculturation process about development of related health - promoting / risking lifestyle patterns over the continuum of medical training is critical to the improvement of the health care delivery system. the aim of this cross - sectional study is to describe the changes in prevalence of health - promoting / risking lifestyle among medical students during their stay in the medical colleges. this cross - sectional study was conducted in two conveniently selected medical colleges in southern india. ethical clearance was obtained from the institutional ethical committee and informed consent was obtained from the participants from both the colleges. fourth year medical students were selected for the study as they have spent over 3 years in the medical college. inclusion criterion was all students of the regular batch of fourth year and exclusion criterion was all the irregular batch students as they may have spent more time in the medical college than the regular batch students. the purpose of the study and all the terms used in the questionnaire were explained to the students. a pre - tested self - administered multiple choice type questionnaire was used to collect data. information was sought on the status of the students regarding the behavioral factors under study, namely smoking, alcohol use, junk food consumption, and physical activity, before joining the medical college and at the time of the study. the students who had reported smoking or using alcohol even once were taken as users. those who were smoking or using alcohol before joining the medical college and have not done so after joining the college were taken as to have quitted the use. the criteria for physical activity were defined as those adults aged 1864 who were doing at least 150 minutes of moderate - intensity aerobic physical activity throughout the week. junk foods were defined as any of various pre - packaged snack foods, high in calories but low in nutritional value. frequencies, proportions, and chi - square test were the statistical methods used to interpret the findings. frequencies, proportions, and chi - square test were the statistical methods used to interpret the findings. there were a total of 185 students studying in the regular batch of fourth year mbbs in the medical colleges under study : 89 in college a and 96 in college b. five students were absent from their class in the college a and four students were absent from their class in the college all of the 176 students who were present at the time of study gave their consent to participate in study and filled up the questionnaire. ninety - four (53%) among them were males and the remaining 82 (47%) were females. the study reported an increase in health - risking behaviors and a decline in health - promoting behaviors among the medical students during their stay in medical college [figure 1 ]. lifestyle risk factors among fourth year medical students before joining the medical college and at the time of study there was no statistically significant difference between the observations from the two colleges. the study revealed that the number of smokers among medical students had almost doubled from 24 (13.6%) to 46 (26.1%) since they joined the medical college [table 1 ]. a statistically significant increase was observed in the prevalence rate of smoking among males as well as females after joining the medical college. though smoking was more prevalent among male medical students than females before joining the medical college as well as after 3 years of their stay in medical college, female medical students were getting into smoking faster than male medical students. peer pressure was mentioned as the reason to get into smoking by majority [24 (48%) ] of the smokers, followed by stress 14 (28%), desire to experiment 10 (20%), and freedom by 2 (4%) of the smokers (both females). peer pressure was observed to be more prevalent among boys (54.3%) as compared to girls (33.3%) ; among girl students, stress (33.3%) was an equally important reason to start smoking. pattern of smoking among fourth year medical students a statistically significant increase was observed in the prevalence of alcohol consumption among respondents since joining the medical college [table 2 ]. reasons to initiate alcohol consumption given by male and female students were quite different and this difference was statistically significant (p = 0.012). majority of male consumers [29 (53.7%) ] cited peer pressure as a reason to start consuming alcohol, followed by stress [11 (20.4%) ], desire to experiment [8 (14.8%) ] and freedom [6 (11.1%) ]. whereas, 6 (26.1%) and 5 (21.7%) of the female consumers mentioned freedom and to experiment as reasons for getting into alcohol consumption, respectively. peer pressure was mentioned as the reason to start alcohol consumption by 10 (43.5%) female consumers and stress was the reason for only 2 (8.7%) females. pattern of alcohol consumption among fourth year medical students a statistically significant decrease in the physical activity by medical students was observed since their joining medical college. lesser proportion of females [17 (20.7%) ] was having any physical activity as compared to 59 (43.2%) male students, and this proportion further declined to 9 (10.9%) during their stay in the medical college. it was observed that only 5 (5%) medical students stared any physical activity after joining the medical college, whereas 39 (51.9%) stopped and 27 (35.5%) reported decrease in their physical activity during the same period of time. long working hours and lack of time was cited as the reason for stopping or decrease in their physical activity by all of the students. at the time of study, 100% students reported eating junk foods. they also reported an increase in the quantity and frequency of eating junk food as well as irregularity in their eating habits after their joining medical college. all the students cited poor quality of food, lack of choice in available foods, and mess timings as the main causes for their unhealthy eating habits. the focus on preventing chronic disease is increasing worldwide, which identifies the importance of promoting healthy lifestyles by addressing risk factors in general practice. however, it has been reported that the physicians vary in their attempts to motivate their patients to change risk behavior. physicians own behaviors and perceptions about lifestyle may have a direct impact on the advice that they provide to their patients. physicians who live healthier lifestyles have been found to be more likely to discuss these lifestyles with their patients and to encourage their patients to behave in healthier ways. the present study revealed an increase in health - risking behaviors and a decline in health - promoting behaviors among medical students during their stay in medical colleges. it was observed that the number of smokers among medical students had increased almost by twofold and the number of alcohol consumers by 2.5-fold since they joined their respective medical colleges, whereas the number of students doing any physical activity had reduced to half during the same period of time and food habits of all of the students had become unhealthier. these results are consistent with the observations made elsewhere that students were healthier prior to beginning medical school and that less healthy behaviors become evident by the time they begin residency. it has been observed that if physicians do not engage in these healthy behaviors, they are less likely to encourage such behaviors in their patients, and patients are less likely to listen to them even if they do talk about it. given the significant potential for negative outcomes to physicians own health as well as the health and safety of their patients, examination of the natural history of this acculturation process about physician self - care and lifestyle development is critical to the improvement of the health care delivery system. this was a cross - sectional study based on recall by medical students ; therefore, no inferences about causation may be made from these data. this was a cross - sectional study based on recall by medical students ; therefore, no inferences about causation may be made from these data. health and health risk behaviors as well as beliefs about self - care represent a significant part of the formation of professional identity for physicians which is not being given due attention at present. therefore, it is recommended to develop such a system in our medical colleges that provides adequate education and support for the development of healthy lifestyle among medical students as part of their training process. | background : behavioral risk factors are responsible for a substantial portion of chronic disease. educating patients is a professional responsibility of medical practitioners. however, it has been observed that physicians did not practice what they preach. to study whether medical colleges inculcate health - promoting lifestyle among medical students during their stay in medical colleges.methods:a cross - sectional study conducted in two conveniently selected medical colleges in southern india. fourth year mbbs students were included in the study. a pre - tested self - administered multiple choice type questionnaire was used to collect data. information was sought on the behavioral factors, namely smoking, alcohol use, junk food consumption, and physical activity, before joining the medical college and at the time of the study. spss version 10.0 was used to analyze the data. frequencies, proportions, chi - square test.results:out of 176 respondents, 94 (53%) were males and 82 (47%) were females. the number of smokers had increased from 24 (13.6%) to 46 (26.1%) and the number of alcohol consumers from 34 (19.3%) to 77 (43.8%) since they joined medical college. the number of students doing any physical activity declined from 76 (43.2%) to 43 (24.4%) and their food habits became unhealthier during the same period.conclusions:the study reported an increase in health - risking behavior and a decline in health - promoting behavior among medical students during their stay in medical college. |
selective serotonin reuptake inhibitors (ssris) are the most widely used drugs for the treatment of depression because their efficacy is similar to that of tricyclic antidepressants (tcas) ; further, they have an advantage over tcas for tolerability.1 despite differences in structure and activity, ssris, including fluvoxamine, share several common features, and there is no evidence for the superior efficacy of one agent over another. in a review of double - blind comparative studies of fluvoxamine vs imipramine for treating major depressive patients, no difference was indicated in 12 trials, while fluvoxamine was found to be the superior antidepressant in 2 trials.2 depression occurs more often in women than in men, and differences are also observed between men and women in terms of the clinical features of depression and response to treatments.3 kornstein suggested that women had an advantage in terms of the response rate to sertraline, while men had a higher response rate to imipramine. in this study, the responder analysis at the end point by menopausal status demonstrated that premenopausal women were significantly more likely to respond to sertraline than imipramine, whereas the response rates to sertraline and imipramine of postmenopausal women were similar. however, hildebrandt demonstrated similar clinical effects with antidepressant (clomipramine vs citalopram, paroxetine, and moclobemide) treatment for male and female patients with major and predominantly melancholic depression. there was also no gender difference in treatment response to sertraline in 6-month treatment of depression.6 in japanese depressive patients, there was no significant difference in treatment response to fluvoxamine and paroxetine between males and females;7 however, we recently revealed that fluvoxamine was more effective in younger female patients than in older female and male patients in a preliminary report dealing with 66 patients. a major limitation of this study was the small number of patients, particularly the number of younger females.8 therefore, we recruited new patients in order to increase the total number of patients to 100. in the present study, we re - analyzed the effects of gender differences and age on the treatment response to fluvoxamine in 100 major depressive patients. this study included a total of 100 japanese patients who fulfilled the diagnostic and statistical manual of mental disorders (dsm)-iv criteria for the diagnosis of a major depressive disorder and whose montgomery and sberg depression rating scale (madrs)9 scores at pretreatment were 21 or higher. patients suffering from other axis i disorders (such as dementia, substance abuse, dysthymia, panic disorder, obsessive - compulsive disorder, and generalized anxiety disorder) and those with axis ii disorders as determined by a clinical interview were excluded from the study. patients with a past history of childhood disorders, and those with severe non - psychiatric medical disorders were also excluded. the subjects were patients aged between 20 and 69 years who had not used any psychotropic drug for at least 14 days before participating in the study. informed consent was obtained from the subjects after providing them with a complete description of the study. well trained psychiatrists made diagnoses and provided antidepressant treatment for the patients. the same dose of fluvoxamine was administered twice daily, after dinner and at bedtime, for 6 weeks. the initial daily dose was 50 mg, which was increased to 100 mg after 1 week. after another week, the dose was set to 100, 150, or 200 mg, depending on the clinical judgment of the treating psychiatrists ; this fixed dose was maintained until the end of the study. patients with insomnia were prescribed 0.25 or 0.5 mg of brotizolam, a benzodiazepine sedative hypnotic, at bedtime. the patients were divided into 3 groups : females aged < 44 years, females aged 44 years, and males. for the female patients, we set the cut - off point at 44 years of age because females aged < 44 years have a high potential for the intact gonadotropin - releasing hormone (gnrh) pulse pattern and functioning ovulation cycle.10 the normal pattern of pulsatile gnrh secretion changes during the early 40s as a consequence of irregularity in hypothalamic pacemaker function ; therefore, both phases of the ovarian cycle are affected during the perimenopausal period.11,12 several endocrine changes precede clinical menopause, such as changes in the pattern of pulsatile gnrh secretion;11 therefore, we used 44 years as the cut - off point to distinguish the fertile period from the peri-/postmenopausal periods. assessments were conducted at the baseline and at 1, 2, 4, and 6 weeks after the initiation of fluvoxamine treatment. a decrease of 50% or higher in the baseline madrs score was defined as a clinical response. we used an intent - to - treat last - observation - carried - forward analysis. the clinical characteristics of the patients, responders, and non - responders were analyzed by a chi - square test or an unpaired t - test where appropriate. the distribution of responders and non - responders in the 3 groups was analyzed by a chi - square test. the changes in the time course of madrs scores (the mean score at each evaluation point minus the mean score at the baseline) among females aged < 44 years, females aged 44 years, and males were analyzed by a repeated - measures analysis of variance (anova). the changes in madrs scores at 1, 2, 4, and 6 weeks among the 3 groups were analyzed by an unpaired t test. all the tests were two - tailed, and a p value 0.05 was regarded as significant. this study included a total of 100 japanese patients who fulfilled the diagnostic and statistical manual of mental disorders (dsm)-iv criteria for the diagnosis of a major depressive disorder and whose montgomery and sberg depression rating scale (madrs)9 scores at pretreatment were 21 or higher. patients suffering from other axis i disorders (such as dementia, substance abuse, dysthymia, panic disorder, obsessive - compulsive disorder, and generalized anxiety disorder) and those with axis ii disorders as determined by a clinical interview were excluded from the study. patients with a past history of childhood disorders, and those with severe non - psychiatric medical disorders were also excluded. the subjects were patients aged between 20 and 69 years who had not used any psychotropic drug for at least 14 days before participating in the study. informed consent was obtained from the subjects after providing them with a complete description of the study. the same dose of fluvoxamine was administered twice daily, after dinner and at bedtime, for 6 weeks. the initial daily dose was 50 mg, which was increased to 100 mg after 1 week. after another week, the dose was set to 100, 150, or 200 mg, depending on the clinical judgment of the treating psychiatrists ; this fixed dose was maintained until the end of the study. patients with insomnia were prescribed 0.25 or 0.5 mg of brotizolam, a benzodiazepine sedative hypnotic, at bedtime. the patients were divided into 3 groups : females aged < 44 years, females aged 44 years, and males. for the female patients, we set the cut - off point at 44 years of age because females aged < 44 years have a high potential for the intact gonadotropin - releasing hormone (gnrh) pulse pattern and functioning ovulation cycle.10 the normal pattern of pulsatile gnrh secretion changes during the early 40s as a consequence of irregularity in hypothalamic pacemaker function ; therefore, both phases of the ovarian cycle are affected during the perimenopausal period.11,12 several endocrine changes precede clinical menopause, such as changes in the pattern of pulsatile gnrh secretion;11 therefore, we used 44 years as the cut - off point to distinguish the fertile period from the peri-/postmenopausal periods. assessments were conducted at the baseline and at 1, 2, 4, and 6 weeks after the initiation of fluvoxamine treatment. a single person rated each patient. a decrease of 50% or higher in the baseline madrs score we used an intent - to - treat last - observation - carried - forward analysis. the clinical characteristics of the patients, responders, and non - responders were analyzed by a chi - square test or an unpaired t - test where appropriate. the distribution of responders and non - responders in the 3 groups was analyzed by a chi - square test. the changes in the time course of madrs scores (the mean score at each evaluation point minus the mean score at the baseline) among females aged < 44 years, females aged 44 years, and males were analyzed by a repeated - measures analysis of variance (anova). the changes in madrs scores at 1, 2, 4, and 6 weeks among the 3 groups were analyzed by an unpaired t test. all the tests were two - tailed, and a p value 0.05 was regarded as significant. among the 100 patients, 3 stopped visiting our hospitals after the first visit without providing an explanation. therefore, the remaining 93 patients constituted the subjects, who included 50 females and 43 males (mean age sd = 48.8 13.7 years). the final daily dose of fluvoxamine was 50 mg for 7, 100 mg for 15, 150 mg for 8, and 200 mg for 64 patients. the number of intent - to - treat responders and non - responders was 55 and 38 patients, respectively. no significant difference was observed in average age, number of previous depressive episodes, proportion of melancholia and non - melancholia, and severity in madrs scores at pretreatment between the responders and non - responders (table 1). table 2 shows the distributions and intent - to - treat response rates to fluvoxamine between responders and non - responders. although the response rate of the females of age group < 44 years was high (75%), there was no significant difference in the distributions of the responders and non - responders between females aged < 44 years and those aged 44 years (= 2.79, p = 0.09), females aged < 44 years and males (= 1.08, p = 0.30), and females aged 44 years and males (= 0.84, p = 0.36). there was a significant difference in the changes in the time course of the madrs score between the females aged < 44 years and those aged 44 years (f = 2.97, p = 0.02). however, there was no significant difference between females aged < 44 years and males (f = 1.50, p = 0.20), and females aged 44 years and males at 6 weeks (f = 0.96, p = 0.43) (figure 1). figure 2 depicts changes in the madrs scores in the 3 groups. there was a significant difference in the madrs score at each evaluation point between females aged 44 years and those aged < 44 years (1 week : t = 3.45, p = 0.001 ; 2 weeks : t = 2.71, p = 0.009 ; 4 weeks : t = 2.11, p = 0.04 ; and 6 weeks : t = 2.17, p = 0.04). although a significant difference was observed between females aged 44 years and males for the madrs scores in the first week (t = 2.50, p = 0.015), there was no significant difference at 2 weeks (t = 1.55, p = 0.13), 4 weeks (t = 1.12, p = 0.27), and 6 weeks (t = 1.11, p = 0.27). there was no significant difference between females aged < 44 years and males for the madrs score at each evaluation point (1 week : t = 0.70, p = 0.49 ; 2 weeks : t = 0.47, p = 0.64 ; 4 weeks : t = 0.48, p = 0.63 ; and 6 weeks : t = 0.68, p = 0.50). the results of this study reveal that the fluvoxamine treatment significantly improved the changes in the time course of madrs score and changes in the madrs scores at each evaluation point in younger female depressive patients (females aged < 44 years) compared with older female patients (females aged 44 years). our results were in agreement with those of a recent study in which the menopause status negatively affected the ssri treatment response of caucasian female depressive patients treated in primary care.13 kornstein hypothesized a mechanism for the effect of gender differences ; the female gonadal hormones, particularly estrogen, may play an important role in antidepressant activity, thereby enhancing the response to ssris in younger women. halbreich have shown that estrogen enhances monoaminergic activity and augments serotoninergic postsynaptic responsiveness. several studies have suggested that estrogen augments the response to ssris in female postmenopausal major depressive patients.15,16 therefore, we suggested that the augmentation therapy using estrogen was useful for postmenopausal patients who did not response to ssris. on the other hand, sex differences in depressive response during monoamine depletions in remitted depressive patients were recently reported. in the study, women experienced greater depressive responses than men during tryptophan depletion inducing hyposerotonengic function, but not during catecholamine depletion.17 similar results were obtained in another recent study on healthy people.18 these findings suggest that differential sex effects in serotonengic function may be related to gender differences in the clinical effects of ssris. in conclusion, the present study suggests that fluvoxamine is more effective in younger female patients than in older female and male patients. this limitation leads to the possibility of a false negative in the distributions of the responders and non - responders between younger females and older females. | the effects of gender differences and age on the treatment response to fluvoxamine were investigated in major depressive japanese patients. a total of 100 japanese patients participated in this study. the daily dose of fluvoxamine was fixed to 100, 150 or 200 mg in the fourth week. this fixed dose was maintained until the end of the 6-week study. the patients were divided into 3 groups : younger females, older females, and males. depressive symptoms were evaluated using the montgomery and sberg depression rating scale (madrs) at pretreatment and at 1, 2, 4, and 6 weeks after the commencement of the study. seven of the 100 patients were excluded, and the remaining 93 patients constituted the subjects (50 females, 43 males). the number of intent - to - treat responders and non - responders was 55 and 38, respectively. there was a significant difference in the changes in the time course of the madrs score and changes in the madrs scores at each evaluation point between the younger and older females. younger females demonstrated a significantly better response than older females. the results suggest that fluvoxamine is more effective in younger female patients than in older female patients. |
coronary artery bypass surgery is the treatment of choice in patients with left main coronary artery disease or triple - vessel disease.1)2) however, graft occlusion remains a challenge for interventional cardiologists, particularly in patients who develop occlusion at the site of anastomosis. recently, a paclitaxel - eluting balloon catheter (sequent please, b. braun, melsungen, germany) was introduced and showed favorable results in the management of in - stent restenosis, small vessel disease, and bifurcation lesions.3 - 5) we herein report two cases of percutaneous intervention for the management of coronary artery bypass graft (cabg) anastomosis using paclitaxel - eluting balloon catheters. a 77 year - old male patient was admitted through the outpatient clinic with a 1-month history of chest pain on exertion. his medical history included hypertension for 15 years. a saphenous vein graft (svg) was anastomosed with the ascending aorta and the distal left anterior descending artery (lad). post - operatively, he had been taking aspirin, clopidogrel, atorvastatin, and nitrates. five months prior to his presentation, percutaneous balloon angioplasty was performed with a 3.020 mm ryujin balloon (terumo, tokyo, japan) at 10 atmospheres because coronary angiography showed significant stenosis of the anastomosis between the svg and the distal lad. on admission, electrocardiography (ecg) showed sinus bradycardia, left ventricular hypertrophy with strain, and q waves in leads iii and avf. the levels of creatinine kinase - mb (ck - mb) and troponin - i were within the normal limits. left coronary angiography showed significant stenosis of the left main coronary artery, total occlusion of the proximal lad and the proximal left circumflex artery (lcx). right coronary angiography showed significant diffuse stenosis from the proximal - to - distal right coronary artery (rca). svg angiography showed significant restenosis of the anastomosis between the svg and the distal lad, and significant stenosis in the distal lad. a 6-fr judkins right guiding catheter (cordis, johnson & johnson, bridgewater, nj, usa) was engaged in the svg through the right femoral artery. a 0.014-inch fielder fc wire (ashahi intecc, nagoya, japan) was placed in the svg to the lad. we performed angioplasty with a 2.515 mm ryujin balloon (terumo) at 8 atmospheres, and a 2.7515 mm sequent please paclitaxel - eluting balloon catheter (b. braun, melsungen, germany) at 7 atmospheres. post - intervention angiography showed minimal residual stenosis and thrombolysis in myocardial infarction (timi) grade iii blood flow. four months later, repeat coronary angiography showed no restenosis at the site of the anastomosis (fig. a 71-year - old female patient was admitted through the outpatient clinic with a 3-month history of chest pain on exertion. she underwent coronary artery bypass surgery for myocardial infarction secondary to triple - vessel disease 2 years prior to admission. she was managed by anastomosing the left internal mammary artery (lima) with the distal lad. post - operatively, she was commenced on aspirin, clopidogrel, pitavastatin, and nitrates. on admission, her blood pressure and pulse rate were 124/70 mm hg and 75/min, respectively. left coronary angiography showed total occlusion of the proximal lad and diffuse stenosis of the proximal lcx. right coronary angiography showed diffuse stenosis of the proximal - to - mid rca and total occlusion of the distal rca. lima angiography showed significant stenoses of the mid lad and anastomosis between the lima and the distal lad. the following day she underwent myocardial perfusion scan, which confirmed perfusion defect of the anteroseptal wall in the adenosine stress phase. we considered mid lad stenosis, and the anastomosis between the lima and lad as the cause of her clinical state, and decided to perform percutaneous intervention at these sites. a 6-fr judkins right guiding catheter (cordis) was engaged in the svg through the right femoral artery. a 0.014 inch fielder fc wire (ashahi intecc) was placed in the lima to mid lad. we performed angioplasty of the anastomosis with a 2.015 mm maverick balloon (boston scientific, natick, ma, usa) at 6 atmospheres. we attempted to deliver a drug - eluting stent into the lesion, but it would not advance beyond a site just before the lesion. therefore, we replaced a drug - eluting stent with a 2.517-mm sequent please paclitaxel - eluting balloon catheter (b. braun) at 7 atmospheres. we performed angioplasty of the mid lad with a 2.015 mm maverick balloon (boston scientific) at 8 atmospheres. post - intervention angiography showed minimal residual stenosis and blood flow of timi grade iii. a 77 year - old male patient was admitted through the outpatient clinic with a 1-month history of chest pain on exertion. his medical history included hypertension for 15 years. a saphenous vein graft (svg) was anastomosed with the ascending aorta and the distal left anterior descending artery (lad). post - operatively, he had been taking aspirin, clopidogrel, atorvastatin, and nitrates. five months prior to his presentation, percutaneous balloon angioplasty was performed with a 3.020 mm ryujin balloon (terumo, tokyo, japan) at 10 atmospheres because coronary angiography showed significant stenosis of the anastomosis between the svg and the distal lad. on admission, electrocardiography (ecg) showed sinus bradycardia, left ventricular hypertrophy with strain, and q waves in leads iii and avf. the levels of creatinine kinase - mb (ck - mb) and troponin - i were within the normal limits. left coronary angiography showed significant stenosis of the left main coronary artery, total occlusion of the proximal lad and the proximal left circumflex artery (lcx). right coronary angiography showed significant diffuse stenosis from the proximal - to - distal right coronary artery (rca). svg angiography showed significant restenosis of the anastomosis between the svg and the distal lad, and significant stenosis in the distal lad. a 6-fr judkins right guiding catheter (cordis, johnson & johnson, bridgewater, nj, usa) was engaged in the svg through the right femoral artery. a 0.014-inch fielder fc wire (ashahi intecc, nagoya, japan) was placed in the svg to the lad. we performed angioplasty with a 2.515 mm ryujin balloon (terumo) at 8 atmospheres, and a 2.7515 mm sequent please paclitaxel - eluting balloon catheter (b. braun, melsungen, germany) at 7 atmospheres. post - intervention angiography showed minimal residual stenosis and thrombolysis in myocardial infarction (timi) grade iii blood flow. four months later, repeat coronary angiography showed no restenosis at the site of the anastomosis (fig. a 71-year - old female patient was admitted through the outpatient clinic with a 3-month history of chest pain on exertion. she underwent coronary artery bypass surgery for myocardial infarction secondary to triple - vessel disease 2 years prior to admission. she was managed by anastomosing the left internal mammary artery (lima) with the distal lad. post - operatively, she was commenced on aspirin, clopidogrel, pitavastatin, and nitrates. on admission, left coronary angiography showed total occlusion of the proximal lad and diffuse stenosis of the proximal lcx. right coronary angiography showed diffuse stenosis of the proximal - to - mid rca and total occlusion of the distal rca. lima angiography showed significant stenoses of the mid lad and anastomosis between the lima and the distal lad. the following day she underwent myocardial perfusion scan, which confirmed perfusion defect of the anteroseptal wall in the adenosine stress phase. we considered mid lad stenosis, and the anastomosis between the lima and lad as the cause of her clinical state, and decided to perform percutaneous intervention at these sites. a 6-fr judkins right guiding catheter (cordis) was engaged in the svg through the right femoral artery. a 0.014 inch fielder fc wire (ashahi intecc) was placed in the lima to mid lad. we performed angioplasty of the anastomosis with a 2.015 mm maverick balloon (boston scientific, natick, ma, usa) at 6 atmospheres. we attempted to deliver a drug - eluting stent into the lesion, but it would not advance beyond a site just before the lesion. therefore, we replaced a drug - eluting stent with a 2.517-mm sequent please paclitaxel - eluting balloon catheter (b. braun) at 7 atmospheres. we performed angioplasty of the mid lad with a 2.015 mm maverick balloon (boston scientific) at 8 atmospheres. post - intervention angiography showed minimal residual stenosis and blood flow of timi grade iii. patency (stenosis < 20%) rates of a lima at 5 and 10 years have been reported to be 98% and 95%, respectively, while the patency rates of a svg are 95% and 71%, respectively.6) patency rate of a radial artery graft at 5 years is 95% in younger patients, (< 70 years) and 86% in older patients (70 years).7) treatment options of graft occlusion include percutaneous balloon angioplasty, percutaneous stenting, and redo - cabg. because redo - cabg is associated with high mortality rate,8) percutaneous intervention is preferred. because cardiac surgeons have been using svg for a long time, and because svg has a relatively low patency rate, interventional cardiologists should occasionally perform svg intervention. however, svg intervention is challenging because of the high rate of periprocedural myocardial infarction and restenosis.9) in addition, a stent in the svg can fracture due to movement of the svg.10) recent reports have demonstrated that drug - eluting stents and embolic protection devices lead to good results in the management of svg disease.11)12) because a lima graft has high patency rate, lima intervention is not performed frequently. for lima intervention, favorable results can be obtained by stenting.13) intervention of distal anastomosis is more challenging, because distal anastomosis is bifurcating, and different vessel response to intraluminal ballooning exists between the graft and native coronary artery. standard treatment modalities of distal anastomosis disease have not been established. the recently - released paclitaxel - eluting balloon catheter, which is based on a new matrix - coating technique, is polymer - free and can deliver paclitaxel homogeneously into the targeted vessel wall. because there are no polymers on the paclitaxel - eluting balloon, the risk of intracoronary thrombosis is lower than that with a drug - eluting stent. therefore, the need for long - term dual anti - platelet therapy should be reduced. according to experienced operators, deliverability of a paclitaxel - eluting balloon is better than a drug - eluting stent. it has been reported that paclitaxel - eluting balloon catheters can be a treatment option of in - stent restenosis, small vessel disease, and bifurcating lesions.3 - 5) in the pepcad ii trial, paclitaxel - eluting balloons had comparable binary restenosis rate to paclitaxel - eluting stents for the treatment of coronary in - stent restenosis at 6-month follow - up.14) however, in the piccoleto study, paclitaxel - eluting balloon catheters failed to show superior angiographic and clinical outcomes for the intervention of small coronary arteries compared to paclitaxel - eluting stents.15) the potential use and long - term restenosis rate of paclitaxel - eluting balloon catheters have not been studied. we successfully managed two cases of percutaneous intervention of distal anastomoses of cabgs with paclitaxel - eluting balloon catheters. in the cases, because of difference in the proximal and distal reference diameters and because of the acute angles between the proximal and distal arteries, risks of stent mal - apposition and fracture is high following stenting of the anastomosis sites. good deliverability and comparable restenosis rate,3 - 5) intervention with a paclitaxel - eluting balloon catheter can be an option for the treatment of cabg distal anastomosis. long - term randomized studies are required to evaluate paclitaxel - eluting balloon catheters for the intervention of cabg. | coronary artery bypass graft (cabg) intervention, particularly anastomosis site intervention, is challenging for interventional cardiologists. a paclitaxel - eluting balloon catheter (sequent please) is a recently - introduced device capable of delivering paclitaxel homogeneously into the targeted vessel wall. we herein report our experience with two cases. in the first case, coronary angiography showed significant stenosis at the site of anastomosis between the saphenous vein graft and the left anterior descending artery (lad). in the second case, coronary angiography showed significant stenosis at the site of anastomosis between the left internal mammary artery and the lad. we performed percutaneous intervention of these cabg anastomoses using paclitaxel - eluting balloon catheters, and obtained favorable angiographic and clinical outcomes. |
in sub - saharan africa, malaria remains a major cause of morbidity and mortality. its transmission is driven by a complex interaction of the vector, host, parasite, and the environment, and is governed by different ecological and social determinants [2, 3 ]. the survival and bionomics of malaria vectors are affected by climate variability, that is, rainfall, temperature, and relative humidity. in this light, even minute spatial variations and temporal heterogeneities in the mosquito population can result in significant malaria - risk [5, 6 ] and its endemicity [79 ]. since malaria distribution is not homogeneous, much effort needs to be expended towards defining local spatial distribution of the disease precedent to deployment of interventions. in resource constrained environments, monitoring, and evaluation is often incomprehensive and irregular and tend to lack the actual spatial and temporal distribution patterns. if transmission determining parameters are to be harnessed effectively for decision - making and objectively plan, implement, monitor, and evaluate viable options for malaria vector control, they must be well organized, analyzed, and managed in the context of a geographical - information - system- (gis-) based decision support system (dss) [3, 12 ]. while vector control interventions are being deployed according to the world health organization - led integrated vector management straandtegy [10, 13, 14 ], prompt availability of relevant spatial and attribute data is vital to support malaria surveillance, management research, and policy initiatives. different strategies coupled with new technologies such as mapping, gis, and dss, and spatial and temporal modelling are being harnessed to more effectively target limited surveillance, prevention, and control at research scale. however, potential utilization of these approaches and their incorporation in the operational malaria vector control programmes remains a significant constraint and continues to receive limited attention [16, 17 ]. until recently, very few malaria endemic countries had incorporated the gis technology into operational malaria control programmes, that is, in south africa and the lubombo spatial development in mozambique in southern africa, where it has been harnessed for case mapping and monitoring of vector control coverage [18, 19 ]. in india, it has been used to monitor malaria transmission attributes as well as social - economical and social cultural aspects of malaria. to achieve enhanced utilization of mapping and gis technologies in operational malaria control, sharing of experiences with gis and emerging technologies by malaria control programmes is critical [15, 17 ]. herein is provided a review of data related to the operational use of a gis - based dss [12, 20 ] for optimal deployment, monitoring, and evaluation of entomological interventions for malaria control in zambia. the integration of operational and logistical data for malaria control program planning with epidemiological data will serve to strengthen both the epidemiological analysis and the planning and execution of control programs. gis facilitate the integration of quantitative malaria determination and control data with data obtained from maps, satellite images, and aerial photos. a comprehensive review of data collected through nationally representative malaria indicator surveys and insecticide resistance data in major malaria vectors : an. funestus, including the comparative impact of main stream vector control interventions, has been conducted in zambia. the intervention consists of scaled - up indoor residual spraying (irs) in urban and periurban areas and insecticide treated nets (itns) in rural areas [14, 2123 ]. indoor residual spraying is implemented through annual campaigns with 85% coverage of eligible households using pyrethroids at 25 mg / m (syngenta and bayer) and ddt at 2 g / m (avima) at the beginning of the peak malaria transmission period. pyrethroid - impregnated itns, that is, permanet (verstargaard frandsen) and olyset (sumitomo corporation), are deployed through antenatal and child clinics, equity programme, community mass distribution, and commercial sector and strive towards attaining 100% coverage in eligible areas. this effort is coupled with effective case management by provision of definitive diagnosis, using rapid diagnostic tests (rdts) and microscopy, and treatment with artemisinin - based combination therapy (act), and intermittent preventive treatment (ipt) to expecting mothers. this is further augmented with interactive information, education, and communication (iec) and behavioural change and communication (bcc) strategies to enhance utilization of interventions. zambia is situated in the southern african region with a population of approximately 12 million, 45% of whom are below the age of fifteen. malaria is endemic country - wide and transmission is throughout the year with peak in rain season. the disease is the leading cause of morbidity and mortality accounting for 40% of outpatient attendances, 45% of hospital admissions with 47% and 50% of disease burden among pregnant women, and children under five years of age, respectively. current trends in the country indicate that malaria is responsible for at least 3 million clinical cases and about 6,000 recorded deaths annually, including up to 40% of the under five deaths and 20% of maternal mortality [28, 29 ]. malaria stratification aids in the development of community - based malaria control programs, by accumulating past experiences with and solutions to different factors associated with malaria outbreaks. stratification can also point to the existing inequalities in resources, allowing for a more equal and homogeneous distribution of available resources. in this regard, to allow for adaptation of intervention policy, procedures and methods to better outcomes, nineteen gis - based sentinel sites, distributed amongst nine districts within a 350 km radius of the capital lusaka (figure 1), were established for the continual monitoring and collation of key malaria data such as parasitaemia risk, insecticide resistance profiles in vectors and impact of interventions on malaria prevalence. the study region is characterized by reduced seasonality of transmission with extensive vector control through irs at 6 sites and itns in all sites from 2003 to 2010 by the national malaria control programme (figure 1). the spatial and temporal impact of irs and itns on human parasite prevalence and insecticide resistance status in major malaria vectors was monitored. at each sentinel site annual household surveys were carried out annually from 2008 to 2010 to measure plasmodium falciparum prevalence in children aged 1 to 14 [31, 32 ]. in zambia, three nationally representative malaria indicator surveys (miss) were also conducted in children under five years of age in 2006, 2008, and 2010. the mis have been used (i) to estimate an empirical high - resolution parasitological risk map in the country and (ii) to assess the relation between malaria interventions and parasitaemia risk. by standard who protocol, spatiotemporal insecticide resistance profiles of major malaria vectors : anopheles gambiae s.s, an. funestus were determined at sentinel sites and were extended to other regions of the country [32, 35, 36 ]. more data on spatial distribution of insecticide resistance to bendiocarb (0.01%), ddt (4%), deltamethrin (0.05%), lambda - cyhalothrin (0.05%), malathion (5%), and permethrin (0.75%) have been collected by different partners and collated by the national malaria control programme (figures 4 and 5). plasmodium falciparum accounts for 98% of all malaria infections in the country, causing the severest form of disease, with a low frequency of infections from p. malariae and p. ovale, and no transmission of p. vivax. the national malaria indicator survey for 2010 in children under the age of five years shows great spatial heterogeneity in prevalence of infection. this has resulted in stratification of the country in three epidemiological categories : type 1 areas with very low transmission and parasite prevalence of < 1%, type 2 areas with low transmission and prevalence of under 10%, and type 3 areas with persistent high transmission and prevalence exceeding 20% at peak transmission season. cross - sectional surveys at sentinel sites (type 2 areas) in children between 1 and 14 years across the study area (figures 2 and 3) showed a combined prevalence of infection with p. falciparum to be below 10% albeit with great heterogeneity between irs and itn areas. by standard who protocol, suspected and overt resistance to insecticides being harnessed for vector control, pyrethroids, and ddt, has been detected in all the key vectors in operational settings of both irs and itns (figures 4 and 5). there is great variation in the level of resistance between irs and itns localities, with exceptionally higher level resistance being detected in irs areas compared to itns areas (p < 0.0001). the west form of knockdown resistance (kdr) mutation has been detected in an. the overall prevalence of infection in children whose house had not been sprayed in the past year and did not sleep under a net the night before the survey was 6.8%. children who slept under a net, but whose house had not been sprayed during the past year, had a prevalence of infection of 5.2%. children whose house had been sprayed during the past year, but did not sleep under a net, had a significantly lower prevalence of infection of 3.2%. children who slept under a net in a dwelling that had been sprayed had the lowest risk of infection with a prevalence of 2.6%. thus incremental effect was observed for combined use of irs and itns (figure 6). given the spatial heterogeneity in the distribution of malaria vectors and variations in the inherent malaria risk, gis has potential applications in deployment and monitoring of interventions. for resource - constrained malaria - endemic sub - saharan african countries, like zambia, the need for a gis - based malaria information system can not be overemphasized. until recently, decisions in the malaria control programmes were taken on an ad hoc basis driven by limited empirical evidence and undoubtedly resulting in misdirection of the limited resources available. following the increased funding for malaria control particularly in sub - saharan africa [39, 40 ], insecticide - based malaria vector control interventions are being scaled up in most endemic countries albeit with limited empirical evidence on their impact and amenability to local settings. invariable monitoring, evaluation, and continuous surveillance of vector species abundance, infectivity, insecticide resistance status, and parasite prevalence in the population are imperative to ensure effective deployment of interventions and optimal utilization of limited resources. the gis - based decision support system is proving to be an invaluable tool to optimize impact assessment of malaria control interventions and thus rationalize resource utilization. the use of gis in zambia has enabled detection of spatial trends of parasite prevalence following extensive deployment of front line vector control interventions. cross - sectional prevalence surveys show continuous prevalence increase in children from 2008 to 2010 in chongwe district. in kapiri mposhi, mumbwa, mazabuka and kafue districts, prevalence dropped between 2008 and 2009 but increased in 2010. however, progressive reduction in malaria prevalence was detected in monze, kabwe, and chibombo districts from 2008 through to 2010 (figure 3). the gis has introduced new dimensions to the understanding, prediction, analysis, and dissemination of spatial relations between disease, time, and space [43, 44 ]. it allows the integration of geographical referenced data, together with local knowledge in relational databases to accurately display complex interactions in simple formats. the use of these data sets in a gis provides an opportunity to integrate up - to - date information, local knowledge, and historical trends in a manner that draws attention to areas of change - associated problems and options for action. this makes gis a tool not only for data analysis, but also for information management and decision - making thus facilitating policy formulation. there was great heterogeneity in prevalence of malaria at sentinel sites relative to detected insecticide resistance in malaria vectors. at chibombo, prevalence has been reducing despite high pyrethroid resistance detected in an. funestus. at myooye and chimoto, gambiae s.s, prevalence was reducing and remained at a low level across the three years. funestus, exhibited constant increase in parasitaemia despite high coverage of itns (figure 2). the usefulness of a gis - based dss for planning and managing control programmes is dependent on the availability of accurate and raw data on malaria transmission - related parameters. monitoring and evaluation of malaria interventions and understanding of their true impact on disease burden is essential for measuring performance of a control programme. an effective system for monitoring and evaluation and continuous surveillance requires integration of spatially and temporally explicit data for entomological and epidemiological outcome indicators. this allows for identification of disease prevalence, planning of effective interventions, assessments of reduction of vector exposure and malaria burden resulting from implemented control measures. continuous surveillance capturing real time data enables routine monitoring and evaluation of programme to demonstrate goals and impact on malaria burden. the effective control of malaria requires programme managers to have access to the most up - to - date information on the disease in order to best direct interventions efforts against the vectors. effective implementation and monitoring and evaluation of malaria control interventions have resulted in redefinition of stratification of the country in three epidemiological zones for malaria transmission potential in zambia [33, 45 ]. this necessitates appropriate targeting of interventions guided by entomological and epidemiological evidence of active malaria transmission. although the quality of data collection and archiving kept on improving, most data bases have been vertical. an excel spread sheet contained itn data base capturing quantities distributed by district and year. the irs database captured quantities of commodities and equipment, and spraying coverage per district and year. the itns have been monitored through a two component system : (1) compilation of information on number of itns distributed and (2) tracking itn coverage and/or ownership and utilization rates by householders. since 2000, irs has been monitored based on generic reporting forms for formal spraying management introduced by world health organization. this set comprised daily spray operator record, team leaders record, supervisors report, a weekly report, and a spraying completion report. by 2005, a computerized data base developed by booman. was adopted. in this case, the gis - based dss has not only streamlined evidence - based implementation of interventions, but has improved the tracking of entomological indicators : species characterization and insecticide resistance status, including parasite prevalence and impact assessment of itns and irs. it has been greatly valuable in enabling the display of heterogeneities in malaria risk areas within low transmission intensities [42, 46, 47 ]. the marked insecticide resistance problem in irs (mufweshya, kabulongo, kafue estates, and mukobeko) and llin (rufunsa, myooye, chipepo, chibombo, and chiawa) deploying sites (figure 2), confirms other findings of resistance developing in the wake of extensive vector control [4850 ]. this allows the malaria control programme manager to better utilize the limited resources on insecticides to which the malaria vectors are still susceptible. detection of high resistance levels has facilitated the planning of rational insecticide resistance management strategies and introduction of alternative noninsecticide - based vector control interventions. malaria vector control interventions amenable to focalized implementation, such as larval source management using larvicides [51, 52 ] in the context of integrated vector management [13, 14 ], are being implemented. the impact of main thrust vector control interventions on parasite prevalence in children between 114 years of age has been monitored through annual malaria surveys for three consecutive years at 19 sentinel sites (figure 2). the use of the gis - based dss has facilitated for the assessment of the efficacy of irs and itns either in combination or singly (figure 6). in areas with high parasitaemia, this has allowed for the identification of areas that require replenishment of torn nets or areas that may require irs instead of itns. therefore, the value of any surveillance system for infectious disease is measured by its ability to provide timely, accurate data for action to people responsible for effective prevention and control activities and its ability to provide ongoing feedback to the primary gatherers of information [53, 54 ]. although routine surveillance data have proved inadequate for monitoring control programmes, and have presently been supplanted by parasite prevalence surveys, vector - borne diseases demonstrate decided geographical heterogeneities and therefore require special tools for analysis. the gis with an inherent ability to manage spatial data provides an exceptional tool for continuous surveillance [57, 58 ] and provides a framework for harmonizing surveillance data and parasitaemia survey data. at a regional level, the ability of gis to display data in an intuitively understandable manner has been harnessed to establish a continental database in africa of spatial distribution of malaria the dss has been used to collate data on insecticide resistance in africa. the ability of gis - based dss to deal with large data sets and to incorporate satellite images increases the feasibility of studying transmission determinants of malaria and has resulted in prompt availability of data to support surveillance and policy formulation. the epidemiological mapping of high - risk areas of malaria transmission and insecticide resistance profiles of major vectors has facilitated the recognition of those populations and geographic areas where it is possible to identify the main determinants of malaria morbidity and mortality. the revealed trends and interrelationships have allowed the identification of high risk areas and facilitated decision making and rational utilization of limited resources in a cost - effective manner. in zambia, an evidence - based decision support has created a more focused and purposeful approach to directing resources to areas of most need with reasonable returns for effort and resources invested. monitoring the impact of malaria vector control interventions through the gis - based dss on relative change in prevalence of infection and vector susceptibility to insecticides over time has enabled measurement of spatial heterogeneity of trend or impact. the revealed trends and interrelationships have allowed the identification of areas with reduced parasitaemia and increased insecticide resistance thus demonstrating the impact of vector control. targeting interventions based on entomological and epidemiological evidence have not only contributed markedly to the success of the zambian malaria control programme, but also have provided opportunity for rational decision making in deployment of interventions and cost effective utilization of limited resources for enhanced malaria control. | geographic information systems (giss) with emerging technologies are being harnessed for studying spatial patterns in vector - borne diseases to reduce transmission. to implement effective vector control, increased knowledge on interactions of epidemiological and entomological malaria transmission determinants in the assessment of impact of interventions is critical. this requires availability of relevant spatial and attribute data to support malaria surveillance, monitoring, and evaluation. monitoring the impact of vector control through a gis - based decision support (dss) has revealed spatial relative change in prevalence of infection and vector susceptibility to insecticides and has enabled measurement of spatial heterogeneity of trend or impact. the revealed trends and interrelationships have allowed the identification of areas with reduced parasitaemia and increased insecticide resistance thus demonstrating the impact of resistance on vector control. the gis - based dss provides opportunity for rational policy formulation and cost - effective utilization of limited resources for enhanced malaria vector control. |
myeloperoxidase (mpo) is a well - known enzyme, mainly released by activated neutrophils, characterised by powerful pro - oxidative and proinflammatory properties. recently, myeloperoxidase has been proposed as a useful risk marker and diagnostic tool in acute coronary syndromes and in patients admitted to emergency room for chest pain. oxidative stress and inflammation play important roles in the pathogenesis of destabilization of coronary artery disease (cad) leading to acute coronary syndromes (acs). infiltrating macrophages and neutrophils participate in the transformation of stable coronary artery plaques to unstable lesions [1, 2 ]. recently, there has been a renewed interest in mpo, a proinflammatory enzyme that is abundant in ruptured plaque and can be measured in peripheral blood. mpo is a hemoprotein that is stored in azurophilic granules of polymorphonuclear neutrophils and macrophages. mpo catalyzes the conversion of chloride and hydrogen peroxide to hypochlorite and is secreted during inflammatory condition. in addition, mpo consumes endothelial - derived no, thereby reducing no bioavailability and impairing its vasodilating and anti - inflammatory properties. major evidence for mpo as enzymatic catalyst for oxidative modification of lipoproteins in the artery wall has been suggested in a number of studies performed with low - density lipoprotein. in contrast to low - density lipoprotein, plasma levels of high - density lipoprotein (hdl)-cholesterol and apoai, the major apolipoprotein of hdl, inversely correlate with the risk of developing coronary artery disease. there is now strong evidence that hdl is a selective in vivo target for mpo - catalyzed oxidation, that may represent a specific molecular mechanism for converting the cardioprotective lipoprotein into a dysfunctional form, raising the possibility that the enzyme represents a potential therapeutic target for preventing vascular disease in humans. zhou. showed that atorvastatin reduced serum mpo and crp concentrations in patients with acs. mpo activity can be measured in blood and tissues by spectrophotometric assays using hydrogen peroxide and o - dianisidine dihydrochloride as substrates. in addition, mpo content can be measured in neutrophils as an index of degranulation with the coulter counter and flow cytometry and circulating mpo by elisa. very recently, commercial methods allowing low - cost and high - volume measurements have been proposed. the introduction of these methods of measurement might make mpo a new and useful cardiac biomarker. there have been a few but important clinical studies examining the role of mpo as a marker of risk for cad. using an enzyme assay, zhang. showed that blood and leukocyte mpo activity were higher in patients with cad than angiographically verified normal controls, and that this increased activity was significantly associated with presence of cad (odds ratio, 11.9 ; 95% confidence interval (ci), 5.525.5). results were independent of the patient 's age, sex, hypertension, smoking, or diabetes status, ldl concentration, leukocyte count, and framingham global risk score. more recently, meuwese., in the epic- (european prospective investigation into cancer and nutrition-) norfolk prospective population study, have evaluated the association of mpo levels with the risk of future cad in apparently healthy individuals. mpo was measured in baseline samples of a case - control study nested in the prospective epic - norfolk population study : case subjects (n = 1138) were apparently healthy men and women who developed cad during 8 years of follow - up ; control subjects (n = 2237) matched for age, gender, and enrollment time, remained free of cad. the mpo levels were significantly higher in case subjects than in control subjects and correlated with c - reactive protein (crp) and white blood cell count. risk of future cad increased in consecutive quartiles of mpo concentration, with an odds ratio (or) of 1.49 in the top versus bottom quartile. after adjustment for traditional risk factors, the or in the top quartile remained significant at 1.36 (95% ci 1.07 to 1.73). of interest in this study, serum mpo levels were associated with the risk of future development of cad in apparently healthy individuals, but the association was weaker than that of traditional risk factors and crp. however mpo, at variance from crp, was largely independent from classical risk factors. in acs, mpo has been consistently found to be associated with the presence of instability and risk of future events in the studies that have explored these topics. first observed that circulating neutrophils in patients with acute myocardial infarction (ami) and unstable angina (ua) have a low mpo content, and therefore high mpo levels in the circulation, as compared with those with chronic stable angina and variant angina. this is indicative of a significant release of mpo from neutrophils related to their activation. the lack of neutrophil activation in patients with variant angina, and after stress test suggests that this phenomenon may occur independently of ischemic episodes. therefore, mpo is prevalently a marker of instability and not simply a marker of oxidative stress and damage. furthermore, in this study mpo did not correlate with ck - mb and troponin t release ; this observation is clinically important as an extremely sensitive and specific marker of damage already exists (troponin), but no definite markers of instability exists so far. in this study, mpo content was determined on the coulter counter, which measures the neutrophil count by flow cytometry and subsequently calculates the mean mpo content in that population. using the same method, buffon the mpo content of the leukocytes collected from the arterial circulation and the coronary sinus effluent were compared. the authors found a gradient of mpo across the coronary circulation in patients with acs and this gradient was present even when the culprit lesion involved with the acs was in the distribution of the right coronary artery, which does not drain into the coronary sinus. in this study, as in the previous one, a significant correlation was found between systemic levels of c - reactive protein and either the aortic and coronary sinus neutrophil mpo. the potential usefulness for risk stratification of blood concentrations of mpo was examined in 2 recent studies. in the capture trial, rates of death and myocardial infarction (mi) were determined at 6months of follow - up. an mpo cutoff of 350 g / l was associated with an adjusted hazard ratio was 2.25 (95% ci, 1.323.82). the effects were particularly impressive in patients with undetectable cardiac troponin t (ctnt < 0.01 g / l), in whom the hazard ratio was 7.48 (95% ci, 1.9828.29). of interest, the increase in risk was already evident after 72 hours, increasing only slightly thereafter (figure 1). this observation is in keeping with the data by biasucci. who had shown return of mpo to baseline levels in all patients, including those with myocardial infarction, within one week. this point is important, as suggests a peculiar characteristic of mpo, at variance from other inflammatory markers commonly used (as crp, fibrinogen) and from other proposed inflammatory markers that remain elevated for relatively long time or have an extremely short and unreliable half - life (such as interleukins). the predictive value of mpo was independent by c - reactive protein and high mpo serum levels indicated increased cardiac risk both in patients with medium c - reactive protein serum levels (20.0% versus 5.9% ; p <.001) and in those with low c - reactive protein serum levels (17.8% versus 0% ; p <.001), suggesting that recruitment and degranulation of neutrophils is a primary event and is followed by release of other systemic mediators and acute - phase proteins such as c - reactive protein. at variance from crp levels, levels of mpo were not influenced by troponin, suggesting a prognostic role of mpo independent from troponin and confirming that inflammation is a primary phenomenon in acs. in a study of 604 consecutive patients presenting to the emergency department with chest pain, brennan. demonstrated a progressive increase in odds ratios for major adverse events at 30 days and 6 months with each quartile increase in mpo concentration in patients with negative troponin. the 6-month outcomes were similar to the results of the capture trial : corresponding odds ratios were 1.6 (95% ci, 1.02.7), 3.6 (2.25.8), and 4.7 (2.97.7) for the second, third, and fourth quartiles, respectively (cut offs of 119, 198, and 394 pmol / l, resp.). it is of interest that, even in the absence of myocardial necrosis and in negative cardiac troponin patients, baseline measurements of mpo significantly enhanced the identification of patients at risk, although this study included the need for revascularization within the definition of major adverse cardiac events and used an inappropriately high ctnt cutoff of 0.10 g / l. furthermore, while troponin t takes three to six hours to rise to measurable circulating levels after myocardial injury, mpo levels were significantly elevated at baseline (even within two hours after the onset of symptoms) in patients who were initially negative for troponin t. these findings suggest that measurement of mpo levels may be useful in triage in the emergency department and that elevated plasma mpo levels may be a marker of unstable angina preceding myocardial necrosis and therefore, a predictor of vulnerable plaque. interestingly, c - reactive protein levels predicted the risk of myocardial infarction at presentation for the entire cohort but were not predictive of major adverse cardiac events in the group that was negative for troponin t. in the cohort that was consistently negative for troponin t, the areas under the curve for mpo were significantly higher than those for troponin t, ck - mb, and c - reactive protein. taken together, these data suggest that crp and mpo may be complementary and explore different fields : crp is a marker of disease activity and vascular inflammation, and is useful for long - term risk stratification while mpo is a marker of plaque instability and neutrophil activation and may be associated with short - term stratification, in particular in patients with troponin negative levels. more recently, several studies have also investigated the value of mpo in predicting long - term outcomes. the patients were divided into four groups according to the quartile of mpo level. they have found that : (1) acs rate (36.2%) in the fourth quartile group of mpo level was 6 times higher than that in the first quartile group of mpo level, p <.01. (2) gensini score in the fourth quartile group of mpo level was significantly higher than that in the first quartile group (p <.01). wbc in the fourth quartile group was also significantly higher than that in the first quartile group, p <.05. in addition, kaplan - meier event rate curve showed that there was a significant difference in outcome (death, ami, revascularization) between the groups with mpo 62.9 auu / l and 62.9 auu / l of mpo serum level at 6-month follow - up visit (chi(2) = 13.5, p =.01). furthermore, cavusoglu. have investigated the long - term prognostic significance of baseline mpo levels in a well - characterized cohort of 193 men with acs. all patients were followed prospectively for the development of death and mi, and follow - up data were available for all patients at 24 months. using the median mpo value of the entire cohort of patients (20.34 ng / ml) as a prespecified cutoff, the mi - free survival at 24 months for the group with mpo values below cutoff were significantly lower than in those with values above cutoff. have investigated the relationship between plasma mpo and clinical outcome after ami [15, 16 ]. they have studied 512 ami patients at hospital admission and have measured plasma mpo in ami patients and found a significant association of mpo with follow - up events. importantly, mpo was of incremental prognostic value on the top of ejection fraction and bnp, a finding observed also by khan in a similar population of patients with stemi. mpo is a marker of inflammation and oxidative stress that has been consistently demonstrated to be elevated in patients with acs. however, the data so far available are relatively few ; therefore, more studies are requested to precisely define the role of mpo. in particular all studies involving mpo assessment have used different methods, thus a standardization effort is needed. furthermore, increased mpo is not likely to be specific for cardiac diseases, as activation of neutrophils and macrophages can occur in any infectious, inflammatory, or infiltrative process, therefore, more studies should address and clarify these points. so far, as reported in table 1, mpo seems to have, among the inflammatory markers, a role superior to that of pregnancy - associated plasma protein - a (papp - a), cd40 ligand (cd40l), and cytokines, but still inferior to crp. | myeloperoxidase (mpo) is an enzyme stored in azurophilic granules of polymorphonuclear neutrophils and macrophages and released into extracellular fluid in the setting of inflammatory process. the observation that myeloperoxidase is involved in oxidative stress and inflammation has been a leading factor to study myeloperoxidase as a possible marker of plaque instability and a useful clinical tool in the evaluation of patients with coronary heart disease. the purpose of this review is to provide an overview of the pathophysiological, analytical, and clinical characteristics of mpo and to summarize the state of art about the possible clinical use of mpo as a marker for diagnosis and risk stratification of patients with acute coronary syndrome (acs). |
the development of new imaging studies, such as magnetic resonance imaging (mri), and the improvement of the quality of those already available have made it possible to identify new potential etiologies for degenerative arthritis of the hip. femoroacetabular impingement (fai) has been increasingly recognized as a cause for hip pain and degenerative hip arthritis with an estimated prevalence of 10% to 15%. initially described as a complication of hip osteotomies, fai corresponds to an anatomical alteration either in the femoral head - neck junction (cam - type impingement) or in the acetabular rim (pincer - type impingement), which causes a conflict between the two corresponding anatomical structures during articular motion. the third and most common form of femoroacetabular impingement is the combination of cam and pincer types, which is found in around 86% of cases. either type of impingement causes pain and abnormal hip biomechanics with high pressure points at extreme range of motion. it has been demonstrated that these biomechanical alterations cause lesions of the labrum and/or the articular cartilage. cam impingement occurs when the femoral head has an oversized radius, resulting in an insufficient head - neck offset. a widening of the femoral neck reduces its concavity, creating an anterior impingement between a triangular - shaped extension of bone and articular cartilage and the anterior undersurface of the acetabular rim, resulting in delamination of cartilage secondary to a shearing injury. the cross - table lateral view allows quantifying the anterior femoral head - neck offset (ao). the method described by eijer. is performed as follows : a line bisecting the longitudinal axis of the femoral neck, using the narrowest portion of the neck, is drawn ; then, parallel lines tangent to the anterior border of the femoral head and neck are drawn. 1). (a) anterior head border. (b) anterior neck border. a value under 9 mm for the ao is considered abnormal according to eijer. these authors described the offset ratio as well, which is the ao divided by the diameter of the femoral head. this ratio was established as 0.21 0.03 in asymptomatic patients and 0.13 0.05 in symptomatic patients with cam impingement. goodman. consider that the main deformity in the head - neck junction, secondary to slipped capital femoral epiphysis (scfe), is in the sagittal plane. considering this, the deformity might not be seen at all in the anteroposterior (ap) views of the hip and pelvis. even if the bump is seen, it is only a partial impression and can not be precisely measured unless a lateral view is assessed. notzli. described the angle, which evaluates the femoral head - neck junction considering the angle at which the femoral head reduces its convexity. kassarjian reported abnormal angles in 39 of 42 patients with fai (mean of 69.7 degrees). a minimal slipped capital femoral epiphysis is recognized as one of the main etiologies for cam - type femoroacetabular impingement. in this pathology, a minimal posterior displacement of the femoral head epiphysis would exist, altering the ao. we believe that this phenomenon should increase the posterior femoral head - neck offset (po) as well, creating an inverse relation between the ao and the po. we are not aware of any reports regarding the normal values for the posterior offset ; the purpose of this article is to determine if the po and the ao / po ratio may be additional parameters for the evaluation of radiographs of patients with suspected cam impingement. preoperative radiographs of 15 symptomatic patients (6 women, 9 men) with a mean age of 41 years (range, 30 - 62) with a diagnosis of isolated cam - type fai were assessed. inclusion criteria for the cases were a positive impingement test during the physical examination and an altered angle (greater than 55 degrees in all cases) measured on magnetic resonance imaging arthrogram (mria). more important, hip arthroscopy was performed in all the cases, and findings were supposed to be compatible with cam impingement lesions to be included in this study. exclusion criteria included radiographic findings compatible with a pincer - type impingement such as coxa profunda, protrusio acetabuli, and acetabular retroversion (suggested by a positive crossover sign and/or posterior wall sign) ; a center - edge angle less than 25 degrees or greater than 35 degrees ; and patients with hip pain suspected of being from any other source than a cam impingement. the control group consisted of 15 asymptomatic volunteers, age and gender matched with the symptomatic group. none of the patients included in the control group recalled any form of pain related to the hip ; on the physical examination, all of them had a negative impingement test. for obvious reasons, the control group did not undergo surgical confirmation. the radiographic evaluation included an ap view of the pelvis, as well as an ap view and a cross - table lateral view of the involved hip. the ap views were used mainly to rule out a pincer - type impingement, dysplasia, and/or osteoarthritis. the cross - table lateral radiographs were certified as technically correct by members of the radiology department. in the cross - table view, the ao was measured with the method described by eijer. (described above) and the po in the same fashion, considering the posterior border of the femoral neck and the posterior border of the femoral head. differences between groups were tested using the t test or wilcoxon mann - whitney u test according to data distribution. significant tests were all 2-tailed (p < 0.05). for all statistical analyses, a normal distribution was found for the ao and the ao / po ratio ; as a result, the student t test was applied. for the po values, intraobserver and interobserver reliability for calculating the ao / po ratio was measured with the intraclass correlation coefficient test. the median angle for the cases was 68 (range, 55 - 81). the mean sd ao in the symptomatic group was 7 1.7 mm, which is comparable to the values reported by eijer. for symptomatic patients. the control group presented a mean sd ao of 9.5 1.8, which was comparable to the 11 mm (mean) reported by eijer. four asymptomatic volunteers presented ao values under 9 mm and would be considered abnormal according to eijer. the symptomatic group had a median po of 13.5 mm (range, 9 - 18), whereas the control group median po was 12 mm (range, 8 - 16). the mean sd ao / po ratio was 0.56 0.1 for the symptomatic group and 0.9 0.2 for the asymptomatic group ; this difference was statistically significant (p < 0.01 ; table 1). anterior offset, posterior offset, and offset ratio intraobserver correlation coefficient for ao / po ratio was 0.8. primary or idiopathic degenerative arthritis is still the most common etiological classification for hip osteoarthritis. evidence has been mounting in the past few years that point toward subtle undetected and underdiagnosed femoroacetabular pathologies and that in fact primary idiopathic osteoarthritis would be rare. among them, fai has been increasingly recognized as a cause for hip pain and degenerative hip arthritis. tanzer and noiseux studied a group of 125 patients with hip osteoarthritis (formerly classified as idiopathic by classic radiographic standards) who presented signs of fai in 100% of the cases. in this group of patients, 22% showed anterolateral signs of degenerative joint disease, and the rest presented a diffuse compromise of the joint. in the series analyzed by tanzer and noiseux, 62% were classified as having a pistol grip deformity and fai, 5% had developmental hip dysplasia, and 2% had protrusio acetabuli. the angle measured on mri has been very helpful since its introduction by notzli. for the identification of bony abnormalities in the proximal femur suggestive of a cam impingement. nevertheless, mri arthrograms are not always available, and its costs may sometimes be a matter of concern ; more affordable and available methods for the screening and diagnosis of patients with a suspected fai are valued. defined the values for ao in symptomatic patients with cam impingement and the normal values for asymptomatic people. absolute values for ao are potentially dependent on the anthropometric variations of the patients, and thus normal values may vary according to the population being assessed. in our study, a volunteer in the control group presented an ao of 7.2 mm, which is abnormal according to values published by ganz., and this patient s ao / po ratio was 0.8, within the normal limits according to our results, and compatible with an individual from the control group. a symptomatic patient presented an ao of 11 mm, which is normal according to eijer., and a large po of 18 mm (according to us), which results in an ao / po ratio of 0.56, which is altered and compatible with cam femoroacetabular impingement according to our study (table 2). results of anterior offset (ao), posterior offset (po), and ao / po ratio according to eijer. and nemtala. (current study) note how the ao / po relationship is altered in the femoroacetabular impingement in the presence of a normal ao according to ganz. also described the offset ratio (defined in the introduction) to prevent anthropometrical variations. for this method,, the correct identification of the femoral head center sometimes can be difficult, especially in the presence of pathologies grossly compromising the femoral head contour, such as a scfe or a legg - calv - perthes disease, making the offset ratio somehow difficult to estimate. the po in our study showed no statistical difference between the cases and the control subjects, which may reflect that this parameter tends to remain without significant variations in the presence of a cam impingement. furthermore, if the cam impingement was due to a minimal scfe, which has been postulated as a common cause for this disease, the displacement of the epiphysis posteriorly would tend to increase the posterior offset instead of reducing it, thus reducing even more the ao / po ratio. the ao / po ratio is a parameter independent from anthropometry that, according to our study, is altered in the presence of a cam impingement. although the intraobserver correlation is very good, the interobserver variations may be an issue, requiring the results of the ao / po ratio to be interpreted with caution. a potential bias of our study is the limited number of cases and controls ; however, the study in which the ao and the offset ratio were described had even fewer patients. on the basis of the results obtained in our study, we conclude that the anterior / posterior offset ratio can be effectively used as a new parameter in the screening of patients with suspected cam - type fai ; it is independent from anthropometry and has a fair intra-/interobserver correlation. nevertheless, many other factors must be considered when assessing these patients, including more sophisticated imaging studies (i.e., mria). | purpose : the purpose of this study was to determine if the anterior - posterior offset ratio is altered in patients with symptomatic cam impingement.study design : preoperative radiographs of 15 symptomatic patients with isolated cam - type impingement diagnosed by physical examination and magnetic resonance imaging arthrogram (mria) and confirmed by hip arthroscopy findings were assessed. fifteen asymptomatic volunteers made up the control group. the anterior offset (ao), posterior offset (po), and ao / po ratio were calculated.results:the mean sd ao / po ratio was 0.56 0.1 for the symptomatic group and 0.9 0.2 for the asymptomatic group. this difference was statistically significant. intra- and interobserver correlation factor for calculating the ao / po ratio was 0.8 and 0.5, respectively, and differences were not statistically different.conclusions:the ao / po ratio in this study was a useful radiological parameter for the assessment of patients with a cam - type impingement. |
search for new materials for advanced medical applications, the so called biomaterials, is at present within the range of interest of a large number of scientists : physicists, chemists, biologists, for whom the coincident interdisciplinary approach evolves. hydrogel materials are modern polymer materials used for making contact lenses, and their chemical and biological properties must be bio - compatible with human organism [14 ]. the objective of the work is making an assessment of physical properties of the polymer material used for making contact lenses by using the positron annihilation lifetime spectroscopy (pals) method and uv vis nir spectroscopy. in spite of constant improvement of the materials used for making contact lenses, many patients keep suffering from pathological changes in their cornea. there is a need for conducting new studies on properties of polymer materials used for making contact lenses, so as to provide patients with good moistening and appropriate optical parameters, as well as comfort by ensuring their long time use. proclear family of contact lenses made by means of the pc technology contain omafilcon a. the pc technology is based on the assumption that the external layer of the red blood cell membrane is hemocompatible, whereas the internal layer is thrombogenic. the internal, thrombogenic surface has negatively charged phospholipids, while the outer surface is made of closely arranged positively and negatively charges phospholipids, which are, consequently, electrically neutral. 80 % of the external surface of the erythrocyte cell membrane contains phosphoryl - choline, whereas the remaining part is composed of other phospholipids. the smooth pc coating on the surface of contact lenses imitated the bipolar nature of the physiological film and increases bio- and hemo - compatibility. water molecules also have bipolar structure and this is why many of them are loosely bound on the surface of phosphoryl - choline contained in the contact lenses. consequently, water molecules bound on the pc surface inhibit binding other molecules and thus reduce the friction factor, which minimize irritation of an eyeball. coopervision informs that the proclear family lenses maintain 98 % of water even after 12-h - use. pc covered lenses inhibit formation of bio - film on the surface of the contact lenses. very little deposit and few bacteria accumulate on the surface (fig. 1). the biofilm contains diverse microorganisms and extracellular matrix, therefore, it is considered to be as a permanent reservoir of bacteria contributing to development of inflammation of eyeballs. 1contact lens with a visible reservoir of bacteria contact lens with a visible reservoir of bacteria free positron annihilation is a process involving change of the entire mass of both particles and of their kinetic energy into photon energy of electromagnetic radiation. this is why studying photons formed in the process of annihilation provides information about the condition of the annihilating electron positron pair. apart from free annihilation, there is also the bounded state annihilation when a positron forms with an electron a hydrogen - like atom called a ps positronium. the high energy positron annihilation in the matter is preceded by the phenomenon of thermalization, which involves quick loss of positron energy due to scattering and excitation of the medium and thermalization. when losing the last 1050 ev of its energy a positron covers the distance of the same order and then there may occur a reaction of positronium formation with one of the liberated electrons accompanying, as it were, the positron. due to different placement of spins, we can distinguish two different types of positronium : para - positronium p - ps of anti - parallel placement of spins (the 2 annihilation) and ortho - positronium o - ps of parallel placement of spins (the 3 annihilation). physical properties of positronium change due to its interaction with the surrounding medium. one of the observed phenomena is shortening of three - photon orthopositronium mean annihilation lifetime, which is also called o - ps annihilation [5, 6 ]. it involves the ability of the positron, which is part of orthopositronium, to perform two - photon annihilation with one of the atoms that can be found in the surroundings of the positronium [7, 8 ]. the existence of free volume an area of zero electron density is necessary for the positronium to survive in a condensed medium without being quenched with a mean lifetime shorter by two orders of magnitude. in this paper, the tao eldrup model was used to describe the relations between the o - ps orthopositronium lifetime and the size of a free volume. the model has fully accomplished its task and it has been used for years to calculate the dimensions of free volumes in polymer materials by numerous research centers throughout the world. it assumes that a positronium is trapped within a spherical free volume, is capable of spontaneous annihilation with emission of three quanta, or as a result of the pick off process into two quanta [912 ]. modern technologies make it possible to produce lenses of better and better parameters that allow using them by children and adolescents by solving problems of dry eyes and tissue reactions to anoxia that occur in hydrogel lenses. they provide possibility for safe use in the case of ageing eyes [13, 14 ]. the aim of this paper is making an attempt at comparative analysis between individual hydrogel lenses of the proclear family by means of pals and uv vis nir spectroscopy. table 1 shows the family of proclear contact lenses used in the study. table 1family proclear contact lenses used in the studysamplebrandoxygen permeability : dk / twater content (%) material omafilcon a (%) 1proclear asphere2860402proclear sphere4262383proclear ep1660404proclear multifocal toric d4259415proclear multifocal toric n425941 family proclear contact lenses used in the study as described in the preceding paper, measurements of pals positron lifetimes were taken in room temperature with ortec spectrometer, based on the start stop principle. the temporary peak resolution of the measuring system was fwhm = 270 ps. each sample was formed by 8 layers of contact lenses of 10 mm diameter and 1.2 mm thickness. the examined sample, together with the positron source, which was na sodium isotope of 4 10 bq activity, formed a sandwich harmful effect of uv radiation to eses and the necessity to use appropriate protection have been discussed for years. protecting eyes from solar radiation is very important, and this is why additional uv vis nir spectroscopy has been carried out. the uv vis nir spectroscopy is an instrumental technique in which energetic transitions taking place in molecules is used. the transitions are caused by absorption of the electromagnetic radiation in the uv range (200400 nm), visible range (400800 nm) or near - infrared range (8001,000 nm). tao and eldrup. [10, 11 ] developed the model in 1972 for free volumes in polymers. they pondered over a simple model in which the ps atom is located in a finite, spherical potential trough. in order to simplify calculation, replacement of the finite potential trough by an infinite potential trough, extended by the r value. it is necessary for reconstruction of the values of probability of finding o - ps outside the potential trough of finite depth and radius r. successive theoretical deliberations showed that 3 o - ps lifetime expressed as a function of radius r free volume is expressed by the formula:1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tau _ { 3 } \, (ns) = 0.5\left [{ 1 - \frac{\text{r}}{{{\text{r } } + \updelta { \text{r } } } } + \frac{1}{2\pi } { \sin } \left ({ \frac { { 2\pi { \text { r}}}}{{{\text{r } } + \updelta { \text{r } } } } } \right) } \right]^ { - 1 } $ $ \end{document}where r = 0.166 nm is the fitted empirical electron layer thickness. by fitting the above equation with the measured 3 values, r and free volume size vf as:2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\text{v}}_{\text{f } } = \frac{4}{3}\pi { \text{r}}^{3 } $ $ \end{document}can be evaluated. the relative intensity of the longest component i3, is generally correlated to the density of the holes, which can be considered as a kind of trapping centres for ps. a semi - empirical relation may be used to determine the fraction of free volume (fv) in polymers as:3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\text{f}}_{\text{v } } \, = \,{\text{cv}}_{\text{f } } { \text{i } } _ { 3 } $ $ \end{document}where : vf is the free volume calculated from 3, using eq. (1) with a spherical approximation, i3(in %) is the intensity of long - lived component, c is empirically determined to be 0.0018 from the specific volume data. in the further part of the study, a two - state model of positron annihilation in this model, a positron annihilates from the free state and from one state localized in a defect, without the detrapping process. after calculating, with the use of the lt program, the major annihilation parameters mean positron lifetimes 1, 2 and their intensities, we can also calculate : mean positron lifetime av illustrating defectiveness of the dominant medium in the examined lenses;4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tau_{av } \, = \,\frac{{\tau_{1 } i_{1 } \, + \,\tau_{2 } i_{2 } } } { { i_{1 } \, + \,i_{2 } } } $ $ \end{document } mean positron lifetime in an unidentified structure b;5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tau_{b } \, = \,\frac{{i_{1 } \, + \,i_{2 } } } { { \frac{{i_{1 } } } { { \tau_{1 } } } \, + \,\frac{{i_{2 } } } { { \tau_{2 } } } } } $ $ \end{document } capture speed of positrons by traps (defects) d;6\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\kappa_{d } \, = \,\frac{{i_{2 } } } { { i_{1 } } } \,\left ({ \frac{1}{{\tau_{b } } } - \frac{1}{{\tau_{2 } } } } \right) $ $ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tau_{2 } - \tau_{b}$$\end{document}the magnitude connected with means size of defects in which annihilation occurs, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tau_{2 } /\tau_{b}$$\end{document}illustrating the type of volume defects. a model experimental curve of the spectrum of positron lifetime in a contact lens sample is shown in fig. 2. 2the spectrum of positron lifetime in a contact lens sample the spectrum of positron lifetime in a contact lens sample the uv vis nir spectrometric testing was carried out with an oceanoptics high - resolution hr 4000cg - uv nir spectrometer in the spectrum range 2001,000 nm. a deuterium tungsten - halogen lamp, covering the spectrum range of the spectrometer used in the study was used as a source of light. the power of the source of light was 3.8 w (deuterium lamp) and 1.2 w (tungsten - halogen lamp). 3uv vis nir transmission spectra for the examined contact lenses uv vis nir transmission spectra for the examined contact lenses results of calculation of values of mean lifetimes of positrons in the examined samples revealed existence of three components, 1, 2 and 3, in the positron lifetime spectra. in our earlier publications on the topic we concentrated on analyzing only 3, the third component of positron lifetimes. this time we additionally analyse the components 1 and 2, which concern the two - state model of positron annihilation. table 2mean positron lifetime values 1, 2, 3 and their intensitiessample1 (ns)i1 (%) 2 (ns)i2 (%) 3 (ns)i3 (%) 1 asphere0.207 0.00676.03 1.560.580 0.03418.23 1.601.800 0.1805.75 0.352 sphere0.180 0.00663.06 0.970.476 0.01031.12 0.971.803 0.0245.80 0.193 ep0.211 0.00768.05 1.450.533 0.02224.90 1.531.822 0.0287.07 0.304 multifocal toric d0.230 0.00571.34 1.340.517 0.05424.01 1.431.853 0.0754.66 0.175 multifocal toric n0.235 0.00576.17 1.430.564 0.08920.07 1.471.893 0.0663.79 0.26 mean positron lifetime values 1, 2, 3 and their intensities the obtained errors are the result of mathematical analysis. positron lifetime values 3 o - ps (the pick - off process) and their intensity i3, as well as the dimensions of free volumes r are displayed in table 3., their intensity i3 and sizes of free volumessample3 (ns)i3 (%) r (nm)vf (10 m)fv (a.u.)1 asphere1.8005.750.26577.92448.042 sphere1.8035.800.26678.80457.043 ep1.8227.070.26880.59569.774 multifocal toric d1.8534.660.27183.33388.325 multifocal toric n1.8933.790.27587.08330.03 mean positron lifetime values 3, their intensity i3 and sizes of free volumes the free volumes dimensions vf and the fraction of free volumes vf i3 = fv / c for all the examined samples are displayed in figs. 4 and 5 respectively. 4the average size of free volume vf for samples of contact lenses : 1 proclear asphere, 2 proclear sphere, 3 proclear ep, 4 proclear multifocal toric d, 5 proclear multifocal toric nfig. 5the fractional of free volume fv / c for samples of contact lenses : 1 proclear asphere, 2 proclear sphere, 3 proclear ep ; 4 proclear multifocal toric d, 5 proclear multifocal toric n the average size of free volume vf for samples of contact lenses : 1 proclear asphere, 2 proclear sphere, 3 proclear ep, 4 proclear multifocal toric d, 5 proclear multifocal toric n the fractional of free volume fv / c for samples of contact lenses : 1 proclear asphere, 2 proclear sphere, 3 proclear ep ; 4 proclear multifocal toric d, 5 proclear multifocal toric n the fraction of free volume fv is proportional to vf i3, because c in the eq. (4) is constant. there are values of 1 and 2 positron lifetimes and their intensities, as well as the determined parameters of positron capture for the examined samples of contact lenses presented in table 4. table 4calculated values of positron lifetime and positron capture of the examined samples of contact lensessamplefitting parametersparameters of positron capture1 (ns)i1 (%) 2 (ns)i2 (%) 3 (ns)i3 (%) av (ns)b (ns)d (ns)2b(ns)2/b (ns)10.20776.030.58018.231.8005.750.2790.2360.6030.3442.45820.18063.060.47631.121.8035.800.2780.2261.1460.2502.10630.21168.050.53324.901.8227.070.2970.2520.7660.2812.11540.23071.340.51724.011.8534.660.3020.2670.6100.2501.93650.23576.170.56420.071.8933.790.3040.2680.5150.2962.104 calculated values of positron lifetime and positron capture of the examined samples of contact lenses changes of 3 life time values and their intensities i3 are presented as changes of free volumes vf and numbers of free volumes fv (figs. 4, 5). it results from fig. 4 that each sample is characterized by a different vf value. as for the fv parameter, responsible for changes of free volume numbers occurring in the examined contact lenses, there are certain similarities. there are values close to fv in the asphere and sphere lenses, another pair are the multifocal toric d and multifocal toric n lenses. the ep lens, on the other hand, is distinguished by an indirect value for both vf and fv parameters. it is possible to look for connections with the optical properties of the contact lens. according to coopervision, the lens is something in between ordinary lenses for correcting far - sightedness and multifocal lenses, and it is generally used to correct presbyopia in young people. when analyzing results of the study (table 4), it is noticeable that there is a relation between the percentage of omafilcon content and 1 responsible for free annihilation of positrons and the annihilation with electrons of point defects of vacancy type. two contact lenses, multifocal toric d and multifocal toric n stand out as having the highest 1 values and the highest omafilcon content (41 %). the same relation can also be found for the mean positron lifetime av reflecting defectiveness of the medium for the same contact lenses. apart from that the lenses multifocal toric d and multifocal toric n also stand out because of highest values of b analysing subsequent parameters of positron capture, one can observe that the highest speed of positron capture by the d traps (defects) is demonstrated by the sphere lens, whereas the lowest speed is demonstrated by the multifocal toric n lens. the parameter 21, i.e. mean defect sizes is the highest for the asphere lens and the lowest for the sphere and multifocal toric d lenses. differences for the 2/1 parameter can be observed, which is evidence of geometric variability for individual contact lenses. this paper deals with a study on determining occurrence of free volume vacancies in the structure of polymer hydrogel contact lenses made by means of pc technology by coopervision and on the degree of defectiveness of the structure by means of the pals method. as a result of the conducted studies and calculations of positron lifetime spectra values of three components (1 short - lived, 2 average - lived, and long - lived 3 o - ps) and their intensities i have been obtained. the results of these measurements show that formation of free volume vacancies takes place in all contact lenses of the proclear family. in spite of the fact that all the examined contact lenses are made of the same polymer material (omafilcon), the designated parameters vary. the calculated values of dimensions of vf free volumes and the fraction of fv free volumes show that the highest values of vf occur in multifocal toric d and multifocal toric n lenses, and at the same time the lowest fv values are observed for the same contact lenses. as for the positron capture parameters (the degree of defectiveness of the structure), multifocal toric d and multifocal toric n contact lenses show the highest defectiveness of the medium, but at the same time a low speed of defecting positrons. however, the same type of positron trapping forms in all cases, and the differences in results confirm structural changes in contact lenses resulting from their technological modification. there arises one basic conclusion from the uv vis nir study, which indicates that all the examined contact lenses from the coopervision proclear family stop harmful ultraviolet radiation. | a study has been conducted in order to determine presence of free volume gaps in the structure of structure of polymer hydrogel contact lenses made in phosphoryl choline technology and of the degree of defect of its structure. the study was made by means of positron annihilation lifetime spectroscopy. as a result of the conducted measurements, curves were obtained, which described numbers of counts of the acts of annihilation in the time function. the conducted studies revealed existence of three components 1, 2 and 3. the 3 component is attributed to the pick - off annihilation of o - ps orthopositronium trapping by free volume gaps and provides information about geometrical parameters of the volumes. at the same time, the uv vis nir spectrometry examination was conducted on the same samples in the spectral range 2001,000 nm. |
a term boy was born by cesarean section for fetal distress after an uncomplicated pregnancy. apgar scores were 6 and 6 at 1 and 5 minutes, respectively. at 30 minutes after birth a diagnosis of congenital heart disease was suspected, and treatment with prostaglandin e1 was initiated. the neonate was intubated and ventilated, and he was transferred to our neonatal intensive care unit. on admission, he had tachypnea and cyanosis, and his oxygen saturation was 94% with a fraction of inspired oxygen of 70%. physical examination showed a loud systolic cardiac murmur and prominent venous neck pulsations, decreased peripheral pulsations, and hepatomegaly. neurological examination showed an agitated neonate with a head circumference of 37 cm (90th percentile), a fontanel in level, a cephalohematoma, and a generalized hypertonia. an aortic coarctation was suspected, prostaglandin e1 was continued, and circulatory support with dobutamine was started. echocardiography showed a structurally normal heart with a dilated right ventricle with suprasystemic pulmonary arterial pressures based on doppler velocity measurements of a moderate tricuspid valvular insufficiency. an open ductus arteriosus was present with systolic right - to - left ductal flow. within the proximal descending aorta, finally, a dilated superior caval vein was observed (figure 1). based upon these findings, a low resistance cerebral arteriovenous malformation was suspected because of the diastolic steal phenomenon observed in the descending aorta. two - dimension echocardiographic recording of the distal portion of the aortic arch with color flow mapping. still - frame showing retrograde flow during diastole (in red) caused by diastolic steal toward the cerebral arteriovenous malformation. at the site of the arrow, a pulsed - wave doppler signal was recorded (inset at the lower right portion of the figure) which documents the holodiastolic retrograde flow in the descending aorta. magnetic resonance imaging showed a vein of galen aneurysmal malformation, with a maximal diameter of 2.3 cm and ischemic lesions in the left hemisphere and a hydrocephalus (figure 2). the option of endovascular treatment, which is the preferred method of treatment in vein of galen aneurysmal malformation, was discussed. after having discussed the case in the medical team, the authors refrained from endovascular treatment and further intensive care unit treatment because the neurologic prognosis was considered infaust based on the clinical condition and the findings on electroencephalography and magnetic resonance imaging. ischemic lesions in the left hemisphere and as a result of the venous congestion, there is a resorption disorder of the cerebrospinal fluid resulting in a hydrocephalus. the authors report a case that illustrates the dramatic outcome of a neonate presenting with heart failure and finally being diagnosed with a vein of galen aneurysmal malformation. the vein of galen aneurysmal malformation in our patient was of the choroidal type, which can be classified as type i by the classification criteria of lasjaunias. this type is the most severe form of the disease, causing high - output cardiac failure in the newborn. furthermore, the symptoms of our patient started at age of less than 5 months, which is also known to be a poor prognostic factor. the unfavorable combination of a lasjaunias type 1 vein of galen aneurysmal malformation with severe heart failure and presentation of symptoms at age less than 5 year explains why the clinical course of this patient quickly deteriorated and lead to his death. in the fetus, high - output cardiac failure may not occur because the low vascular resistance in the cerebral arteriovenous malformations is balanced by the low resistance of the uteroplacental unit. the exclusion of the low resistance placental circulation and the increase in systemic vascular resistance after birth favor flow through the cerebral arteriovenous fistulas, leading to decreased systemic circulation and high output cardiac heart failure. the size of the fistulas determine the amount of arteriovenous shunting and consequently the time course to the development of cardiac failure. due to a low vascular resistance in the head, the majority of left ventricular output is directed toward the head. these 2 mechanisms lead to reduced systemic blood flow, severe lactic acidosis, potentially ischemic multiple organ failure, and persistent pulmonary hypertension of the newborn. during diastole, the systemic perfusion is further reduced by circulatory steal to the vein of galen aneurysmal malformation, which produces the characteristic reversed diastolic flow in the descending aorta. coronary blood flow is reduced by this diastolic steal phenomenon and combined with increased ventricular pressures can lead to reduced myocardial perfusion or even ischemia, further compromising cardiac function. our patient also presented with a hydrocephalus as a result of systemic venous hypertension in the presence of an arteriovenous shunt causing cerebrospinal fluid malabsorption. the clinical picture is dominated by findings suggestive of a congenital cardiac disease such as cyanosis, compromised peripheral pulses, and a cardiac murmur. often cyanosis is present suggestive of persistent pulmonary hypertension of the newborn with signs of right - to - left arterial shunting at the ductal level. a continuous murmur can be heard over the scalp, but this is not routinely checked as our case illustrates. chest x - ray imaging may show an enlarged cardiac silhouette with right chamber preponderance, superior mediastinal widening, retrosternal fullness with posterior displacement of the upper trachea, and retropharyngeal soft tissue thickening. echocardiography often shows a structural normal heart and great vessels but a dilated and noncompliant right ventricle with signs of suprasystemic pulmonary arterial pressure and right - to - left ductal shunting. the left ventricle on the contrary is often hyperdynamic and not enlarged. finally, an enlarged aortic arch and superior systemic veins, especially the superior caval vein, can be observed. computer tomography and magnetic resonance imaging give a clear demonstration of the intracranial arteriovenous malformation. cerebral angiography will eventually provide an accurate anatomical definition of all the vessels feeding the fistula. the treatment of vein of galen aneurysmal malformation involves initial cardiovascular stabilization directed toward improvement in the noncerebral systemic cardiac output. this is achieved by reducing systemic and pulmonary vascular resistance and improving the systemic output and myocardial function of the heart. in most situations, a vasodilator agent, either alone or in combination with low to moderate dose inotropic agents, is needed. milrinone and inhaled nitric oxide have been found suitable for use in vein of galen aneurysmal malformation. right - to - left ductal shunt facilitating an adequate systemic circulation is also maintained. however, in about half of patients, cardiovascular stabilization is not achieved. in the modern treatment of vein of galen aneurysmal malformation embolization by endovascular treatment is the preferred treatment modality, preferably after the age of 5 to 6 months. a good outcome is to be expected when treatment is performed before significant brain injury has occurred in patients who have been selected carefully. a recent systematic meta - analysis of 667 patients revealed that 68% of the patients, including neonates 44%, infants 41%, children and adults 12%, had a good outcome after endovascular embolization. complications such as cerebral hemorrhage, cerebral ischemia, hydrocephalus, leg ischemia, or vessel perforation were seen in 37%. similar results were found in a review by khullar, which reported 84% good to fair outcome and a 15% mortality rate among 337 patients undergoing endovascular treatment during the time period of 2001 and 2010. a good outcome was defined as neurologically normal or mild developmental delays, and a fair outcome was defined as moderate or severe developmental delays. however, untreated patients with vein of galen malformation revealed a mortality rate of 77% underlying the great importance of embolization as preferred treatment modality. in conclusion, vein of galen aneurysmal vein of galen aneurysmal malformation is a rare disorder and is often diagnosed late. considering vein of galen aneurysmal malformation in the differential diagnosis of neonatal congestive heart failure initial treatment is targeted at improving noncerebral systemic circulation and reduction in pulmonary vascular resistance. when circulatory stabilization can be achieved, endovascular treatment of the arteriovenous malformation is the preferred method of intervention. | the authors report the case of a neonate presenting with signs of a congenital cardiac disease. echocardiography showed a structural normal heart, right - to - left ductal flow, a dilated superior caval vein, and reversed diastolic flow in the proximal descending aorta. brain magnetic resonance imaging showed a vein of galen arteriovenous malformation. this highlights the importance of considering an intracranial cause in the differential diagnosis of neonatal congestive heart failure. |
mouse preadipocyte cells 3t3-l1 obtained from atcc (manassas, va) were maintained in high glucose dulbecco s modified eagle s medium (invitrogen, carlsbad, ca) supplemented with 10% fbs and antibiotics. for adipocyte differentiation, confluent cells were treated with 10 g / ml insulin, 0.25 mol / l dexamethasone, and 0.5 mmol / l isobutyl-1-methylxanhine for 2 days followed by 3- to 5-day treatment with insulin alone. differentiation of adipocytes was confirmed by lipid staining with red oil and mrna expression of ppar- and adiponectin as described earlier (15). human primary subcutaneous adipocytes were obtained from zenbio (research triangle park, nc) and cultured following the instruction of the manufacturer in subcutaneous adipocyte medium am-1 (zenbio). uric acid solution for cell treatments (ultrapure, 115 mg / dl ; sigma) was prepared in prewarmed cell culture medium as previously described (15). if treatments with uric acid were continued for more than 3 days, medium was replaced with freshly prepared identical medium. by the end of the 3-day treatment, there was no difference in ph between cell culture medium containing uric acid and control medium. in some experiments, conditioned medium was collected and stored at 80c for measuring the levels of adipokines. the animal care and use committee of the university of florida approved protocols for all animal experiments. to test the effect of lowering uric acid, we used the pound mouse, a recently established prediabetes / metabolic syndrome model (charles river, wilmington, ma). these mice have a mutation in the leptin receptor (a deletion of the exon 2), and they develop obesity, insulin resistance, dyslipidemia, and fatty liver disease but do not progress to type 2 diabetes (39). in preliminary experiments we showed that these mice develop hyperuricemia (results). obese (c57bl/6ncrl - lepr / crl) and lean (c57bl/6ncrl) 6-week - old mice were maintained on a standard purina 5001 diet (pmi, richmond, in) under a 12-h light/12-h dark cycle with access to food and water ad libitum. to assess the effects of lowering uric acid levels, obese and lean animals (810 animals per group) were either administered for 8 weeks with allopurinol, an inhibitor of xor (125 mg / l in the drinking water ; sigma, st. every 3 weeks, samples of blood were obtained through facial vein to monitor the level of uric acid. animals were fasted for 4 h and then injected intraperitoneally with insulin (humulin r ; eli lilly, indianapolis, in) at 1 unit / kg body wt. glucose in tail vein blood was measured immediately before injection (time 0) and at 15, 30, 60, 90, and 120 min after injection using onetouch ultra glucometer (lifescan, milpitas, ca). at the end of the treatment period, the animals were killed by the intraperitoneal injection of 150 mg / kg sodium pentobarbital. blood was collected via cardiac puncture, and serum was stored in aliquots at 80c. for the mean arterial blood pressure (map) measurements, animals were placed under anesthesia (13% isoflurane), and arterial blood pressure was measured via the carotid artery by inserting a 27-gauge needle connected to a pressure transducer as described (40,41) followed by blood collection. while measurement of blood pressure under anesthesia may not recapitulate values in the conscious, unstressed state, we have previously reported that differences in map between experimental groups are maintained (40). samples of visceral adipose tissue were collected on ice and rinsed in ice - cold pbs. for rna isolation, tissue samples were preserved in rnalater solution (applied biosystems / ambion, austin, tx) at 80c. trace dna was removed using a dna - free kit (applied biosystems / ambion, austin, tx), and 0.5 g of total rna was converted to cdna using iscript cdna synthesis kit (bio - rad, hercules, ca). quantitative real - time rt - pcr was performed using ssofast evagreen supermix (bio - rad) with primers spanning two or more exons and optimized for real - time pcr, which were designed using geneious pro software (v. 4.8 ; biomatters ltd., auckland, new zealand) and beacon designer (v. 7.70 ; premier biosoft international, palo alto, ca). real - time pcr was performed using cfx96 real - time pcr detection system as follows : 50c for 2 min, then 95c for 2 min, then 40 cycles of denaturation at 95c for 15 s, annealing at 55c for 30 s, and extension at 72c for 30 s. reaction specificity was confirmed by 1) electrophoresis of products in 2% agarose gel after real - time rt - pcr to check if bands of expected size were detected ; 2) melting curve analysis ; and 3) sequencing amplified fragments. relative gene expression was analyzed by c(t) method using the cfx manager software (v. 1.6 ; bio - rad, hercules, ca). primers for gene expression analysis by quantitative rt - pcr to assess the macrophage infiltration in the adipose tissue, samples of the visceral adipose tissue were collected in 10% buffered formalin, processed, embedded in paraffin, and sectioned. macrophages were stained as described (42) with the f4/80 antibody from invitrogen / caltag laboratories (carlsbad, ca), secondary horseradish peroxidase conjugated antibody, diaminobenzidine chromogen, and a hematoxylin counterstain. levels of mcp-1 and adiponectin in the cell culture medium were measured using the multiplex mouse adipocyte lincoplex kit (linco research, st. charles, mo) and the luminex 100 system (luminex, austin, tx). serum levels of mcp-1 and adiponectin were measured with the milliplex map mouse single - plex kit and the mouse serum adipokine milliplex map kit (millipore, billerica, ma), correspondingly using the luminex 100 system. uric acid was measured by an enzymatic assay with urate oxidase (diagnostic chemicals limited, oxford, ct). thiobarbituric acid reactive substances were measured with the assay from cayman chemical (ann arbor, mi). at least three independent experiments were performed in the case of in vitro studies, and/or 610 animals per group were used for the in vivo studies. data were analyzed by one - way anova followed by fisher least significant test, unpaired student t test, or mann - whitney u test, with a value of p 50% while inducing a threefold increase in adiponectin mrna level with an increase in the level of the circulating adiponectin. thus, hyperuricemia in the obese mice with metabolic syndrome might be partially responsible for the proinflammatory generation of mcp-1 by adipose. in addition, elevated levels of uric acid might contribute to the decrease in adiponectin. because lowering urate blood levels with allopurinol improved the balance of the production of mcp-1 and adiponectin the effect of lowering uric acid with allopurinol on the mcp-1 and adiponectin production in the adipose tissue of the obese pound mice. obese pound mice and lean (+ / ?) control mice were treated with allopurinol for 8 weeks as described in the research design and methods section. mrna abundance (a and c) as well as blood levels (b and d) for mcp-1 (a and b) and adiponectin (c and d) were measured at the end of the experiment. the values are mean sem (n = 810, performed in duplicate). p 50% while inducing a threefold increase in adiponectin mrna level with an increase in the level of the circulating adiponectin. thus, hyperuricemia in the obese mice with metabolic syndrome might be partially responsible for the proinflammatory generation of mcp-1 by adipose. in addition, elevated levels of uric acid might contribute to the decrease in adiponectin. because lowering urate blood levels with allopurinol improved the balance of the production of mcp-1 and adiponectin the effect of lowering uric acid with allopurinol on the mcp-1 and adiponectin production in the adipose tissue of the obese pound mice. obese pound mice and lean (+ / ?) control mice were treated with allopurinol for 8 weeks as described in the research design and methods section. mrna abundance (a and c) as well as blood levels (b and d) for mcp-1 (a and b) and adiponectin (c and d) were measured at the end of the experiment. the values are mean sem (n = 810, performed in duplicate). p < 0.05 and p < 0.001 (u test), correspondingly, for the effect of obesity. & p < 0.05 for the effect of allopurinol. in order to check whether lowering uric acid reduced signs of inflammation in the adipose tissue in addition to mcp-1 production, we examined the visceral adipose tissue for the presence of macrophages, a key step in proinflammatory changes in obesity (42,44). the number of the f4/80-stained macrophages was negligent in lean mice and increased dramatically in obese mice (fig. 6a, b, and d) while it significantly decreased in obese mice treated with allopurinol (fig. 6c and d). because expression of the proinflammatory cytokine tnf- in the adipose tissue is elevated in obesity and it is macrophage - specific (44), we measured the abundance of mrna for tnf- in the adipose tissue. as expected, the tnf- mrna was induced dramatically in the pound mice while allopurinol treatment reduced it (fig. 6e), consistent with changes in macrophage infiltration and changes in the mcp-1 production. we assessed the effect of lowering uric acid on the level of oxidative stress by measuring malondialdehide (mda), a product of lipid peroxidation. mda levels in the visceral adipose tissue and serum of obese mice was increased while allopurinol treatment substantially reduced it (fig. 6f and g). the effect of lowering uric acid levels on the macrophage infiltration, tnf- expression, and oxidative stress in the adipose tissue of the obese pound mice. samples of the adipose tissue from lean (a), obese (pound) (b), and obese mice treated with allopurinol for 8 weeks (c) were stained with f4/80 antibody and counterstained with hematoxylin. because diaminobenzidine was used as a chromogen, macrophages (f4/80-positive cells) are stained in brown as indicated with arrowheads (see also magnified rectangular region). d : the percentage of f4/80-positive macrophages within the adipose tissue is greatly induced by obesity and reduced by allopurinol treatment. counting positive and negative cells were performed in a blind fashion at least in three fields per animal. e : effect of allopurinol treatment on the relative expression of mrna for tnf- in the visceral adipose tissue of the obese (pound) mice. in addition, the level of the oxidative stress in the serum (f) and in the visceral fat (g) in these mice was assessed by measuring the product of lipid peroxidation mda using thiobarbituric acid reactive substance assay. the values are mean sem (n = 56, performed in triplicate). p < 0.01 and p < 0.001 (u test), correspondingly, for the effect of obesity. (a high - quality digital representation of this figure is available in the online issue.) the proinflammatory endocrine imbalance and low - grade inflammation in the visceral adipose tissue is an underlying mechanism in the insulin resistance and cardiovascular abnormalities in subjects with metabolic syndrome (18,19,29,30). to test whether lowering uric acid can improve signs of metabolic syndrome, we performed insulin tolerance tests and measured blood pressure in obese mice treated with allopurinol. lowering uric acid levels in obese (pound) mice improves signs of metabolic syndrome (insulin resistance, hypertension). a : to assess level of insulin sensitivity, the insulin tolerance test was used. insulin (1 unit / kg) was injected, and blood level was measured at 0, 15, 30, 60, 90, and 120 min after injection. sensitivity to insulin was normal in lean mice but dramatically reduced in obese mice, which was partially improved by treatment with allopurinol. b : obesity induced increase in the map in mice, which was attenuated by allopurinol treatment. c : representative recordings of blood pressure averaged in b. the values are mean sem (n = 56, performed in triplicate). p < 0.01 and p < 0.001 (u test), correspondingly, for the effect of obesity. (a high - quality color representation of this figure is available in the online issue.) we found that uric acid can increase expression and release of mcp-1 and reduce production of adiponectin in cultured adipocytes. we further showed that an increase in mcp-1 with a reduction in adiponectin occurs in both adipose tissue and serum of the obese, hyperuricemic pound mice, and that lowering uric acid can attenuate these changes. obesity is associated with an increase in mcp-1 production (27,28) and a decrease in the adiponectin production (19,46) in the adipose tissue. these changes contribute substantially to the obesity - related low - grade inflammation and metabolic syndrome, including insulin resistance and hypertension (17,25). a dramatic increase in macrophage infiltration with expression of the proinflammatory cytokine tnf- was observed in visceral adipose tissue in obese mice and is consistent with reported results (42,44). importantly, lowering uric acid caused a reduction in macrophage infiltration and tnf- expression as well as improved insulin sensitivity and blood pressure. this study may provide a mechanism linking hyperuricemia, obesity, and metabolic syndrome that has been shown previously in clinical studies (4,5,12). the observation that lowering uric acid improved but did not reverse the changes in mcp-1 and adiponectin, inflammation, and insulin resistance in the pound mouse is consistent with uric acid being a modifying factor but not the sole causal factor in driving these changes. we observed an increase in serum uric acid in the pound mice and other animal models of obesity in comparison with the corresponding lean controls. hyperuricemia progressed as animals gained weight, and the level of uric acid correlated with the body weight. as these mice are obese and insulin - resistant because of the mutation in the leptin receptor (39), hyperuricemia can not be considered as a causal factor of obesity in this case. however, an increase in uric acid contributed to the low - grade inflammation and metabolic syndrome via its direct effect on the production of mcp-1 and adiponectin in the adipose tissue. in mouse 3t3-l1 adipocytes and human primary adipocytes, uric acid can induce a direct dose - dependent increase in the production of mcp-1 and a decrease in the production of adiponectin at the level of the mrna expression and protein release. for mice, cells responded to uric acid beginning at concentrations of 5 mg / dl, which is similar to the levels of uric acid in the obese pound mice. in the case of human adipocytes, cells responded to 7.515 mg / dl uric acid, which encompass the range observed in subjects with asymptomatic hyperuricemia to severe gout (3). the ability of soluble uric acid to induce mcp-1 expression was first demonstrated in rat vascular smooth muscle cells (31). the effect of uric acid was mediated by map kinases erk1/2 and p38 and nuclear factor-b in a redox - dependent fashion (31). in our previous work with 3t3-l1 adipocytes (15), we showed that uric acid induced ros production via activation of nadph oxidase followed by phosphorylation of p38. the urate - induced increase in the mcp-1 expression in adipocytes was downregulated by a superoxide scavenger and a nox inhibitor. collectively, these data suggest that uric acid induced mcp-1 production in adipocytes via the redox - dependent signaling initiated by nox activation. furukawa. (45) reported that obesity was associated with oxidative stress in adipose tissue, which, in turn, caused an overexpression of a variety of proinflammatory cytokines including mcp-1. we observed an increase in oxidative stress in our mouse model of obesity by measuring mda in the adipose tissue and serum. this was reduced by allopurinol, suggesting that hyperuricemia induced by obesity is another mechanism triggering oxidative stress followed by induction of the mcp-1 expression and inflammation in the adipose tissue. the uric acid induced increase in the mcp-1 production observed in adipocytes was prevented not only by antioxidants but also by activation of ppar- with rosiglitazone. an activation of ppar- is known to block the proinflammatory effects of tnf- in adipocytes by affecting the proinflammatory branch of the nuclear factor-b xor in adipocytes is thought to be a crucial upstream regulator of ppar- activity (38). treating adipocytes with exogenous uric acid reduced expression of xor, which could be a cause of downregulated anti - inflammatory activity of ppar- and facilitated redox - dependent mcp-1 production. the mechanism of downregulation of the adiponectin production in obesity is not completely understood but appears to involve proinflammatory pathways (48,49), oxidative stress (45), and a deficiency in the ppar- activity (48,50). the uric acid induced decrease in adiponectin expression and secretion in our experiments was preventable by the ppar- agonist, rosiglitazone, suggesting an involvement of the same kind of proinflammatory mechanism that was responsible for the induction of mcp-1. on the other hand, antioxidants could not improve adiponectin production affected by uric acid. we can suggest therefore that the deficiency of ppar- but not oxidative stress was a primary trigger of this downregulation. because rosiglitazone prevented the uric acid induced induction of mcp-1 and downregulation of adiponectin, these effects of uric acid might depend on ppar-. xor - dependent regulation of the adipocyte differentiation via control of ppar- activity is known to be a finely orchestrated mechanism, which could be affected by manipulating the expression of xor (38). our data suggest that elevated concentrations of uric acid may reduce expression of xor and attenuate ppar-dependent endocrine regulation in adipocytes. in summary, uric acid can affect adipocytes directly by inducing effects resembling those observed in obesity : upregulation of proinflammatory factors and downregulation of the production of the insulin sensitizer and anti - inflammatory factor adiponectin via redox - dependent mechanisms, which can be prevented by an agonist of ppar- (fig. 8). in the mouse model of the metabolic syndrome, lowering uric acid by inhibiting xanthine oxidoreductase in obese mice with the metabolic syndrome could improve the proinflammatory endocrine imbalance in the adipose tissue by lowering production of mcp-1 and increasing production of adiponectin. this model summarizes the results of the current and the previous (15) studies. activation of nox by uric acid occurs via unknown mechanism, and it was localized on the plasma membrane as well as on intracellular membranes. ros generated from superoxide produced by nox is followed by ros - dependent activation of the proinflammatory signaling via p38. an activation of this mechanism in response to uric acid is followed by an increase in the production of mcp-1 and a decrease in the production of adiponectin. in addition, uric acid entering the adipocyte may downregulate expression of xor, which (xanthine dehydrogenase but not xanthine oxidase) is known as a crucial upstream regulator of activity of ppar-, a master - regulator of adipogenesis, expression of adiponectin, and an anti - inflammatory factor in adipocytes (38). upregulation of mcp-1 in response to uric acid can be prevented by a superoxide scavenger or by inhibiting nox. the effect of hyperuricemia might be partially responsible for the low - grade inflammation and insulin resistance in the adipose tissue and for increased risk of cardiovascular disease induced by obesity. | objectivehyperuricemia is strongly associated with obesity and metabolic syndrome and can predict visceral obesity and insulin resistance. previously, we showed that soluble uric acid directly stimulated the redox - dependent proinflammatory signaling in adipocytes. in this study we demonstrate the role of hyperuricemia in the production of key adipokines.research design and methodswe used mouse 3t3-l1 adipocytes, human primary adipocytes, and a mouse model of metabolic syndrome and hyperuricemia.resultsuric acid induced in vitro an increase in the production (mrna and secreted protein) of monocyte chemotactic protein-1 (mcp-1), an adipokine playing an essential role in inducing the proinflammatory state in adipocytes in obesity. in addition, uric acid caused a decrease in the production of adiponectin, an adipocyte - specific insulin sensitizer and anti - inflammatory agent. uric acid induced increase in mcp-1 production was blocked by scavenging superoxide or by inhibiting nadph oxidase and by stimulating peroxisome - proliferator activated receptor- with rosiglitazone. downregulation of the adiponectin production was prevented by rosiglitazone but not by antioxidants. in obese mice with metabolic syndrome, we observed hyperuricemia. lowering uric acid in these mice by inhibiting xanthine oxidoreductase with allopurinol could improve the proinflammatory endocrine imbalance in the adipose tissue by reducing production of mcp-1 and increasing production of adiponectin. in addition, lowering uric acid in obese mice decreased macrophage infiltration in the adipose tissue and reduced insulin resistance.conclusionshyperuricemia might be partially responsible for the proinflammatory endocrine imbalance in the adipose tissue, which is an underlying mechanism of the low - grade inflammation and insulin resistance in subjects with the metabolic syndrome. |
immune responses to gut flora are thought to play an important role in the multifactorial pathogenic process of crohn s disease (cd). differences in serologic responses to intestinal microbiota have been associated with disease location and behavioral phenotypes, further implicating the interaction between host immunity and enteric antigens in the etiopathogenesis of cd.1 anti - saccharomyces cerevisiae antibodies (asca) were the first serologic marker identified for cd in the 1980s.2 asca immunoglobulins a and g (iga and igg), antibodies to escherichia coli outer membrane porin - c (anti - omp - c), antibodies to clostridial flagellin (anti - cbir-1) and to pseudomonas fluorescens (i2) have been associated with small bowel disease location and complicated (fibrostenotic or perforating) disease behavior.39 post - operative complication risk has also been associated with the presence of anti - cbir-1 and anti- omp - c.1013 perinuclear anti - neutrophil cytoplasmic antibodies (p - anca) are common in patients with ulcerative colitis (uc). however, existing literature has demonstrated that cd patients with isolated colonic location and non - stricturing, non - penetrating phenotype are more likely to have positive p - anca serology.14 it is estimated that approximately 15% of patients with cd are diagnosed > 40 years of age, and that the incidence of inflammatory bowel disease (ibd) diagnosis in this older age group appears to be increasing over time.15 retrospective studies have demonstrated that patients with older age at diagnosis are less likely to have a complicated disease course, and more often have isolated colonic disease.16,17 conversely, younger patients are more likely to have complicated disease behavior and small bowel disease location.16 similar findings were shown in a retrospective analysis, which also evaluated those diagnosed at age 60 and over. older patients with cd in this study were also less likely to develop complicated disease behavior and were more likely to have isolated colonic disease location.17 in pediatric onset ibd, differences in serologic expression to gut microbial antigens is variable depending on age at diagnosis;14,18 however, little information is available regarding serologic response to gut microbial antigens in older patients with cd. in the current study, we compared the levels of asca igg, asca iga, anti - omp - c, anti - cbir-1, and p - anca by age of diagnosis. we hypothesized that based on the decreased prevalence of small bowel involvement and complicated disease behavior in older cd patients, those diagnosed at age 60 years or older would be less likely to have positive responses to microbial antigens and to have lower quartile scores to the cd - specific antigens than younger cd patients. patients with cd from the university of maryland, baltimore ibd program were eligible to participate from january 2010 to february 2011. the diagnosis of cd was confirmed using standard clinical, endoscopic, radiologic, and histologic criteria.19 patients with uc, ibd unknown type, or other forms of colitis were excluded. sera collected from cd patients were tested during a routine clinical visit for the presence of asca iga, asca igg, anti - omp - c, anti - cbir-1, and p - anca using the prometheus laboratories inc. a patient was considered positive for a serology marker if the result was above the reference range values. demographic and clinical data were extracted from an institutional review board (irb)-approved clinical data repository. the proportion of patents with positive serologic responses to each microbial antigen was compared among the following age - at - diagnosis groups : < 40 years, 4059 years, and 60 years of age, using the chi - square test. mean titers to each microbial antigen were compared among the three groups using the kruskal wallis test. the proportion of patients with positive antibodies to multiple antigens was also compared between groups, using the chi - square test. scatter plots were generated to compare the distribution of antibody positivity for each cd - specific antigen in the cohort, using the kruskal for each cd - specific microbial antigen, patients with detectable antibody levels in the first, second, third, and fourth quartile of distribution were assigned a quartile score of 1, 2, 3, or 4, respectively.8 individual quartile scores for each microbial antigen were then added to create a sum quartile score for each patient to represent cumulative quantitative immune response toward cd - specific antigens.8 mean quartiles scores between groups were compared using analysis of variance (anova), as these scores were noted to have a normal distribution. statistical analyses were performed using sas (cary, nc, usa) statistical analysis software, version 9.2. this study was carried out with the approval of the university of maryland at baltimore human research protections office. at recruitment, this study was carried out with the approval of the university of maryland at baltimore human research protections office. at recruitment, eighteen, 17, and 12 patients were diagnosed at ages < 40 years, between ages 40 and 59 years, and at age 60 years or over, respectively, with ages ranging from 20 to 79 years. fifty - three percent of the total patient population was female, and 81% was caucasian. there were no differences in sex, race, smoking status, or extra - intestinal manifestations between the age - at - diagnosis groups (table 1). while no difference was detected in behavioral phenotype between the age - at - diagnosis groups, there was a difference in disease location noted, with 71% of patients < 40 years of age at diagnosis demonstrating ileocolonic disease location compared to 36% ileocolonic disease location in patients diagnosed at age 60 years and over (p<0.01). in addition, 47% of patients diagnosed at ages 4059 years had ileal disease location compared to 0% of patients diagnosed at < 40 years of age (p<0.01). in addition, there was a trend detected for perianal involvement in patients diagnosed at age < 40 years ; however, this trend was not statistically significant (p=0.15). fifty percent, 18%, and 50% of patients diagnosed at < 40 years, 4059 years, and 60 years of age had elevated levels of asca iga (p=0.09 ; table 2). the mean titer of asca iga was 44.627.7 eu / ml in patients diagnosed < 40 years of age, 42.623.2 in patients diagnosed at ages 4059, and 24.03.6 in patients diagnosed 60 years of age (p=0.19 ; figure 1). fifty percent, 18%, and 17% of patients diagnosed at ages < 40, 4059, and 60 years had elevated levels for asca igg (p=0.07 ; table 2). similarly, the mean titer of asca igg was elevated at 55.324.6 in patients diagnosed at < 40 years of age, compared to 36.414.8 in patients diagnosed between ages 40 and 59 years, and 33.417.7 in patients diagnosed at 60 years of age, though not statistically significantly (p=0.13 ; figure 1). fifty percent, 18%, and 33% of patients aged < 40 years, 4059 years, and 60 years at diagnosis had abnormal anti - omp - c levels (p=0.16 ; table 2). there was also no difference in mean titers to anti - omp - c between the three different age - at - diagnosis groups (p=0.69 ; figure 1). forty - four percent, 29%, and 50% of patients < 40, 4059, and 60 years of age at diagnosis had abnormal anti - cbir-1 levels (p=0.49 ; table 2). there was also no difference in mean titers to anti - cbir-1 between the three different age - at - diagnosis groups (p=0.09 ; figure 1). twenty - eight percent, 24%, and 42% of participants in the youngest, middle, and oldest age - at - diagnosis groups had an abnormal p - anca level (p=0.56 ; table 2). there was also no difference in mean titers of p - anca between age groups (p=0.96 ; figure 1). overall, 81%, 45%, 30%, and 13% of all patients had an abnormal response to at least one, at least two, at least three, and at least four microbial antigens, respectively. the proportion of patients with an abnormal response to any microbial antigen overall did not differ between age - at - diagnosis groups (p=0.19 ; table 3). mean quartile sum scores for antibody levels were higher in the youngest age - at - diagnosis group : 7.22.8 in those patients diagnosed at < 40 years of age, 4.92.9 in those diagnosed between ages 40 and 59 years, and 5.62.6 in those diagnosed at age 60 years or over (p=0.06 ; figure 2). eighteen, 17, and 12 patients were diagnosed at ages < 40 years, between ages 40 and 59 years, and at age 60 years or over, respectively, with ages ranging from 20 to 79 years. fifty - three percent of the total patient population was female, and 81% was caucasian. there were no differences in sex, race, smoking status, or extra - intestinal manifestations between the age - at - diagnosis groups (table 1). while no difference was detected in behavioral phenotype between the age - at - diagnosis groups, there was a difference in disease location noted, with 71% of patients < 40 years of age at diagnosis demonstrating ileocolonic disease location compared to 36% ileocolonic disease location in patients diagnosed at age 60 years and over (p<0.01). in addition, 47% of patients diagnosed at ages 4059 years had ileal disease location compared to 0% of patients diagnosed at < 40 years of age (p<0.01). in addition, there was a trend detected for perianal involvement in patients diagnosed at age < 40 years ; however, this trend was not statistically significant (p=0.15). fifty percent, 18%, and 50% of patients diagnosed at < 40 years, 4059 years, and 60 years of age had elevated levels of asca iga (p=0.09 ; table 2). the mean titer of asca iga was 44.627.7 eu / ml in patients diagnosed < 40 years of age, 42.623.2 in patients diagnosed at ages 4059, and 24.03.6 in patients diagnosed 60 years of age (p=0.19 ; figure 1). fifty percent, 18%, and 17% of patients diagnosed at ages < 40, 4059, and 60 years had elevated levels for asca igg (p=0.07 ; table 2). similarly, the mean titer of asca igg was elevated at 55.324.6 in patients diagnosed at < 40 years of age, compared to 36.414.8 in patients diagnosed between ages 40 and 59 years, and 33.417.7 in patients diagnosed at 60 years of age, though not statistically significantly (p=0.13 ; figure 1). fifty percent, 18%, and 33% of patients aged < 40 years, 4059 years, and 60 years at diagnosis had abnormal anti - omp - c levels (p=0.16 ; table 2). there was also no difference in mean titers to anti - omp - c between the three different age - at - diagnosis groups (p=0.69 ; figure 1). forty - four percent, 29%, and 50% of patients < 40, 4059, and 60 years of age at diagnosis had abnormal anti - cbir-1 levels (p=0.49 ; table 2). there was also no difference in mean titers to anti - cbir-1 between the three different age - at - diagnosis groups (p=0.09 ; figure 1). twenty - eight percent, 24%, and 42% of participants in the youngest, middle, and oldest age - at - diagnosis groups had an abnormal p - anca level (p=0.56 ; table 2). there was also no difference in mean titers of p - anca between age groups (p=0.96 ; figure 1). overall, 81%, 45%, 30%, and 13% of all patients had an abnormal response to at least one, at least two, at least three, and at least four microbial antigens, respectively. the proportion of patients with an abnormal response to any microbial antigen overall did not differ between age - at - diagnosis groups (p=0.19 ; table 3). mean quartile sum scores for antibody levels were higher in the youngest age - at - diagnosis group : 7.22.8 in those patients diagnosed at < 40 years of age, 4.92.9 in those diagnosed between ages 40 and 59 years, and 5.62.6 in those diagnosed at age 60 years or over (p=0.06 ; figure 2). in this tertiary referral population of patients with cd, we found a trend toward increased proportion of patients diagnosed at < 40 years of age with positive asca iga and asca igg antibodies, compared to those diagnosed at 40 years and older, although this trend did not reach statistical significance. further, we noted a trend toward higher titers of asca iga, asca igg, and overall quartile scores for all cd - associated antibodies in younger patients, compared to older patients with cd. studies in adults have shown that in cd, age is associated with disease location and behavior.16,17,20,21 information on serologic responses to gut microbial antigens by age at diagnosis is lacking in adults. however, differential serologic responses to microbial gut antigens have been evaluated in pediatric cd populations.18,2224 markowitz compared the presence of asca iga and igg, anti - omp - c antibody, anti - cbir-1 antibody, and ibd - associated p - anca among children diagnosed with cd at # 7 years of age and between 8 and 15 years of age.14 children diagnosed between 8 and 15 years of age were more likely to have elevated asca iga and igg compared to patients diagnosed at 7 years or younger. conversely, children diagnosed at 7 years or younger were more likely to have elevated anti - cbir-1 antibody responses. interestingly, there were also differences noted in the distribution of disease location in these children. children diagnosed at 7 years or younger were more likely to have isolated colonic disease, whereas children diagnosed between 8 and 15 years of age were more likely to have ileocolonic disease location. these results suggest an alternate genetic and environmental pathophysiologic process as the etiology of cd at extremes of age. in our present study, a trend toward higher titers of asca iga and igg was noted among patients diagnosed at < 40 years of age. positivity for asca has been associated with small bowel disease location and complicated behavioral phenotypes including stricturing and perforating disease.2527 complicated cd phenotypes are also more often seen in patients diagnosed at younger ages ; therefore, these trends are not surprising. prior epidemiologic studies comparing younger and older cd patients point to an alternate pathway for the development of cd in older patients.2527 the lower prevalence of asca in this older population may point to a pathogenic process that does not typically involve the small bowel or result in complicated phenotypes. conversely, it is possible that the development of ibd in older patients is in fact due to dysregulated immune responses to gut microbiota that are not yet identified and not on the ibd 7 test. analyzing the potential differences in gut microflora in cd patients with younger and older ages at diagnosis would help to further elucidate the meaning of our observed serologic responses. our present study was limited by small sample size, particularly in the oldest age - at - diagnosis group. this small sample size may have impaired our ability to detect differences in responses to other microbial antigens in the ibd 7 test. further, when interpreting our results, one must consider the role of immune senescence and how it may impact the ibd 7 test analysis. currently, there are no studies that elucidate the possible effect of immune senescence on the sensitivity and specificity of the ibd 7 serologic test. this natural decline in the production of nave, antigen - specific lymphocytes and t cell dysregulation with aging may be the underlying explanation for the reduced asca level seen in older patients.28 the most accurate method to clarify the role of asca in the etiopathogenesis of cd in the elderly would be to follow this patient cohort (particularly the youngest age - at - diagnosis group) in a long - term, prospective study where serologic analyses are performed at regular intervals and assessed for declines over time. given the understanding that asca positivity does not correlate with disease duration or activity,29 a decline over time in this serologic response would suggest an effect of immune senescence. while medical therapy data was not included in the present analysis, there is currently no evidence in the literature to suggest that medical therapy impacts detection of serologic markers in ibd. to the best of our knowledge, this is the first study to focus on serologic responses to gut microflora in young, middle - aged, and older adults with cd. it also addresses the clinically relevant question of whether cd - specific serologic testing is beneficial in patients diagnosed at older ages. in summary, our present study demonstrated an increased proportion of patients diagnosed with cd at < 40 years of age with a positive asca compared to patients diagnosed at older ages. this finding is congruent with studies that have shown that patients with cd diagnosed at older ages are less likely to develop complicated cd, given that positive asca has been associated with complicated cd.2527 our findings also suggest that the same disease may have differing etiopathogenesis depending on the age at diagnosis. further research is needed to identify differences in bacterial populations in different age - at - diagnosis groups and how these changes, if present, affect the pathogenesis of cd. in addition, clinicians should be aware that cd diagnosed in older ages may represent a distinct phenotype, one in which serologic responses to microbial antigens are less common. | purposefifteen percent of incident crohn s disease (cd) cases are diagnosed at older ages and demonstrate colonic location and inflammatory behavior. serologic responses to gut microbial antigens are associated with specific phenotypes, and may differ by age at diagnosis. our aim was to identify an association between age at diagnosis of cd and responses to gut microbial antigens.patients and methodslevels of anti - saccharomyces cerevisiae antibodies (asca) immunoglobulins a and g (iga and igg), antibodies to escherichia coli outer membrane porin - c (anti - omp - c), antibodies to clostridial flagellin (anti - cbir-1), and perinuclear anti - neutrophil cytoplasmic antibodies (p - anca) were compared in patients by age in three diagnosis groups : patients diagnosed at ages of < 40, 4059, and 60 years. for each antigen, patients with antibody levels in the first, second, third, and fourth quartile were assigned a score of 1, 2, 3, or 4, respectively. individual scores were added to create a quartile sum score representing cumulative quantitative immune response.resultseighteen, 17, and 12 patients were diagnosed at ages < 40, 4059, and 60 years, respectively. the majority (71%) had ileocolonic disease in the youngest group, compared to 36% in the oldest group (p=0.001). mean asca iga and igg titers were increased in the youngest age group compared to the older groups (p=0.19 and p=0.13, respectively). mean quartile sum scores for antibody levels were 7.22.8 in those patients diagnosed at ages < 40 years, 4.92.9 in the 4059-year - old age group, and 5.62.6 in the 60-year - old age group (p=0.06).conclusiona trend toward decreased cumulative immune responses to cd - associated gut antigens was observed in cd patients diagnosed at older ages compared to younger patients. host responses to microbial antigens may be less important in older onset ibd and may contribute to the distinct phenotype in this group. |
in japan s super - aging society, social demands for rehabilitation are increasing. in line with this, the numbers of physical (pt) and occupational (ot) therapist training schools have rapidly increased ; as of 2013, there were 248 and 182, respectively, and the total numbers of certified pts and ots were 110,000 and 65,000, respectively. the rapid increase in the number of therapists has consequently reduced the overall years of experience of therapists in clinical environments, suggesting a decrease in the quality of education provided by them to novices and students. under these circumstances, it is necessary to place more importance on postgraduate education in workplaces, in addition to improving school education systems. however, at present, postgraduate education is independently provided by associations, prefectural societies, or privately, and it would be difficult for therapists to establish systems to provide such education in their own workplaces by themselves. furthermore, although a number of technical seminars for clinical therapists have been held, their contents are not applicable to some actual situations, as most of them solely involve healthy individuals. considering such a situation, school education should enable students to sufficiently learn about the basic items necessary to achieve more specialized knowledge and skills after graduation, rather than only the application of skills. in line with this, some researchers point out the necessity of considering pre and postgraduate education separately to determine their appropriate contents and subsequently establishing comprehensive education systems1. to conduct consistent education at the undergraduate to postgraduate levels, we teach novice therapists using the same objective structured clinical examination (osce) items. the contents taught include how to use the knowledge and skills acquired through use of the undergraduate osce in clinical practice. until now, diverse approaches to postgraduate training have been implemented in clinical environments ; however, such approaches have not been unified, and appropriate methods to assess novice therapists clinical skills, as well as educational methodologies, have not yet been fully examined. in addition, clinical skill training is rarely provided by faculty members in clinical environments after graduation. therefore, unification of postgraduate and clinical education methods, based on the results of objective assessment of clinical skills in early stages, may be the key to novice therapists development. at our university, physical and occupational therapies are regarded as a domain of therapeutics or a clinical science, and specialized education for students, in other words, education to nurture specialists with clinical skills, has been provided, focusing on clinical demands, since our department was founded in 2004. in short, however, in the current therapist education system, students clinical skills are assessed by supervisors of clinical training facilities, rather than the faculty members of training schools ; in therapist education to nurture clinical professionals, this should be a critical issue. our faculty members are also engaged in clinical treatment and are engaged in ongoing discussions concerning appropriate methods to train students by making use of therapists working in clinical environments. at our university, osce system has been adopted, with a view to specifying standards for clinical education in training schools and standardizing methods of assessment by clinical supervisors1. the osce is a method of clinical skill assessment proposed by harden2 in 1975 and has been reported to be appropriate for assessment of learning achievement levels in the psychomotor and emotional domains, which are difficult to evaluate with written examinations. we have reported an association between osce scores, academic achievement levels, and clinical training outcomes, as well as inter - rater agreement rates (0.767 to 0.935) between faculty members, in our previous studies4,5,6. furthermore, in a recent study, we examined the appropriateness of adopting the osce in postgraduate education by confirming the consistency between osce scores, representing the results of assessment by faculty members, and clinical training outcomes assessed by clinical supervisors. as a result, favorable agreement rates were obtained in behavioral aspects, while challenges were revealed in the technical aspects. the present study examined the applicability of the osce to postgraduate education systems for novice and mid - career therapists in workplaces. the study involved pts and ots working in clinical environments for 1 to 5 years after graduating from training schools as osce examinees, and a physical or occupational therapy faculty member and a clinical supervisor as raters. another clinical supervisor acted as a simulated patient. before examination, the examiners (the faculty member and clinical supervisor) and simulated patient agreed upon the latter s condition, in addition to the points of assessment. the osce consisted of items to assess skills to assist and guide patients performing the following level 3 activities : < elevating the upper body and sitting on the edge of the bed, standing up, wheelchair - to - bed transfer, dressing, toileting and walking. it was conducted before (examination 1) and after (examination 2) learning sessions consisting of 60-minute lectures regarding each item, practice, and practical guidance in clinical settings, focusing on the applicability of the osce items to clinical environments. the osce was conducted in 3 rooms (stations), within each of which a task was presented, and the examinees made the rounds of these stations to implement all the tasks in accordance with the instructions. at each station, 2 examiners (a physical or occupational therapy faculty member and a clinical supervisor) and 1 simulated patient (another clinical supervisor) were present. the time to implement each task was 5 minutes, and, immediately after its completion, 2 minutes of feedback was provided. comparison between before and after osce - based learning was performed by conducting a paired two sample (wilcoxon signed - rank) test, and that between therapists with 1 to 2 years (novice) and 3 to 5 years (mid - career) of clinical experience was performed by conducting an unpaired two sample (mann - whitney u) test. the study was approved by the ethics committee of the fujita health university (10 - 121). prior to data collection, all subjects gave written informed consent after thorough explanation of the study. on comparison between before and after osce (level 3)-based learning, no significant differences in behavioral aspects were observed in novice or mid - career therapists. behavioral differences between novice and mid - career therapists were nonsignificant both before and after learning. in contrast, in the technical aspects, both novice and mid - career therapists showed significantly higher scores after learning than before learning. significant differences were also observed between them after learning (table 1table 1.comparisons of scores between before and after osce - based learning and between novice and mid - career therapistsnovicetherapistsmid - careertherapistsnovice therapistsvs. mid - career therapistspreparatory itemsbefore learning96.63.496.82.8after learning97.11.397.40.9before learning vs. after learningtechnical itemsbefore learning48.68.158.315.0after learning64.08.976.38.5before learning vs. after learning p<0.05). among the six level 3 osce items, scores representing behavioral skills to assist / guide patients for elevating the upper body and sitting on the edge of the bed markedly increased after learning in both novice and mid - career therapists. regarding technical skills, a significant increase in scores for elevating the upper body and sitting on the edge of the bed, toileting and dressing was observed after learning in both novice and mid - career therapists. such an increase was also observed for transfer in novice and for standing up in mid - career therapists. furthermore, scores for transfer were significantly higher in mid - career therapists than novice therapists after learning. scores for dressing were also significantly higher in mid - career therapists than novice therapists before learning (table 2table 2.comparisons of scores for the six level 3 osce items between before and after learning and between novice and mid - career therapistsnovice and mid - careertherapistsnovicetherapistsmid - careertherapistsnovice therapistsvs. mid - career therapistspreparatory items standing upbefore learning99.42.799.03.5100.00.0after learning99.42.799.03.5100.00.0before learning vs. after learningsitting up on a bedbefore learning86.714.285.31688.911.8after learning99.22.598.73.1100.00.0before learning vs. after learningtoilet activitybefore learning98.93.799.03.598.64.2after learning98.93.799.03.598.64.2before learning vs. after learningtransferbefore learning97.74.997.15.598.64.2after learning98.93.798.14.7100.00.0before learning vs. after learningwalkingbefore learning98.93.799.03.598.64.2after learning86.92.786.53.587.50.0before learning vs. after learningdressing activitybefore learning98.95.398.16.9100.00.0after learning100.00.0100.00.0100.00.0before learning vs. after learningtechnical itemsstanding upbefore learning67.015.963.117.672.811.5after learning73.917.664.214.187.812.0before learning vs. after learningsitting up on a bedbefore learning41.614.640.813.842.816.4after learning59.314.555.414.265.013.7before learning vs. after learningtoilet activitybefore learning42.815.139.914.747.015.5after learning69.118.463.319.877.412.8before learning vs. after learningtransferbefore learning57.219.653.513.862.525.8after learning74.614.869.614.581.912.5before learning vs. after learningwalkingbefore learning42.815.139.914.747.015.5after learning48.918.742.918.357.416.6before learning vs. after learningdressing activitybefore learning64.117.856.214.775.616.1after learning88.29.286.59.490.68.8before learning vs. after learning p<0.05). on comparison between before and after learning, mid - career therapists showed markedly higher scores for the following 3 subitems of standing up after learning : being able to confirm the appropriate height of an adjustable bed, being able to confirm the position of a patient s buttocks and move them forward with guidance / assistance, and being able to appropriately control hip flexion, shift the axis of rotation to the knee joint, and move the patient s knees slightly forward using the legs based on the principle of leverage when leaving the bed. such an increase in scores was also observed for the following 2 subitems of elevating the upper body and sitting on the edge of the bed in both novice and mid - career therapists : being able to remove and keep blankets and clothes on the bed in order and being able to confirm sufficient space for the lateral position. novice therapists also showed significantly higher scores for the following three subscales after learning : being able to provide appropriate explanations before assessing movements to elevate the upper body and sit on the edge of the bed, being able to appropriately confirm the patient s consent, and being able to stand in an appropriate position for guiding the patient. on the other hand, mid - career therapists showed significantly higher scores for the following 2 sub - items after learning : being able to appropriately guide the head upward and being able to appropriately guide the lower body toward the lateral position. regarding toileting, both novice and mid - career therapists showed markedly higher scores after than before learning for the following 6 subitems being able to provide guidance for appropriately standing up from the bed, being able to provide guidance for a change of direction and stable standing position, being able to confirm the appropriate posture when pulling down clothes, being able to provide guidance for appropriately sitting on the toilet seat, being able to provide guidance for appropriately standing up from the toilet seat, and being able to confirm the appropriate posture when pulling up clothes. mid - career therapists also showed a significant increase in scores for the following 3 subitems being able to give advice for smoothly and appropriately handling clothes when pulling them down, being able to give advice for smoothly and appropriately handling clothes when pulling them up, and being able to provide guidance for a change of direction and sitting on the edge of the bed. regarding transfer, novice therapists showed a significant increase in scores for the following 4 subitems after learning being able to appropriately place the wheelchair by the bed being able to adjust the height of the bed to ensure safety being able to advise or assist the patient to take the feet off the foot plates, place them on the ground and raise them and, being able to support the patient s chest and abdomen with the forearms from behind before standing up. regarding dressing, scores for being able to provide appropriate guidance / assistance for drawing the garment over the head (while maintaining the pelvis in a neutral position) markedly increased after learning in both novice and mid - career therapists. the novices therapist scores for the following 8 subitems also significantly increased : being able to take appropriate safety measures, being able to confirm the maintenance of a stable sitting position and assist / guide for appropriate anteroposterior pelvic tilts being able to confirm the reach movements of the non - paralyzed upper limb being able to provide appropriate guidance for maintaining the paralyzed upper limb straight by the trunk and using the weight of the upper limb, being able to provide appropriate guidance for placing the paralyzed upper limb in the sleeves, being able to provide appropriate guidance for placing the non - paralyzed upper limb in the sleeves of a garment (while ensuring a stable sitting position), being able to provide appropriate guidance / assistance for the elbow and shoulder when placing the paralyzed upper limb in the sleeves of a garment and to prepare for pulling, and prepare for pulling an upper garment over the head and being able to correct the shoulder line and bottom edge of the garment on the paralyzed side. no significant differences between before and after learning were observed in scores for walking (table 3table 3.comparison of scores for the six level 3 osce items between before and after learning (in novice and mid - career therapists)novice therapists vs. mid - career therapistsstanding upnovice therapistsmid - career therapistspreparatory items appropriately groomed being able to appropriately greet being able to provide appropriate explanations before assessing movements to stand up being able to appropriately confirm the patient s consenttechnical items being able to confirm the appropriate height of an adjustable bed being able to confirm the position of the patient s buttocks, and move them forward with guidance / assistance being able to move the patient s legs at an appropriate angle, confirm the appropriate width between his / her feet and condition on the heels on the floor, and correct them with guidance / assistance being able to correct the posture, alignment, and center of balance with guidance / assistance being able to confirm and give advice for anterior pelvic tilt and participation as a preparatory step for leaving the bed being able to support the patient s pelvis with the hands and his / her trunk with the chest, while standing with the knees bent when leaving the bed being able to move the patient s knees forward using the legs when leaving the bed being able to bend the patient s head and trunk forward and shift the center of balance at a safe speed with guidance / assistance being able to support the patient s knees and ensure appropriate hip, knee, and trunk extension with guidance / assistance after leaving the bed being able to correct the alignment with guidance / assistance to maintain a stable standing positionnovice therapists vs. mid - career therapistssitting up on the bednovice therapistsmid - career therapistspreparatory items appropriately groomed being able to appropriately greet being able to provide appropriate explanations before assessing movements to elevate the upper body and sit on the edge of the bed being able to appropriately confirm the patient s consent being able to appropriately remove a bed rail near the patient s leg being able to remove and keep blankets and clothes on the bed in ordertechnical items being able to motivate the patient to rise and enhance his / her activeness being able to confirm sufficient space for the a lateral position being able to roughly determine the patient s remaining functions being able to provide appropriate explanations regarding procedures from elevating the upper body to sitting on the edge of the bed being able to appropriately guide the head upward being able to appropriately guide the upper body toward a lateral position being able to appropriately guide the lower body toward a lateral position being able to give instructions at appropriate times throughout the activity being able to stand in an appropriate position for guiding the patient being able to take preventive measures against falls while maintaining an appropriate sitting positionnovice therapists vs. mid - career therapiststoilet activitynovice therapistsmid - career therapistspreparatory items appropriately groomed being able to appropriately greet being able to provide patients with appropriate explanations before toilet training being able to appropriately confirm the patient s consenttechnical items being able to confirm appropriate environments for the activity being able to provide appropriate explanations regarding procedures being able to provide guidance for appropriately standing up from the bed being able to provide guidance for a change of direction and stable standing position being able to confirm the appropriate posture when pulling down clothes being able to give advice for smoothly and appropriately handling clothes being able to provide guidance for appropriately sitting on the toilet seat being able to provide guidance for maintenance of an appropriate sitting position being able to give advice for appropriately cleaning the anus / genitals being able to provide guidance for appropriately standing up from the toilet seat being able to confirm the appropriate posture when pulling up clothes being able to give advice for smoothly and appropriately handling clothes when pulling them up being able to provide guidance for a change of direction and sitting on the edge of the bednovice therapists vs. mid - career therapiststransfernovice therapistsmid - career therapistspreparatory items appropriately groomed being able to appropriately greet being able to provide appropriate explanations before assessing transfer being able to appropriately confirm the patient s consentnovice therapists vs. mid - career therapiststechnical itemsnovice therapistsmid - career therapists being able to appropriately place the wheelchair by the bed being able to adjust the height of the bed to ensure safety being able to advise or assist the patient to raise the foot plates being able to advise the patient to participate in the activity as much as possible being able to provide guidance for moving the buttocks forward and sitting on the anterior part of the wheelchair seat being able to place the non - paralyzed foot slightly ahead of the paralyzed one being able to support the patient s knee on the paralyzed side by maintaining it between the distal parts of both thighs being able to give advice for bending the trunk forward while placing the hands by the waist or on the knees being able to support the patient s chest and abdomen with the forearms before standing up being able to give instructions to stand up together at an appropriate time being able to guide / assist the patient to turn his / her buttocks to the bed and slowly sit on it being able to correct the posture when sitting on the edge of the bed and make it stablewalkingpreparatory items appropriately groomed being able to appropriately greet being able to provide appropriate explanations before performing gait analysis being able to appropriately confirm the patient s consenttechnical items being able to take safety measures being able to ensure appropriate walking environments being able to confirm appropriate clothes, shoes, and aids for walking being able to advise the patient to participate in the activity as much as possible being able to confirm the ability to move in a standing position being able to appropriately instruct to start walking being able to appropriately provide guidance / assistance during the swing phase on the paralyzed side being able to appropriately provide guidance / assistance during the initial contact to loading response phases on the paralyzed side being able to appropriately provide guidance / assistance during the mid - stance phase on the paralyzed side being able to appropriately provide guidance / assistance during the terminal stance phase on the paralyzed side being able to confirm appropriate walking rhythms being able to appropriately guide the patient after walkingdressing activitypreparatory items appropriately groomed being able to appropriately greet being able to provide appropriate explanations before assessing dressing being able to appropriately confirm the patient s consenttechnical items being able to take appropriate safety measures being able to confirm the maintenance of a stable sitting position being able to confirm the reach movements of the non - paralyzed upper body being able to confirm appropriate procedures before placing the arms in the sleeves of a garment being able to provide appropriate guidance for maintaining the paralyzed arm straight by the trunk and using the weight of the upper body being able to provide appropriate guidance for placing the paralyzed arm in the sleeves of a garment being able to provide appropriate guidance for placing the non - paralyzed arm in the sleeves of a garment (while ensuring a stable sitting position) being able to provide appropriate guidance / assistance for the elbow and shoulder when placing the paralyzed arm in the sleeves of a garment and to prepare for pulling an upper garment over the head being able to provide appropriate guidance / assistance for drawing the garment over the head (while maintaining the pelvis in a neutral position) being able to correct the shoulder line and bottom edge of the garment on the paralyzed side p<0.01, p<0.05). on comparison between novice and mid - career therapists, significant differences were observed in scores for standing up and transfer after learning and for dressing. among the sub - items of standing up, significant differences were observed in the 4 after learning. among those of transfer, significant differences were observed in being able to advise the patient to participate in the activity as much as possible after confirming the patient s ability after learning. among those of dressing, significant differences were observed in the following 3 before learning being able to take safety measures, being able to confirm the reach movements of the non - paralyzed upper limb, and being able to provide appropriate guidance for maintaining the paralyzed upper limb straight by the trunk and using the weight of the upper limb (table 4table 4.differences in scores for the level-3 osce items between novice and mid - career therapistsnovice therapists vs. mid - career therapistsstanding up : technical itemsnovice therapistsmid - career therapists being able to confirm the appropriate height of an adjustable bed being able to confirm the position of the patient s buttocks, and move them forward with guidance / assistance being able to move the patient s legs at an appropriate angle, confirm the appropriate width between his / her feet and condition on the heels on the floor, and correct them with guidance / assistance being able to correct the posture, alignment, and center of balance with guidance / assistance being able to confirm and give advice for anterior pelvic tilt and participation as a preparatory step for leaving the bed being able to support the patient s pelvis with the hands and his / her trunk with the chest while standing with the knees bent when leaving the bed being able to move the patient s knees forward using the legs when leaving the bed being able to bend the patient s head and trunk forward and shift the center of balance at a safe speed with guidance / assistance being able to support the patient s knees and ensure appropriate hip, knee, and trunk extension with guidance / assistance after leaving the bed being able to correct the alignment with guidance / assistance to maintain a stable standing positionnovice therapists vs. mid - career therapiststransfer : technical itemsnovice therapistsmid - career therapists being able to appropriately place the wheelchair by the bed being able to adjust the height of the bed to ensure safety being able to advise or assist the patient to raise the foot plates being able to advise the patient to participate in the activity as much as possible being able to provide guidance for moving the buttocks forward and sitting on the anterior part of the wheelchair seat being able to place the non - paralyzed foot slightly ahead of the paralyzed one being able to support the patient s knee on the paralyzed side by maintaining it between the distal parts of both thighs being able to give advice for bending the trunk forward while placing the hands by the waist or on the knees being able to support the patient s chest and abdomen with the forearms before standing up being able to give instructions to stand up together at an appropriate time being able to guide / assist the patient to turn his / her buttocks to the bed and slowly sit on it being able to correct the posture when sitting on the edge of the bed and make it stabledressing activity : technical items being able to take appropriate safety measures being able to confirm the maintenance of a stable sitting position being able to confirm the reach movements of the non - paralyzed upper body being able to confirm appropriate procedures before placing the arms in the sleeves of a garment being able to provide appropriate guidance for maintaining the paralyzed arm straight by the trunk and using the weight of the upper body being able to provide appropriate guidance for placing the paralyzed arm in the sleeves of a garment being able to provide appropriate guidance for placing the non - paralyzed arm in the sleeves of a garment (while ensuring a stable sitting position) being able to provide appropriate guidance / assistance for the elbow and shoulder when placing the paralyzed arm in the sleeves of a garment and to prepare for pulling an upper garment over the head being able to provide appropriate guidance / assistance for drawing the garment over the head (while maintaining the pelvis in a neutral position) being able to correct the shoulder line and bottom edge of the garment on the paralyzed side p<0.05). in recent years, the osce has been used as an educational approach to objective assessment of clinical skills mainly in the field of medicine8,9,10. compared with conventional written examinations, the osce enables examiners to assess clinical skills in the psychomotor, emotional, and cognitive domains, and clarify points to improve7. reported that adoption of the osce in medical education is effective for training of medical students by developing necessary basic skills in both technical and behavioral aspects, and it enables educators to guide students toward the appropriate integration of knowledge, skills, and behavior11. in fact, the osce adopted for clinical education in training schools has provided a certain effect4,5,6 ; however, education methods in training schools and clinical training facilities have not yet been unified. although a diverse range of training seminars has been held by the japanese physical therapy association for postgraduate skill improvement, not all novice therapists have necessarily participated in them. in addition, the quality of education for novices in workplaces widely varies and remains unclear. considering such a situation, this study examined the applicability of the osce to postgraduate education systems by performing comparisons of skills between before and after osce - based learning and between novice and mid - career therapists. as a result, no changes in the behavioral aspects of the osce items were observed ; the favorable scores before learning support the appropriateness of the current education method in the behavioral aspects. however, as this result was obtained in a single facility, further studies involving multiple facilities may be necessary to confirm it. in contrast, in the technical aspects, scores for all items increased after learning ; the increase was particularly marked in mid - career therapists, suggesting the possibility of developing basic clinical skills that every therapist has to acquire through daily clinical practice, regardless of the field. furthermore, considering that the increase in scores after learning was generally more marked in mid - career therapists than in novice therapists, their participation in training seminars, as well as self - directed learning and skill improvement, may have positively influenced osce - based learning as postgraduate education. on comparison of scores for the osce subitems, scores increased for similar subitems in novice and mid - career therapists. particularly, in novice therapists, scores for a large number of the sub - items of transfer and dressing significantly increased, highlighting the necessity of providing them with education focusing on these domains. regarding dressing, mid - career therapists exhibited markedly higher scores than novices before learning ; this may be explained by novice pts having insufficient experience in this domain, dressing is frequently considered a learning item for ot students in schools, and most pts learn about it through clinical practice after graduation. similarly, the novices scores for basic skills, such as being able to adjust the height of the bed to ensure safety and being able to confirm the reach movements of the non - paralyzed upper limb, and technical skills, such as being able to provide appropriate guidance for maintaining the paralyzed upper limb straight by the trunk and using the weight of the upper limb, were generally lower than those of mid - career therapists, suggesting the importance of providing education related to this activity in consideration of the contents of all these sub - items. mid - career therapists showed such higher scores, presumably due to their basic abilities, which had improved through adl training, such as fim - based approaches, in addition to typical physical therapy approaches. after learning, scores for standing up were markedly higher in mid - career therapists than novice therapists ; as standing up is one of the main rehabilitation tasks and mid - career therapists had accumulated their knowledge of it through daily clinical experiences for a longer period of time, the effect of osce - based learning in reviewing the points of clinical skills may have been more marked in them compared with novices. mid - career therapists scores for the subscales of standing up also markedly increased, particularly in the technical aspects, supporting the appropriateness of the osce - based learning method for improving therapists skills even after completing basic education programs. the results of this study suggest that the osce may be sufficiently applicable to postgraduate education systems in workplaces, in addition to school education systems. rehabilitation medicine is a practical system, in which medical services are provided based on learning achievements. therefore, in therapist education aiming to nurture learning specialists, it may be important to review the significance of osce - based skill education from a viewpoint of rehabilitation medicine in order to systematize it with new approaches, rather than simply adopting medical education systems. further studies may be necessary to examine the learning effects of the osce at different levels. | [purpose ] the aim of this study was to examine the applicability of the objective structured clinical examination (osce) to postgraduate education systems for novice and mid - career therapists in workplaces. [subjects ] physical and occupational therapists with 1 to 5 years of clinical experience took the osce to assess their learning, with a physical or occupational therapy faculty member and a clinical supervisor as examiners. another clinical supervisor acted as a simulated patient. [methods ] a wilcoxon signed - rank test was performed to compare skills between before and after osce - based learning, and a mann - whitney u test was used to compare them between therapists with 1 to 2 years (novice) and 3 to 5 years (mid - career) of clinical experience. [results ] while no experience - related differences were observed in behavioral aspects, mid - career therapists exhibited markedly higher scores compared with novices in technical aspects, such as skills to guide patients for standing up, transfer, and dressing. [conclusion ] the osce may be sufficiently applicable to postgraduate education systems in workplaces. |
despite health improvement being an explicit purpose of housing programmes in indigenous communities,13 the housing conditions and health of many indigenous australians continue to be very poor.4 australian government policy initiatives have consistently attempted to improve hygiene and living conditions through infrastructure programmes.5 attention has been focused on the components of infrastructure required for the effective conduct of healthy living practices (hlps).6 there is mounting evidence that the focus on infrastructure and relative neglect of hygiene and other interventions has failed to produce improvements in health in australia711 and internationally.1214 internationally, the contribution of poor domestic hygiene to poor health is well recognised,15 16 as is household crowding.1624 these factors may have a significant influence at the general community level as well as at the household level. however, the tendency for epidemiological research to focus on individual child or household level exposures and outcomes of housing improvements means there is little understanding of the extent to which reduced risks to health may be moderated by the extent to which housing programmes impact at the general community level. this moderating effect is likely to be an important influence on the potential for health improvement where there is a high level of interaction between children from different households and mobility of children between households. this paper addresses this issue of community level impact of housing programmes by reporting on three potentially important housing - related factors affecting child health : (1) overcrowding ; (2) the functional state of the house infrastructure ; and (3) the hygienic condition of houses. this work is part of a wider study of housing and child health called the housing improvement and child health (hich) study. the hich study was conducted in 10 northern territory (nt) communities in which there was the greatest construction of new houses by the australian government 's national aboriginal health strategy (nahs) environmental health program and other large infrastructure programmes over the period 20045. the communities were spread across the nt, and their distance from the nearest regional town ranged between 1 and 500 km. four of the 10 communities were isolated by floodwaters for at least part of most years. indigenous people living in these communities generally experience poor health, educational, employment and other social outcomes.25 the mean population of the 10 communities was 730 (range 2501450) and the average number of houses in each community was 66 (range 33103). the average of 11 people per house in these communities is markedly higher than the national average for indigenous households of 3.5 and the average for all australian households of 2.6.26 the high levels of crowding and multi - family households means that, when new houses become available in these remote communities, there is a shift from pre - existing to new houses and between pre - existing houses. the extended nature of indigenous families means that this shift can not be simply described in terms of rehousing of clearly - defined family units. allocation of new houses is reportedly based on need and residential history,26 but the definition of need and the way the allocation criteria are applied is unclear. some members of a pre - existing household may move to a new house, and/or some may move to another pre - existing house, and/or some may stay in the same house. these moves may or may not be associated with a change in a child 's primary carer. the intervention in this study was the construction of additional housing to specific housing standards for these communities. these standards are significantly more rigorous than standards applied in these communities over past decades.27 the average number of new houses to be constructed in each of the 10 communities was 11 (range 715) there were no concurrent renovation programmes or hygiene promotion activities conducted over the study period, so the housing intervention essentially consisted of the construction of a defined number of new houses. the hich study was conducted in 10 northern territory (nt) communities in which there was the greatest construction of new houses by the australian government 's national aboriginal health strategy (nahs) environmental health program and other large infrastructure programmes over the period 20045. the communities were spread across the nt, and their distance from the nearest regional town ranged between 1 and 500 km. four of the 10 communities were isolated by floodwaters for at least part of most years. indigenous people living in these communities generally experience poor health, educational, employment and other social outcomes.25 the mean population of the 10 communities was 730 (range 2501450) and the average number of houses in each community was 66 (range 33103). the average of 11 people per house in these communities is markedly higher than the national average for indigenous households of 3.5 and the average for all australian households of 2.6.26 the high levels of crowding and multi - family households means that, when new houses become available in these remote communities, there is a shift from pre - existing to new houses and between pre - existing houses. the extended nature of indigenous families means that this shift can not be simply described in terms of rehousing of clearly - defined family units. allocation of new houses is reportedly based on need and residential history,26 but the definition of need and the way the allocation criteria are applied is unclear. some members of a pre - existing household may move to a new house, and/or some may move to another pre - existing house, and/or some may stay in the same house. these moves may or may not be associated with a change in a child 's primary carer. the intervention in this study was the construction of additional housing to specific housing standards for these communities. these standards are significantly more rigorous than standards applied in these communities over past decades.27 the average number of new houses to be constructed in each of the 10 communities was 11 (range 715). there were no concurrent renovation programmes or hygiene promotion activities conducted over the study period, so the housing intervention essentially consisted of the construction of a defined number of new houses. the conceptual framework and methods used in the hich study have been described elsewhere.28 the main methods relevant to this paper include : (1) structured interviews with the main householder in all houses with children aged 7 years ; (2) infrastructure surveys of these houses ; and (3) interviews with staff of the community housing office and/or council and inspection of their housing records. local community residents were employed to work with survey teams and played a key role in the identification of houses where there were children aged < 7 years. the baseline householder interviews and infrastructure surveys were completed in each study community on average 6 months (range 118) before occupation of new houses. follow - up interviews and surveys were completed on average 10 months (range 712) after occupation of new houses. the total population in each study community did not change significantly between baseline and follow - up. our assessment of the impact of the building programme on the housing conditions for young children is based on comparison of conditions in houses that were the usual place of residence for children for whom we were able to collect data at both baseline and follow - up. the infrastructure surveys involved an inspection (and, where appropriate, testing) of the functional state of each house in relation to the effective conduct of hlps, including required infrastructure items based on the nt minimum housing standards29 and national indigenous housing guidelines.30 in order to provide more specific measures of infrastructure related to risks to child health, the nine hlps specified in the national guidelines were refined for the purpose of this study. the hlp of reducing negative contact between people and animals, insects and vermin was split into two measures : separate dogs and people and control pests and vermin. additional measures introduced were : control mould, as an observable effect of heat and humidity related to the hlp of control temperature ; and sleeping and bedding, as a measure of the key requirement for a space for children to get adequate rest. the first method, the failed healthy living practices (fhlps) score, was based on an assessment of all items of infrastructure required for conducting each hlp as described in our previous work20 and in table 1 in the online supplement. for any hlp for which an item of infrastructure was not functioning at an adequate level (fully functional or requiring minor maintenance only), the infrastructure required for that hlp was scored as failed. the overall assessment of house function using this method is then based on the number of failed hlps. the overall fhlp score reflects the number of hlps for which the score was fail (potential range 08). the technical function approach of the fhlp score allows for an objective assessment of function for some hlps. this has limitations in terms of practical function for the purposes of conducting healthy living practices and is not suited to assessment of all hlps. we therefore used a complementary method to assess infrastructure function, the surveyor function score (sfs). this relied on the surveyor using a 7-point likert scale to provide a score of their perception of the functional state of the house in relation to the ability to conduct each hlp. the intention of this method was to address some of the limitations associated with the approach used for the fhlp score, to provide a finer measure of functional state (using a 17 score rather than a pass / fail assessment) and to include assessment of availability of other requirements (such as furnishings and fixtures) for the effective conduct of hlps. a limitation of the sfs is that it is possibly a more subjective measure, hence its use in addition to rather than in place of the fhlp score. to assess the hygienic condition of houses, surveyors were required to provide a surveyor condition score (scs) for the hygienic condition of the areas or infrastructure items required for specific hlps and for the overall house using a 7-point likert scale (see table 2 in the online supplement). the repeatability of the sfs and scs was examined through repeat surveys of 15 houses at least 1 week after the initial survey.25 household hygiene was expected to vary over time in relation to cleaning and other household activities. repeatability of the scs provides an indication of the extent to which the scs reflects the general state of household hygiene over time. on testing repeatability of the exact scores on the 17 scale, 9 of the 13 scs measures recorded fair to moderate agreement (0.20<0.60) while 5 (wash people, wash clothes and bedding, house surrounds, control pests and vermin, and overall house condition) showed slight agreement (0.01<0.20). across all of the scs measures in the repeat survey, the scores were within one point of the original score in over 85% of cases. repeatability across sfs measures would be expected to be relatively stable over a short time frame. eleven of the 13 measures recorded fair to substantial agreement, with food preparation and storage, remove human waste and remove rubbish only showing a slight agreement. across all of the sfs measures in the repeat survey, scores were within one point of the original score in over 87% of cases (see table 3 in online supplement). crowding, sfs, scs and fhlp scores were compared between baseline and follow - up. the crowding data were not normally distributed and a natural logarithm transformation was applied prior to testing for a change over time using a t test. the kruskal wallis test of equality of proportions was used to test for a change in overall sfs, scs and fhlp scores. for individual sfss and scss the non - parametric kruskal wallis test was used to compare between baseline and follow - up, while a fisher exact test was used for individual fhlps. at the time of the baseline surveys, 326 houses (51%) were identified from a total of 644 houses in the 10 communities as being the main place of residence of at least one child aged 7 years. the main householder in 285 (88%) of these houses houses the main householder declined to be interviewed and in 31 (11%) houses the main householder was away on each of three consecutive visits by the project team. a total of 618 children living in these 285 houses were recruited to the study. at the follow - up surveys we were able to identify 208 houses where children recruited at baseline were now living. these houses were the main place of residence for 418 (68%) of the children originally recruited to the study. they had been living in 185 houses at the time of the baseline surveys (figure 1). of the 208 houses where these children were living at follow - up, 44 (21%) were new houses completed between the baseline and follow - up surveys. crowding data were available at baseline and follow - up for 327 (78%) children. at follow - up there was a small non - significant decrease in the mean number of people per bedroom sleeping in the house on the night before the survey (3.4, 95% ci 3.1 to 3.6 at baseline to 3.2, 95% ci 2.9 to 3.4 at follow - up ; natural logarithm transformed t test, t=1.3, p=0.102). at baseline and follow - up surveys there was no significant change in the composition of households in terms of the numbers of younger and older children and adults. fhlp data were available at both baseline and follow - up for 315 children (75%). the baseline distribution of the fhlp scores is skewed, peaking at scores of 6 for follow - up and 7 for baseline (a score of 7 means 7 out of the 8 infrastructure components scored using this method were scored as failed). there is also a secondary peak at the score of 2 for follow - up (figure 2). the distribution shows a shift to improvement in scores, with no houses scoring 0 (a score of 0 means no failed infrastructure components) at baseline, and just under 5% scoring 0 at follow - up. wallis test showed a significant difference between baseline (mean 5.6, 95% ci 5.3 to 6.0) and follow - up (4.4, 95% ci 4.1 to 4.8) for the fhlp score (=22.8, p<0.001). distribution of failed healthy living practices score (with standard errors) for houses at baseline and follow - up. sfs data were available at both baseline and follow - up for 349 children (83%) in the study cohort. the distribution of the sfs scores shows a bimodal pattern with peaks at scores of 2 and 4 for both time periods, suggesting some digit preference by the surveyors (figure 3). the distribution shows a shift to improvement in scores, with about 10% of houses receiving a score of 1 (best function) at follow - up compared with none at baseline. wallis test showed a marginally statistically significant difference between baseline (mean 3.8, 95% ci 3.5 to 4.0) and follow - up (3.4, 95% ci 3.1 to 3.6) for the sfs (=3.9, p=0.047). distribution of surveyor function score (with standard errors) for houses at baseline and follow - up. changes between baseline and follow - up in the proportion of houses that failed specific hlps or in the mean sfs score for specific hlps are shown in tables 1 and 2. hlps for which there was consistent evidence of improvement on both the fhlp score and sfs included : wash people, functioning toilet/remove human waste (toilet) and wash clothes and bedding. hlps which were assessed by either the fhlp score or the sfs only and which showed evidence of improvement included : safe electrical, separate animals and humans, control pests and vermin, control mould, control temperature and sleeping and bedding. hlps which were assessed on both the fhlp score and the sfs, but for which there was inconsistent evidence of improvement, included : prepare and store food and control dust. reduce trauma, house surrounds and overall house sfs were assessed on the sfs only and did not show evidence of improvement. remove waste water was assessed on the fhlp only and did not show evidence of improvement. percentage of houses failing to meet the infrastructure requirements for specific healthy living practices (hlps) included in the failed healthy living practice (fhlp) score for children at baseline and follow - up fisher exact test p value for one - tailed test (improvement in hlps between baseline and follow - up). mean score at baseline and follow - up for specific healthy living practices (hlps) included in the surveyor function score (sfs) and surveyor condition score (scs) data are shown as mean (se). there is no evidence of improvement of the overall scs at follow - up compared with baseline (figure 4). wallis test (baseline mean 4.1, 95% ci 3.9 to 4.4 ; follow - up mean 4.1, 95% ci 3.9 to 4.4 ; =0.3, p=0.605). kruskal wallis tests indicated a significant improvement in scs scores for control mould (=10.3, p=0.001) and control pests and vermin (=4.9, p=0.028), and deterioration in prepare and store food (=6.3, p=0.012 ; table 2). distribution of surveyor condition scores (with standard errors) for houses at baseline and follow - up. fhlp data were available at both baseline and follow - up for 315 children (75%). the baseline distribution of the fhlp scores is skewed, peaking at scores of 6 for follow - up and 7 for baseline (a score of 7 means 7 out of the 8 infrastructure components scored using this method were scored as failed). there is also a secondary peak at the score of 2 for follow - up (figure 2). the distribution shows a shift to improvement in scores, with no houses scoring 0 (a score of 0 means no failed infrastructure components) at baseline, and just under 5% scoring 0 at follow - up. wallis test showed a significant difference between baseline (mean 5.6, 95% ci 5.3 to 6.0) and follow - up (4.4, 95% ci 4.1 to 4.8) for the fhlp score (=22.8, p<0.001). distribution of failed healthy living practices score (with standard errors) for houses at baseline and follow - up. sfs data were available at both baseline and follow - up for 349 children (83%) in the study cohort. the distribution of the sfs scores shows a bimodal pattern with peaks at scores of 2 and 4 for both time periods, suggesting some digit preference by the surveyors (figure 3). the distribution shows a shift to improvement in scores, with about 10% of houses receiving a score of 1 (best function) at follow - up compared with none at baseline. wallis test showed a marginally statistically significant difference between baseline (mean 3.8, 95% ci 3.5 to 4.0) and follow - up (3.4, 95% ci 3.1 to 3.6) for the sfs (=3.9, p=0.047). distribution of surveyor function score (with standard errors) for houses at baseline and follow - up. changes between baseline and follow - up in the proportion of houses that failed specific hlps or in the mean sfs score for specific hlps are shown in tables 1 and 2. hlps for which there was consistent evidence of improvement on both the fhlp score and sfs included : wash clothes and bedding. hlps which were assessed by either the fhlp score or the sfs only and which showed evidence of improvement included : safe electrical, separate animals and humans, control pests and vermin, control mould, control temperature and sleeping and bedding. hlps which were assessed on both the fhlp score and the sfs, but for which there was inconsistent evidence of improvement, included : prepare and store food and control dust. reduce trauma, house surrounds and overall house sfs were assessed on the sfs only and did not show evidence of improvement. remove waste water was assessed on the fhlp only and did not show evidence of improvement. percentage of houses failing to meet the infrastructure requirements for specific healthy living practices (hlps) included in the failed healthy living practice (fhlp) score for children at baseline and follow - up fisher exact test p value for one - tailed test (improvement in hlps between baseline and follow - up). mean score at baseline and follow - up for specific healthy living practices (hlps) included in the surveyor function score (sfs) and surveyor condition score (scs) data are shown as mean (se). there is no evidence of improvement of the overall scs at follow - up compared with baseline (figure 4). wallis test (baseline mean 4.1, 95% ci 3.9 to 4.4 ; follow - up mean 4.1, 95% ci 3.9 to 4.4 ; =0.3, p=0.605). kruskal wallis tests indicated a significant improvement in scs scores for control mould (=10.3, p=0.001) and control pests and vermin (=4.9, p=0.028), and deterioration in prepare and store food (=6.3, p=0.012 ; table 2). distribution of surveyor condition scores (with standard errors) for houses at baseline and follow - up. these empirical findings add to the australian and international literature711 13 14 which indicates that programmes which are largely limited to improving the functional state of infrastructure have limited impact at the community level on major housing - related risks to health such as domestic hygiene. there was no systematic programme to achieve hygiene behaviour change in association with the building programme that was the subject of our study. other possible contributing reasons for the lack of observable improvement in hygiene include : (1) the pattern of improvements in infrastructure function shows the improvements resulted largely from changes in the proportions of houses rated worst and best (figures 2 and 3). thus, although there was a marked improvement in the functional state of houses for some children, at a population / community level there appears to have been little change ; (2) the continuing high levels of crowding may have been a barrier to hygiene improvements despite improvements in infrastructure. however, data from the study show only a weak association between crowding and hygiene (data not reported here), indicating that some households were able to achieve relatively good hygiene despite high levels of crowding ; (3) the building programmes were based on reasonably rigorous standards30 and improvements were achieved across a wide range of components of housing infrastructure. although these components of infrastructure are all recognised as important aspects of housing, their relative importance to health, and specifically to maintenance of domestic hygiene, is unclear. consideration of these issues raises two clear sets of implications for policy and further research. first, there is a need for better quality evidence on the relative health benefits of different components of housing infrastructure to support the design of houses that will result in the greatest potential health gain. the emphasis on achieving improvement across the full scope of building standards may limit the potential to achieve improvements in those components of infrastructure that may be most important to health. housing programmes may need either to be massively scaled - up to provide housing to the current standard for new houses to a large proportion of the population in these communities or to take a more population - based approach, with a stronger emphasis on equity and achieving adequate standards for as many people as possible in aspects of infrastructure that are most essential to health improvement. high mobility of children between houses and/or households provides further justification for this approach, and is the rationale for the ecological analysis presented in this paper. the communities included in this study were the focus of the largest scale building programmes in 20045. even with the large recent funding commitments to community housing programmes,31 significant increases in the proportion of the population of all communities beyond policy makers may therefore need to compromise on standards of some aspects of infrastructure in order to build the large numbers of houses required to reduce crowding in communities and provide and maintain the essential infrastructure to enable achievement of domestic hygiene for all community residents. second, the lack of association between quality of infrastructure and hygiene, and the weak association between crowding and hygiene, highlight the need for hygiene promotion initiatives regardless of the quality of housing infrastructure or levels of crowding. hygiene interventions such as hand washing have been shown to be effective in reducing the incidence of a range of common infectious conditions among children.32 33 in resource - poor countries, an ecological approach to hygiene improvement that takes account of infrastructure, hygiene promotion and which provides an enabling environment has been recommended.34 however, evidence on the effectiveness of broader hygiene interventions in indigenous australian communities9 and internationally35 36 is deficient, and there is a need for rigorous development and evaluation of such interventions. potential limitations of the study include : (1) reliability of infrastructure function and hygiene measures, although similar results from the different methods used and our repeatability analysis provides some reassurance on this point ; (2) variation in the time between the surveys and the occupation of new houses. we aimed to conduct baseline surveys at least a few months before handover of completed houses to ensure that housing conditions were not allowed to deteriorate in anticipation of moving to a new house, and to conduct follow - up surveys at least several months after occupation of new houses to allow residents time to settle into routine living arrangements so housing conditions would reflect the impact of these arrangements. these time frames were usually achieved, although there were four communities where baseline surveys were conducted only 12 months before handover of new houses ; (3) seasonal effects : baseline and follow - up surveys were staggered over the years and tended to be conducted around the same time each year, limiting the potential for any seasonal effects. the scope and history of disadvantage of indigenous people in australia means there are substantial political challenges to achieve agreement on the design and implementation of housing programmes. regardless of house design, it is increasingly clear that the benefits of improved infrastructure are unlikely to be fully realised without concurrent hygiene promotion programmes as an integral part of a broader ecological approach to housing improvement. an ecological approach to housing - related interventions would use a multifaceted programme targeting priorities in key areas of influence : infrastructure, behaviour, access to cleaning equipment and providing a supportive policy environment.8 37 from a research / evaluation perspective, failure to demonstrate improvements in the health of individual children living in houses with improved infrastructure may be the result of failure to achieve improvements at the community level (as demonstrated here) rather than the lack of a causal association between quality of housing and child health. we will be reporting on this association at the individual child level in a separate paper, and the findings in relation to the community level impact will be important in interpretation of this analysis. the general acceptance of the importance of housing to human health and well - being is reflected in the enshrinement of the right to adequate housing in international instruments. for decades, housing conditions have been identified as a major contributor to the poor health status of australia 's indigenous people. despite health improvement being an explicit purpose of indigenous housing programmes, this study 's findings show that housing programmes that are largely limited to improving the functional state of infrastructure have limited impact at the community level on major housing - related risks to health such as domestic hygiene. there was a marked improvement in the functional state of houses for some children but, at a population level, there appears to have been little change. the building programmes were based on reasonably rigorous standards and improvements were achieved across a wide range of components of housing infrastructure, all of which are believed to be important to health. this study highlights the need to gain a clearer understanding of the relative importance to health, and specifically to maintenance of domestic hygiene, of different components of housing infrastructure, of community level impacts of housing programmes and of the need for a stronger evidence base on interventions to create healthy household environments in disadvantaged settings. | background and aimhousing programmes in indigenous australian communities have focused largely on achieving good standards of infrastructure function. the impact of this approach was assessed on three potentially important housing - related influences on child health at the community level : (1) crowding, (2) the functional state of the house infrastructure and (3) the hygienic condition of the houses.methodsa before - and - after study, including house infrastructure surveys and structured interviews with the main householder, was conducted in all homes of young children in 10 remote australian indigenous communities.resultscompared with baseline, follow - up surveys showed (1) a small non - significant decrease in the mean number of people per bedroom in the house on the night before the survey (3.4, 95% ci 3.1 to 3.6 at baseline vs 3.2, 95% ci 2.9 to 3.4 at follow - up ; natural logarithm transformed t test, t=1.3, p=0.102) ; (2) a marginally significant overall improvement in infrastructure function scores (kruskal wallis test, 2=3.9, p=0.047) ; and (3) no clear overall improvement in hygiene (kruskal wallis test, 2=0.3, p=0.605).conclusionhousing programmes of this scale that focus on the provision of infrastructure alone appear unlikely to lead to more hygienic general living environments, at least in this study context. a broader ecological approach to housing programmes delivered in these communities is needed if potential health benefits are to be maximised. this ecological approach would require a balanced programme of improving access to health hardware, hygiene promotion and creating a broader enabling environment in communities. |
oligonucleotides are becoming increasingly important in the postgenomic era, as molecular tools for basic research as well as potential therapeutic and diagnostic reagents for the treatment and detection of genetic diseases [14 ]. however, for many of the in vivo applications, it is not sufficed just to be able to design oligonucleotide reagents that can recognize and bind sequence specifically to dna or rna. these reagents would also need to be able to get into cells and withstand enzymatic degradation by nucleases in the cellular milieu. to date, diverse classes of oligonucleotide analogues have been developed, but none possesses all the characteristic features [58 ]. it is, therefore, important to be able to modify the structures and/or chemical functionalities of these reagents further, with ease and flexibility, so that many of these desired features could be augmented and/or improved upon [9, 10 ] and undesired attributes, such as nonspecific binding and toxicity, could be further minimized [11, 12 ]. a particular class of oligonucleotide analogue endowed with such synthetic flexibility is peptide nucleic acids (pnas). pnas are nucleic acid mimics, comprised of n-(2-aminoethyl) glycine backbone and dna / rna nucleobases that are connected through a flexible carboxymethylene linker. despite the structural departure from the natural biopolymers, pnas maintain the ability to hybridize to complementary dna and rna strands through watson - crick base - pairing, just as their natural counterparts, but with higher affinity and sequence selectivity. the improvement in binding affinity has been attributed in part to the lack of electrostatic repulsion in the backbones, while the enhancement in sequence selectivity has been attributed in part to the increased backbone rigidity upon hybridization as the result of solvation [15, 16 ]. unlike dna or rna, which are prone to nucleolytic degradation, pnas are resistant to both proteases and nucleases. these properties, together with the ease and flexibility of synthesis, make pnas an attractive commodity for in vivo applications. so far, a large number of structural modifications have been made to the backbone of pnas [1721 ]. among them, modifications made at the -position show the most promise because of the simplicity and flexibility in synthesis and the benefits that they confer on the hybridization properties of pnas [2228 ]. recently, we showed that randomly folded, single - stranded pnas can be preorganized into either a right - handed or left - handed helix by installing an appropriate stereogenic center at the -backbone position [2729 ]. pnas derived from l - amino acids adopted a right - handed helix, while those derived from d - amino acids adopted a left - handed helix (unpublished data) ; however, only the right - handed helical pnas are able to hybridize to dna and rna with high affinity and sequence selectivity. although a number of amino acid side chains including alanine [22, 23 ], serine, cysteine, and lysine [24, 26, 30 ] have been incorporated at this position, a systematic study aimed at assessing the effect of steric hindrance on the conformations and hybridization properties of pnas has not yet been established. knowledge of this information is crucial to the future design of pnas with improved hybridization properties, water solubility, cellular uptake, biodistribution, and pharmacokinetics all of which are essential for their in vivo applications. in an attempt to address this issue, we synthesized a series of thymine - containing pna monomers and corresponding oligomers with different amino acid side chains at the -backbone position and characterized their conformations and hybridization properties using a combination of cd and uv - vis spectroscopic techniques. all chemicals were purchased from aldrich except for boc - val - oh, boc - phe - oh, boc - ala - oh, and boc - ile - oh, which were purchased from novabiochem. h - nmr and c - nmr spectra were recorded on a bruker avance av-300 nmr spectrometer using standard bruker software. maldi - tof experiments were performed on a perseptive biosystems voyager str maldi - tof mass spectrometer using a 10 mg / ml solution of -hydroxycinnamic acid in acn - water (1 : 1) with 0.1% tfa. mass spectra were recorded on a finnigan lcq esi / apci ion trap mass spectrometer by electrospray ionization. cd experiments were performed on a jasco j-715 spectropolarimeter equipped with a thermoelectrically controlled single - cell holder. uv - vis measurements were taken on a varian cary 300 bio spectrophotometer equipped with a thermoelectrically controlled multicell holder. the oligomers were purified by reverse - phase hplc and characterized by maldi - tof. all pna stock solutions were prepared using nanopure water, and the concentrations were determined at 95c using the following extinction coefficients for pna monomers : 13,700 m cm (a), 6,600 m cm (c), 11,700 m cm (g), and 8,600 m cm (t). the dna oligomers were purchased from integrated dna technologies, inc. and used without further purification. the samples were prepared in buffer containing 0.1 mm edta, 100 mm nacl, 10 mm sodium phosphate at ph 7.4. the uv absorption at 260 nm was carefully adjusted at 95c so that each sample contained the same concentration. all spectra represent an average of at least twelve scans between 200320 nm, measured at the rate of 100 nm per min in a 1-cm path - length cuvette at 25c. the spectra were baseline corrected and then smoothed using an eight - point adjacent averaging algorithm. uv - vis absorbance at 260 nm was recorded every 0.5c as the samples were cooled from 95c to 20c and then heated to 95c at a rate of 1.0c / min. 5a : hrms (esi / msm / z) mcalc for c17h26n4nao4 421.17, found 412.1344. boc - val - ol (1b)boc - val - oh (501 mg, 2.31 mmol) was dissolved in a cold (15c) solution of dimethoxyethane (dme) (5 ml). n - methyl morpholine (nmm) (0.25 ml, 2.27 mmol) and isobutyl chloroformate (0.30 ml, 2.30 mmol) were added dropwise to the mixture. the precipitated n - methyl morpholine hydrochloride was removed by vacuum filtration and washed with dme (3 2 ml). the filtrate and washing solutions were recombined, cooled to 15c in a methanol ice bath. to this mixture, a solution of nabh4 (0.130 g, 3.45 mmol) in water (0.5 ml) was added dropwise (producing evolution of gas). the mixture was allowed to stir for 1 min, and approximately 100 ml of water was added to quench the reaction. the solution was then transferred to a separatory funnel and extracted with ethyl acetate (etoac) (3 50 ml) and then brine (1 100 ml). the solvent was then removed under reduced pressure to give 450 mg (2.21 mmol, 97% yield) of the desired product 1b as pale yellow oil. tlc : rf = 0.65 (6 : 4 etoac : hexane). h - nmr (cdcl3, 300 mhz) : = 0.801.06 (6h, m, ch(ch3)2) ; 1.49 (9h, s, c(ch3)3) ; 1.85 (1h, m, ch(ch3)2) ; 2.25 (1h, br, ch2oh) ; 3.50 (1h, m, nchco) ; 3.76 (2h, m, ch2oh) ; 4.65 (1h, br, conh). minor solvent impurities were found in the h - nmr : etoac (1.30, 2.10, and 4.12), h2o (1.60), acetone (2.20), and dme (3.40 and 3.60). (esi - ms m / z) mass calculated 203.28 for c10h21no3, found 226.07 (203.28 + na). boc - val - oh (501 mg, 2.31 mmol) was dissolved in a cold (15c) solution of dimethoxyethane (dme) (5 ml). n - methyl morpholine (nmm) (0.25 ml, 2.27 mmol) and isobutyl chloroformate (0.30 ml, 2.30 mmol) were added dropwise to the mixture. the precipitated n - methyl morpholine hydrochloride was removed by vacuum filtration and washed with dme (3 2 ml). the filtrate and washing solutions were recombined, cooled to 15c in a methanol ice bath. to this mixture, a solution of nabh4 (0.130 g, 3.45 mmol) in water (0.5 ml) was added dropwise (producing evolution of gas). the mixture was allowed to stir for 1 min, and approximately 100 ml of water was added to quench the reaction. the solution was then transferred to a separatory funnel and extracted with ethyl acetate (etoac) (3 50 ml) and then brine (1 100 ml). the solvent was then removed under reduced pressure to give 450 mg (2.21 mmol, 97% yield) of the desired product 1b as pale yellow oil. tlc : rf = 0.65 (6 : 4 etoac : hexane). h - nmr (cdcl3, 300 mhz) : = 0.801.06 (6h, m, ch(ch3)2) ; 1.49 (9h, s, c(ch3)3) ; 1.85 (1h, m, ch(ch3)2) ; 2.25 (1h, br, ch2oh) ; 3.50 (1h, m, nchco) ; 3.76 (2h, m, ch2oh) ; 4.65 (1h, br, conh). minor solvent impurities were found in the h - nmr : etoac (1.30, 2.10, and 4.12), h2o (1.60), acetone (2.20), and dme (3.40 and 3.60). (esi - ms m / z) mass calculated 203.28 for c10h21no3, found 226.07 (203.28 + na). boc - ile - ol (1c)synthesis is analogous to that of 1b, starting from boc - ile - oh (499.6 mg, 2.08 mmol), nmm (0.23 ml, 2.09 mmol), isobutyl chloroformate (0.27 ml, 2.08 mmol), and nabh4 (0.12 g, 3.17 mmol). 430 mg (1.99 mmol, 95% yield) of 1c was obtained as colorless oil. tlc : rf = 0.65 (6 : 4 etoac : hexane). h - nmr (cdcl3, 300 mhz) : = 0.801.00 (6h, m, chch3, chch2ch3) ; 1.201.40 (2h, m, chch2ch3) ; 1.49 (9h, s, c(ch3)3) ; 1.60 (1h, m, chch2ch3) ; 2.25 (1h, br, ch2oh) ; 3.50 (1h, m, nchco) ; 3.76 (2h, m, ch2oh) ; 4.65 (1h, br, conh). minor impurities were found in the h - nmr : isobutyl chloroformate (0.9, 2.0, 3.90), etoac (1.30, 2.10, and 4.12), and h2o (1.60). (esi - ms m / z) mass calculated 217.31 for c11h23no3, found 240.31 (217.31 + na). synthesis is analogous to that of 1b, starting from boc - ile - oh (499.6 mg, 2.08 mmol), nmm (0.23 ml, 2.09 mmol), isobutyl chloroformate (0.27 ml, 2.08 mmol), and nabh4 (0.12 g, 3.17 mmol). 430 mg (1.99 mmol, 95% yield) of 1c was obtained as colorless oil. tlc : rf = 0.65 (6 : 4 etoac : hexane). h - nmr (cdcl3, 300 mhz) : = 0.801.00 (6h, m, chch3, chch2ch3) ; 1.201.40 (2h, m, chch2ch3) ; 1.49 (9h, s, c(ch3)3) ; 1.60 (1h, m, chch2ch3) ; 2.25 (1h, br, ch2oh) ; 3.50 (1h, m, nchco) ; 3.76 (2h, m, ch2oh) ; 4.65 (1h, br, conh). minor impurities were found in the h - nmr : isobutyl chloroformate (0.9, 2.0, 3.90), etoac (1.30, 2.10, and 4.12), and h2o (1.60). (esi - ms m / z) mass calculated 217.31 for c11h23no3, found 240.31 (217.31 + na). boc - phe - ol (1d)synthesis is analogous to that of 1b, starting from boc - phe - oh (500 mg, 1.89 mmol), nmm (0.21 ml, 1.91 mmol), isobutyl chloroformate (0.25 ml, 1.93 mmol), and nabh4 (0.11 g, 2.91 mmol). 410 mg (1.63 mmol, 86% yield) of 1d was obtained as colorless oil. tlc : rf = 0.65 (6 : 4 etoac : hexane). h - nmr (cdcl3, 300 mhz) : = 1.49 (9h, s, c(ch3)3) ; 2.25 (1h, br, ch2oh) ; 2.85 (2h, m, chch2ph) ; 3.50 (1h, m, nchco) ; 3.76 (2h, m, ch2oh) ; 4.75 (1h, br, conh) ; 7.257.40 (5h, m, ch2phh26). minor impurities were found in the h - nmr : isobutyl chloroformate (0.90, 2.00, 3.90), etoac (1.30, 2.10, and 4.12), h2o (1.60), and acetone (2.20). (esi - ms m / z) mass calculated 251.32 for c14h21no3, found 252.0 and 274.13 (251.32 + na). synthesis is analogous to that of 1b, starting from boc - phe - oh (500 mg, 1.89 mmol), nmm (0.21 ml, 1.91 mmol), isobutyl chloroformate (0.25 ml, 1.93 mmol), and nabh4 (0.11 g, 2.91 mmol). 410 mg (1.63 mmol, 86% yield) of 1d was obtained as colorless oil. tlc : rf = 0.65 (6 : 4 etoac : hexane). h - nmr (cdcl3, 300 mhz) : = 1.49 (9h, s, c(ch3)3) ; 2.25 (1h, br, ch2oh) ; 2.85 (2h, m, chch2ph) ; 3.50 (1h, m, nchco) ; 3.76 (2h, m, ch2oh) ; 4.75 (1h, br, conh) ; 7.257.40 (5h, m, ch2phh26). minor impurities were found in the h - nmr : isobutyl chloroformate (0.90, 2.00, 3.90), etoac (1.30, 2.10, and 4.12), h2o (1.60), and acetone (2.20). (esi - ms m / z) mass calculated 251.32 for c14h21no3, found 252.0 and 274.13 (251.32 + na). ethyl n-(o - nitrophenylsulfonyl) glycinate (nos - gly - oet) it was prepared following the reported procedures. boc - val[ch2n(nos)]gly - oet (2b) 1b (450 mg, 2.21 mmol), nos - gly - oet (475 mg, 1.65 mmol), and triphenylphosphine (ph3p) (653 mg, 2.49 mmol) were dissolved in freshly distilled dry thf (25 ml). the solution was stirred in an ice bath under nitrogen. diisopropyl azodicarboxylate (diad) (0.5 ml, 2.54 mmol) was added dropwise over 10 min. the oily product was purified by column chromatography (3 : 7 etoac : hexane, rf = 0.65) to give 700 mg (1.48 mmol, 90% yield) of 2b. h - nmr (cdcl3, 300 mhz) : = 0.901.00 (6h, m, ch(ch3)2) ; 1.25 (3h, t, och2ch3, j = 7.1 hz) ; 1.49 (9h, s, c(ch3)3) ; 1.80 (1h, m, ch(ch3)2) ; 3.453.80 (3h, m, nchch2n) ; 4.004.20 (2h, m, och2ch3) ; 4.404.65 (2h, m, nch2co) ; 6.30 (1h, br, nh) ; 7.607.80 (3h, m, arh46) ; 8.10 (1h, m, arh3). minor impurities were found in the h - nmr : diad byproduct (1.30, 5.00, 6.30), h2o (1.60), and acetone (2.20). (esi - ms m / z) mass calculated 473.54 for c20h31n3o8s, found 496.13(473.54 + na). 1b (450 mg, 2.21 mmol), nos - gly - oet (475 mg, 1.65 mmol), and triphenylphosphine (ph3p) (653 mg, 2.49 mmol) were dissolved in freshly distilled dry thf (25 ml). diisopropyl azodicarboxylate (diad) (0.5 ml, 2.54 mmol) was added dropwise over 10 min. the oily product was purified by column chromatography (3 : 7 etoac : hexane, rf = 0.65) to give 700 mg (1.48 mmol, 90% yield) of 2b. h - nmr (cdcl3, 300 mhz) : = 0.901.00 (6h, m, ch(ch3)2) ; 1.25 (3h, t, och2ch3, j = 7.1 hz) ; 1.49 (9h, s, c(ch3)3) ; 1.80 (1h, m, ch(ch3)2) ; 3.453.80 (3h, m, nchch2n) ; 4.004.20 (2h, m, och2ch3) ; 4.404.65 (2h, m, nch2co) ; 6.30 (1h, br, nh) ; 7.607.80 (3h, m, arh46) ; 8.10 (1h, m, arh3). minor impurities were found in the h - nmr : diad byproduct (1.30, 5.00, 6.30), h2o (1.60), and acetone (2.20). (esi - ms m / z) mass calculated 473.54 for c20h31n3o8s, found 496.13(473.54 + na). boc - ile[ch2n(nos)]gly - oet (2c)synthesis is analogous to that of 2b, starting from 1c (430 mg, 1.99 mmol), nos - gly - oet (436 mg, 1.51 mmol), and ph3p (604 mg, 2.30 mmol). the solvent was removed, and the oily product mixture was purified by column chromatography (3 : 7 etoac : hexane, rf = 0.68) to give 649 mg (1.33 mmol, 88% yield) of 2c. h - nmr (cdcl3, 300 mhz) : = 0.801.00 (6h, m, chch3, chch2ch3), 1.10 (2h, m, chch2ch3) ; 1.20 (3h, t, och2ch3, j = 7.1 hz) ; 1.49 (9h, s, c(ch3)3) ; 1.60 (1h, m, chch2ch3) ; 3.453.80 (3h, m, nchch2n) ; 4.004.20 (2h, m, och2ch3) ; 4.404.65 (2h, m, nch2co) ; 6.30 (1h, br, nh) ; 7.607.80 (3h, m, arh46) ; 8.10 (1h, m, arh3). minor impurities were found in the h - nmr : diad byproduct (1.30, 5.00, 6.30), h2o (1.60), and acetone (2.20). (esi - ms m / z) mass calculated 487.57 for c21h33n3o8s, found 510.57 (487.57 + na). synthesis is analogous to that of 2b, starting from 1c (430 mg, 1.99 mmol), nos - gly - oet (436 mg, 1.51 mmol), and ph3p (604 mg, 2.30 mmol). the solvent was removed, and the oily product mixture was purified by column chromatography (3 : 7 etoac : hexane, rf = 0.68) to give 649 mg (1.33 mmol, 88% yield) of 2c. h - nmr (cdcl3, 300 mhz) : = 0.801.00 (6h, m, chch3, chch2ch3), 1.10 (2h, m, chch2ch3) ; 1.20 (3h, t, och2ch3, j = 7.1 hz) ; 1.49 (9h, s, c(ch3)3) ; 1.60 (1h, m, chch2ch3) ; 3.453.80 (3h, m, nchch2n) ; 4.004.20 (2h, m, och2ch3) ; 4.404.65 (2h, m, nch2co) ; 6.30 (1h, br, nh) ; 7.607.80 (3h, m, arh46) ; 8.10 (1h, m, arh3). minor impurities were found in the h - nmr : diad byproduct (1.30, 5.00, 6.30), h2o (1.60), and acetone (2.20). (esi - ms m / z) mass calculated 487.57 for c21h33n3o8s, found 510.57 (487.57 + na). boc - phe[ch2n(nos)]gly - oet (2d)synthesis is analogous to that of 2b, starting from 1d (410 mg, 1.63 mmol), nos - gly - oet (402 mg, 1.39 mmol), and ph3p (528 mg, 2.01 mmol). the solvent was removed, and the oily product mixture was purified by column chromatography (3 : 7 etoac : hexane, rf = 0.70) to give 560 mg (1.07 mmol, 77% yield) of 2d. h - nmr (cdcl3, 300 mhz) : = 1.20 (3h, t, och2ch3 j=7.1 hz) ; 1.49 (9h, s, c(ch3)3) ; 2.85 (2h, m, chch2ph) ; 3.503.70 (3h, m, nchch2n) ; 4.02 (1h, m, chch2ph) ; 4.30 (2h, m, nch2co) ; 6.30 (1h, br, nh) ; 7.107.40 (5h, m, ch2phh26) ; 7.657.80 (3h, m, onbs arh46) ; 7.90 (1h, m, onbs arh3). minor impurities were found in the h - nmr : diad byproduct (1.30, 5.00, 6.30), h2o (1.60), and acetone (2.20). (esi - ms m / z) mass calculated 521.58 for c24h31n3o8s, found 544.13 (521.58 + na). synthesis is analogous to that of 2b, starting from 1d (410 mg, 1.63 mmol), nos - gly - oet (402 mg, 1.39 mmol), and ph3p (528 mg, 2.01 mmol). the solvent was removed, and the oily product mixture was purified by column chromatography (3 : 7 etoac : hexane, rf = 0.70) to give 560 mg (1.07 mmol, 77% yield) of 2d. h - nmr (cdcl3, 300 mhz) : = 1.20 (3h, t, och2ch3 j=7.1 hz) ; 1.49 (9h, s, c(ch3)3) ; 2.85 (2h, m, chch2ph) ; 3.503.70 (3h, m, nchch2n) ; 4.02 (1h, m, chch2ph) ; 4.30 (2h, m, nch2co) ; 6.30 (1h, br, nh) ; 7.107.40 (5h, m, ch2phh26) ; 7.657.80 (3h, m, onbs arh46) ; 7.90 (1h, m, onbs arh3). minor impurities were found in the h - nmr : diad byproduct (1.30, 5.00, 6.30), h2o (1.60), and acetone (2.20). (esi - ms m / z) mass calculated 521.58 for c24h31n3o8s, found 544.13 (521.58 + na). boc - val(ch2n)gly - oet (3b) 2b (700 mg, 1.48 mmol) was dissolved in acetonitrile (25 ml) under nitrogen. dried potassium carbonate (440 mg, 3.13 mmol) was added to the solution. while stirring thiophenol (0.450 ml, 4.40 mmol) the resulting mixture was gravity - filtered to remove excess potassium carbonate and the solvent removed under reduced pressure. the crude product was dissolved in 100 ml etoac, washed with water (2 100 ml), followed by brine (1 50 ml). the crude product was purified by column chromatography (20 : 1 etoac : etoh, rf = 0.15) to give 200 mg (0.69 mmol, 47% yield). h - nmr (cdcl3, 300 mhz) : = 0.901.00 (6h, m, ch(ch3)2) ; 1.25 (3h, t, och2ch3, j = 7.1 hz) ; 1.49 (9h, s, c(ch3)3) ; 1.80 (1h, m, ch(ch3)2) ; 2.65 (2h, m, chch2nh) ; 3.40 (2h, ab, nch2co, j=17.4 hz) ; 3.50 (1h, m, nchch2) ; 4.104.30 (2h, q, och2ch3, j = 7.2 hz) ; 4.60 (1h, c nmr (cdcl3, 75 mhz) : = 172.5, 156.2, 77.2, 60.7, 55.5, 50.9, 30.5, 28.4, 19.3, 18.2, 14.2. (esi - ms m / z) mass calculated 288.38 for c14h28n2o4, found 289.07. 2b (700 mg, 1.48 mmol) was dissolved in acetonitrile (25 ml) under nitrogen. dried potassium carbonate (440 mg, 3.13 mmol) was added to the solution. while stirring thiophenol (0.450 ml, 4.40 mmol) the resulting mixture was gravity - filtered to remove excess potassium carbonate and the solvent removed under reduced pressure. the crude product was dissolved in 100 ml etoac, washed with water (2 100 ml), followed by brine (1 50 ml). the crude product was purified by column chromatography (20 : 1 etoac : etoh, rf = 0.15) to give 200 mg (0.69 mmol, 47% yield). h - nmr (cdcl3, 300 mhz) : = 0.901.00 (6h, m, ch(ch3)2) ; 1.25 (3h, t, och2ch3, j = 7.1 hz) ; 1.49 (9h, s, c(ch3)3) ; 1.80 (1h, m, ch(ch3)2) ; 2.65 (2h, m, chch2nh) ; 3.40 (2h, ab, nch2co, j=17.4 hz) ; 3.50 (1h, m, nchch2) ; 4.104.30 (2h, q, och2ch3, j = 7.2 hz) ; 4.60 (1h, c nmr (cdcl3, 75 mhz) : = 172.5, 156.2, 77.2, 60.7, 55.5, 50.9, 30.5, 28.4, 19.3, 18.2, 14.2. (esi - ms m / z) mass calculated 288.38 for c14h28n2o4, found 289.07. boc - ile(ch2n)gly - oet (3c)synthesis is analogous to that of 3b, starting from 2c (649 mg, 1.33 mmol), potassium carbonate (426 mg, 3.08 mmol), and thiophenol (0.420 ml, 4.11 mmol). the crude product was purified by column chromatography (etoac, rf = 0.15) to give 300 mg (0.99 mmol, 75% yield) of oily product. h - nmr (cdcl3, 300 mhz) : = 0.801.00 (6h, m, chch3, chch2ch3), 1.15 (2h, m, chch2ch3) ; 1.30 (3h, t, och2ch3, j = 7.1 hz) ; 1.49 (9h, s, c(ch3)3) ; 1.60 (1h, m, chch2ch3) ; 2.65 (2h, m, chch2nh) ; 3.40 (2h, ab, nch2co, j = 17.4 hz) ; 3.70 (1h, m, nchch2) ; 4.104.30 (2h, q, och2ch3, j = 7.2 hz) ; 4.60 (1h, br, boc nh). c nmr (cdcl3, 75 mhz) : = 172.5, 156.1, 77.2, 60.7, 54.5, 50.9, 50.3, 37.2, 28.4, 25.4, 15.2, 14.2, 11.6. (esi - ms m / z) mass calculated 302.41 for c21h33n3o8s, found 303.00. synthesis is analogous to that of 3b, starting from 2c (649 mg, 1.33 mmol), potassium carbonate (426 mg, 3.08 mmol), and thiophenol (0.420 ml, 4.11 mmol). the crude product was purified by column chromatography (etoac, rf = 0.15) to give 300 mg (0.99 mmol, 75% yield) of oily product. h - nmr (cdcl3, 300 mhz) : = 0.801.00 (6h, m, chch3, chch2ch3), 1.15 (2h, m, chch2ch3) ; 1.30 (3h, t, och2ch3, j = 7.1 hz) ; 1.49 (9h, s, c(ch3)3) ; 1.60 (1h, m, chch2ch3) ; 2.65 (2h, m, chch2nh) ; 3.40 (2h, ab, nch2co, j = 17.4 hz) ; 3.70 (1h, m, nchch2) ; 4.104.30 (2h, q, och2ch3, j = 7.2 hz) ; 4.60 (1h, br, boc nh). c nmr (cdcl3, 75 mhz) : = 172.5, 156.1, 77.2, 60.7, 54.5, 50.9, 50.3, 37.2, 28.4, 25.4, 15.2, 14.2, 11.6. (esi - ms m / z) mass calculated 302.41 for c21h33n3o8s, found 303.00. boc - phe(ch2n)gly - oet (3d)synthesis is analogous to that of 3b, starting from 2d (560 mg, 1.07 mmol), potassium carbonate (398 mg, 2.88 mmol), and thiophenol (0.400 ml, 3.91 mmol). after workup and column purification, 300 mg (0.89 mmol, 83% yield) of 3d (20 : 1 etoac : etoh, rf = 0.15) was obtained. h - nmr (cdcl3, 300 mhz) : = 1.20 (3h, t, och2ch3, j = 7.1 hz) ; 1.49 (9h, s, c(ch3)3) ; 2.65 (2h, m, chch2nh) ; 2.85 (2h, m, chch2ph) ; 3.40 (2h, ab, nch2co, j = 17.4 hz) ; 3.80 (1h, m, nchch2) ; 4.104.30 (2h, q, och2ch3, j = 7.2 hz) ; 4.60 (1h, br, boc nh) ; 7.107.40 (5h, m, ch2phh26). c nmr (cdcl3, 75 mhz) : = 172.4, 155.6, 138.0, 129.4, 128.4, 126.4, 77.2, 60.8, 51.6, 51.0, 39.1, 28.4, 14.2. (esi - ms m / z) mass calculated 336.43 for c18h28n2o4, found 337.07. synthesis is analogous to that of 3b, starting from 2d (560 mg, 1.07 mmol), potassium carbonate (398 mg, 2.88 mmol), and thiophenol (0.400 ml, 3.91 mmol). after workup and column purification, 300 mg (0.89 mmol, 83% yield) of 3d (20 : 1 etoac : etoh, rf = 0.15) was obtained. h - nmr (cdcl3, 300 mhz) : = 1.20 (3h, t, och2ch3, j = 7.1 hz) ; 1.49 (9h, s, c(ch3)3) ; 2.65 (2h, m, chch2nh) ; 2.85 (2h, m, chch2ph) ; 3.40 (2h, ab, nch2co, j = 17.4 hz) ; 3.80 (1h, m, nchch2) ; 4.104.30 (2h, q, och2ch3, j = 7.2 hz) ; 4.60 (1h, br, boc nh) ; 7.107.40 (5h, m, ch2phh26). c nmr (cdcl3, 75 mhz) : = 172.4, 155.6, 138.0, 129.4, 128.4, 126.4, 77.2, 60.8, 51.6, 51.0, 39.1, 28.4, 14.2. (esi - ms m / z) mass calculated 336.43 for c18h28n2o4, found 337.07. boc - val[ch2n(thyac)]gly - oet (4b)thymin-1-ylacetic acid (154 mg, 0.84 mmol), n, n, dicyclohexylcarbodiimide (dcc) (176 mg, 0.85 mmol), and 3-hydroxy-1,2,3-benzotriazin-4(3h)-one (dhbtoh) (137 mg, 0.84 mmol) were dissolved in 10 ml dry n, n - dimethylformamide (dmf). then, 3b (200.0 mg, 0.69 mmol), which was dissolved in dry dmf (2 2.5 ml), was added dropwise into the mixture. the crude mixture was dissolved in 100 ml etoac and then washed with 100 ml of saturated nahco3 solution. the organic layer was set aside, and the nahco3 layer was washed with etoac (2 100 ml). the organic layers were recombined and washed with 10% khso4 (3 50 ml), followed by saturated nahco3 (3 50 ml) and then brine (1 100 ml). the organic layer was dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. the product was purified by column chromatography (etoac, rf = 0.45) to give 100 mg (0.22 mmol, 32% yield) of the desired product. h - nmr (dmso, 300 mhz) : = 0.901.00 (6h, m, ch(ch3)2) ; 1.20 (3h, t, och2ch3, j = 7.1 hz) ; 1.49 (9h, s, c(ch3)3) ; 1.70 (1h, m, ch(ch3)2) ; 1.80 (3h, s, thymine ch3) ; 2.903.60 (2h, m, chch2nco) ; 3.453.60 (1h, m, nchch2) ; 3.70 and 3.90 (2h, m, nch2co) ; 4.144.22 (2h, q, och2ch3, j = 7.2 hz) ; 4.40 and 4.70 (2h, 2ab, ncoch2, j = 7.6 and 16.6 hz) ; 6.60 and 6.80 (1h, 2d, boc - nh, j = 9.4 and 9.7 hz) ; 7.20 (1h, s, thymine h) ; 11.20 (1h, 2s, thymine - nh). minor impurities were found in the h - nmr : etoac (1.20, 2.00, 4.00), h2o (3.30), and dhbtoh byproduct (5.50). (esi - ms m / z) mass calculated 454.52 for c21h34n4o7, found 477.20 (454.52 + na). thymin-1-ylacetic acid (154 mg, 0.84 mmol), n, n, dicyclohexylcarbodiimide (dcc) (176 mg, 0.85 mmol), and 3-hydroxy-1,2,3-benzotriazin-4(3h)-one (dhbtoh) (137 mg, 0.84 mmol) were dissolved in 10 ml dry n, n - dimethylformamide (dmf). then, 3b (200.0 mg, 0.69 mmol), which was dissolved in dry dmf (2 2.5 ml), was added dropwise into the mixture. the crude mixture was dissolved in 100 ml etoac and then washed with 100 ml of saturated nahco3 solution. the organic layer was set aside, and the nahco3 layer was washed with etoac (2 100 ml). the organic layers were recombined and washed with 10% khso4 (3 50 ml), followed by saturated nahco3 (3 50 ml) and then brine (1 100 ml). the organic layer was dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. the product was purified by column chromatography (etoac, rf = 0.45) to give 100 mg (0.22 mmol, 32% yield) of the desired product. h - nmr (dmso, 300 mhz) : = 0.901.00 (6h, m, ch(ch3)2) ; 1.20 (3h, t, och2ch3, j = 7.1 hz) ; 1.49 (9h, s, c(ch3)3) ; 1.70 (1h, m, ch(ch3)2) ; 1.80 (3h, s, thymine ch3) ; 2.903.60 (2h, m, chch2nco) ; 3.453.60 (1h, m, nchch2) ; 3.70 and 3.90 (2h, m, nch2co) ; 4.144.22 (2h, q, och2ch3, j = 7.2 hz) ; 4.40 and 4.70 (2h, 2ab, ncoch2, j = 7.6 and 16.6 hz) ; 6.60 and 6.80 (1h, 2d, boc - nh, j = 9.4 and 9.7 hz) ; 7.20 (1h, s, thymine h) ; 11.20 (1h, 2s, thymine - nh). minor impurities were found in the h - nmr : etoac (1.20, 2.00, 4.00), h2o (3.30), and dhbtoh byproduct (5.50). (esi - ms m / z) mass calculated 454.52 for c21h34n4o7, found 477.20 (454.52 + na). boc - ile[ch2n(thyac)]gly - oet (4c)synthesis is analogous to that of 4b, starting with thymin-1-ylacetic acid (223 mg, 1.21 mmol), dcc (251 mg, 1.22 mmol), and dhbtoh (196 mg, 1.20 mmol) dissolved in 10 ml dry dmf. after stirring for 1 hr at room temperature, 3c (300 mg, 0.99 mmol) was dissolved in dry dmf (2 2.5 ml) and added dropwise. the mixture was heated at 50c for 48 hrs. after workup and purification by column chromatography (20 : 1 etoac : etoh, rf = 0.45), 300 mg (0.64 mmol, 65% yield) of the desired product was obtained. h - nmr (dmso, 300 mhz) : = 0.801.00 (6h, m, chch3, chch2ch3) ; 1.10 (2h, m, chch2ch3) ; 1.20 (3h, t, och2ch3, j = 7.1 hz) ; 1.49 (9h, s, c(ch3)3) ; 1.60 (1h, m, chch2ch3) ; 1.80 (3h, s, thymine ch3) ; 2.903.70 (2h, m, chch2nco) ; 3.95 and 4.00 (1h, 2 m, nchch2) ; 4.054.12 (2h, m, nch2co) ; 4.144.22 (2h, q, och2ch3, j = 7.2 hz) ; 4.40 and 4.70 (2h, 2ab, ncoch2, j = 7.6 and 16.6 hz) ; 6.65 and 6.82 (1h, 2d, boc - nh, j = 9.4 and 9.7 hz) ; 7.20 (1h, s, thymine h) ; 11.20 (1h, 2s, thymine - nh). minor impurities were found in the h - nmr : etoac (1.20, 2.00, 4.00), dmf (2.70 and 2.90), h2o (3.30), and dhbtoh byproduct (5.50). (esi - ms m / z) mass calculated 468.54 for c22h36n4o7, found 491.13 (468.54 + na). synthesis is analogous to that of 4b, starting with thymin-1-ylacetic acid (223 mg, 1.21 mmol), dcc (251 mg, 1.22 mmol), and dhbtoh (196 mg, 1.20 mmol) dissolved in 10 ml dry dmf. after stirring for 1 hr at room temperature, 3c (300 mg, 0.99 mmol) was dissolved in dry dmf (2 2.5 ml) and added dropwise. the mixture was heated at 50c for 48 hrs. after workup and purification by column chromatography (20 : 1 etoac : etoh, rf = 0.45), 300 mg (0.64 mmol, 65% yield) of the desired product was obtained. h - nmr (dmso, 300 mhz) : = 0.801.00 (6h, m, chch3, chch2ch3) ; 1.10 (2h, m, chch2ch3) ; 1.20 (3h, t, och2ch3, j = 7.1 hz) ; 1.49 (9h, s, c(ch3)3) ; 1.60 (1h, m, chch2ch3) ; 1.80 (3h, s, thymine ch3) ; 2.903.70 (2h, m, chch2nco) ; 3.95 and 4.00 (1h, 2 m, nchch2) ; 4.054.12 (2h, m, nch2co) ; 4.144.22 (2h, q, och2ch3, j = 7.2 hz) ; 4.40 and 4.70 (2h, 2ab, ncoch2, j = 7.6 and 16.6 hz) ; 6.65 and 6.82 (1h, 2d, boc - nh, j = 9.4 and 9.7 hz) ; 7.20 (1h, s, thymine h) ; 11.20 (1h, 2s, thymine - nh). minor impurities were found in the h - nmr : etoac (1.20, 2.00, 4.00), dmf (2.70 and 2.90), h2o (3.30), and dhbtoh byproduct (5.50). (esi - ms m / z) mass calculated 468.54 for c22h36n4o7, found 491.13 (468.54 + na). boc - phe[ch2n(thyac)]gly - oet (4d)synthesis is analogous to that of 4b, starting with thymin-1-ylacetic acid (198 mg, 1.08 mmol), dcc (228 mg, 1.11 mmol), and dhbtoh (186 mg, 1.14 mmol) dissolved in 10 ml dry dmf. after stirring for 1 hr at room temperature, a solution of 3d (300.0 mg, 0.60 mmol) in dry dmf (2 2.5 ml) was added dropwise. the mixture was heated at 50c for 24 hrs. after workup and purification by column chromatography (20 : 1 etoac : etoh rf = 0.55), 280 mg (0.56 mmol, 93% yield) of 4d was obtained. h - nmr (dmso, 300 mhz) : = 1.20 (3h, t, och2ch3, j = 7.1 hz) ; 1.49 (9h, s, c(ch3)3) ; 1.80 (3h, s, thymine ch3) ; 2.602.85 (2h, m, chch2ph) ; 3.053.50 (2h, m, chch2nco) ; 3.80 and 3.95 (1h, 2 m, nchch2) ; 4.004.15 (2h, m, nch2co) ; 4.20 (2h, q, och2ch3, j = 7.2 hz) ; 4.50 and 4.70 (2h, 2ab, ncoch2 j = 16.1 and 16.6 hz) ; 6.75 and 6.90 (1h, 2d, boc - nh, j = 8.7 and 9.4 hz) ; 7.107.30 (6h, m, ch2phh26 and thymine h)) ; 11.20 (1h, 2s, thymine - nh). minor impurities were found in the h - nmr : etoac (1.20, 2.00, 4.00), etoh (1.10 and 3.60), h2o (3.30), and dhbtoh byproduct (5.50). (esi - ms m / z) mass calculated 502.56 for c25h34n4o7, found 530.47 (502.56 + na). synthesis is analogous to that of 4b, starting with thymin-1-ylacetic acid (198 mg, 1.08 mmol), dcc (228 mg, 1.11 mmol), and dhbtoh (186 mg, 1.14 mmol) dissolved in 10 ml dry dmf. after stirring for 1 hr at room temperature, a solution of 3d (300.0 mg, 0.60 mmol) in dry dmf (2 2.5 ml) was added dropwise. the mixture was heated at 50c for 24 hrs. after workup and purification by column chromatography (20 : 1 etoac : etoh rf = 0.55), 280 mg (0.56 mmol, 93% yield) of 4d was obtained. h - nmr (dmso, 300 mhz) : = 1.20 (3h, t, och2ch3, j = 7.1 hz) ; 1.49 (9h, s, c(ch3)3) ; 1.80 (3h, s, thymine ch3) ; 2.602.85 (2h, m, chch2ph) ; 3.053.50 (2h, m, chch2nco) ; 3.80 and 3.95 (1h, 2 m, nchch2) ; 4.004.15 (2h, m, nch2co) ; 4.20 (2h, q, och2ch3, j = 7.2 hz) ; 4.50 and 4.70 (2h, 2ab, ncoch2 j = 16.1 and 16.6 hz) ; 6.75 and 6.90 (1h, 2d, boc - nh, j = 8.7 and 9.4 hz) ; 7.107.30 (6h, m, ch2phh26 and thymine h)) ; 11.20 (1h, 2s, thymine - nh). minor impurities were found in the h - nmr : etoac (1.20, 2.00, 4.00), etoh (1.10 and 3.60), h2o (3.30), and dhbtoh byproduct (5.50). (esi - ms m / z) mass calculated 502.56 for c25h34n4o7, found 530.47 (502.56 + na). boc - val[ch2n(thyac)]gly - oh (5b) 4b (100 mg, 0.22 mmol) was dissolved in tetrahydrofuran (thf) (3 ml). the solution was stirred and cooled to 0c in an ice bath and followed by dropwise addition of 2 m naoh (3 ml). after confirming by tlc that the reaction has gone to completion, water (25 ml) the aqueous layer was then acidified with 1 m hcl (aq) solution to ph 3. the solution was then extracted with etoac (3 100 ml) and then dried over anhydrous sodium sulfate. the white precipitate was filtered off and purified by column chromatography (8 : 2 dcm : meoh, rf = 0.40) to give 51 mg (0.12 mmol, 54% yield) of white crystalline product. h - nmr (dmso, 500 mhz) : = 0.901.00 (6h, m, ch(ch3)2) ; 1.49 (9h, s, c(ch3)3) ; 1.70 (1h, m, ch(ch3)2) ; 1.80 (3h, s, thymine ch3) ; 3.00, 3.20, 3.45, 3.55 (2h, 4 m, chch2nco) ; 3.50 and 3.60 (1h, 2 m, nchch2) ; 3.70 and 3.85 (2h, 2ab, nch2co, j = 17.4 and 18.2 hz) ; 4.45 and 4.70 (2h, 2ab, ncoch2, j = 16.3 and 17.1 hz) ; 6.60 and 6.90 (1h, 2d, boc - nh, j = 9.4 and 9.7 hz) ; 7.20 (1h, s, thymine h) ; 11.20 (1h, 2s, cooh). c nmr (dmso, 75 mhz) : = 172.1, 168.2, 167.3, 164.9, 156.4, 156.2, 151.5, 142.6, 142.3, 108.5, 78.3, 77.9, 54.3, 54.1, 52.1, 49.5, 48.7, 48.0, 39.1, 30.6, 30.3, 28.7, 19.9, 19.0, 18.3, 12.4. (esi - ms m / z) mass calculated 426.46 for c19h30n4o7, found 449.20 (426.46 + na). hrms (esi / msm / z) mcalc for c19h30n4nao7 449.20, found 449.1682. 4b (100 mg, 0.22 mmol) was dissolved in tetrahydrofuran (thf) (3 ml). the solution was stirred and cooled to 0c in an ice bath and followed by dropwise addition of 2 m naoh (3 ml). after confirming by tlc that the reaction has gone to completion, water (25 ml) the aqueous layer was then acidified with 1 m hcl (aq) solution to ph 3. the solution was then extracted with etoac (3 100 ml) and then dried over anhydrous sodium sulfate. the white precipitate was filtered off and purified by column chromatography (8 : 2 dcm : meoh, rf = 0.40) to give 51 mg (0.12 mmol, 54% yield) of white crystalline product. h - nmr (dmso, 500 mhz) : = 0.901.00 (6h, m, ch(ch3)2) ; 1.49 (9h, s, c(ch3)3) ; 1.70 (1h, m, ch(ch3)2) ; 1.80 (3h, s, thymine ch3) ; 3.00, 3.20, 3.45, 3.55 (2h, 4 m, chch2nco) ; 3.50 and 3.60 (1h, 2 m, nchch2) ; 3.70 and 3.85 (2h, 2ab, nch2co, j = 17.4 and 18.2 hz) ; 4.45 and 4.70 (2h, 2ab, ncoch2, j = 16.3 and 17.1 hz) ; 6.60 and 6.90 (1h, 2d, boc - nh, j = 9.4 and 9.7 hz) ; 7.20 (1h, s, thymine h) ; 11.20 (1h, 2s, cooh). c nmr (dmso, 75 mhz) : = 172.1, 168.2, 167.3, 164.9, 156.4, 156.2, 151.5, 142.6, 142.3, 108.5, 78.3, 77.9, 54.3, 54.1, 52.1, 49.5, 48.7, 48.0, 39.1, 30.6, 30.3, 28.7, 19.9, 19.0, 18.3, 12.4. (esi - ms m / z) mass calculated 426.46 for c19h30n4o7, found 449.20 (426.46 + na). hrms (esi / msm / z) mcalc for c19h30n4nao7 449.20, found 449.1682. boc - ile[ch2n(thyac)]gly - oh (5c)synthesis is analogous to that of 5b, starting with 4c (300 mg, 0.68 mmol) dissolved in thf (9 ml). the solution was cooled to 0c, and 2 m naoh (9 ml) was added dropwise. to quench the reaction 75 ml of water was added. after extraction and precipitation, the product was purified by column chromatography (8 : 2 dcm : meoh, rf = 0.40) to give 156 mg (0.36 mmol, 52% yield) of 5c as white crystals. h - nmr (dmso, 500 mhz) : = 0.801.00 (6h, m, chch3, chch2ch3) ; 1.201.40 (2h, m, chch2ch3) ; 1.49 (9h, s, c(ch3)3) ; 1.60 (1h, m, chch2ch3) ; 1.80 (3h, s, thymine ch3) ; 3.00, 3.30, 3.50, 3.60 (2h, 4 m, chch2nco) ; 3.55 and 3.65 (1h, 2 m, nchch2) ; 3.90 and 3.95 (2h, 2ab, nch2co, j = 16.5 and 18.7 hz) ; 4.45 and 4.70 (2h, 2ab, ncoch2, j = 16.2 and 16.7 hz) ; 6.60 and 6.90 (1h, 2d, boc - nh, j = 9.2 and 9.5 hz) ; 7.20 (1h, s, thymine h) ; 11.20 (1h, 2s, cooh). c nmr (dmso, 75 mhz) : = 171.8, 168.1, 167.5, 164.8, 156.2, 156.1, 151.5, 142.5, 142.2, 108.5, 78.3, 77.9, 53.3, 52.9, 51.7, 49.3, 48.9, 48.4, 48.0, 37.3, 28.7, 25.5, 25.2, 15.7, 12.4, 11.9, 11.5. (esi - ms m / z) mass calculated 440.49 for c20h32n4o7, found 463.13 (440.49 + na). hrms (esi / msm / z) mcalc for c20h32n4nao4 463.22, found 463.1957. synthesis is analogous to that of 5b, starting with 4c (300 mg, 0.68 mmol) dissolved in thf (9 ml). the solution was cooled to 0c, and 2 m naoh (9 ml) was added dropwise. to quench the reaction 75 ml of water was added. after extraction and precipitation, the product was purified by column chromatography (8 : 2 dcm : meoh, rf = 0.40) to give 156 mg (0.36 mmol, 52% yield) of 5c as white crystals. h - nmr (dmso, 500 mhz) : = 0.801.00 (6h, m, chch3, chch2ch3) ; 1.201.40 (2h, m, chch2ch3) ; 1.49 (9h, s, c(ch3)3) ; 1.60 (1h, m, chch2ch3) ; 1.80 (3h, s, thymine ch3) ; 3.00, 3.30, 3.50, 3.60 (2h, 4 m, chch2nco) ; 3.55 and 3.65 (1h, 2 m, nchch2) ; 3.90 and 3.95 (2h, 2ab, nch2co, j = 16.5 and 18.7 hz) ; 4.45 and 4.70 (2h, 2ab, ncoch2, j = 16.2 and 16.7 hz) ; 6.60 and 6.90 (1h, 2d, boc - nh, j = 9.2 and 9.5 hz) ; 7.20 (1h, s, thymine h) ; 11.20 (1h, 2s, cooh). c nmr (dmso, 75 mhz) : = 171.8, 168.1, 167.5, 164.8, 156.2, 156.1, 151.5, 142.5, 142.2, 108.5, 78.3, 77.9, 53.3, 52.9, 51.7, 49.3, 48.9, 48.4, 48.0, 37.3, 28.7, 25.5, 25.2, 15.7, 12.4, 11.9, 11.5. (esi - ms m / z) mass calculated 440.49 for c20h32n4o7, found 463.13 (440.49 + na). hrms (esi / msm / z) mcalc for c20h32n4nao4 463.22, found 463.1957. boc - phe[ch2n(thyac)]gly - oh (5d)synthesis is analogous to that of 5b, starting with 4d (280 mg, 0.56 mmol) dissolved in thf (12 ml). the solution was cooled to 0c and 2 m naoh (12 ml) was added dropwise. to quench the reaction 75 ml of water was added. after extraction and precipitation, the product was purified by column chromatography (8 : 2 dcm : meoh, rf = 0.50) to give 150 mg (0.32 mmol, 56% yield) of 5d as white crystals. h - nmr (dmso, 500 mhz) : = 1.49 (9h, s, c(ch3)3) ; 1.80 (3h, s, thymine ch3) ; 2.55, 2.65, 2.75, 2.85 (2h, 4ab, chch2ph, j = 5.4 and 10.0 some peaks are obscured by h2o peak) ; 3.00, 3.35, 3.40, 3.60 (2h, 4ab, chch2nco, j~6.5 hz) ; 3.75 and 3.95 (1h, 2 m, nchch2) ; 3.90 (2h, m, nch2co) and 4.0 (2h, ab, nch2co, j = 17.8 hz) ; 4.45 (2h, m, ncoch2) and 4.70 (2h, ab, ncoch2, j = 16.4 hz) ; 6.80 and 6.90 (1h, 2d, boc - nh, j = 9.0 and 9.4 hz) ; 7.107.30 (6h, m, ch2phh26 and thymine h) ; 11.20 (1h, 2s, cooh). c nmr (dmso, 75 mhz) : = 171.1, 168.3, 167.6, 164.8, 155.7, 151.5, 142.6, 139.6, 139.2, 129.5, 128.5, 128.4, 126.5, 126.3, 108.4, 78.4, 78.0, 51.7, 50.8, 48.7, 48.1, 46.0, 38.1, 28.7, 12.4, 10.3. (esi - ms m / z) mass calculated 474.51 for c23h30n4o7, found 497.07 (474.51 + na). hrms (esi / msm / z) mcalc for c23h30n4nao7 497.20, found 497.1580. synthesis is analogous to that of 5b, starting with 4d (280 mg, 0.56 mmol) dissolved in thf (12 ml). the solution was cooled to 0c and 2 m naoh (12 ml) was added dropwise. to quench the reaction 75 ml of water was added. after extraction and precipitation, the product was purified by column chromatography (8 : 2 dcm : meoh, rf = 0.50) to give 150 mg (0.32 mmol, 56% yield) of 5d as white crystals. h - nmr (dmso, 500 mhz) : = 1.49 (9h, s, c(ch3)3) ; 1.80 (3h, s, thymine ch3) ; 2.55, 2.65, 2.75, 2.85 (2h, 4ab, chch2ph, j = 5.4 and 10.0 some peaks are obscured by h2o peak) ; 3.00, 3.35, 3.40, 3.60 (2h, 4ab, chch2nco, j~6.5 hz) ; 3.75 and 3.95 (1h, 2 m, nchch2) ; 3.90 (2h, m, nch2co) and 4.0 (2h, ab, nch2co, j = 17.8 hz) ; 4.45 (2h, m, ncoch2) and 4.70 (2h, ab, ncoch2, j = 16.4 hz) ; 6.80 and 6.90 (1h, 2d, boc - nh, j = 9.0 and 9.4 hz) ; 7.107.30 (6h, m, ch2phh26 and thymine h) ; 11.20 (1h, 2s, cooh). c nmr (dmso, 75 mhz) : = 171.1, 168.3, 167.6, 164.8, 155.7, 151.5, 142.6, 139.6, 139.2, 129.5, 128.5, 128.4, 126.5, 126.3, 108.4, 78.4, 78.0, 51.7, 50.8, 48.7, 48.1, 46.0, 38.1, 28.7, 12.4, 10.3. (esi - ms m / z) mass calculated 474.51 for c23h30n4o7, found 497.07 (474.51 + na). hrms (esi / msm / z) mcalc for c23h30n4nao7 497.20, found 497.1580. the oligomers were cleaved from the resin with tfa / tfmsa / m - cresol / thioanisole mixture (6 : 2 : 1 : 1), precipitated with ethyl ether (4x), and then air - dried. the oligomers were purified by reverse - phase hplc and characterized by maldi - tof mass spectrometry. the following amino acid side chains, alanine (ala), valine (val), isoleucine (ile), and phenylalanine (phe), were incorporated at the -backbone position of pna. the corresponding thymine monomers were prepared from their respective boc - protected l - amino acids according to the procedures outlined in scheme 1. mitsunobu coupling reaction was chosen over reductive amination in the preparation of the backbone intermediates because it is less prone to racemization. these chiral building blocks were then individually incorporated into the pna oligomers using standard solid - phase synthesis procedures. after cleavage from the resin, the oligomers were purified by reverse - phase hplc (see figures s1a s9a, in supplementary material available online at doi : 10.4061/2011/652702) and characterized by maldi - tof mass spectrometry (figures s1b s9b). cd measurements were recorded at 5 m strand concentration each in sodium phosphate buffer (10 mm sodium phosphate, 0.1 mm edta and 100 mm nacl, ph 7.4) at room temperature. inspection of figure 1 reveals that all pna oligomers containing -backbone modifications (pna2 through 5) exhibited biphasic exciton coupling patterns, characteristic of a right - handed helix. no noticeable cd signals were observed in the nucleobase absorption regions (220300 nm) for the unmodified pna (pna1). this is expected since unmodified, single - stranded pna does not have a well - defined helical conformation. we ruled out the possibility of pna adopting an equimolar ratio of a right - handed and left - handed helix as suggested by md simulations, based on multinuclear and multidimensional nmr analyses. the similarity in the cd profiles of pna2 through 5 suggests that variations in the amino acid side chains at the -position have little effect on the overall conformation of pnas. the subtle differences in the 240 nm minima generally indicate variations in the helical pitch, with the positive minima characteristic of a more relaxed pna - pna and the negative minima characteristic of a more tightly wound pna - dna duplex. the variations in the degree of winding are likely to be the result of steric clash, with the largest phenylalanine side - chain expected to induce the greatest effect. to better understand how these side chains affect the hybridization properties of pnas, we measured their melting transitions (tms) following hybridization with an antiparallel, complementary dna strand. our data showed that incorporation of each -building block resulted in an increase in the tm of the hybrid duplex by ~4c for all four amino acid side chains examined (table 1), consistent with the cd data. next, we examined the effects of backbone spacing on the conformations and hybridization properties of pnas. we selected phenylalanine (phe) because it is the most sterically hindered side - chain among this group, which is likely to cause the greatest steric clash. pna6 contained three phe groups placed in alternate positions with the unmodified pna units, while pna7 also contained three phe groups but they were placed consecutively next to one another. the two oligomers showed similar cd profiles (figure 2) but with varied signal strengths, with pna6 (phe 3alt) exhibiting stronger signals than pna7 (phe 3con). it is interesting to note that pna5, which contained one phe group, showed similar cd profile as that of pna7, which beared three phe groups. this result indicates that one phe side - chain is sufficient to preorganize pna into a helical motif. uv melting data showed that pna6 binds less tightly to a complementary dna strand than pna7, with the tm of + 11 and + 13c, respectively, compared to that of the unmodified pna - dna. this result is unexpected because pna6 seems more organized (or better base - stacked) based on the cd data. the three phe rings placed in the consecutive arrangement might be able to interact with the adjacent dna bases better than those placed in the alternate positions. a second possibility may come from the fact that the phe rings are able to stack better with one another in the consecutive than in the alternate arrangement upon hybridization with the complementary dna strand. this provides a more favorable solvophobic driving force for pna7-dna to remain in the duplex form rather than dissociate into individual strands. a third possibility may come from the difference in the rigidity of the two helices. since pna6 is more rigid than pna7, as inferred from the cd data, it is less accommodating to the dna strand which may explain the lower tm. to delineate these three possible effects, we synthesized another set of pna oligomers (pna8 and pna9) with valine side - chain at the -position. val and phe side chains are similar in size but they differ in their ability to interact with one another. the phe side chains can -stack with one another whereas the val side chains can not. therefore, comparing the cd and tm profiles of the two sets of oligomers should provide insights into the role of - interaction between the phe side chains on the conformations and stability of the hybrid duplex. our result shows that both pna8 (val pna alt) and pna9 (val pna con) have the same tm (55c) (figure 3, table 1) suggesting that the extra binding affinity of pna6 (phe alt) and pna7 (phe con) comes from - interaction. however, it is not clear at this point whether the phe ring stacking occurs intramolecularly with the adjacent pna bases or intermolecularly with the dna bases both of which produce similar results. the same cd patterns were observed for the second set of oligomers, with the alternating pna8 exhibiting greater cd signal than the consecutively modified pna9 (figure 4). it is not obvious why the alternate arrangement displayed greater helical character than their consecutive counterparts. one possible explanation is that the helical directors, in this case the stereogenic centers at the -backbone position, are more spread out in the alternate than in the consecutive arrangement. the alternate arrangement, therefore, should enable the helical directors to induce and propagate the helical sense of the oligomer more effectively than the consecutive arrangement, since each chiral director has fewer achiral units in front of them to direct. this explanation is consistent with the sergeants and soldiers concept proposed by green and coworkers [36, 37 ] to explain helical induction in polymers. when comparing the two arrangements it should be noted that although they both start out at the same position (the third unit from the c - terminus), the alternate sequence contained -building blocks that are spread out further toward the n - terminus. this should give them greater advantage in organizing the remaining achiral, n - terminal residues since our previous study showed that helical induction occurs in a unidirectional fashion from c- to n - terminus. this may explain why pnas with the alternate placement exhibit greater cd signals, hence more helical character, than those with the consecutive arrangement. detailed explanations for this phenomenon will await further structural studies. nevertheless, this result shows that the chiral backbone units can be placed in the consective or alternative position with minimal effect on the oligomer 's binding affinity, unless the side - chain contains an aromatic group which slightly favors the consecutive arrangement. in addition to the perfect match, we have determined the melting transitions for the pna - dna hybrid duplexes containing single - base mismatched binding sites (see table 2). our result shows that conformationally preorganized pnas can discriminate between related sequences, with similar degree of specificity as that of the unmodified pna oligomer. the tms for mismatched duplexes range from 11 to 19c, depending on the mismatch pair with t - t mismatch being the least discriminating. though incorporation of additional -backbone modified units further improved the binding affinity of pnas towards complementary dna strands, it does not significantly affect their ability to discriminate single - base mismatched sequences. this result shows that the binding affinity of pna can be improved by installing an appropriate stereogenic center at the -backbone position without compromising sequence specificity. in summary, we have shown that a number of amino acid side chains with varying degree of steric hindrance can be placed at the -position of the n-(2-aminoethyl) glycine backbone of pna without inducing adverse affect on the hybridization properties of pnas. spectroscopic measurements showed that these pna oligomers adopted a right - handed helix and hybridized to a complementary dna strand with higher affinity than their unmodified counterpart, with tm ~ + 4c per unit incorporated. despite their strong binding affinities, these conformationally preorganized pnas can discriminate related sequences, with similar level of specificity as that of the unmodified pna. placement of the chiral -units (consecutive versus alternating) has subtle effects on the confirmations and hybridization properties of pna oligomers depending on whether the side chains are involved in intramolecular - stacking ; but overall, these effects are negligible. our results confirm that pnas are true hybrids of peptides and nucleic acids, capable of binding dna (and rna), and can be functionalized with a number of amino acid side chains without inducing adverse affects on the hybridization properties of the oligomers. the ability to modify the structures and chemical functionalities of oligonucleotide analogues is important because it allows other functional properties beside hybridization, such water solubility, cellular uptake, biodistribution, and pharmacokinetics to be augmented and/or further improved upon and undesired features, such nonspecific binding and toxicity, to be further minimized. the ease and flexibility of synthesis, along with superior hybridization properties and enzymatic stability, make pnas an attractive nucleic acid platform for various biological and medical applications as molecular tools as well as therapeutic and diagnostic reagents. | conformationally preorganized peptide nucleic acids (pnas) have been synthesized through backbone modifications at the -position, where r = alanine, valine, isoleucine, and phenylalanine side chains. the effects of these side - chains on the conformations and hybridization properties of pnas were determined using a combination of cd and uv - vis spectroscopic techniques. our results show that the -position can accommodate varying degrees of sterically hindered side - chains, reaffirming the bimodal function of pnas as the true hybrids of peptides and nucleic acids. |
neuroendocrine tumors (nets) consist of a heterogeneous group of neoplasms of varying presentation and prognosis. while a complete list of this family of tumors includes dozens of distinct histopathologic subtypes from multiple different organ systems, the majority of nets are carcinoid tumors of the gastrointestinal tract and endocrine tumors of the pancreatic islet cells [13 ]. primary liver nets have been reported but are unusual and will not be discussed in this paper. nets are relatively rare, with an incidence ranging from 2.5 to 5.3 per 100,000 [3, 5 ]. the prevalence is significantly higher at about 35 per 100,000, indicating that many patients are alive with disease [3, 6 ]. however, these figures may not capture the full burden of net disease, since conflicting nomenclature systems exist, making them difficult to classify and quantify [1, 3, 57 ]. although some nets are more aggressive in their behavior than others, all have the potential for distant metastases and should be considered malignant. in patients with resectable tumors without metastatic disease, surgery is considered the gold standard and is the only curative option. 5-year survival rates in patients with localized, nonmetastatic nets undergoing curative resection range from 80% to 100% [2, 6 ]. while many nets are nonfunctioning, these tumors are traditionally categorized by their classic patterns of symptoms arising from the secretion of various peptides and hormones. for example, patients with gastrin hypersecretion from a gastrinoma tumor may present with severe peptic ulcer disease refractory to treatment. other nets include vipoma, characterized by diarrhea and hypokalemia, and somatostatinoma, presenting with cholelithiasis, diabetes, and steatorrhea. carcinoid tumors of the gi tract frequently produce serotonin (5-ht), which can manifest as skin flushing, severe diarrhea, abdominal cramping, and electrolyte abnormalities. the symptoms associated with functional nets may be severe and debilitating and detract significantly from the quality of life of the patient ; therefore, aggressive treatments to reduce symptoms have an important role in therapy. even in the setting of an unresectable primary tumor or widely metastatic disease, most nets have an indolent course ; 510-year survival with stage iv disease is not uncommon and makes the treatment of symptoms a fundamental component of patient care [3, 914 ]. because the majority of nets arise from the gastrointestinal tract and the pancreas, the liver is the most common site of metastases. in patients with liver metastases, 7580% will present with these metastases at the time of diagnosis (synchronous), while 2025% of patients develop liver metastases during the course of treatment (metachronous). an estimated 8090% of patients with liver metastases are inoperable at presentation. many primary nets are small in size, and it is not unusual for the liver metastases to be of greater volume than the primary tumor. given the high tumor burden often associated with metastases, symptoms can become significantly worse as the disease advances. in addition, many of the peptides and hormones produced by nets are eliminated by metabolism through the liver. therefore, it is only after liver metastases are present, and these compounds spill directly into the systemic circulation, that the phenotype of the tumor is fully expressed. as a result, surgical resection of hepatic metastases has been shown to reduce symptoms and is indicated for this purpose alone [2, 12 ]. in addition, some data indicate an improvement in overall survival as well, and therefore metastasectomy should be considered for resectable disease even in patients with nonfunctional tumors [10, 12, 17 ]. meaningful improvements in symptom control and overall survival can be achieved even if complete resection of metastatic disease is not possible. however, available data suggest that debulking should only be considered if greater than 90% of the tumor burden can be resected [11, 18 ]. while resection is preferred, excessive tumor bulk, tumor location, and other biological factors often preclude surgery. even with resection, recurrence is common (5060% at 5 years) and repeat hepatectomy may not be feasible [20, 21 ]. in these cases where resection is not possible or would not be tolerated by the patient 's physiology, interventional radiology alternatives to surgery have been proposed, including radiofrequency ablation, hepatic arterial radioembolization with y, and hepatic arterial bland or chemoembolization. the goals of these therapies are twofold : to increase overall survival by stabilizing tumor growth, and to reduce the morbidity in symptomatic patients. the radiologic appearance of liver metastases from nets is distinct and has important ramifications for treatment. compared to liver metastases that are of gastrointestinal origin, metastases from nets derive a greater amount of their blood supply from the hepatic artery. as a result, when imaged during the arterial phase, metastatic nets will typically appear brighter than the surrounding liver ; and during the venous phase when the normal liver parenchyma is filled with contrast, net metastases will appear darker than the surrounding liver. in other words, net metastases typically light up and wash out (figure 1). this pattern of enhancement is similar to that seen with hepatocellular carcinoma and is ideally suited for the arterial embolization techniques more commonly associated with that disease. treatment response can be assessed using radiographic measures by examining the degree of enhancement of the lesions following embolization procedures (figure 2). since surgical metastasectomy is the most effective treatment, only patients with unresectable liver disease or who are unable to undergo surgery should be considered for embolization procedures. previous resection of the primary tumor is not necessary, although the disease should be stable and not at risk for complications such as bleeding or obstruction. liver involvement greater than 75% is considered a relative contraindication to embolization, since these patients tend to have less response to treatment coupled with greater rates of complications [13, 23 ]. the presence of main portal vein thrombosis is a strict contraindication since hepatic arterial embolization relies upon the portal venous blood supply to rescue the nontumorous liver parenchyma. therefore, hepatic arterial embolization in patients with complete portal vein thrombosis risks severe liver ischemia. embolization in patients with bilirubin levels greater than 2 - 3 mg / dl has also been reported to be unsafe [13, 24, 25 ]. even though the liver parenchyma is relatively spared with arterial embolization patients with already borderline liver function may be tipped over into frank liver failure following embolization. accordingly, patients with ascites should be carefully considered for embolization procedures since its presence suggests poor liver function. finally, patients with general contraindications to angiography, intolerance of contrast media, peripheral vascular disease, or coagulopathies should not be considered for embolization. occlusion of the hepatic artery causes selective ischemia to the tumor, while the remainder of the liver parenchyma is rescued by the portal venous flow. as a result, the tumor is disproportionately affected by the ischemic insult, with relative sparing of the normal parenchyma. while not curative, hepatic arterial embolization procedures slow tumor growth and prolong progression - free survival, until the eventual revascularization from collateral angiogenesis resupplies the tumor. one lobe of the liver is treated per session to minimize the risk of liver failure. if both lobes of the liver are involved with tumor, the contralateral side can be treated approximately one month after the initial embolization. three types of hepatic arterial embolization techniques are currently in use : transarterial bland embolization (tae), transarterial chemoembolization (tace), and embolization using drug - eluting beads (deb - tace). these procedures all involve percutaneous access to the femoral artery, followed by selective cannulation of the hepatic artery and its derivatives to the affected lobe. prior to embolization, an arteriogram is performed to identify the vascular anatomy supplying the tumor (figure 3). if femoral access is not available, the brachial artery can be used as an alternative, although this route is more technically challenging. in bland embolization, catheterization is typically followed by the injection of 50 m polyvinyl alcohol (pva) particles, with or without ethiodized oil. these particles physically occludes blood flow through the selected hepatic artery, thereby inducing ischemic injury ; if stasis remains unachieved, then larger 200500-m pva particles can be used [2629 ]. other embolic agents currently employed include gelfoam, cyanoacrylate, tris - acryl particles, and embospheres [26, 28, 29 ]. previous studies established that tae is effective at reducing tumor size as well as decreasing tumor hormone production for palliation of symptoms [11, 2534 ]. systemic adjuvant chemotherapy following tae was noted to prolong the duration of symptom relief, prompting the development of embolization coupled with chemotherapy. tace combines the use of embolic material with an initial infusion of a chemotherapeutic agent. however, it is unclear whether the addition of intrahepatic chemotherapy improves the efficacy of embolization techniques. the literature has not consistently shown a clear benefit of tace over tae, and no randomized head - to - head studies have been performed. while select reports have found that patients treated with tace experienced slightly longer progression - free survival (pfs) and greater overall survival (os), other reports have not found any benefit of tace over tae [28, 31, 32 ]. it is likely that the efficacy of these techniques is largely due to the ischemia produced by the embolization itself, with only secondary benefits derived from the addition of chemotherapeutic agents. in addition, there is no consensus on which chemotherapeutic agents for tace are the most efficacious in the treatment of liver metastases from nets. doxorubicin, mitomycin c, streptozocin, vinblastine, gemcitabine, fluorouracil (5-fu), and cisplatin have all been used for tace, and some regimens employ them in combination. the most common regimen described in the literature is a three - drug combination of doxorubicin (2030 mg), cisplatin (50 mg), and mitomycin c (1030 mg) mixed with 10 ml ethiodized oil [13, 27, 36, 37 ]. doxorubicin alone with ethiodized oil is the second most common regimen described [3840 ]. lipiodol is an oily agent which is typically used during tace to enhance chemotherapy retention within the tumor. lipiodol appears bright white on ct imaging and therefore interferes with assessment of tumor viability. surveillance following tace should utilize mri imaging since lipiodol does not appear on mri images. a third chemoembolization technique, deb - tace, uses 500700 m embolic beads that are loaded with a chemotherapeutic drug, usually doxorubicin, which slowly elutes into the liver parenchyma over a period of 714 days [4143 ]. the controlled release of chemotherapy allows for sustained, higher tumor levels of doxorubicin, while maintaining lower levels in the systemic circulation, which may decrease the incidence of systemic side effects. studies have shown the os and pfs in patients undergoing chemoembolization with deb - tace to be similar to tae and tace [41, 42 ]. currently, all three techniques are actively in use, with no clear evidence for superiority of one approach over another. although there is a considerable amount of literature on hepatic arterial embolization treatments for metastatic nets, most are retrospective reviews of smaller case series with historical controls ; there are no randomized controlled trials. importantly, all methods of embolization have greater pfs and os than no treatment or systemic chemotherapy alone [6, 11 ]. most series include patients with both metastatic carcinoid tumors as well as pancreatic islet cell tumors in their cohorts. patients with carcinoid nets in general have longer os and higher response rates than patients with pancreatic nets, and as a result, outcomes should be interpreted accordingly [31, 32, 35 ]. one of the largest trials to date on the effects of embolization procedures is an analysis from bloomston and colleagues on the outcomes of patients with metastatic carcinoid tumors undergoing tace. a cohort of 122 patients underwent 156 tace procedures using a combination of doxorubicin (30 mg), mitomycin c (30 mg), and cisplatin (50 mg). the origin of the primary tumor was predominantly in the small bowel (47%), pancreas (21%), or lung (8%), with 14% of unknown origin. 81% of the patients presented with carcinoid syndrome, and the primary tumor had been previously resected in 75% of the patients. interestingly, resection of the primary tumor did not prove to be predictive of survival. following tace, regression or stabilization of the hepatic metastases was observed in 94% of the patients, with a median duration of 19 months. symptom improvement was reported in 92% of patients and was associated with a benefit in os (41 months versus 8 months). pfs for the entire cohort was 10 months, and os was reported to be 33.3 months with a 5-year survival of 28% from the date of the first tace procedure. 106 of the patients had resection of the primary tumor, as well as prophylactic cholecystectomy. repeat tae was performed in the setting of progressive disease, as demonstrated by two consecutive ct scans at least 6 months apart and a two fold increase in urinary 5-hiaa, a metabolite of 5-ht. symptomatic improvement following tae was reported in 71% of the patients, and an os of 56 months from the date of embolization was shown for the group. importantly, swrd. were able to demonstrate a relationship between biochemical markers and survival benefit. compared to patients who demonstrated no reduction in urinary 5-hiaa, patients with greater than 50% reduction experienced a 6-month gain in estimated survival, with an additional 6-month gain if the reduction was increased to 75%. in addition, increases in liver enzymes or chromogranin a both significantly correlated with reduced survival. a more recent study by pitt. compared the outcomes of 100 patients with carcinoid or islet cell tumors undergoing either tace (n = 49) or tae (n = 51). particle embolization was performed with pva, gelfoam, or embospheres ; the chemotherapeutic agents used in tace were cisplatin, doxorubicin, and mitomycin c. both cohorts were similar with respect to age, gender, tumor type, and tumor burden. 67% of the tace cohort underwent resection of the primary tumor, compared with 49% in the tae cohort, although this difference was not significant. response rates were 86% in the tace cohort and 83% in patients treated with tae and were not statistically different. median overall survival from the date of the first procedure was also similar between the tace and tae groups, at 25.5 months and 25.7 months, respectively. in addition, 5-year survival rates for the tace and tae groups were not statistically different at 19% and 13%, respectively. furthermore, the tace and tae groups exhibited similar complication rates (2.4% versus 6.6%, resp.) and mortality rates (0.8% versus 1.8%, resp.). in contrast to the study by bloomston., resection of the primary tumor was significantly associated with an increase in overall survival : 73 months versus 28 months, respectively, from the time of diagnosis of metastatic disease. no other factors were found to be significantly predictive of survival, including tumor type, tumor burden, embolization type, and resection of liver metastases. patients with extensive liver tumor burden experience poorer response to embolization, as well as a greater rate of major complications. a major complication rate of 29% following embolization has been reported in patients with large volume disease [23, 31 ] however, hepatic arterial embolization can still be of benefit in this group of patients. demonstrated a median overall survival of 17.9 months and a pfs of 9.2 months using either tae or tace in patients with greater than 75% liver involvement. while only 44% of the patients demonstrated radiologic response, 65% showed improvement of symptoms, indicating that symptom relief can be used to guide therapy irrespective of radiologic findings despite embolization, most patients will exhibit disease progression as determined through both radiologic measures and the resumption of symptoms. repeat embolizations are generally spaced 46 weeks apart to allow for the liver to recover fully. the outcomes of patients undergoing repeat tace have been reported in the literature, including a study by varker.. although both radiologic and symptomatic responses were found to be slightly lower than for patients having their initial tace (61% versus 82% and 77% versus 92%, resp.), this did not reach statistical significance. in addition, os and pfs were similar between patients having initial versus repeat tace. of note, patients undergoing repeat embolization better tolerated the procedure and had a lower complication rate (11% versus 23%) than patients undergoing initial embolization. these results indicate that repeat tace is safe and effective in patients with progressive disease after initial embolic therapy and should be aggressively pursued to maintain disease control. both minor and serious complications as defined by the society of interventional radiology standard criteria are not uncommon among patients undergoing embolization. a review of the literature has shown the incidence of serious complications to range from 3% to 17% in most series [11, 13, 17, 2534, 3638, 45 ]. postembolization syndrome (fever, nausea, vomiting, abdominal pain, and elevated liver enzymes) has been found to occur in the majority of patients but typically subsides within three days post - procedurally [11, 16, 27, 2932, 38, 40 ]. there are anecdotal reports that the occurrence of postembolization syndrome correlates with the amount of tumor insult and that a robust physical response to embolization is in fact a positive prognostic indicator. hepatic failure, hepatic abscess, hepatorenal syndrome, sepsis, and severe hypertension occurring during embolization can all result from the local ischemia induced by arterial embolization [11, 13, 16, 30, 31, 33, 36 ]. patients with bilioenteric anastomoses or large tumors (greater than 5 cm) are especially at risk for hepatic abscess formation after embolization [47, 48 ]. due to the risk of abscess formation, many physicians advise prophylactic antibiotic administration prior to the procedure [25, 27, 29, 36, 38 ]. patients who develop a hepatic abscess can be treated with percutaneous drain placement and parenteral antibiotics ; rarely liver resection may be indicated for persistence. these events are thought to be due to reflux of embolic material into the cystic artery or pancreaticoduodenal artery respectively, causing ischemic injury to these organs. careful positioning of the catheter tip into the intrahepatic portion of the hepatic artery, along with gentle infusion techniques, is thought to limit the incidence of these potentially serious complications. mortality following embolization procedures is rare in high - volume centers. in a study of 26 patients undergoing 62 tace procedures, kress. reported fatal hepatic failure in 2 patients (3.2%) within 30 days after embolization. similar 30-day mortality rates have been found by other investigators, ranging from 0% to 6%, the majority of which were caused by hepatic failure, acute renal failure, sepsis, and myocardial infarction [17, 27, 30, 31, 36 ]. as to be expected, both acute and chronic renal failure secondary to contrast media administration during arteriogram have been noted as a severe complication from tae and tace [23, 27, 38 ]. finally, all hepatic arterial embolization procedures carry the potential complications which accompany femoral arterial catheterization, including groin hematoma, peripheral embolization, and arterial dissection [27, 30, 33 ]. of note, additionally, deb - tace procedures have demonstrated comparable morbidity rates, with 30%60% of patients experiencing elements of postembolization syndrome. both gaur. reported mortality rates of 5% in patients undergoing deb - tace [41, 42 ]. patients with nets metastatic to the liver are often afflicted with debilitating symptoms that severely affect quality of life, and most patients with nets ultimately die from progression in the liver. as a result, control of the hepatic tumor burden should be the primary goal in the management of patients with metastatic nets. surgical metastasectomy is considered preferable, although there are no randomized controlled trials comparing resection to nonsurgical therapies. in cases where resection is not feasible, interventional radiologic therapies such as hepatic arterial embolization can be used to control disease progression. the characteristic arterial enhancement of nets can be taken advantage of, allowing selective embolization of the tumor while sparing normal parenchyma. although not curative, hepatic arterial embolization can improve symptoms and reduce or stabilize tumor progression, prolonging pfs and os, and should be the mainstay of treatment of patients with liver metastases from nets. | neuroendocrine tumors (nets) have a high predilection for metastasizing to the liver and can cause severe debilitating symptoms adversely affecting quality of life. although surgery remains the treatment of choice, many liver metastases are inoperable at presentation. hepatic arterial embolization procedures take advantage of the arterial supply of net metastases. the goals of these therapies are twofold : to increase overall survival by stabilizing tumor growth, and to reduce the morbidity in symptomatic patients. patients treated with hepatic arterial embolization demonstrate longer progression - free survival and have 5-year survival rates of nearly 30%. the safety of repeat embolizations has also been proven in the setting of recurrent symptoms or progression of the disease. despite not being curative, hepatic arterial embolization should be used in the management of nets with liver metastases. long - term survival is not uncommon, making aggressive palliation of symptoms an important component of treatment. |
complete regeneration of own tissue is the ideal treatment for tissue loss in the oral and maxillofacial region. therefore, various surgical reconstructions have been developed for functional restoration, which sometimes results in the morbidity of the unnecessary donor site and long - term complications. moreover, mechanical devices can not always restore the function of an organ completely1. to overcome such disadvantages, new reconstructive modalities using tissue engineering tissue engineering is an interdisciplinary field that applies the principles of engineering and life science to the development of biologically functional constituents that restore, maintain, or improve tissue function2. many investigations and clinical trials have been attempted to engineer virtually every type of mammalian tissue. since autologous keratinocyte sheets were used to treat burns in 19813, several improved methods have been established to make bioengineered skin substitutes using only extracellular matrix, mainly cells, or combination of cells and matrices4,5. several types of composite grafts of cultured oral mucosal keratinocytes (omks) and fibroblasts of the lamina propria of the oral mucosa or dermal analog have been developed10 - 12. note, however, that the preparation procedure of a composite tissue is complicated and time - consuming. there are a few reports on the successful grafting of epi - thelial sheets from the oral mucosa for large intraoral wounds and skin repair9,13. nonetheless, using only the epidermal component without the dermal matrix has several disadvantages such as fragility, wound contracture, and varying graft " take " rate12,14. to overcome these disadvantages, the cell suspension technique is less time - consuming than the preparation of the sheet grafts, and it is now possible to transfer the proliferating single keratinocytes actively with a simplified application method7. cultured keratinocytes exhibit a hyperproliferative phenotype and release cytokines that are not found in stable condition15,16. the cytokines produced by the activated keratinocyte play a role in immunity, inflammatory reaction, control of cell growth and differentiation, and wound healing17,18. the main objective of this investigation is to assess the role of suspension of cultured omks in skin wounds of athymic nude mice for the previous study19. human oral mucosal tissue was obtained from non - inflamed gingival tissue of 5 donors in their 20s who underwent surgical extraction of the third molar. primary cultures of omks were performed on a collagen - coated plate (iwaki, japan) as described previously20. to trace the grafted cells by brdu labeling, 20 mol / l of brdu each was added to the culture medium 3 days and 1 hour before cell transplantation. the cultured omks were examined under phase contrast microscope, and the labeled cells were detected using anti - brdu immunohistochemistry. a total of 30 adult female nude mice (balb / c slc - nu, japan slc, inc.) 8 weeks of age and weighing 202 g were used for the omk transplantation, with 15 mice for the control (ctl) group and the remaining 15 for the omk group. all mice were injected with 25 mg of ketamine hcl (hankookunite, korea), pentobarbital sodium (choongwae, korea), and flumoxef sodium (ildong, korea) intraperitoneally. we created a 1.51.5 cm full - thickness wound and sutured the normal skin ends and underlying muscles of the defect wound with 5/0 mersilk (johnson & johnson intl, uk) at the corners and centers of each side to prevent wound contracture. primary cultured omks labeled with brdu were suspended in culture medium at a density of 110 cells/40 l onto skin defects in the omk group, and the culture medium without cells was placed in the ctl group. for the wound dressing, three pieces of lens cleaning tissue (whatman, uk) coated with vaseline gauze (daeshin, korea) were placed over the skin defect area in both groups ; the wound areas were then secured with elatex (alcare co., japan). vaseline gauze could prevent the skin wound from drying, and whatman lens cleaning tissue absorbs the excess grease from the vaseline gauze. five mice for each group were sacrificed at the 3rd day, 1st week, and 2nd week from the date of omk transplantation by being divided into groups named ctl-3rd day, 1st week, 2nd week, omk-3rd day, 1st week, and 2nd week groups, respectively. as a trace marker for grafted cells, double immunohistochemical staining using anti - cytokeratin (ck) ae1/3 and anti - brdu was performed in the paraffin section of the omk-3rd day groups. anti - ck ae1/3 was used to detect epithelial cells, and anti - brdu immunohistochemistry, to detect the labeled cells. anti - brdu (sigma, usa) was treated first, and color development was done with 3,3'-diaminobenzidine (dab ; vector, usa). the sections were reblocked and treated with monoclonal mouse anti - human ck, clones ae1/ae3 (anti - ck ae1/3 ; dako). 3-amino-9 ethyl carbazole (aec ; vector) was reacted for color development for ck ae1/3. phosphate buffered saline (pbs) was substituted for the primary antibody. at the 3rd day, 1st week, and 2nd week since the omk transplantation date, wounds were totally excised, including the visible surgical defect area and normal tissue surroundings. one piece was fixed and embedded in paraffin for the histological examination and histomorphometric analysis of the center of the wound ; the other piece was used for western blot analysis to investigate cytokine expression. serial sections 4 m thick were used for h&e staining for the histological examination. in the h&e slides, the length of the regenerating epithelia was measured using image - pro plus (media cybernetics, md, usa). each value was recorded in the dbase and analyzed using the sas program (sas institute inc.). serial five data per section were statistically analyzed using one - way anova followed by paired t - test to compare the length of the regenerating epithelial front. cytokine expression and growth factors (keratinocyte growth factor [kgf ], interleukin [il]-1, and il-6) were determined using western blot analysis. the proteins were extracted from the excised tissues of both ctl and omk groups after the 3rd day, 1st week, and 2nd week by ripa buffer (roche applied science, indianapolis, in, usa). 15 g of proteins was separated in 10% sds polyacrylamide gel, and then transferred onto a nitrocellulose membrane. the membrane was blocked with 10% dry milk in pbs and incubated with monoclonal anti - kgf (santa cruz biotechnology), il-1 (santa cruz biotechnology), and il-6 antibody (santa cruz biotechnology). -actin was used as loading control. to verify the effect of cytokine, rhil-1 (r&d) as previously described, 1.51.5 cm skin wounds were made in the dorsal surface of the mice and sutured. 10 ng / ml of rhil-1 was scattered onto the skin defect area in the il-1 treatment group, with only the culture medium placed in the ctl group. they were sacrificed 5 days after the treatment and divided into ctl-5rd day and rhil-1-5rd day groups. human oral mucosal tissue was obtained from non - inflamed gingival tissue of 5 donors in their 20s who underwent surgical extraction of the third molar. primary cultures of omks were performed on a collagen - coated plate (iwaki, japan) as described previously20. to trace the grafted cells by brdu labeling, 20 mol / l of brdu each was added to the culture medium 3 days and 1 hour before cell transplantation. the cultured omks were examined under phase contrast microscope, and the labeled cells were detected using anti - brdu immunohistochemistry. a total of 30 adult female nude mice (balb / c slc - nu, japan slc, inc.) 8 weeks of age and weighing 202 g were used for the omk transplantation, with 15 mice for the control (ctl) group and the remaining 15 for the omk group. all mice were injected with 25 mg of ketamine hcl (hankookunite, korea), pentobarbital sodium (choongwae, korea), and flumoxef sodium (ildong, korea) intraperitoneally. we created a 1.51.5 cm full - thickness wound and sutured the normal skin ends and underlying muscles of the defect wound with 5/0 mersilk (johnson & johnson intl, uk) at the corners and centers of each side to prevent wound contracture. primary cultured omks labeled with brdu were suspended in culture medium at a density of 110 cells/40 l onto skin defects in the omk group, and the culture medium without cells was placed in the ctl group. for the wound dressing, three pieces of lens cleaning tissue (whatman, uk) coated with vaseline gauze (daeshin, korea) were placed over the skin defect area in both groups ; the wound areas were then secured with elatex (alcare co., japan). vaseline gauze could prevent the skin wound from drying, and whatman lens cleaning tissue absorbs the excess grease from the vaseline gauze. five mice for each group were sacrificed at the 3rd day, 1st week, and 2nd week from the date of omk transplantation by being divided into groups named ctl-3rd day, 1st week, 2nd week, omk-3rd day, 1st week, and 2nd week groups, respectively. as a trace marker for grafted cells, brdu was injected into the culture media before cell transplantation. double immunohistochemical staining using anti - cytokeratin (ck) ae1/3 and anti - brdu was performed in the paraffin section of the omk-3rd day groups. anti - ck ae1/3 was used to detect epithelial cells, and anti - brdu immunohistochemistry, to detect the labeled cells. anti - brdu (sigma, usa) was treated first, and color development was done with 3,3'-diaminobenzidine (dab ; vector, usa). the sections were reblocked and treated with monoclonal mouse anti - human ck, clones ae1/ae3 (anti - ck ae1/3 ; dako). 3-amino-9 ethyl carbazole (aec ; vector) was reacted for color development for ck ae1/3. at the 3rd day, 1st week, and 2nd week since the omk transplantation date, wounds were totally excised, including the visible surgical defect area and normal tissue surroundings. one piece was fixed and embedded in paraffin for the histological examination and histomorphometric analysis of the center of the wound ; the other piece was used for western blot analysis to investigate cytokine expression. serial sections 4 m thick were used for h&e staining for the histological examination. in the h&e slides, the length of the regenerating epithelia was measured using image - pro plus (media cybernetics, md, usa). each value was recorded in the dbase and analyzed using the sas program (sas institute inc.). serial five data per section were statistically analyzed using one - way anova followed by paired t - test to compare the length of the regenerating epithelial front. cytokine expression and growth factors (keratinocyte growth factor [kgf ], interleukin [il]-1, and il-6) were determined using western blot analysis. the proteins were extracted from the excised tissues of both ctl and omk groups after the 3rd day, 1st week, and 2nd week by ripa buffer (roche applied science, indianapolis, in, usa). 15 g of proteins was separated in 10% sds polyacrylamide gel, and then transferred onto a nitrocellulose membrane. the membrane was blocked with 10% dry milk in pbs and incubated with monoclonal anti - kgf (santa cruz biotechnology), il-1 (santa cruz biotechnology), and il-6 antibody (santa cruz biotechnology). to verify the effect of cytokine, rhil-1 (r&d) was applied instead of omk transplantation. as previously described, 1.51.5 cm skin wounds were made in the dorsal surface of the mice and sutured. 10 ng / ml of rhil-1 was scattered onto the skin defect area in the il-1 treatment group, with only the culture medium placed in the ctl group. they were sacrificed 5 days after the treatment and divided into ctl-5rd day and rhil-1-5rd day groups. the phase contrast microscopic findings revealed that cultured omks predominantly consist of uniform, small basaloid cells and intersperse with larger differentiated cells when confluent.(fig. a) the human omks in culture were positive for anti - brdu before grafting.(fig. b) the cultured cells can be successively pursued after the graft with anti - brdu. double immunohistochemical analysis for the regenerating epithelium and grafted omks was performed with anti - ck ae1/3 and anti - brdu in omk-3rd day groups. ck ae1/3 appeared in the cytoplasm of the entire epithelium except the basal and suprabasal cells. the transitional epithelium of the wound margin was thickened and was positive for anti - ck ae1/3 but negative for anti - brdu. there was active re - epithelialization, but grafted omks were absent in the marginal zone of the wound. scattered in the mid - portion of the wound were epithelial cell nests showing both cytoplasmic ck ae1/3 and nuclear brdu expression.(fig. c) cultured and transplanted omks labeled with brdu were present in the skin wound of omk-3rd day groups, predominantly in the central zone. the histological analysis showed the effect of the grafted omks on re - epithelialization. within the 3rd day, the regeneration of epithelium from the wound margin toward the central defect significantly increased in the omk group compared to the ctl group.(figs. c) a few acute inflammatory cells and scattered cell nests were found in the central defect area of the omk-3rd day group. a) after the 2nd week, complete epithelialization was observed in the omk group, but not in the ctl group.(figs. d) in the regenerating epithelium, polarity of normal basal cells and differentiation of squamous cells were observed, similar to normal skin. d) the histologically observed length between the margin of the wound and regenerating epithelial front was measured and calculated using image - pro plus. the average length of the regenerating epithelium in the ctl-3rd day, ctl-1st week, and ctl-2nd week groups was 0.73 mm (9.7%), 1.88 mm (25.1%), and 2.99 mm (39.9%), respectively. the average length of the regenerating epithelium in the omk-3rd day, omk-1st week, and omk-2nd week groups was 3.76 mm (50.1%), 6.64 mm (88.5%), and 7.5 mm (100%), respectively. the regenerating epithelial length increased in a time - dependent fashion, with the rate of re - epithelialization significantly faster in the omk groups than the ctl groups (p<0.05). we found that wound beds undergo staged response to the wound healing procedure by producing pro - inflammatory cytokines and growth factors. the expressions of kgf, il-6, and il-1 at various times after the graft of cultured omks were investigated. kgf produced by fibroblasts affects the step of epithelial proliferation in wound healing and stimulates keratinocyte migration, proliferation, and differentiation. as the cytokines produced by leucocytes, il-1 and il-6 are the most important mediators of the acute - phase inflammatory reaction. il-1 can induce fibroblast proliferation, which is also chemotactic for fibroblast, stimulating the synthesis of collagen. in the omk groups of our study, the western blot analysis demonstrated superior expression of kgf, il-6, and il-1 over the ctl groups. the kgf level was maintained highly through the 3rd day to the 1st week but decreased on the 2nd week. the expression of il-1 was at its maximum on the 3rd day and gradually decreased in the omk groups but showed continual increase in the ctl groups.(fig. 3) the average length of the regenerating epithelium of the ctl-5rd day and rhil-1-5rd day group was 1.17 mm and 2.29 mm, respectively. the marginal portion of the rhil-1 treatment group showed faster re - epithelialization than the ctl group (p<0.0.5).(fig. the phase contrast microscopic findings revealed that cultured omks predominantly consist of uniform, small basaloid cells and intersperse with larger differentiated cells when confluent.(fig. a) the human omks in culture were positive for anti - brdu before grafting.(fig. b) the cultured cells can be successively pursued after the graft with anti - brdu. double immunohistochemical analysis for the regenerating epithelium and grafted omks was performed with anti - ck ae1/3 and anti - brdu in omk-3rd day groups. ck ae1/3 appeared in the cytoplasm of the entire epithelium except the basal and suprabasal cells. the transitional epithelium of the wound margin was thickened and was positive for anti - ck ae1/3 but negative for anti - brdu. there was active re - epithelialization, but grafted omks were absent in the marginal zone of the wound. scattered in the mid - portion of the wound were epithelial cell nests showing both cytoplasmic ck ae1/3 and nuclear brdu expression.(fig. c) cultured and transplanted omks labeled with brdu were present in the skin wound of omk-3rd day groups, predominantly in the central zone. the histological analysis showed the effect of the grafted omks on re - epithelialization. within the 3rd day, the regeneration of epithelium from the wound margin toward the central defect significantly increased in the omk group compared to the ctl group.(figs. c) a few acute inflammatory cells and scattered cell nests were found in the central defect area of the omk-3rd day group. a) after the 2nd week, complete epithelialization was observed in the omk group, but not in the ctl group.(figs. d) in the regenerating epithelium, polarity of normal basal cells and differentiation of squamous cells were observed, similar to normal skin. d) the histologically observed length between the margin of the wound and regenerating epithelial front was measured and calculated using image - pro plus. the average length of the regenerating epithelium in the ctl-3rd day, ctl-1st week, and ctl-2nd week groups was 0.73 mm (9.7%), 1.88 mm (25.1%), and 2.99 mm (39.9%), respectively. the average length of the regenerating epithelium in the omk-3rd day, omk-1st week, and omk-2nd week groups was 3.76 mm (50.1%), 6.64 mm (88.5%), and 7.5 mm (100%), respectively. the regenerating epithelial length increased in a time - dependent fashion, with the rate of re - epithelialization significantly faster in the omk groups than the ctl groups (p<0.05). we found that wound beds undergo staged response to the wound healing procedure by producing pro - inflammatory cytokines and growth factors. the expressions of kgf, il-6, and il-1 at various times after the graft of cultured omks were investigated. kgf produced by fibroblasts affects the step of epithelial proliferation in wound healing and stimulates keratinocyte migration, proliferation, and differentiation. as the cytokines produced by leucocytes, il-1 and il-6 are the most important mediators of the acute - phase inflammatory reaction. il-1 can induce fibroblast proliferation, which is also chemotactic for fibroblast, stimulating the synthesis of collagen. in the omk groups of our study, the western blot analysis demonstrated superior expression of kgf, il-6, and il-1 over the ctl groups. the kgf level was maintained highly through the 3rd day to the 1st week but decreased on the 2nd week. the expression of il-1 was at its maximum on the 3rd day and gradually decreased in the omk groups but showed continual increase in the ctl groups.(fig. the average length of the regenerating epithelium of the ctl-5rd day and rhil-1-5rd day group was 1.17 mm and 2.29 mm, respectively. the marginal portion of the rhil-1 treatment group showed faster re - epithelialization than the ctl group (p<0.0.5).(fig. the reconstruction of the oral innate deficiency and maxillofacial surgical defects has been a primary target for regenerative medicine. there are several advantages of using oral keratinocytes for wound healing instead of skin keratinocyte : first, oral keratinocyte has shorter turnover time with a resulting shorter culture time over skin keratinocyte. second, cells can be maintained longer under culture conditions without terminal differentiation, whereas skin keratinocyte easily becomes keratinized in vitro, thereby losing its viability. third, donor site morbidity is minimal because the biopsy scar of the oral cavity is inconspicuous9. therefore, the autologous graft of cultured oral keratinocytes can avoid immunological problems and promote efficiency in the preparation and maintenance of cultured cells. recently, several trials for the three - dimensional reconstruction of the omks have been done21, revealing the following unsolved problems in clinical use : rolling of composites due to lack of physical property ; difficulty of manipulation, and ; graft contraction. the cell suspension technique is less time - consuming in cell culture ; carrying the cultured cells into the recipient site is also simpler than the preparation of sheet grafts or three - dimensional reconstruction. horch.22 developed a technique of single cell suspensions of cultured human keratinocytes in fibrin - glue reconstitute and reported a successful result of reproducible, standardized full - thickness wounds in an athymic nude mouse model compared to cultured epidermal sheet grafts. in this study, we examined the acceleration of re - epithelialization and inducement of skin epithelium by cultured and suspended omks in the skin wound regions of athymic nude mice. the histomorphometric evaluation revealed that the omk groups showed more rapid re - epithelialization than the ctl groups. such differences have statistically significant meaning, suggesting that primary cultured omks promote the re - epithelialization of skin wound defects. cultured and transplanted omks may provide factors that help the ingrowth of recipient skin keratinocytes from the wound edges unlike when they are implanted, since the differentiation of new forming epithelium shows mainly the orthokeratinization of cornified layer as the character of skin epithelium. therefore, the grafted omks labeled with brdu were present in the skin wound at immediate post - graft status but may undergo apoptosis later. inflammation remained longer in the ctl groups than the omk groups, and such may be related to the retardation of re - epithelialization.(figs. d) mackenzie and hill suggested the epitheliomesenchymal interaction (emi), i.e., the differentiation and maintenance of epithelial specificity are directly dependent on the influence of the underlying mesenchymal tissue23,24. based on emi, the specificity of recipient epithelial cells is reproducible by grafting only epithelial cells without underlying connective tissue9. in this study, the maintenance of skin epithelial specificity of wound beds was enabled by grafting only omks without underlying connective tissue to the skin wound defect area. therefore, the cell suspension technique has an additional advantage in functional reconstruction. in normal mucosa and skin, there is balance between the number of keratinocytes being produced in the basal cell layer and the number of cells shed at the surface. gottlieb.15 and kupper16 demonstrated that keratinocyte, when injured or cultured, releases a number of cytokines not found in stable epidermis and described it as " activated keratinocyte ". we hypothesized that grafted omks may induce cytokine production for accelerated wound healing. to prove this hypothesis, we analyzed the expression of kgf, il-6, and il-1 at different moments, such as 3rd day, 1st week, and 2nd week after the grafting of cultured omks. the omk groups showed higher expression levels of kgf, il-6, and il-1 in comparison with the ctl groups. the expression patterns of kgf, il-6, and il-1 were quite different, suggesting that kgf, il-6, and il-1 appear at different times through the process of wound healing. note, however, that cytokines can also be influenced by the extent of inflammation. the gradual increase of il-1 level in the ctl groups may have been caused by continual inflammation due to retarded wound healing. to verify the role of the cytokine for re - epithelialization, rhil-1 was added externally to the skin wound of the nude mouse instead of the omk graft. the marginal portion of the rhil-1 treatment group showed faster re - epithelialization than the ctl group. this result suggests that the released cytokines produced by the graft of the omks play a main role in the acceleration of re - epithelialization of the wound. in conclusion, grafted omks accelerate the epithelial regeneration of skin wounds through the stimulation of cytokine expression. maas - szabowski.25,26 demonstrated that keratinocyte growth regulation in organotypic co - cultures is mainly regulated by keratinocyte - derived il-1 signaling, which induces kgf expression in co - cultured fibroblasts. the increased cytokine expression of the omk groups in our experiment may depend on the reciprocal activation between recipient cells and grafted omks. the cell suspension has a limitation in terms of the wound site for application because it is affected by gravity. the glue has high concentration of fibrinogen and growth factors, inducing rapid wound healing. to eliminate the effect of fibrin glue on wound healing, horch.22 suspended the cultured cell pellet in fibrin glue with heterogenous human plasma protein fractions. recently, good clinical records for cell graft with autologous fibrin glue were reported27. autologous fibrin glue contains large numbers of platelets releasing significant quantities of growth factors that are known to promote wound healing28. note, however, that this technique has a drawback, i.e., requires an additional process in making autologous fibrin glue from the patient 's own blood. the use of an appropriate carrier could be helpful in overcoming the limitation of cell suspension technique, inducing more rapid wound healing not only by the retention of more grafted cells in the wounds but also through the release of more cytokines and growth factors. we successfully confirmed that the graft of primary cultured omks promoted regeneration of skin defects. the mechanism of accelerated wound healing by primary cultured omks was attributed to inducement of cytokine expression as required for re - epithelialization. | objectivesthe purpose of this study was to investigate the wound healing effect of primary cultured oral mucosal keratinocytes (omks) and to assess their roles in skin wounds.materials and methodsomk labeled with bromodeoxyuridine were scattered onto 1.51.5 cm skin defects of adult female nude mice (omk group, n=15). for the control, culture media were placed on the wound (control group, n=15). mice in both groups were sacrificed at three days (n=5), one week (n=5), and two weeks (n=5), and histomorphometric and immunoblot analyses with keratinocyte growth factor (kgf), interleukin (il)-6, and il-1 antibody were performed for the biopsied wound specimen. to verify the effect of the cytokine, rhil-1 was applied instead of omk transplantation, and the omk and control groups were compared with regard to re-epithelialization.resultshistomorphometric analyses demonstrated faster re - epithelialization in the graft group than in the control group at the third day, first week, and second week. newly forming epithelium showed maintenance of the histological character of the skin epithelium. the graft group showed superior expression of kgf, il-6, and il-1 protein, compared with the control group. similar faster re - epithelialization was observed after treatment with rhil-1 instead of omk transplantation.conclusionwe successfully confirmed that the graft of primary cultured omks promoted regeneration of skin defects. the mechanism of accelerated wound healing by primary cultured omks was attributed to inducement of cytokine expression as required for re - epithelialization. |
glaucoma is a progressive optic neuropathy characterized by death of retinal ganglion cells (rgc) and degeneration of the retinal nerve fiber layer (rnfl), resulting in a distinct appearance of the optic nerve head (onh) and concomitant visual field (vf) loss. however, in the early stages of the disease, structural changes may precede vf defects. some studies have shown that as many as half of rgc can be lost before a vf defect is detected by standard automated perimetry (sap) [2, 3 ]. therefore, detecting structural changes in the neuroretinal rim and rnfl is important for early glaucoma detection. several techniques have been introduced over the past years aiming at detecting morphologic glaucomatous abnormalities earlier than functional tests. 1991, optical coherence tomography (oct) has been widely accepted in glaucoma management. dublin, ca) spectral domain oct (sd - oct) offers greater image axial resolution (5 m) and faster acquisition speeds (27,000 a - scans per second) when compared to previous generations time domain octs (td - octs). however, even with a faster image acquisition (approximately 2 seconds), rnfl thickness (rnflt) measurements may be affected by artifacts and misalignments (mas), which may occur as a result of eye or head movements. in a recent study, moreno - montas. evaluated the frequency of mas in normal individuals and individuals with glaucoma imaged with the cirrus sd - oct and observed whether these misalignments resulted in changes in rnflt measurements. the authors found a high frequency of mas and no difference in rnflt measurements when they compared scans showing complete mas (cmas) and scans without cmas. however, the effects of the cma were analyzed in all patients regardless of the site of ma and exams were obtained in different days. we hypothesize that mas detected in a given sector will be more prone to affect measurements obtained in that same sector. hence, the purpose of the present study was to investigate the effects of mas on rnflt measurements according to the site of the ma. the study was approved by the university of campinas medical institutional review board and followed the tenets of the declaration of helsinki. one randomly selected eye from 34 patients with glaucoma and 32 healthy individuals was included in that study. all subjects were recruited from the glaucoma service, university of campinas (unicamp), brazil. subjects underwent a complete ophthalmic evaluation that included medical history, best corrected visual acuity (bcva), slit lamp biomicroscopy, measurement of intraocular pressure (iop) with goldmann tonometry, gonioscopy, slit lamp fundus examination with a 78-diopter lens, sap using the standard 30 - 2 or 24 - 2 swedish interactive threshold algorithm (sita) or standard full threshold mode (humphrey field analyzer ii, carl zeiss meditec inc., dublin, ca), and imaging with cirrus sd - oct (carl zeiss meditec inc., dublin, ca). in the present study, we included all patients who had at least 1 oct exam showing only one ma and another exam without ma acquired in the same session. only one eye per patient was included in the study. in patients with more than one exam with ma, the first image obtained was selected for comparison. among the 66 patients examined in our previous study, we found 56 patients (29 patients with glaucoma and 27 healthy individuals) with 1 aligned and 1 misaligned exam. the inclusion criteria for healthy eyes (selected among the staff or patients ' spouses) were intraocular pressure (iop) 21 mmhg with no history of elevated iop or glaucoma cases in the family, reliable normal visual field, open angle at gonioscopy and normal optic disc appearance based on clinical stereoscopic examination, no history of ocular or systemic disease or surgery that might interfere with rnflt measurements, ability to perform the tests, and willingness to participate as a subject in the study. subjects in the glaucoma group included those with any form of chronic glaucoma, defined as the presence of optic disc abnormalities consistent with glaucomatous optic neuropathy with or without visual field loss. two of the following optic disc abnormalities had to be present for the disc to be characterized as glaucomatous : cup / disc ratio > 0.6, localized rim loss, optic disc hemorrhage, or cup / disc asymmetry > 0.3. exclusion criteria for both groups included age 33%, false - negative errors > 33%, and fixation losses > 20%, significant cataract according to the criteria of lens opacification classification system iii (locsiii), defined as nuclear opacity equal to or greater than nc3 or no3, cortical equal to or greater than c3, and/or subcapsular equal to or greater than p3, corneal diseases, contact lens use, history of posterior segment intraocular surgery, and systemic or ocular diseases that can cause visual field loss. rnflt measurements were obtained with the cirrus sd - oct (software version 3.0.0.64) (carl zeiss meditec inc., dublin, ca). the onh mode, which consists of a 3-dimensional dataset of 200 a - scans that are derived from 200 b - scans and analyze a 6 mm area centered on the optic disc, was utilized. the software generates a rnfl thickness map from the 3-dimensional cube data set centered on the disc. subsequently, it also extracts rnflt measurements from a circumpapillary circle of 1.73 mm of radius. rnflt measurements are generated : 4 quadrants (superior from 45 to 135, inferior from 225 to 315, nasal from 315 to 45, and temporal from 135 to 225), 12 clock - hours and average thickness (clock - hour 1 from 75 to 45, clock - hour 2 from 45 to 15, clock - hour 3 from 15 to 345, clock - hour 4 from 345 to 315, clock - hour 5 from 315 to 285, clock - hour 6 from 285 to 255, clock - hour 7 from 255 to 225, clock - hour 8 from 225 to 195, clock - hour 9 from 195 to 165, clock - hour 10 from 165 to 135, clock - hour 11 from 135 to 105, and clock - hour 12 from 105 to 75) and average thickness. hence, clock - hour 3 of the circumpapillary scan represented the nasal side of the optic disc for both eyes. we excluded all poor - quality scans analyzed at printouts with incorrect identification of the vitreoretinal surface. only well - centered scans, with signal strength greater than 6, all images were acquired with undilated pupils by a single, well - trained ophthalmologist (fernanda cremasco), who was masked to the patient 's diagnosis. mas were defined as improper alignment of the vertical vessels in the pdf printout file. to better detect the presence of mas, the sd - oct scan (en face image) mas were classified by two examiners (kleyton a. barella and fernanda cremasco) as partial (pma), when they affected only part of the scanning line, and complete (cma), when the entire line was affected. we also classified the site of the ma in 4 sectors, as illustrated in figure 1(c). we compared 12 clock - hour, global, and quadrant rnflt between scans with mas (cmas + pmas) and scans without mas. we then investigated eyes with mas situated in the superior area (sectors 1 and 2) and compared their rnflt measurements with the scans without mas. the same analysis was undertaken for eyes with mas situated in the inferior area (sectors 3 and 4) and scans of eyes with mas within the measurement ring (sectors 2 and 3). the direction of the mas was classified in nasal, temporal, superior, and inferior according to the direction of the shift of the vertical vessels on the sd - oct scan (en face image). a post hoc power analysis was performed to determine the power of the study to detect differences in rnflt between aligned and misaligned tests, given our sample size and effect size. the sample size of 56 allowed us to detect a 2.6 m difference in rnflt between the exams, with a standard deviation (sd) of 4 m, at a power of 97% for misalignment in any of the 4 sectors. a sample size of 29 with misalignments in sectors 1 and 2 allowed us to detect a 4.3 m difference (sd = 5.7 m) at a power of 0.79. a sample size of 25, with misalignments in sectors 3 and 4, gave us a power of 0.79 to detect an 8.7 m difference (sd = 10.8 m) and a sample size of 37 with misalignments in sectors 2 and 3 gave us a power of 0.97 to detect 1.5 m difference (sd = 2.3 m). normal distribution of data was evaluated by the kolmogorov - smirnov test (with 5% significance level). rnflt parameters and signal strength were analyzed using the paired student 's t - test. wald - wolfowitz runs test was applied to evaluate whether there was a tendency for misalignment in a particular direction. the f - test was employed to compare the percentage of change in rnflt between normal and glaucomatous eyes. fifty - six eyes of 56 patients were analyzed in this study, 27 of them were healthy eyes and 29 were glaucomatous. of these, 40 eyes had a cma and 16 eyes had a pma. overall, the mean age was 49.1 13.5, 20 (36%) subjects were male, mean bcva logmar equivalent was 0.04 0.096, mean spherical equivalent was 0.25 1.18 d, mean iop was 14 2.6 mmhg, and mean vf mean defect (md) was 3.2 5.4 db. table 2 displays rnflt measurements of sd - oct exams with and without mas (cma or pma) in any of the 4 sectors. there were no statistically significant differences in rnflt measurements between aligned and misaligned exams. table 3 displays the mean sd - oct parameters of exams with and without mas (cma or pma) in the superior area (sectors 1 + 2). mean sd - oct measurements were not significantly different between aligned and misaligned exams, except for the 12 clock - hour, which showed lower mean rnflt in misaligned exams compared to aligned exams (101.7 39.7 m versus 111.4 34.5 m, p = 0.043). table 4 displays mean sd - oct parameters of exams with and without mas (cma or pma) in the inferior area (sectors 3 + 4) and table 5 displays the mean of sd - oct parameters of exams with and without mas (cma or pma) within the measurement ring (sectors 2 + 3). none of the sd - oct parameters showed statistically significant differences between aligned and misaligned exams (tables 4 and 5). in all previous comparisons, there were no statistically significant differences between signal strengths of eyes with and without mas (p > 0.05). a subgroup analysis was also performed in order to investigate whether exams with cma would present more pronounced rnflt measurements changes. only exams with cma (n = 40) were compared to aligned exams and the same results were observed : cma exams in the superior area (sectors 1 + 2) showed lower mean rnfl thickness in the 12 clock - hour compared to aligned exams (p = 0.026). it is important to consider that it is likely that eyes with thinner rnflt may be more prone to the effect of mas. when we applied the f - test, we found that the percentage change was similar in glaucomatous (n = 29) and normal eyes (n = 27) at the 12 clock - hour position (p = 0.094). regarding the direction of the 56 misalignments, 1 ma was inferior, 5 mas were superior, 14 mas were temporal, and the majority of mas (n = 36) were nasal (p = 0.020) (figure 1(a)). exams with mas in the rnflt deviation map have been excluded from octs studies, even without scientific evidence for this. in this study we found that the presence of cmas or pmas on superior sectors (sectors 1 + 2) of the cirrus sd - oct scan (en face image) was associated with lower rnflt in the 12 clock - hour (p = 0.043). this finding has clinical importance, since glaucomatous damage is frequently detected at the inferior and/or superior locations [12, 13 ]. hence, the presence of misalignments on sd - oct scans may affect the detection of rnfl damage in glaucoma patients. it is important to emphasize that the differences we observed can not be explained by differences in mean signal strength between eyes with and without mas (tables 25), which could potentially interfere with rnflt measurements. to our knowledge, this is the first study to assess the effects of single misalignments on rnflt measurements, acquired on the same day from normal and glaucomatous patients. examined 26 patients with one aligned exam and one cma exam obtained up to one month apart. however, they found no statistical difference in global and quadrant rnflt between aligned and misaligned exams. in fact, in accordance with their findings, when we compared rnflt measurements between aligned and misaligned exams regardless of the location of the ma, we were not able to detect significant differences (table 2). however, only when we analyzed the effects of superior mas on superior rnflt measurements differences were noticed. this confirms our hypothesis and is probably explained by the fact that the capture of the cube of 6 6 2 mm (200 lines) happens from the top to the bottom. hence, it is possible that superior mas do not affect rnflt measurements obtained below that sector. vizzeri. performed a similar study with the stratus td - oct (carl zeiss meditec inc., dublin, ca) and suggested that peripapillary scan circle misplacement secondary to motion artifacts produced significant changes in rnfl assessment : measurements for a given sector of the scan circle tended to increase for displacement toward the optic disc margin and to decrease when the displacement occurred in the opposite direction. different from stratus, the cirrus oct, used in our study, automatically places the measurement ring around the optic disc, reducing the errors of rnfl measurements caused by decentralization. recently, taibbi. evaluated the effect of scan circle displacements on rnflt measurements generated from cirrus sd - oct scans with mas passing through the optic disc. in this cross - sectional study, 70 scans from 18 healthy eyes and 100 scans from 26 glaucomatous eyes were divided into 85 pairs, each composed of a scan with one ma affecting the optic disc and a scan from the same eye without ma. interestingly, they found no difference in average and quadrant rnflt between scans with and without motion artifacts. however, in healthy eyes, scans with motion artifacts had greater rnflt in clock - hours 4 and 5 (p = 0.03), while in glaucomatous eyes scans with motion artifacts had greater rnflt in clock - hours 4, 5, 10, and 11 (p < 0.05) and lower rnflt in clock - hour 7 (p = 0.05), compared to scans without motion artifacts. this is not in accordance with our findings, since we found no statistical difference in scans with mas passing through the measurement ring (table 5). this difference may be due to the fact that taibbi. included patients with mas passing through the optic disc, whereas we evaluated patients with mas passing through the 3.46 mm diameter rnfl thickness measurement ring (sectors 2 + 3), an area greater than the optic disc (figure 1(c)). we also demonstrated that the direction of the misalignments occurs significantly more often to the nasal region (p = 0.020) (figure 1(a)). although eye movements may not be detectable during steady fixation, small movements, which are undetected visually but are commonly found in healthy persons, may explain mas. one hypothesis is that the capitation lines, which are always located temporally to the fixation target (large green fixation asterisk), may cause these small eye movements in the nasal direction. in 2006, ishikawa. proposed two solutions to improve image acquisition with octs : that the examination be carried out quickly and that an eye - motion tracking system be used to correct for eye movements. eye - tracking is used in some octs such as the spectralis oct (heidelberg engineering, heidelberg, germany) and the new cirrus hd - oct 5000 (carl zeiss meditec inc., dublin, ca), which features the fasttrac, that allows users to capture high - resolution b - scans in identical locations from visit to visit, and possibly eliminates motion artifacts during scan acquisition. our study has some limitations, such as being retrospective in nature, with a relatively small sample size (without statistical power to analyze healthy and glaucoma subgroups separately). furthermore, we have not quantified the mas, as performed by taibbi. this may explain why mas in the inferior sector or within the measurement ring were not found to cause significant changes in rnflt measurements in the correspondent sectors. our study showed that cmas or pmas in the superior area (sectors 1 + 2) of the rnfl deviation thickness map of the sd - oct lead to lower rnflt measurements at the 12 clock - hour and that the direction of misalignment is usually to the nasal region. further studies are needed using instruments equipped with eye - tracking to evaluate the occurrence of mas during image acquisition. | purpose. to investigate misalignments (mas) on retinal nerve fiber layer thickness (rnflt) measurements obtained with cirrus sd - oct. methods. this was a retrospective, observational, cross - sectional study. twenty - seven healthy and 29 glaucomatous eyes of 56 individuals with one normal exam and another showing ma were included. mas were defined as an improper alignment of vertical vessels in the en face image. mas were classified in complete ma (cma) and partial ma (pma), according to their site : 1 (superior, outside the measurement ring (mr)), 2 (superior, within mr), 3 (inferior, within mr), and 4 (inferior, outside mr). we compared rnflt measurements of aligned versus misaligned exams in all 4 sectors, in the superior area (sectors 1 + 2), inferior area (sectors 3 + 4), and within the measurement ring (sectors 2 + 3). results. rnflt measurements at 12 clock - hour of eyes with mas in the superior area (sectors 1 + 2) were significantly lower than those obtained in the same eyes without mas (p = 0.043). no significant difference was found in other areas (sectors 1 + 2 + 3 + 4, sectors 3 + 4, and sectors 2 + 3). conclusion. sd - oct scans with superior mas may present lower superior rnflt measurements compared to aligned exams. |
reports suggesting an increasing incidence of male genitourinary anomalies such as hypospadias, possibly related to environmental factors such as environmental estrogen - like compounds, have recently received considerable publicity. these reports are based on birth defects registry data, and there may be variation in the completeness of the registries used. we analyzed temporal trends in the prevalence of hypospadias in finland to assess the previously reported low overall prevalence and to detect any possible increasing tendencies during the past decade. we identified all patients who were surgically treated for hypospadias before the age of 9 years among boys born 1970 - 1986 in the national hospital discharge registry. we calculated the cumulative prevalence by dividing the number of patients by the number of male births, and we used poisson regression analysis. out of 549,176 boys born in finland in 1970 - 1986, 1,543 were treated for hypospadias by the age of 8 years (28.1 surgically treated patients per 10,000 male live births ; 95% confidence interval, 26.7 - 29.5). the prevalence of hypospadias in finland remained constant throughout the study period and appears to have been approximately three times higher than previously reported. changes in completeness of registration may account for a substantial proportion of the reported increases in the prevalence of hypospadias in finland and possibly also elsewhere.imagesfigure 1figure 2 |
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since becoming a nationally notifiable disease in the united states in 1995, chlamydia has experienced consistent annual increases (averaging 5.8%) to its 2009 prevalence rate of 409.2 per 100,000 inhabitants (figure 1(a)), making it the most common bacterial sexually transmitted infection (sti) in this country. data from the united states centers for disease control and prevention (cdc) have revealed that chlamydia rates are highest in late adolescents and young adults (, figure 1(b)). african americans and native americans demonstrate higher rates of chlamydia compared to other races or ethnicities (figure 1(c)). sexually active asymptomatic populations have been implicated in widespread transmission of the chlamydia trachomatis etiology. selective screening of sexually active women has yielded infection rates ranging from 8% to 40% (typical mean of 15%,), while approximately 10% of sexually active asymptomatic males are infected [3, 4 ]. in contrast to neisseria gonorrhoeae infection in which most patients develop symptoms and seek care promptly, hook. reported that most females and males with c. trachomatis infection were asymptomatic or mildly symptomatic upon clinical presentation. further evidence that chlamydia is a prevalent disease rather than an incident disease comes from extrapolations of sti agent acquisition rates.while past studies have suggested that gonorrhea sexual transmission can be more efficient than chlamydia transmission [6, 7 ], recent data utilizing c. trachomatis molecular diagnostics report less of a disparity between transmission rates. katz. estimated a 0.320.39 chlamydia transmission rate when using culture as a detection modality, while quinn. it is important to note that these extrapolations were performed on the basis of historical average frequency of intercourse between pairs, rather than single sexual encounter, which provided the basis for studies of gonorrhea transmission [9, 10 ]. ocular trachoma, lymphogranuloma venereum, perinatal infections, and adult oculogenital disease outline four clinical categories of c. trachomatis infections described by stamm.. we briefly summarize selected important clinical manifestations of urogenital disease as they pertain to subsequent laboratory diagnosis of the disease etiology. c. trachomatis is thought to be responsible for 3050% of cases of nongonococcal urethritis (ngu) in men. appropriate laboratory diagnostics in male urethritis are important for at least four reasons : (1) symptom overlap with clinical gonococcal urethritis and ngu may exist (table 1) ; (2) prevalence of ngu in the united states exceeds that of gonococcal urethritis ; (3) c. trachomatis may be detected from a substantial proportion of patients with gonococcal urethritis, and concomitantly, (4) dually infected males who are treated solely for gonococcal urethritis are likely to develop post gonococcal urethritis, manifested as persistence or recurrence. risk factors for chlamydial urethritis have included heterosexual orientation, african american race, and age younger than 20 years. mucopurulent cervicitis caused by c. trachomatis is said to be the female counterpart of male ngu, as approximately 70% of women are asymptomatic or experience only mild symptoms such as bleeding, discharge, mild abdominal pain, and dysuria. being a sex partner of a male with ngu or gonococcal urethritis has been reported to confer an infection risk of greater than 30% [2, 12 ]. additional factors such as younger age, african american race, unmarried status, new or multiple sex partners, oral contraceptive use, and residence in the southeast united states promote higher rates of chlamydia in sexually active females [2, 19 ]. symptoms relative to mucopurulent cervicitis can also characterize conditions such as cystitis and vaginitis. as such, diagnosis of chlamydia may be masked by diagnosis and treatment of concomitant n. gonorrhoeae and trichomonas vaginalis infection. a milwaukee, wisconsin laboratory used highly sensitive molecular methods for the detection of these agents [20, 21 ] to determine the sti profile of 272 female healthcare encounters that proved to be positive for at least one sti. in this populace that ranks second in the united states in both chlamydia and gonorrhea prevalence, 17% of patients with detectable c. trachomatis - specific nucleic acid had concomitant n. gonorrhoeae and/or t. vaginalis nucleic acid detection. the development of accurate laboratory diagnostics for c. trachomatis bears additional importance in light of data associating chlamydial cervicitis with acquisition of human immunodeficiency virus in women [23, 24 ]. for many years, the accepted gold standard for c. trachomatis detection was culture. culturing techniques in mccoy cell lines are rather complex and time consuming, with the necessity for experienced laboratory technologists for accurate follow - up staining and microscopy. sensitivity of culture methodology is much less compared to nucleic acid amplification testing (naat), allowing for false negative results to potentiate the spread of infection [2528 ]. in 2002, the cdc recommended routine laboratory screening for individuals at high risk of acquiring stis, particularly with effective treatment regimens for c. trachomatis and n. gonorrhoeae being both accessible and inexpensive. the detection of c. trachomatis by rapid screening or point - of - care methods includes nonamplification methods such as direct fluorescent antibody testing (dfa), optical immunoassay (oia), and rapid solid - phase enzyme immunoassay (eia). these methods were developed to provide a level of service to the community, enabling clinicians to begin treating patients on the day of the detection (a test and treat strategy) and consequently reducing the risk of inflammatory sequelae and the spread of infection. this represents a significant public health issue as 20% of patients that are diagnosed with c. trachomatis do not return to follow - up medical attention within a one - month interval, with 3% of these patients subsequently developing pid within this timeframe [30, 31 ]. the dfa procedure begins with fixing epithelial cells from the conjunctiva, urethra, or cervix to a microscope slide. monoclonal antibodies specific for c. trachomatis major outer membrane protein, conjugated with fluorescein isothiocyanate (fitc), bind to intracellular inclusions if the organism is present. the dfa procedure is considered rapid or point - of - care testing due to its capacity to be performed within 30 minutes, although expertise in reading fluorescence microscopy is required and the method exhibits low sensitivity when compared to naat. in one study, boyadzhyan. reported that c. trachomatis culture and dfa failed to detect 28% and 0%, respectively, of specimens determined to be positive by naat. the rapid oia consists of an optical reading, which has a very subjective color change as its basis. c. trachomatis antigens present within specimens will react with specific antibodies impregnated on a silicon wafer. the biostar oia has been evaluated in urogenital specimens from women at an sti clinic [25, 26 ]. evaluated 1,385 women for c. trachomatis infection using the dfa, oia, and culture methods. sensitivity and specificity for testing methods were 73.6% and 99.9%, respectively, for dfa, 64.2% and 99.1% for oia, 56.1% and 100% for culture, and 95.3% and 99.8% for pcr. during the study, these modalities were referenced against an expanded gold standard, which included naat testing if the culture was negative. the researchers concluded that a universal screening program utilizing rapid testing for laboratory diagnosis of c. trachomatis was not recommended. the decision analysis the investigators provided did show that the poorly sensitive rapid testing is potentially useful in clinics where patients do not comply to follow - up treatments. the true sensitivity of nonmolecular diagnostic testing for c. trachomatis is predicated on the quality of the reference standard. those assays that are compared to culture or analogous rapid or point - of - care testing in terms of sensitivity could demonstrate falsely elevated performance characteristics. bandea and colleagues showed the sensitivity of the biostar chlamydia oia as 78.6% and specificity as 97.2% with culture as the reference standard. this is in contrast to another study, utilizing a reference standard based on concordant results from two naat modalities that reported sensitivity and specificity for the biostar oia at 59.4% and 98.4%, respectively. moreover, c. trachomatis detection by point - of - care eia methodology has also shown to have decreased sensitivity compared to naat. van dommelen. demonstrated that sensitivity of three rapid eias was extremely low (17.1% to 25%) compared to naat. using a test with a low sensitivity may result in patients being falsely reassured by a negative test result, potentiating spread of infection and progression of disease to pid or other infertility sequelae. in assessing the proper rapid test to use for c. trachomatis diagnosis, the world health organization has recently released the assured criteria for rapid sti assays ; affordable, sensitive, specific, user - friendly, rapid and robust, equipment - free and deliverable. the optimal context for utilization of point - of - care testing is reflexive followup with naat. until sensitivity of rapid point - of - care testing improves, one has to be very cautious in using nonamplification methods alone, especially in low - prevalence populations in which assays yield positive predictive value of < 90%. nucleic acid hybridization technologies employ oligonucleotide sequences that are designed to anneal to complementary sequences within target nucleic acid. because of analytical sensitivity issues inherent to nucleic acid hybridization, this paradigm is generally reserved for clinical conditions with a high organism burden. in a study set of 201 cervical specimens, lebar. determined the sensitivity of pace 2 (gen - probe, incorporated, san diego, calif, usa) for the detection of c. trachomatis - specific 16s ribosomal rna to be 82.8% compared to a c. trachomatis cell culture reference. specificity of c. trachomatis - specific pace 2 was documented at 98.899.4% [37, 38 ]. in a study of male urethral specimens, kluytmans. developed two off - label modifications to the c. trachomatis pace 2 assay that yielded assay sensitivity of 89.5%. within 398 endocervical specimens, limberger. reported that 19 of 20 c. trachomatis cell culture - positive specimens were also pace 2-positive. this high frequency of concordance was not noted with specimens that were mailed to the laboratory for analogous nucleic acid hybridization for n. gonorrhoeae and may therefore reflect the susceptibility of the latter sti agent to conditions of specimen transport. this may be particularly true in light of past data reporting a 99.4% n. gonorrhoeae - specific pace 2 sensitivity in a sampling of 436 cervical or urethral swabs. taken together, these data begin to portend that decreased sensitivity of nucleic acid hybridization testing for c. trachomatis may be linked with a suboptimal cell culture reference method, rather being limited by mitigating factors such as specimen transport. neither target nor oligonucleotide probe nucleic acid concentrations change in the signal amplification paradigm. instead, the concentration of reporter molecules is increased at the site of target / probe hybridization. commercially available signal amplification methods detecting c. trachomatis alone, or in concert with n. gonorrhoeae, utilize hybrid capture technology (hybrid capture ii (hc2) product line ; digene corporation (qiagen), gaithersburg, md, usa). schachter. evaluated the hc2 ct - id test using a combined c. trachomatis cell culture and direct fluorescent antibody reference method and demonstrated 97.7% sensitivity for detection of c. trachomatis from endocervical specimens. within the 129 true - positive specimens, the utilization of the antecedent hc2 ct / gc test to screen for the presence of either n. gonorrhoeae or c. trachomatis exhibited 95% sensitivity. a two - center study reported 96.6% sensitivity of the hc2 ct - id test on endocervical specimens from high - risk female populations in relation to a culture reference. however, upon the adjudication of discrepancies with pcr, sensitivity of the hc2 ct - id test was 97.2% compared to a culture sensitivity of 80.6%. greater than 98% specificity was noted in both studies [42, 43 ]. modarress. evaluated the hc2 ct / gc test using genital swab specimens collected for pace 2 testing and demonstrated approximately 87% and 100% sensitivity of pace 2 and hc2 ct / gc, respectively, for the detection of either c. trachomatis or n. gonorrhoeae, although only the difference in c. trachomatis detection rate was significant between the two modalities (p < 0.016). the long - standing pcr [45, 46 ] dna target amplification method is the basis of the multiplex amplicor ct / ng product line (including the cobas fully automated platform), heretofore, referred to as amp (roche molecular systems, incorporated, branchburg, nj). c. trachomatis target for this assay is a 207-nucleotide sequence within a cryptic plasmid that is highly conserved within all serotypes of the organism. livengood iii and wrenn demonstrated a disparity in the rate of c. trachomatis detection from endocervical specimens by amp (93.3%) versus c. trachomatis culture (65.0%)far greater than that disparity (3.7%) observed for n. gonorrhoeae. a multicenter evaluation of amp yielded 89.289.7% sensitivity in the detection of c. trachomatis target from female urine and endocervical specimens, respectively, with 88.690.3% sensitivity derived from male urethral and urine specimens when using an infected patient standard. data from a european study demonstrated 92.098.0% amp sensitivity from male specimen sources and female endocervical specimens, yet reported 82.5% sensitivity of c. trachomatis detection from female urine specimens. the aforementioned studies reported 98.4% specificity from all specimen sources [47, 48 ]. from a peripheral and foreshadowing sense, noteworthy from the van doornum. data and an additional study was the apparent deficit of amp to accurately detect n. gonorrhoeae dna from female urine specimens (sensitivity values ranged from 64.866.7%). a summary of manufacturer - published performance characteristics of commercial naat on urine and endocervical / urethral specimens described in this review is presented in table 2 [5052 ]. a multiplex isothermal dna target amplification method constitutes a leading diagnostic assay for the detection of c. trachomatis in the united states (bd probetec et c. trachomatis and n. gonorrhoeae amplified dna assay (heretofore referred to as probetec) ; becton, dickinson and company, sparks, md, usa). in the context of c. trachomatis up to ten copies of which can be found in each cell. a seven - center evaluation utilized c. trachomatis - specific cell culture, dfa, and a since - defunct commercial ligase chain reaction to determine infected patient status and related 92.5% probetec sensitivity in the detection of c. trachomatis from male urethral swabs. sensitivity of the assay on male urine (93.1%) exceeded that of the commercial pcr assay described previously. in spite of reasonable sensitivity for the detection of c. trachomatis derived from endocervical swabs (92.8%), assay of female urine yielded only 80.5% sensitivity. however, combined percentage specificity of the assay was high among both genders (97.3%,) yet it is noteworthy that the specificity of urine specimens among 124 symptomatic males with positive infection status was 92.6%. a third commonly utilized nucleic acid target amplification method in the united states for the detection of c. trachomatis has its basis in isothermal tma. a 10-fold rate of rna amplification is reported to occur in two hours via tma in contrast to a 10-fold dna amplification rate in three to four hours. the multiplex gen - probe aptima combo 2 assay (heretofore referred to as ac2) targets c. trachomatis - specific 23s ribosomal (r)rna which is present in high copy number. a seven - site evaluation of 1391 females demonstrated 94.2% c. trachomatis assay sensitivity from endocervical specimens. sensitivity of c. trachomatis detection from female urine (94.7%) was markedly higher than those values derived from amp or probetec. in this study, specificity of ac2 ranged from 97.6% for endocervical specimens to 98.9% for urine specimens. in response to commercial systems, especially amp, demonstrating nonspecific amplification in the context of n. gonorrhoeae naat [5759 ], direct challenges of ac2 with nonpathogenic neisseria spp. and furthermore, lowe. noted a 10% greater sensitivity of ac2 than amp for detecting c. trachomatis in urine specimens. the increased clinical sensitivity exhibited by ac2 may reflect a phenomenon specific to tma. a 34.6% serum detection rate of hepatitis c virus (hcv)-specific nucleic acid via tma was demonstrated in disease relapse patients who had apparent virus clearance according to conventional qualitative and quantitative pcr assays. sarrazin. reported a 51.1% residual serum hcv detection rate by tma versus conventional qualitative pcr assays, including a 36.4% rate compared to an assay with a lower detection limit of 100 nucleic acid copies / ml.. prepared mock swab specimens containing propagated c. trachomatis elementary bodies and showed that the analytical sensitivity of ac2 was 1000-fold greater than that of probetec and 10-fold greater than that of amp. ac2 exhibited 100-fold greater sensitivity than the two comparators with analogous mock urine specimens. dispensed standardized amounts of c. trachomatis elementary bodies into mock specimens and showed within a subsequent dilution series that ac2 analytical sensitivity was 1000-fold greater than that of amp. wood. demonstrated a lower limit of detection of n. gonorrhoeae via ac2 (10 colony forming units / ml) than that rendered by probetec or amp (10 colony forming units / ml). a second contributory factor to the purported increased analytical sensitivity of ac2 is decreased susceptibility of the assay to endogenous inhibitors of nucleic acid amplification. substances suggested to inhibit c. trachomatis naat have included hemoglobin, low - ph cervical mucosa, -chorionic gonadotropin, urine crystals, and urine nitrites [6769 ]. analysis of the first - generation gen - probe chlamydia tma assay using 388 urine specimens revealed an 11.9% rate of amplification inhibition. introduction of the organism - specific nucleic acid target capture protocol with concomitant washing and aspiration (under the auspices of second - generation ac2) has negated this inhibitory effect. subjected mock ac2 and amp specimens containing c. trachomatis near the amp lower limit of detection to increasing concentrations of phosphate and iron and demonstrated that both chemicals only promoted an inhibitory effect on amp performance. reported 75 true - positive c. trachomatis screens from 506 total female and male urine specimens via ac2 plus an additional four specimens that also tested positive by a tma - based assay targeting an alternative sequence. these data compared favorably to the 72 true - positive results from the same study set identified by probetec. in an ex vivo study, rates of c. trachomatis nucleic acid amplification inhibition for ac2 (1.31.7%) were fairly equivalent to that of probetec (2.0%) for female genital swabs but were far less than those derived from amp (10.412.8%). rates of amplification inhibition from urine specimens were exceedingly high for probetec and amp (27.2% and 12.1%, resp.), when compared to ac2 (0.3%). modifications to the probetec urine collection and transport system have addressed issues related to amplification inhibition. a recent canadian study reported 98.0% probetec sensitivity in the detection of c. trachomatis from 500 urine specimens compared to ac2 sensitivity of 99.0%. analogous sensitivity indices for the detection of n. gonorrhoeae were 95.8% for probetec and 100% for ac2. improved performance of naat on urine specimens, and subsequent overall clinical acceptance of this specimen source, has been cited by the cdc as a factor responsible for a larger increase in c. trachomatis detection in males in the united states from 20052009 (37.6%) than that increase observed in females (20.3% ; figure 2). this cell type constitutes the vagina of prepubescent girls and is replaced with stratified squamous epithelium upon increased concentrations of estrogen at puberty. in spite of this histological difference, c. trachomatis has efficiently been recovered from adult vaginal specimens, further promulgating the high analytical sensitivity of naat. schachter. utilized a variety of commercial naat modalities to demonstrate that vaginal swabs had nearly equivalent sensitivity to that of endocervical swabs for the detection of c. trachomatis, with approximately 12% more sensitivity than first - catch urine. moreover, patient - collected vaginal swabs had equal sensitivity as clinician - collected vaginal swabs. a multicenter investigation of ac2 performance for c. trachomatis on vaginal swabs revealed 96.6% and 96.7% sensitivity on patient- and clinician - collected specimens, respectively, extending previous findings. positive c. trachomatis vaginal screening results were in 91% and 95% concordance with those from endocervical and first - void urine collection, respectively. sensitivity of c. trachomatis amp from a vaginal swab was 1822% greater than that of a c. trachomatis eia. c. trachomatis detection via probetec and amp revealed equivalent sensitivity for both vaginal and endocervical specimens. in a study of c. trachomatis detection via ac2, 98.6% of infected women were detected via vaginal swab testing, compared to 89.9% and 81.2% from endocervical swabs and first - void urine, respectively. these ac2 specimen - specific percentages of detection were statistically higher than analogous percentages generated by probetec and amp (p = 0.001). in a limited data set (n = 25 determinations), our laboratory has demonstrated that the transfer of 200-l aliquots of vaginal saline suspensions (originally designated for microscopic examination of vulvovaginitis etiologies) into ac2 specimen transport tubes (lysis medium) results in the detection of c. trachomatis upon performance of ac2 (e. munson, unpublished observations). to date, ac2 is the only commercially available modality that has received an fda indication for vaginal swab collections. in a description of a potential role for naat in laboratory diagnosis of rectal chlamydia, schachter. reported an 36.7% increase of sensitivity between naat modalities and c. trachomatis culture (26.5% sensitivity). in the same males who have sex with males (msm) demographic, sensitivity of c. trachomatis culture from pharyngeal sites was 44.4%. ota. reported sensitivity of c. trachomatis culture and two naat modalities from rectal specimens as being 21.1% and 94.7%, respectively, from an msm demographic. the same group reported a significant proportion of pharyngeal detection of c. trachomatis via naat in the face of a 0% culture - positive rate. using a rotating infected patient status, bachmann. determined c. trachomatis culture sensitivity to be 36.145.7% from rectal swabs in a combined msm and at - risk female demographic. early data suggested pcr utility in the detection of c. trachomatis from ocular specimens [82, 83 ], with one report documenting a 26% increase in overall c. trachomatis detection over that derived from dfa. kowalski. reported 88.1% sensitivity and 100% specificity of amp on adult conjunctival specimens. studies have also spoken to the utility of nasopharyngeal and nasal discharge specimens in both diagnosis and predictive value of antimicrobial therapy in the context of chlamydial conjunctivitis [85, 86 ]. children with a positive c. trachomatis amp result on a nasal discharge at the commencement of macrolide therapy had an odds ratio of 5.15 to yield a positive amp result from an ocular specimen two months after therapy when compared to children with a negative amp result from nasal discharge at baseline. comparisons of commercial naat modalities for the detection of c. trachomatis from non - fda - indicated extragenital sources have ensued. schachter. reported that 32.9% of c. trachomatis - positive vaginal screening results obtained by ac2 could not be replicated via probetec analysis of a corresponding first - void urine specimen. 64.7% sensitivity of amp for the detection of c. trachomatis has been demonstrated from rectal specimens when compared to ac2. the same study reported 33.3% amp sensitivity for the detection of pharyngeal c. trachomatis using a reference infected patient status in what turned out to be a very low - incidence specimen source. furthermore, ac2 was 30% and 33% more sensitive than probetec in detection of c. trachomatis nucleic acid from rectal and pharyngeal sites, respectively. for diagnosis of rectal chlamydia using a rotating infected patient status, bachmann. calculated sensitivity ranges of amp, probetec, and ac2 at 80.795.5%, 92.2100%, and 100%. ota. reported that ac2 outperformed probetec by 1520% in an msm demographic in terms of sensitivity from rectal and pharyngeal specimens, respectively. in further support of this paradigm, in studies of n. gonorrhoeae detection from pharyngeal and rectal sites, bachmann and colleagues [59, 81 ] determined the performance of amp to be inferior to that of probetec or ac2. in a study of an msm demographic, of 86 pharyngeal and 99 rectal specimens that generated a positive ac2 result, only 32.6% and 34.3% were positive by n. gonorrhoeae culture, respectively. of the 102 glans specimens positive for n. gonorrhoeae by ac2, 96100% of these results were confirmed by secondary naat. in contrast, a higher percentage of n. gonorrhoeae amp - positive rectal swabs were positive by n. gonorrhoeae culture when compared to analogous ac2 data. collectively, these findings challenge the overall analytical sensitivity of amp for the detection of sti etiologies from pharyngeal and rectal sources. limited comparative data exist on molecular detection of c. trachomatis from ocular specimens. in an italian study reported by fontana., overall sensitivity of probetec for the detection of c. trachomatis was 76.5% when compared to a laboratory - developed pcr assay targeting 16s rdna which detected all 34 positive specimens. two of three c. trachomatis - positive ocular specimens were detected by probetec (all three were detected by the assay targeting 16s rdna). it was noted that a second laboratory - developed pcr assay targeting c. trachomatis plasmid dna detected 28 of 34 overall positive specimens (2 of 3 positive ocular specimens). a c. trachomatis plasmid dna deletion rate of 17.6% was noted in this study. in a study conducted in ethiopia, yang. demonstrated that a tma - based assay specific solely for c. trachomatis 16s rrna (act ; gen - probe) had a detection rate of 59% which was in contrast to an amp - derived 28% detection rate. increased detection of ocular infection by tma was independent of active clinical disease (p 0.004). seven of 15 tma - positive / amp - negative specimens had detectable rrna subsequent to a 1 : 10 dilution of the original ocular specimen. taken together, these data are relevant because rrna detection can mitigate the possibility of c. trachomatis plasmid deletion, low c. trachomatis burden has been demonstrated in the context of trachoma management [91, 92 ], and rrna concentration far exceeds that of genomic dna and plasmid dna in c. trachomatis. several of the aforementioned findings, plus considerations related to the detection of other sexually - transmitted agents [20, 94 ], may account for increased utilization of commercial tma for the detection of c. trachomatis. surveys of naat modalities employed by clinical laboratories in the united states, conducted by the college of american pathologists laboratory accreditation program, have demonstrated an approximate 30% increase in the utilization of ac2 for c. trachomatis screening from 20032010 (figure 3). overall participant enrollment in these surveys has ranged from 525 laboratories in early 2003 to an average of 925 in 2010. furthermore, a 2004 survey of united states public health laboratories reported that 87% of respondents performed naat for the detection of c. trachomatis, while less than 40% offered nucleic acid hybridization. of the laboratories that offered naat, 50% utilized probetec, while 48% performed ac2. abbott laboratories (des plaines, ill, usa) has recently introduced a testing platform (m2000) to accommodate both automated specimen processing and real - time multiplex pcr for the detection of regions of the c. trachomatis cryptic plasmid and n. gonorrhoeae opacity (opa) gene. limit of the detection was reported at 20 copies of dna for each analyte. when assessed against amp and probetec using residual genital swab material, the m2000 demonstrated 96.399.1% concordance of positive c. trachomatis result, with 98.2100% concordance of negative result. urine testing demonstrated high concordance with ac2 and probetec for c. trachomatis - negative results (98.9%), with 93.7% and 96.8% concordance rates, respectively, for c. trachomatis - positive results. levett. compared automated versions of probetec (viper system) and ac2 (tigris dts system) to the m2000 for c. trachomatis urine testing and reported that sensitivity ranged from 96.9% (m2000) to 99.0% (ac2). a strain of c. trachomatis with a 377-base pair cryptic plasmid deletion is implicated in the purported decreased rates of positive c. trachomatis naat results reported in clinical laboratories in sweden beginning around 2004. interestingly, herrmann. reported a proportional rate for this c. trachomatis variant ranging from 20% to 64% in regions that utilized either m2000 or a commercial real - time pcr system distributed by roche molecular systems. in contrast, in locales that utilized probetec, the proportional rate of the c. trachomatis variant ranged from 7% to 19%. despite these swedish prevalence data, the variant has been identified from clinical specimens in only two neighboring countries. amidst concern that the m2000 demonstrated poor utility in the detection of european plasmid mutant c. trachomatis strains [99, 101 ] this reformulated product (abbott realtime ct) was assessed, along with ac2 and version 2 of the cobas taqman ct test (roche molecular systems), against a panel of 148 c. trachomatis - positive urine specimens. sensitivity of the cobas taqman ct test (83.0%) was outpaced by analogous indices for the abbott laboratories reformulation (95.3%) and ac2 (99.3%). a separate study reported that the reformulated abbott laboratories assay yielded slightly higher sensitivity than probetec. efforts to enhance c. trachomatis naat sensitivity theoretically come at the expense of assay specificity. overall scenarios that could generate such false - positive results include the nucleic acid target of interest being present within other organisms endogenous to a specimen, the detection system generating signal in the absence of target ; iatrogenic contamination, and, clerical errors. in light of this, past literature from the cdc stated that naat assays for c. trachomatis are indeed screening assays and that an initial positive result should be considered strictly as presumptive evidence of infection. as such, the cdc deemed necessary the verification of a positive screen in cases that could have adverse medical or psychosocial impact. furthermore, consideration should be given for the verification of positive naat screens for analyses performed in low - prevalence sti populations that would render positive predictive values on the order of 90% or less. cdc - advocated approaches to additional molecular testing have been four - fold : (1) testing a second primary clinical specimen with an assay that utilizes a different target and a different format, (2) testing the original primary clinical specimen with an assay that utilizes a different target and a different format, (3) repeating the original test on the original primary clinical specimen with a competitive probe, and, (4) repeating the original test on the original specimen. laboratories that choose commercial nucleic acid hybridization as the method of choice for the detection of c. trachomatis can utilize a direct and competitive probe - based nucleic acid hybridization technology [39, 40 ]. however, it is not advisable to utilize less - sensitive signal amplification or nucleic acid hybridization technologies to confirm a positive screen derived by naat. the method advocated first and foremost may not be a reality in certain healthcare environments or in the public health sector due, in part, to the difficulty in successfully contacting a patient to return for specimen recollection. recent literature suggests that the paradigm of repeat testing may introduce difficulties to the final interpretation of naat results. culler. reported that 5.3% of initially positive c. trachomatis screens yielded by probetec failed to duplicate results upon repeat testing. castriciano. demonstrated that 93.1% of initially positive c. trachomatis screens derived by amp remained positive upon repeat testing of original lysates. schachter. reported that only 83.8% of positive c. trachomatis screens derived by probetec retained positive status upon repeat testing. in contrast, 96.7% and 97.7% of amp and ac2 screens, respectively, generated a positive result upon repeat testing. this phenomenon has also been noted in molecular detection of n. gonorrhoeae. in the study of culler., 10.7% of positive n. gonorrhoeae screens obtained via probetec failed to retain positive status upon repeat testing. using multiple specimen sources, moncada. demonstrated that 89.3% of positive n. gonorrhoeae screens derived by probetec retained positive status by repeat testing. this value increased to 92.4% and 93.1% upon a second and third repeat test, respectively. in contrast, repeat testing of positive screens initially derived by amp and ac2 retained positive status 95.7% and 96.4% of the time, respectively. a further delineation of positive naat screens reveals an additional conundrum in terms of a role for confirmatory testing in final result interpretation. 80.8% of low - positive c. trachomatis screens derived from probetec (defined as signal detection method other than acceleration (mota) scores from 2000 to 9999) remained positive upon repeat testing, while only 33.3% of n. gonorrhoeae screens retained the positive status. this paradigm may be of greater consideration when studying a highly sensitive assay such as ac2. two reports [107, 108 ] documented positive status retention rates of 4263% for low - positive c. trachomatis screens in contrast, dunham. determined that only 31.6% of low - positive n. gonorrhoeae screens retested positive. in a high - prevalence population for both c. trachomatis and n. gonorrhoeae, 71.3% and 58.5% of low - positive c. trachomatis and n. gonorrhoeae screens, respectively, yielded positive results upon repeat testing. no significant difference existed between the percentages of low - positive c. trachomatis and n. gonorrhoeae screens that remained positive by repeat testing (p = 0.10). despite the fact that repeat testing potentiates result interpretation challenges, this low - positive phenomenon, even under the auspices of ac2, presents itself in just 2% of c. trachomatis screens and less than 0.1% of n. gonorrhoeae screens performed [107, 109 ]. a fourth cdc - advocated practice, the utilization of an alternative naat system or platform, has met with variable success. schachter. reported that while ac2 confirmed 96.9% of positive probetec screens (95.4% in urine specimens, 98.6% in genital swab specimens), probetec was able to confirm only 82.0% of positive c. trachomatis screens derived from ac2 (85.3% in urine specimens, 78.9% in genital swab specimens). chernesky. reported 69.6% and 80.3% rates of confirmation of positive ac2 urine c. trachomatis screens by amp and probetec, respectively. analogous rates for confirmation of positive ac2 endocervical c. trachomatis screens were 62.9% and 70.9%. in contrast, 98100% of positive urine or genital swab c. trachomatis screens yielded by probetec or amp were confirmed by ac2. in a small subset of specimens that tested equivocal for c. trachomatis via amp or yielded a discrepant result in the context of a combined reference standard, peterson. reported that only 23.1% of specimens yielded a concordant result when subjected to separate pcr assays targeting different sequences. a c. trachomatis concordance rate of 82.1% was demonstrated from initial nucleic acid extracts when commercial ligase chain reaction - positive urine screens were tested by amp.. demonstrated that percentages of positive n. gonorrhoeae screens derived by probetec that were confirmed by ac2 and amp analysis were 85.0% and 78.4%, respectively, and that 84.6% of positive ac2 n. gonorrhoeae screens were confirmed by probetec. yet when similar analysis was restricted to male urine specimens, nearly all positive n. gonorrhoeae screens, independent of modality, were confirmed by secondary naat. aptima ct and gc assays have allowed for detailed analysis of the cdc - advocated practice of confirmatory testing using an alternative nucleic acid target. sensitivity and specificity values greater than 96% were demonstrated for these assays in a multicenter study of msm using both urethral swabs and urine specimens. boyadzhyan. reported complete concordance of ac2 c. trachomatis and aptima ct assay results on 253 urine specimens and 422 genital swab specimens collected from either gender. schachter and colleagues [106, 110 ] demonstrated that the aptima ct assay confirmed 98 - 99% of positive ac2 screens, with just slight differences noted between concordance values from urine and genital swab specimens.. golden. reported that 258 of 265 positive n. gonorrhoeae screens of female urine or endocervical specimens derived by ac2 also yielded a positive aptima gc assay result.. demonstrated that 95.7% of combined gender specimens initially screening positive by ac2 yielded a positive aptima gc result. data from a five - state united states moderate - prevalence chlamydia population (cumulative c. trachomatis infection rate of 312.7 per 100,000 population) revealed that repeat testing versus performance of the aptima ct assay on ac2-positive c. trachomatis screens demonstrated 95% concordance of the final result. schachter. utilized a moderate - prevalence california population (336.7 cases per 100,000 population) to demonstrate an 8498% rate of initial ac2 screen confirmation by repeat testing and a potentially elevated rate (8999%) of initially positive screens confirmed by secondary naat. in a high - prevalence population (684.0 cases per 100,000 population), significantly more low - positive c. trachomatis screens were confirmed by the aptima ct assay than by duplicate repeat testing. however, these authors noted that utilization of alternative target tma for confirmation raised overall ac2 c. trachomatis positive predictive value only 1.8% over that derived from repeat testing.. demonstrated 90% concordance in final n. gonorrhoeae ac2 result derived by repeat testing versus alternative target tma. summarized their side - by - side comparison of the two advocated methods by noting that 8996% of specimens positive by initial naat were confirmed by repeat testing and that 8598% of initial screening results were confirmed via secondary naat. in a high - prevalence gonorrhea population (265.9 cases per 100,000 population), confirmatory testing via the aptima gc assay demonstrated only a 5% increase in the rate of ac2 low - positive result retention. due to very high microbial cure rates exhibited by azithromycin and doxycycline in a recent meta - analysis, the cdc does not advocate c. trachomatis test - of - cure analysis in males or in nongravid females, unless therapeutic compliance is questioned, symptoms persist, or reinfection is suspected. workowski. utilized an in - house pcr to demonstrate a reduction in rate of endocervical detection of c. trachomatis from 50% immediately following completion of doxycycline therapy to 15% seven days later.. reported greater c. trachomatis - specific nucleic acid recovery rates for commercial ligase chain reaction (3773%, interval dependent) over those of amp (2140% for similar intervals) within the first six days following the completion of therapy. in contrast, an in - house pcr detected c. trachomatis nucleic acid from 25% of endocervical swabs collected three weeks after the completion of therapy. in the same study, nucleic acid sequence - based amplification, an rna amplification technology, yielded a c. trachomatis detection rate of only 6.7% and 8.0% from urine and endocervical specimens, respectively, one week after the completion of therapy. bianchi. subjected post treatment urine specimens to amp and the gen - probe first generation tma assay. kinetics of both systems was essentially equivalent in females, demonstrating full clearance within six days. similar results were generated in a smaller sampling of males. according to recently published cdc recommendations, test - of - cure protocols are unnecessary for patients who have completed antimicrobial therapy for n. gonorrhoeae infection because multiple lines of therapy have proven efficacious. exceptions to this paradigm are in the minority and are potentially linked to increasing resistance of n. gonorrhoeae to fluoroquinolone agents [122, 123 ]. however, high prevalence of n. gonorrhoeae infection exists in patients who have had gonorrhea in the preceding months. these data imply that the detection of n. gonorrhoeae post - treatment may actually be reflective of reinfection rather than treatment failure. if symptoms persist in patients following the completion of therapy, clinicians may consider re - testing patients, via culture modalities, for the ultimate purposes of antimicrobial susceptibility testing. this approach is hypothetically far more efficacious in the management of n. gonorrhoeae infection than in c. trachomatis infection because of stark differences in culture sensitivity. a paucity of studies has characterized an auxiliary role for naat in gonorrhea test - of - cure.. demonstrated that nucleic acid hybridization was unable to generate n. gonorrhoeae signal from genital and urine specimens of patients between six and eleven days post - completion of antimicrobial therapy for n. gonorrhoeae infection. bachmann., utilizing a commercial ligase chain reaction, reported the median time to a negative n. gonorrhoeae urine assay being one day for males and two days for females upon completion of therapy. among females, the mean clearance time proved greater for genital specimens (2.8 days) than for urine specimens (1.7 days ; p = 0.008). an intermittent shedding phenomenon was observed in 15% of males and 25% of females during the three - week follow - up period. women who shed n. gonorrhoeae nucleic acid intermittently were twice as likely to have a genital specimen yield detectable n. gonorrhoeae compared to a urine specimen. in one female patient, such detection occurred 19 days after the completion of therapy. the shorter length of the vagina in prepubescent girls, combined with its columnar epithelial cell lining and alkaline environment, can predispose this population to infection with sexually transmitted agents including c. trachomatis. the extrapolation of c. trachomatis detection in children beyond the neonatal period to sexual abuse has some limitations. it has been estimated that 20% of infants born to women with active c. trachomatis infection can acquire the infection in rectal and vaginal sites. persistence of the organism acquired in perinatal fashion may last 2 - 3 years. moreover, retrospective chart reviews [130133 ], subcomponents of which utilized c. trachomatis naat, have outlined very low incidence of c. trachomatis detection (0.53.1%) in the context of child sexual abuse. schachter., within a significant at - risk population for sti acquisition, reported lower c. trachomatis detection rates from oropharyngeal and rectal sites (0.8% and 6.1%, resp.) taken together, it must be noted that the positive predictive value of even very highly specific naat screens can be compromised by low disease prevalence in a given setting. largely as a result, it has been a long - standing axiom that c. trachomatis cultivation has more validity in medicolegal proceedings than results derived from naat. despite this, in a limited sexual abuse victim dataset, matthews - greer. described 11 instances of c. trachomatis culture data being corroborated by a positive pcr result. two rectal specimens were culture positive / pcr negative, while one rectal and three female genital swabs yielded culture - negative / pcr - positive results. girardet. reported that 18 of 215 (8.4%) possible pediatric sexual abuse victims generated a positive naat for c. trachomatis from a noninvasive urine specimen. kellogg. reported 1118% agreement between c. trachomatis culture results and those derived from ligase chain reaction or pcr performance on urine and vaginal specimens collected from girls who reported abusive sexual contact. among 485 girls being evaluated for sexual abuse, sensitivity of urine c. trachomatis naat was 100% when compared to vaginal culture. eight additional patients yielded positive urine c. trachomatis naat in the face of negative culture (p = 0.018). interestingly, in 2001 hammerschlag stated that the advancement of naat evidence in courts of law was hindered by the paucity of appropriate commercially available confirmatory assays. with the advent of the aptima ct assay and defined performance characteristics [32, 112 ], perhaps this scenario warrants additional consideration. 2006 recommendations for the management of sti stated that naat might be a viable alternative in the detection of c. trachomatis if culture systems for the organism are unavailable and if a method of confirmation is available. noted means of confirmation included secondary fda - cleared naat targeting a different sequence than the primary screening method. this nonculture option for the detection was not advocated for laboratory diagnosis of n. gonorrhoeae. remarked that urine naat methodologies (with subsequent confirmatory testing) are adequate as a new forensic standard in children suspected of being sexual abuse victims. the recently published guidelines place primary focus on standardized anal (both genders), urethral discharge (males), and vaginal culture techniques for both c. trachomatis and n. gonorrhoeae (table 3). the paucity of specimen source options available for c. trachomatis culture is related to low source - specific organism prevalence rates and the paradigm of chlamydial persistence following perinatal acquisition. naat for c. trachomatis and n. gonorrhoeae from vaginal and urine specimens is recommended in girls as an alternative to culture. furthermore, in the context of sexual assault in adults and adolescents, the latest recommendations call for the performance of fda - cleared naat for either c. trachomatis or n. gonorrhoeae upon initial examination. the topic of sti detection in the context of medicolegal testing has been reviewed extensively by hammerschlag and guilln. independent of specimen transport conditions, sensitivity of c. trachomatis culture is greatly inferior to those of amplified molecular methods that have since largely replaced signal amplification and nucleic acid hybridization assays. because molecular - based testing for n. gonorrhoeae is simultaneously provided within commercial molecular assays for c. trachomatis, many laboratories subsequently forego sole reliance upon culture methods for n. gonorrhoeae detection from urine and genital sources. sensitivity differences between commercial pcr, sda, and tma have been delineated in the literature, both in clinical and in vitro settings. the aforementioned specimen types are applicable to ac2, with the addition of vaginal swab and gynecological specimens (table 4). a limited role for c. trachomatis culture may be seen in medicolegal settings or for cultivation from specimen sources that are not indicated for fda - approved naat. however, studies have emerged advocating a highly sensitive naat modality, such as commercial tma, to augment culture methodology for accurate detection of c. trachomatis from extragenital sites. validity of results generated by highly sensitive modalities has been addressed with a variety of confirmatory testing algorithms. limitations to follow - up testing include clinicians not routinely providing two specimens for evaluation and the prohibitive expense for some laboratories to either modify an existing molecular assay to target a different nucleic acid sequence or validate secondary naat. even when naat is utilized as a means of confirmation, differences in performance characteristics of these assays, deficiencies in result reproducibility for a given specimen using the same testing modality, and potential differences in sensitivity related to heterologous specimen collection media / transport devices have been reported. even when repeat testing is factored into this paradigm, additional generated data may be difficult to interpret, especially when considering the extremely high rates of sensitivity and specificity already inherent to these screening assays. it must be kept in mind that cdc recommendations related to c. trachomatis screening diagnostics have not been updated since 2002. on the basis of the discussion provided in section 5 of this review while currently not widely accepted as medicolegal evidence due to concerns over specificity, admission of naat results may eventually become standard practice in courts of law. prominent acceptance of alternative target confirmatory testing may have to play a significant role for this to occur, particularly with a highly sensitive method such as commercial tma. viable test - of - cure options are not extensive in the setting of chlamydia due to meager sensitivity of c. trachomatis culture. at the same time, amplified molecular test - of - cure protocols are deemed unnecessary in a majority of settings due to efficacious therapeutic regimens. auxiliary studies utilizing naat demonstrate microbiological cure approximately 714 days after therapy, yet investigations in this vein using latest - generation commercial tma would be compelling. chlamydia prevalence has experienced a significant upswing in the united states over the past 15 years. clinical presentation of male urethritis exhibits overlap with that of nongonococcal urethritis. in females, symptoms of chlamydia with respect to the utilization of the rapid, nonamplification methods for c. trachomatis detection, one must be cautious to the actual point - of - care benefit therein, as many studies have proven these methods to have much lower analytical sensitivity than naat. consequently, as nucleic acid - based diagnostic assays continue to improve, a greater presence for such testing needs to be established in both small- and large - scale clinical laboratory settings. | chlamydia, with its chlamydia trachomatis etiology, is the most common bacterial sexually transmitted infection in the united states and is often transmitted via asymptomatic individuals. this review summarizes traditional and molecular - based diagnostic modalities specific to c. trachomatis. several commercially available, fda - approved molecular methods to diagnose urogenital c. trachomatis infection include nucleic acid hybridization, signal amplification, polymerase chain reaction, strand displacement amplification, and transcription - mediated amplification. molecular - based methods are rapid and reliable genital specimen screening measures, especially when applied to areas of high disease prevalence. however, clinical and analytical sensitivity for some commercial systems decreases dramatically when testing urine samples. in vitro experiments and clinical data suggest that transcription - mediated amplification has greater analytical sensitivity than the other molecular - based methods currently available. this difference may be further exhibited in testing of extragenital specimens from at - risk patient demographics. the development of future molecular testing could address conundrums associated with confirmatory testing, medicolegal testing, and test of cure. |
considered from the standpoint of an experimental system for secretion, one of the most appealing aspects of t. gondii is the polarized organization of its secretory organelles (hager., 1999)a consequence of the parasite 's mechanism of replication, in which two daughter cells are assembled within the mother (hu., 2001). the nucleus is centrally located, essentially bisecting the organism (figs. 1 and 2). the endoplasmic reticulum, although distributed throughout the cell, is concentrated posterior to the nucleus, and is so reduced that the nuclear envelope itself provides a substantial fraction of the er volume. thinly coated vesicles bud from the anterior end of the nucleus / er, destined for the closely juxtaposed golgi stack, which consists of a limited number of cisternae (typically three to five). reporters containing the cooh - terminal er retention signal of t. gondii bip (hdel) localize most prominently to a cup - like region anterior to the apical end of the nucleus, just below the golgi (figs. 1 and 2). the use of the nuclear envelope as an obligatory intermediate between the er and golgi is comparable to other small eukaryotic cells, such as pichia pastoris (rossanese., 1999) but contrasts with mammalian systems, where transitional er elements are dispersed throughout the cell. forward transport from the er to golgi takes advantage of acidic / hydrophobic / acidic motifs in the cytoplasmic tails of secretory proteins, along with upstream tyrosines, likely by recruiting copii coats as observed in mammalian cells and yeast (hoppe and joiner, 2000). both copii and copi coat components (including arf1 and sar1) are present in t. gondii genome and est databases (ajioka., 1998), and copi retrieval motifs have been shown to operate in the parasite (liendo., 2001). protein transport through the golgi is inhibited by low temperature treatment, brefeldin a, and microtubule inhibitors (stokkermans., 1996 ; soldati., 1998). clathrin - coated vesicles are observed at the lateral margins of the trans - most golgi stacks (liendo., 2001). the target for these vesicles is likely to be one or more of the multitude of unusual secretory organelles found in t. gondii and other apicomplexans, as discussed below. in contrast, proteins destined for the parasite surface are delivered via an alternative route (karsten., 1998). many t. gondii surface antigens are gpi anchored, and the parasite may use the gpi anchor itself as a targeting motif. the tachyzoite form of t. gondii contains at least three morphologically distinct secretory organelles : micronemes, rhoptries, and dense granules (figs. 1 and 2). the former two organelles are located in the anterior portion of the cell, whereas dense granules are more broadly distributed. morphologically, dense granules are essentially indistinguishable from the mature secretory granules found in endocrine, neuroendocrine, or exocrine cells (table i and fig. these three organelles discharge sequentially : microneme exocytosis occurs upon host cell binding, rhoptry secretion coincides with invasion, and dense granule secretion is most prominent after parasite entry into the host cell (carruthers and sibley, 1997). micronemes and rhoptries are thought to be critical for host cell invasion, a process that is completed within 1520 s. dense granule proteins are thought to be required for intracellular replication, including establishment of the parasitophorous vacuole within which parasites reside and divide until lysis of the host cell. the requirements for precise temporal regulation of differential organelle secretion are stringent, distinguishing t. gondii parasites from most secretory cells. soluble recombinant proteins (from various sources) are delivered to dense matrix granules by the bulk flow pathway. dense granules are quantitatively secreted in a constitutive, calcium - independent fashion (chaturvedi., 1998 ; karsten., 1998) ; although there is also likely to be a triggered component to the release process (dubremetz., 1993 ; carruthers and sibley, 1997 ; coppens., 1999). even t. gondii proteins from which specific signals for targeting to other organelles have been deleted are routed through the dense granules as soluble proteins (striepen., 1998, 2001 ; it is therefore difficult to invoke the notion that aggregation or retention in dense granules requires specific protein sequence motifs, a low ph / high ca environment, lipid rafts, or other distinguishing characteristics, in contrast to observations in mammalian secretory cells (arvan and castle, 1998 ; tooze., 2001) dense granules appear to be most similar to the post - golgi vesicles involved in constitutive secretion (table i). 1), thought to contain vesicular / membranous material that is secreted via the long, slender neck. labeling with damp (3-[2,4-dinitroanilin]-3amino - n - methyldipropylamine) suggests that rhoptries are the only acidified organelles in t. gondii ; the parasite contains no morphological equivalent of secondary lysosomes (shaw., 1998). unusual organelles designated acidocalciosomes have been reported (moreno and zhong, 1996), but these function in the storage of calcium and pyrophosphate (and possibly other materials as well), and do not appear to be directly related to either exocytic or endocytic trafficking. like multivesicular bodies and late endosomes (bishop and woodman, 2000), rhoptries are enriched in cholesterol, but their cholesterol / phospholipid ratio of 1.5:1 (foussard., 1991) is too high for lipid bilayer stability, suggesting that at least some of this cholesterol may be organized in a crystalline array. secretion from the rhoptries contributes to the formation of a distinctive parasitophorous vacuole, defining the compartment within the host cell where t. gondii parasites reside. the majority of lipids making up the parasitophorous vacuole at the time of invasion are of host cell (rather than rhoptry) origin (suss - toby., 1996), but rhoptry proteins are rapidly incorporated into the vacuolar membrane. the parasitophorous vacuole neither acidifies nor fuses with organelles of the host cell endomembrane system, highlighting the unusual nature of this structure (sibley., 1985 ; joiner., 1990 ; mordue., 1999). inhibitors of parasite actin polymerization (sibley and andrews, 2000) prevent host cell invasion but not rhoptry discharge, producing small vesicular structures in the host cell. these empty e - vacuoles (hkansson., 2001) contain rhoptry markers and associate with host cell mitochondria and er (without fusing), just as seen for parasite - containing vacuoles in the same cell (sinai. rhoptries of the mother cell disappear as morphologically distinct entities during the early phases of cell division (endodyogeny). following division of the golgi apparatus, two distinct rhoptry antigen - positive punctae appear immediately anterior to golgi ; these structures may be regenerated de novo, as precursors to the fully formed rhoptries that will ultimately develop in the two daughter cells. rhoptry protein processing is thought to occur in these immature rhoptries (soldati., 1998), which therefore exhibit some functional similarity to immature secretory granules (table i). protein targeting to the rhoptries has long been a matter of interest, since the evolutionary origin of these unique secretory structures is unknown. soluble rhoptry proteins can harbor multiple independent targeting signals (bradley and boothroyd, 2001 ; striepen., members of the rop2 family, which contain a putative transmembrane domain, display both yxx and ll motifs within the predicted cytoplasmic tail (hoppe., 2000). in higher eukaryotes, both of these motifs mediate binding to adaptor subunits and facilitate clathrin - coated vesicle formation from the trans - golgi (bonifacino and dell - angelica, 1999). deletion or alteration of the yxx motif (hoppe., 2000) or ll motif (unpublished data) abolishes rop2 delivery to mature t. gondii rhoptries, providing the first evidence for tyrosine - dependent sorting machinery in protozoan parasites. t. gondii 1 binds to the cytoplasmic tail of rop2 family members in a tyrosine - dependent fashion and expression of either dominant negative t. gondii 1 or antisense mrna ablation of t. gondii 1 expression impairs rhoptry targeting (unpublished data). alteration of rhoptry targeting motifs leads to protein accumulation in a compartment located just anterior to (but distinct from) the golgi (the precursor compartment noted in table i). combined with the observation that rhoptries are acidic (shaw., 1998), it is tempting to consider the rhoptry a lysosome - like organelle (dell'angelica., 2000) (see table i). a model for rhoptry biogenesis consistent with the existing data protein traffic through the er and golgi likely depends on both copi- and copii - coated vesicles, and is regulated by forward targeting signals, er retrieval and retention motifs, and rab proteins. targeting of soluble proteins from the trans - golgi network to dense granules is signal independent, whereas targeting of membrane proteins to these organelles depends on transmembrane domain length (unpublished data). t. gondii rab6 mediates retrograde transport from dense granules to the parasite golgi (unpublished data). rhoptry proteins appear likely to be transported from the golgi via a precursor compartment, possibly part of the endosomal pathway (robibaro., 2002). transmembrane rhoptry proteins are targeted in a tyrosine-, dileucine-, and adaptor - dependent fashion. targeting of soluble microneme proteins proceeds by association with transmembrane escorters ; transmembrane proteins are capable of using adaptor- and tyrosine - dependent signals, although typical endocytic motifs are not apparent in known microneme proteins. results using dominant negative adaptors suggest that microneme targeting may exploit the same precursor compartment involved in rhoptry targeting. nuclear - encoded proteins destined for the apicoplast exhibit a bipartite nh2-terminal domain (roos., 1999 ; derocher., 2000 ; waller., 2000 ; yung., 2001), mediating transport first into the secretory pathway using a classical secretory signal sequence, and subsequently into the apicoplast using a plastid transit peptide akin to that found in plants. whether all secreted proteins transit this organelle after exit from the golgi remains to be determined, as does the ultimate destination of products produced in the apicoplast (dashed black arrows). a, apicoplast ; dg, dense granule ; e, endosome ; mn, micronemes ; pc, precursor compartment ; rh, rhoptries. microneme proteins typically exhibit one or more of a variety of adhesive domains, and are thought to be involved in host cell adhesion (carruthers., 2000 ; chimeras containing the cytoplasmic tail of a mammalian lysosomal membrane protein are targeted to micronemes in a tyrosine - dependent fashion (hoppe., 2000). no endogenous t. gondii microneme proteins bearing a transmembrane domain and yxx or ll motifs in the cytoplasmic tail have yet been identified, however, and microneme proteins can possess multiple independent targeting domains (striepen., 2001). the transmembrane protein mic6 forms a trimeric complex with the soluble microneme proteins mic1 and mic4, and deletion of mic6 prevents targeting of these molecules (reiss., a similar escort role has been described for mic8 in targeting mic3 (meissner., 2002), and for rhoptry proteins in p. falciparum (baldi., 2000). t. gondii mic2 is predicted to contain a transmembrane domain with cytoplasmic tail tyrosine motifs (syhyy, eieye) that play a role in sorting (di cristina., 2000), and mic2 has been shown to associate with another protein (mic2ap) during transport to and storage in micronemes (rabenau., 2001). genetic deletion of the cytoplasmic domain of the mic2 orthologue trap in p. berghei does not completely abolish microneme targeting (kappe., 1999), however, suggesting functional redundancy in organellar targeting pathways. perhaps the most unusual subcellular organelle in t. gondii (and other apicomplexan parasites) is a relict plastid, acquired by secondary endosymbiosis of a eukaryotic alga and retention of the algal plastid (khler., 1997). the apicomplexan plastid or apicoplastis essential for parasite survival (fichera and roos, 1997 ; he., 2001). this organelle is known to play a role in lipid metabolism (waller., 1998 ;, 1999 ; jelenska., 2001) and possibly other metabolic functions as well. although the apicoplast has a its own genome (wilson., 1996), this 35-kb circular element encodes only a limited protein repertoire ; the majority of apicoplast proteins are synthesized on cytoplasmic ribosomes and posttranslationally imported (waller., 1998 ; roos., 1999). as previously noted in other systems containing complex plastids, nuclear - encoded proteins destined for the apicoplast exhibit a bipartite nh2-terminal domain. molecular genetic manipulation in t. gondii and p. falciparum demonstrates that the extreme nh2 terminus functions as a secretory signal sequence, whereas the subterminal domain (presumed to be exposed after cleavage of the secretory signal) functions as a plastid - targeting signal, directing the cargo protein from the secretory pathway into the apicoplast lumen (roos., 1999 ;, 2000 ; waller., 2000 ; yung., 2001). thus, the combination of two normally distinct targeting processes cotranslational translocation into the endoplasmic reticulum and posttranslational translocation into chloroplasts combine to provide an elegant mechanism for targeting across the four membranes that surround the apicoplast. based on the characteristics of the apicoplast targeting signal, a large number of candidate apicoplast proteins have been identified in the t. gondii est and p. falciparum genome databases (ajioka., 1998 ; 2002), including plastid import machinery of the tic and toc family (mcfadden, g.i., personal communication). although the molecular details of protein targeting to the apicoplast are now clear, precisely how in morphological terms these proteins traffic from the secretory pathway to the apicoplast remains a mystery, as vesicles are never observed fusing with (or budding from) the organelle (compare fig. 1). moreover, treatment with brefeldin a or appending an er retention signal to nuclear - encoded apicoplast proteins fails to inhibit trafficking to the organelle. these observations raise the possibility that the apicoplast lies at a proximal position within the secretory pathway perhaps within the er itself and that all secreted proteins bearing an nh2-terminal signal sequence wash over the apicoplast ! as noted above, the function of the apicoplast is also uncertain, but circumstantial evidence suggests that it may play an important role in establishing the parasitophorous vacuole during host cell invasion (fichera and roos, 1997). protein targeting in t. gondii and related parasites utilizes a combination of conserved and unusual motifs and transport machinery (ng., 2000). the simplified, polarized, and morphologically distinctive organization of this cell readily permits comparison with mammalian cells and yeast, as detailed in table i. where mechanisms are conserved, t. gondii provides an excellent model for eukaryotes in general. for example, studies on the use of copi and copii coats in er golgi transport, or the process of golgi division, should be fruitful areas for study. where mechanisms are different as in the secretion of rhoptry lipids, the targeting across four membranes surrounding the apicoplast, and the trafficking of proteins destined for association with membrane compartments that lie beyond the plasma membrane studies on these parasites are likely to reveal the diversity of eukaryotic evolution and highlight potential targets for antiparasitic drug development. | name a single - celled eukaryote that boasts a small genome size, is easily cultivated in haploid form, for which a wide variety of molecular genetic tools are available, and that exhibits a simple, polarized secretory apparatus with a well - defined endoplasmic reticulum and golgi that can serve as a model for understanding secretion. got it ? now name a cell with all these attributes that contains at least a dozen distinct and morphologically well - defined intracellular organelles, including three distinct types of secretory vesicles and two endosymbiotic organelles. not so sure anymore ? |
natural killer (nk) cells constitute a unique effector lymphocyte lineage, classically defined as part of the innate immune system. their importance is well characterized in the context of health, under normal physiological conditions. for instance, nk cells are involved in tumor surveillance, given their natural cytotoxicity against neoplasms. in pregnancy, nk cells account for 70% of decidual leukocytes ; they are thought to mediate trophoblast invasion into the uterine lining and the modification of maternal spiral arteries. in addition to their implication in these crucial components of homeostasis and normal development, nk cells are well known for their considerable role in defense against infection. infection with a wide variety of pathogens can be limited by the action of nk cells, specifically intracellular microorganisms. indeed, nk cells play a substantial role in the initial control of both bacterial (e.g., listeria monocytogenes) and parasitic (e.g., plasmodium species) spread. yet, in the context of infection, the hallmark of nk cells remains their potent antiviral activity. nk cells participate in the clearance of many different viruses (human immunodeficiency virus, coxsackievirus, influenza or poxviruses, etc.), but their contribution is indispensable with regards to infection with members of the herpesviridae family (herpes simplex virus (hhv-1/2), varicella zoster virus (hhv-3), and cytomegalovirus (hhv-5)). plentiful evidence supports this, namely, the cases of two young patients suffering from numerous and recurrent herpesviral infections due to their nonfunctional nk cells [3, 4 ]. in mice, nk cell depletion and adoptive transfer have long been known to increase, respectively, susceptibility and resistance to mouse cmv (mcmv). the antiviral activity of nk cells relies on the various effector functions induced following their activation. on the one hand, nk cells secrete several cytokines, such as ifn-, mip1-/, rantes, il-10, activation - induced t - cell - derived chemokine - related cytokine (atac, lymphotactin), and others. on the other hand, indeed, they can kill virus - infected cells without prior activation, and they do so via a number of mechanisms, namely exocytosis of perforin - granzymes granules and signaling through members of the tnf death receptor family. yet, cytokine production and cytotoxicity must necessarily be preceded by the recognition of infected cells by nk lymphocytes, a phenomenon that has mostly been studied using a mouse model of infection. given the nonredundant role played by nk cells in resolving infections with these viruses, it is not surprising that cmvs would be perfect candidates for the study of nk cell responses. unfortunately, the strict species specificity of cmvs precludes experimental infection of mice with human cmv (hcmv). yet, mcmv shares many features with its human counterpart, including genome structure and disease manifestations. both are natural pathogens of their respective host and have coevolved with them for eons. moreover, both viruses have developed varied and analogous immune - evasion mechanisms that heavily implicate nk cells. therefore, early mcmv infection has become an established model to study nk cells and, more specifically, their impressive ability to distinguish self from nonself through their germ - line encoded receptors. in addition, at later times post - infection, this model reveled the unforeseen involvement of nk cells in the adaptive immune responses. nk cells discriminate between infected and healthy cells using an extensive panel of cell surface receptors, both activating and inhibitory. among the various receptor families involved in this process, ly49 receptors have proven themselves to be particularly important for mcmv detection by murine nk cells. these polygenic and polymorphic receptors are clustered at the natural killer cell complex (nkc) on mouse chromosome 6. they are stochastically expressed as disulfide - linked homodimers primarily on the surface of nk cells, but also on subsets of monocytes, macrophages, dendritic cells (dcs), and t cells. in terms of ligand specificity, inhibitory ly49 receptors recognize self - mhc class i molecules (mhci, also called h-2 in mice), whereas their activating counterparts can bind to various protein determinants of infection. ly49 receptors are structurally classified as type ii transmembrane, c - type lectin - like proteins. their extracellular domain is comprised of a flexible stalk and a natural killer domain (nkd), which provides ligand binding specificity and is structurally conserved among all members of the ly49 family. yet, activating and inhibitory receptors differ with regards to their intracellular domains. indeed, inhibitory ly49 receptors possess an immunoreceptor tyrosine - based inhibition motif (itim) within their intracellular domain. conversely, activating ly49 receptors lack this itim motif ; instead, a positively charged arginine residue in their transmembrane domain interacts with the dap12/dap10 adaptor proteins, which bears an immunoreceptor tyrosine - based activation motif (itam). during infection, ly49 receptor triggering leads to the initiation of a signaling cascade, the result of which is either inhibition or activation of the nk cell. in the case of both activating and inhibitory receptors, the first step of this cascade is the phosphorylation of the tyrosine residue contained in their respective itam or itim, most likely by a src family kinase. this phosphorylation recruits either ship-1, shp-1, or shp-2 in the case of inhibitory receptors, or syk, zap-70 and pi(3)k or grb2 for activating receptors. in both cases, numerous the end result of inhibitory receptor triggering is the dephosphorylation of itams linked to activating nk receptors and the prevention of ca influx, degranulation, cytokine production, and nk cell proliferation. in opposition, activating ly49 receptor engagement induces the reorganization of the actin cytoskeleton to enable cell polarization and the release of cytolytic granules, as well as the transcription of many cytokine and chemokine genes. of note, inhibition is by far the most common signal received by nk cells over the course of their lifetime. moreover, during nk cell development, binding of inhibitory ly49 receptors to self - mhci molecules renders them functional, that is to say capable of cytokine secretion (e.g., ifn-) and cytotoxic killing. this model, called nk cell education or licensing, postulates that the stronger the inhibitory ly49-self mhci interactions are, the more competent the nk cell will emerge. the immune response which follows mcmv infection relies on the interplay of soluble factors and numerous cell types, among them nk cells. in the early innate response, ly49 receptors are critical for nk cell activation, which in turn controls viral proliferation, due to their ability to recognize various viral and self - ligands. nevertheless, initial nk cell proliferation is ly49-independent and nonselective, while the subsequent preferential nk cell proliferation is driven by activating ly49 receptors. at later stages of the infection, nk cell secreted cytokines and apt virus control allows them to regulate the adaptive response. most surprisingly, ly49-mediated activation of nk cells also allows the generation of nk cells with several characteristics seen in memory t cells (figure 2(a)). all of these aspects of mcmv - ly49-driven nk cell responses will be examined in this paper. most importantly, evidence will be presented to highlight the previously unanticipated sophistication of the nk cell response against infection, redefining the established concepts of innate and adaptive immunity. from its initial peripheral sites of entry, mcmv spreads to the lymph nodes and via the bloodstream to the spleen, and the liver, infecting a broad spectrum of cell types. macrophages, hepatocytes, and reticular fibroblasts are some of its primary targets [11, 12 ]. at 68 hours after infection (p.i.), lymphotoxin / expressed on the surface of b cells interacts with its cognate receptor on splenic stromal cells, triggering type i interferon (ifn-/) secretion by the latter [13, 14 ]. within 24 hours after the initial infection, before the end of the first round of viral division, this first wave of type i ifn secretion wanes. subsequently, a second round of viral infection occurs, this time in many more organs, making the infection systemic. over the course of mcmv infection, several molecular viral byproducts plasmacytoid dcs (pdcs) engulf infected cell debris and recognize them through their endosomal pattern recognition receptors (prrs), that is, tlr3, tlr7 and tlr9 ; this recognition induces a second, much stronger wave of type i ifn secretion (~36 hours p.i) as well as the production of other proinflammatory cytokines (il-12, il-6, il-1, tnf-, etc.) [1517 ]. in parallel, the presence of mcmv particles in the cytoplasm of pdcs can lead to viral recognition by the nod - like and rig - i - like helicase sensors resulting in secretion of more pro - inflammatory cytokines ; these molecules probably also contribute to pdc - independent secretion of type i ifn [1820 ]. conventional dcs (cdcs) are one of the many cell types that are initially infected by mcmv. in response to infection, they secrete il-12, il-18, il-15 (trans - presented), and type i ifn all of which are required for nk cell activation. indeed, different nk cell effector functions require different cytokine inputs for their initiation : while the ifn-/stat1 pathway is necessary for cytotoxicity and initial proliferation, nk cell ifn- production during the first 2 days following infection necessitates il-18 or il-12/stat4 (figure 2(a)i) [2123 ]. moreover, the ifn- secreted by infected cdcs, along with macrophage - secreted mip1-, are critical for the control of viral loads in the liver and lungs. ifn- is also important for perforin - dependent control of viral replication in the spleen. once activated, nk cells start to eliminate infected cdcs and tnf--secreting macrophages, thus reducing the immunopathology caused by this cytokine. in short, the initial nk cell response against mcmv is mediated by a cytokine storm ; it is rapid, nonspecific, and global, involving nk cells with diverse ly49 repertoires. yet, it does not effectively control viral spread. indeed, despite the high levels of antiviral cytokines present in the serum, mcmv replication proceeds unhindered. this is true for all inbred strains at day 1.5 after infection. yet, by day 3 p.i., mouse strains can be segregated according to their nk cell - dependent control of mcmv. these strains include c57bl/6 (b6) and ma / myj mice, whose resistance depends on their strain - specific activating ly49 receptor, as well as pwk / pas and nzw / lacj mice, whose resistance is not yet characterized [2729 ]. mcmv resistance in b6 mice depends on the presence of the activating ly49h receptor on the surface of their nk cells [3032 ]. in this mouse strain, ly49h binds directly to m157, a mcmv - encoded glycoprotein with structural homology to host mhci molecules ; unlike the latter, however, m157 is a gpi - anchored protein, does not associate with 2-microglobulin, or present peptides at the surface of the cell [3335 ]. during the early phase of the infection, ly49h - m157 ligation initiates an activating signaling cascade, resulting in clonal proliferation of ly49h nk cells, killing of infected cells, and secretion of cytokines (ifn-, mip1-, tnf-, etc.), all of which result in the reduction of viral loads (figure 1(a)). the importance of this receptor - ligand pair in the control of mcmv infection was demonstrated by numerous studies. in the case of the virus, resistant mice became susceptible upon infection with a m157-deletant mcmv (m157) (figure 1(b)). as for the host, deletion of ly49h gene or deletion or loss of function mutation of the dap12 gene were shown to render mice susceptible to mcmv infection, as revealed by high viral titers [3739 ]. conversely, transgenesis of ly49h into initially susceptible strains was found to make them resistant to mcmv. once the interaction between ly49h and m157 was established as an essential component of mcmv resistance in b6 mice, the nature of this interaction was investigated. structural analysis, molecular modeling, and targeted mutations of both m157 and ly49h have shown the importance of the receptor homodimerization domain. indeed, ly49h receptors carrying mutations in this region display reduced m157 recognition, while mutations in the putative ligand - binding regions had limited impact on this same interaction. in the same vein, mutation of buried residues required for correct m157 folding (0 : 2 helix interactions) altered the ability of the viral molecule to bind ly49h, while mutations in the exposed and scattered residues across the entire m157 structure did not. furthermore indeed, given the four putative n - glycosylation motifs (nxt / s) of m157, a variety of glycosylated isoforms were found to be expressed on the surface of mcmv - infected cells. although these isoforms are also recognized by ly49h, their binding stability and half - life are increased as compared to the unglycosylated m157. these results reveal a quite tolerant recognition of m157 by ly49h where several amino acid substitutions are necessary in order to disrupt m157-ly49h interactions. such a molecular arrangement could allow ly49h to recognize variable m157 molecules, emerging as the mcmv attempts to escape immune recognition. from an evolutionary standpoint, capture of the h-2 gene by mcmv is thought to have initiated a cascade of events leading to the emergence of the ly49h - m157 interaction. originally, this capture could have allowed the virus to exploit a mhci homologue capable of triggering inhibitory ly49 receptors. since the ly49 locus evolves rapidly, any inhibitory receptor could have been converted to an activating one, including those with binding specificity for m157 [44, 45 ]. in support of this, m157 can be bound by the inhibitory ly49i receptor from 129/j mice ; unfortunately, the involvement of ly49i in susceptibility to mcmv infection could not be assessed due to a defect in nk cell signaling within this strain. recently, additional inhibitory ly49 receptors, namely, ly49c from the b6, balb / c, and nzb strains, have been shown to interact with the m157 obtained from wild - derived mcmv isolates. interestingly, ly49c - specific recognition of nonlaboratory strain - mcmv - infected cells did not seem to interfere with ly49h - mediated nk cell activation and the ensuing antiviral response in b6 mice. the dominant ly49h recognition of m157 is supported by the fact that sequential passage of wild - type (wt) mcmv in b6 mice leads to the emergence of nk cell - escape m157 variants, which evade ly49h - mediated nk cell recognition. nevertheless, the m157 proteins of mcmv strains isolated from wild mice are highly variable, indicating that outside of laboratory settings, both inhibitory and activating ly49 receptors could be targeted by the virus. in any case, the fact that m157, one of many mcmv - encoded proteins, can be recognized by different ly49 receptors, both activating and inhibitory, suggested that other mechanisms of mcmv recognition might exist in other mouse strains. in this second mechanism of mcmv - infected cell recognition, an activating ly49 receptor recognizes altered self - mhci molecules rather than a viral mhci homologue ; this alteration of self - mhci molecules stems from their association with viral protein. this type of recognition could be used by nk cells from ma / myj mice, which are resistant to mcmv even though they lack ly49h. indeed, ma / myj resistance has been associated with a genetic interaction between the nkc and the h-2 loci or the h-2 locus alone [4850 ]. at the cellular level, this receptor can recognize infected cells given the surface expression of two specific elements : the viral m04/gp34 protein in association with h-2d molecules (figure 1(c)). in order to dissect the contribution of h-2 to this recognition, chimeric h-2 molecules were produced, in which domains were swapped between h-2d and h-2d ; these chimeric molecules were used to show that the h-2d - binding platform in particular is required for this recognition. furthermore, the ectopic expression of m04/gp34 alone can not stimulate ly49p - bearing reporter cells in the absence of infection. this stimulation, however, is restored when target cells are infected with a viral deletion mutant lacking m04 (m04 mcmv), suggesting that another host or viral factor is necessary for ly49p / m04/h-2d - mediated recognition of infected cells. m04/gp34 is crucial for the in vivo nk cell response in ma / myj mice, since normally resistant ma / myj mice are unable to control viral replication following infection with m04 mcmv (figure 1(d)). the importance of h-2d in the resistance of ma / myj mice was also demonstrated in vivo : (1) by the creation of an h-2 congenic mouse panel ; (2) by the transgenesis of h-2d into susceptible h-2 mice. in both cases, the mice became capable of controlling the viral infection, as opposed to h-2 animals [50, 52, 53 ].. tested their h-2 congenic mouse panel further and showed that ly49 g nk cells expanded preferentially in these mice at 90 hours p.i. their resistance to mcmv infection was abrogated following depletion of ly49 g nk cells, which suggests that ly49 g is somehow involved [52, 53 ]. since an inhibitory receptor can not directly mediate resistance to mcmv, the authors hypothesized that appropriate licensing via ly49g : h-2d interaction renders nk cells functional, capable of subsequently recognizing and eliminating mcmv through ly49p / m04/h-2d (figure 1(c)). nevertheless, it remains unclear how or if licensing contributes to the ability of nk cells to resolve mcmv infection. indeed, it has recently been shown that unlicensed nk cells respond better to mcmv infection. regardless of the role of licensing, the available evidence suggests that several ly49 receptors might be involved in the control of mcmv infection in ma / myj mice. the contribution of inhibitory ly49 receptors to the nk cell - mediated control of mcmv infection has recently been evaluated. indeed, studies using balb mice show that the presence of ly49 inhibitory receptors negatively impacts early mcmv resistance, that is, it makes mice susceptible to the virus. balb mice are considered susceptible, that is to say that they are unable to control the replication of wt mcmv in the spleen at day 3 p.i. however, infecting these mice with m04 mcmv results in a significant reduction of viral loads in this organ and increased nk cell proliferation in a h-2-dependent manner. indeed, balb mice of the h-2 (balb.k) background showed the greatest reduction in viral load upon infection with m04 mcmv, whereas none was seen in h-2 mice (balb.by) ; the viral load reduction in h-2 mice (balb / c) was an intermediate value between that of h-2 and h-2 mice. this reduction in viral load is due to the absence of m04/gp34, which maintains surface expression of mhci molecules, and the action of activating receptors such as nkg2d. as such, in wt mcmv - infected cells, sufficient levels of mhci molecules are displayed on the cell surface ; this allows inhibitory ly49 receptors to be triggered and to deliver their inhibitory signal, the result of which is the unresponsiveness of nk cells (figure 1(e)). in the case of m04 mcmv infection, the expression of mhci molecules is severely downregulated, allowing nk cell activation through the missing - self mechanism and improved control of viral spread (figure 1(f)). furthermore, viral loads were shown to return to high levels following depletion of certain inhibitory ly49 nk cell fractions during m04 mcmv infection of h-2 balb mice. this effect was more pronounced following ly49 g nk cell depletion than ly49c or ly49a nk cell depletion. in summary altered self - recognition by activating ly49 receptors, requiring the presence of self - mhci molecules associated with viral products, implicates involvement of inhibitory ly49 receptors, which depending on mice strain can have opposite effects. thus, when the predominant signal is m04-dependent activation (e.g., ma / myj via ly49p), m04 ablation allows enhanced viral replication ; however, when the predominant signal is m04-dependent inhibition (e.g., balb via ly49g / a), absence of m04 allows enhanced control of virus replication. in short, these findings suggest that the early, nk cell - dependent control of viral replication is not solely due to activating ly49 receptors, but rather that it involves a delicate interplay between signals generated by both activating and inhibitory receptors. nk cells have classically been thought of as effectors of the antiviral response through their ability to directly eliminate infected cells. however, recent evidence demonstrates that their role extends beyond that of effector lymphocytes into regulators of the inflammatory and adaptive immune response against viral infection. more specifically, ly49h nk cells are essential for the protection and preservation of cdcs, which can themselves stimulate nk cells and potentiate their response [56, 57 ]. nevertheless, infection does occur ; nk cell - dependent elimination of cdcs and, in particular, macrophages, reduces the secretion of tnf-, a major contributor to hemophagocytic lymphohistiocytosis syndrome during mcmv infection. in addition, ly49h - m157-dependent reduction of viral burden decreases the duration and intensity of pdc activation, which limits very high systemic levels of type i ifn and other cytokines that can be detrimental to the host [32, 5860 ]. in doing so, nk cells also accelerate the recruitment and expansion of anti - mcmv specific cd8 t cells, as it was shown that in mice devoid of ly49h, the appearance of cd8 t cells is delayed by ~1 day (figure 2). triggering by m157 is essential for ly49h nk cell amplification and maintenance. in the context of mcmv infection, signaling through ly49h / dap12 induces nk cell clonal proliferation, ultimately leading to an increase in the ly49h nk cell frequency from 50% to 80% and clearance of the virus in the spleen and liver by day 6 p.i.. conversely, in ly49h mice, the overall amount of nk cells declines past uninfected levels, leading to high viral titers in the spleen at the same time point., attempted to dissect the role of ly49h in various aspect of the nk cell response using ly49h or perforin 1 (prf1) knockout mice. for instance, ly49h - expressing prf1 mice were susceptible to mcmv infection, even though ly49h nk cells proliferated substantially. although in these mice the nk cytotoxic function was absent, they survived a dose that killed ly49h prf1 double - knockout mice. therefore, ly49h expression was protective. this was explained by the fact that ly49h nk cells secreted il-10, a potent immunosuppressive cytokine capable of limiting cd8 t cell cytotoxicity as well as ifn- and tnf- secretion (figure 2(a)ii). thus, ly49h is required for the maintenance of nk cells until late time - points after infection, allowing them to exert a regulatory function over cd8 t cells and the immunopathology they can cause. paradoxically, the presence of activating ly49 receptors can also be detrimental to the subsequent adaptive immune response by promoting viral persistence. for example, while ly49h mice could clear infected cdcs better than their ly49h counterparts, killing of these antigen - presenting cells (apcs) prevented them from priming nave, virus - specific t cells. this lack of appropriate t - cell priming in ly49h mice resulted in the reduced activation, cytotoxicity, ifn- secretion, and maintenance of cd8 t cells, as well as the reduced ifn- secretion and maintenance of cd4 t cell, contributing to mcmv persistence in the salivary glands (figure 2(a)ii). however, mcmv - infected, immature cdcs have also been shown to induce anergy, poor effector functions, and inferior recall responses in cd4 and cd8 t cells. further research is needed to determine the precise effect of cdc clearance in the overall response to the virus. nevertheless, ly49h nk cell recognition and removal of mcmv - infected cells can unquestionably shape the course of the adaptive immune response. another unforeseen nk cell role was evidenced by the discovery that, following activation, nk cells acquire features seen in memory t - cell populations. the existence of memory - like nk cells was first hinted at in a study of chemically - induced contact hypersensitivity (chs). in this model, mice lacking t and b lymphocytes, the classical mediators of chs, remained able to elicit an inflammatory response through hepatic nk cells, as measured by dermatitis. sun. followed up on this observation using an mcmv model of infection and adoptive transfer into dap12 mice. the investigators noticed that only ly49h nk cells underwent specific clonal expansion before contraction and persisted for up to 70 days p.i. this was rather astonishing as the generally accepted half - life of mature nk cells is 7 to 17 days. importantly, irrespective of initial transfer numbers the frequency of the mcmv - specific nk - cell response was similar to primary t - cells responses. they also observed that these long - lived, memory - like nk cells were m157-specific and had superior effector responses upon rechallenge with mcmv as compared to nave nk cells. the existence of memory - like nk cells was also confirmed by transferring cytokine - activated nk cells into nave hosts and observing that they could still be detected as far as 22 days after transfer. these memory nk cells were found to have similar cytotoxic activity to that of their nave counterparts. moreover, they were unable to constitutively produce ifn- yet upon restimulation produced significantly more of this cytokine than nave nk cells. more recently, again in the chs model, virus - specific memory - like nk cells were generated in response to influenza virus, vsv or hsv-1 infection. in this case, the adoptive transfer of specific, virus - sensitized hepatic nk cells into naive mice improved their survival following infection with a lethal dose of virus ; this was dependent on the cxcr6 chemokine receptor expressed on nk cells. all of these experiments have shown that innate immune cells can retain an intrinsic memory of prior activation, a function until now restricted to antigen - specific, adaptive immune lymphocytes. however, most of the aforementioned studies were performed with immunodeficient donor and recipient mice. given the adaptability of the immune system, it is possible that donor nk cells, once transferred into these recipients, may have taken up more responsibility (i.e., memory status) than they normally would in wt animals in order to compensate for the absence of t and b cells. as for the work performed by sun., even though their donor nk cells came from wt mice, they were transferred into neonate animals, whose adaptive immune responses are arguably as ineffective as that of immunodeficient mice. since their discovery over 30 years ago, it has become clear that nk cells possess numerous functions, going beyond their original natural killers of tumors role. among these, they act as a first line of defense in the context of infection with a variety of pathogens, in particular viruses. humans have been coevolving for millions of years with some of these viruses, namely cmvs ; therefore, they know us better than we know ourselves, having shaped our immune system and been shaped in return. mouse models of mcmv infection revealed that nk cells are not merely killers of infected targets, but very complex lymphocytes endowed with the molecular machinery necessary to perform a broad spectrum of functions. as part of the innate system, nk cells can immediately react to an infectious insult via fixed, germ - line encoded receptors that recognize pathogen - associated motifs. as the infection progresses, nk cells adapt, modulating the level of inflammatory mediators produced during the initial steps of infection. finally, as adaptive responders, nk cells show clonal expansion, maintenance, and memory of previous insults in the case of reinfection. nevertheless, several questions remain : do nk cells recognize infections other than mcmv with their ly49 repertoire of receptors ? can triggering of other nk receptors by mcmv - infected cells induce the same nk cell outcomes as in the ly49h - m157 model (e.g., clonal expansion, memory, etc.) ? why does the host express so many different nk receptors targeting mcvm, several with opposite effects on nk cell activity (i.e., inhibitory versus activating) ? does the host benefit from the infection ? if so, how ? the possible paths of investigation are plentiful. as more inquiries are resolved, which is currently the case in the thriving field of nk cells in cancer, we will be able to harness the power of these lymphocytes in the treatment of infectious diseases [68, 69 ]. | clinical and experimental data indicate that a subset of innate lymphocytes, natural killer (nk) cells, plays a crucial role in the response against herpesviruses, especially cytomegaloviruses (cmv). indeed, in mice, nk cells, due to the expression of germline encoded ly49 receptors, possess multiple mechanisms to recognize cmv infection. classically, this results in nk cell activation and the destruction of the infected cells. more recently, however, this unique host - pathogen interaction has permitted the discovery of novel aspects of nk cell biology, implicating them in the regulation of adaptive immune responses as well as in the development of immunological memory. here, we will concisely review the newly acquired evidence pertaining to nk cell ly49-dependent recognition of mcmv - infected cell and the ensuing nk cell regulatory responses. |
scaphoid fractures are commonly seen in young, healthy individuals and may occur as a result of a fall on the outstretched arm or a forced dorsiflexion injury of the wrist.1,2 because fractures may disturb the scaphoid s tenuous blood supply, the healing process may be compromised. osteonecrosis is said to occur in 13%50% of cases of fracture of the scaphoid, and the incidence of osteonecrosis is even higher in those with involvement of the proximal one - fifth of the scaphoid.1,35 the blood supply of the scaphoid is primarily from the radial artery via the artery to the dorsal ridge of the scaphoid, whose branches enter the scaphoid via foramina at the dorsal ridge at the level of the waist of the scaphoid.4,6 subsequently, these vessels divide and run proximally and palmarly to supply the proximal pole of the scaphoid.4 other branches provide 20%30% of the blood flow and appear from the distal palmar area of the scaphoid, arising either directly from the radial artery or from the superficial palmar branch.6,7 the proximal pole, therefore, is dependent entirely on intraosseous blood flow. this tenuous blood supply can result in a protracted healing process after fracture, with the average time to healing of an acute proximal pole fracture averaging 36 months. nonunion may occur (in 5%10% of all cases, with an even higher incidence in displaced fractures), and numerous series document progression of nonunion to collapse and arthritis.5,8,9 it is for this reason that diagnosis and appropriate treatment of the acute fracture, and the possible sequelae of nonunion, is essential. three common classifications used for scaphoid fracture include the mayo classification, the russe classification, and the herbert classification. some series have demonstrated limited prognostic value and poor inter- and intraobserver reliability of scaphoid fracture classification schemes10 ; nevertheless, the mayo, russe, and herbert classifications are in common use in clinical practice, and many authorities believe they are helpful for determining treatment options and providing prognostic information. the first two classifications are based on anatomical planes of the scaphoid, whereas the herbert classification defines stable and unstable fractures.1,2,11 thus, the herbert classification may be particularly helpful when determining treatment options. the type a herbert classification fracture is a stable acute fracture, and a type b is an unstable acute fracture. stable fractures include fractures of the tubercle (a1) and an incomplete fracture of the waist (a2). type b (acute unstable fractures) include subtypes b1 (oblique fractures of the distal third) ; b2 (displaced or mobile fractures of the waist) ; type b3 (proximal pole fractures) ; type b4 (fracture dislocations) ; and b5 (comminuted fractures). type c fractures are those that demonstrate delayed union after > 6 weeks of plaster immobilization, and type d fractures are established nonunions, either fibrous (d1) or sclerotic (d2).1 the diagnosis of a scaphoid fracture can sometimes be difficult to establish, as patients may have normal radiographs early in their clinical course. most patients demonstrate tenderness over the anatomic snuff box or over the distal scaphoid tubercle, pain with longitudinal compression of the thumb, and limited range of motion and pain at the end arc of motion, especially with flexion and radial deviation.1113 reduced grip strength may be noted.1113 however, it is important to note that not all patients have pain over the scaphoid even with a well - defined fracture seen on radiographs.11 overall, sensitivity is quite high for the clinical examination, although specificity approaches only 74%80%.12,13 radiographic evaluation of the scaphoid is useful for determining if an acute fracture is present. a lateral radiograph is important in this regard, as it should demonstrate a co - linear capitate and radius, with the pisiform located between the distal pole of the scaphoid and the body of the capitate. classically, patients with clinical findings suspicious for scaphoid fracture but negative initial radiographs were treated with 2 weeks of cast immobilization followed by repeat examination and radiographic studies, with the idea that the time delay allows bony resporption adjacent to the fracture site to occur, making the fracture visible.11,14 although this remains an accepted treatment option, it may result in unnecessary immobilization, with adverse effects upon return to work and the need for repeat radiographs, clinical examinations, and splint or cast changes.1517 alternative imaging techniques may be useful for diagnosing a fracture, and magnetic resonance imaging (mri) is superior to repeat radiographs for detecting an occult scaphoid fracture.18 bone scans are sensitive but not specific for diagnosing a scaphoid fracture.19 bone scintigraphy has demonstrated 100% sensitivity and 98% specificity for a scaphoid fracture compared with approximately 65%70% sensitivity for plain radiography.7,2024 mri is considered by many to be the gold standard for detecting scaphoid fractures, with sensitivity approaching 95%100% and specificity approaching 100%.2527 in addition, one may identify alternative pathology to explain the wrist pain. it is important to recognize that nonunion occurs in up to 12% of patients if an occult fracture is not defined and not immobilized. however, treatment (splinting and casting) can lead to loss of work and economic implications. 16,17,19 the cost of time off from work, serial casting, and office visits easily outstrip the cost of an mri or computed tomography (ct) scan for definitive diagnosis.16 brooks.15 investigated the cost - effectiveness of mri for diagnosing suspected scaphoid fractures in a randomized controlled trial involving 28 patients. those who underwent mri had a shorter duration of immobilization, decreased use of health care resources, but increased cost to treat when compared to patients who were randomized to the non - mri group, in which patients were immobilized and evaluated by serial clinical and radiographic examination. however, these costs do not take into account absenteeism from work and leisure activities.15 pillai and jain17 reported a needless immobilization rate of > 80% when all clinically suspected scaphoid fractures with negative radiographs were immobilized in the traditional treatment algorithm. they found that only 6.7% of the 90 initially radiographically negative wrists actually had a scaphoid fracture, and 10 additional patients had other injuries of the wrist not involving the scaphoid.17 the findings suggest that the cost of needless immobilization, with further clinical and radiographic studies, would have exceeded early alternative investigations, such as mri or bone scan, which frequently were ultimately required anyway.17 thorpe.28 reported on a series of 59 patients with clinical symptoms suggestive of scaphoid fracture but in whom radiographs were negative. clinically, 4 scaphoid fractures, 10 other fractures, and 3 significant ligamentous injuries were identified. all scaphoid fractures were identified by both mri and bone scan ; however, mri was noted to have better interobserver agreement and fewer false positives. likewise, other sources of pathology could be diagnosed on mri, such as a ligamentous injury or carpal instability, whereas these findings were not diagnosable by bone scan. the authors further noted that the costs were similar.28 in a study comparing the sensitivity and specificity of mri and bone scintography for diagnosis of occult scaphoid fractures, 43 patients with wrist trauma and normal radiographs underwent mri and bone scans an average of 19 days after injury. of these 43 patients, 6 (14%) ultimately were diagnosed with a scaphoid waist fracture. mri was noted to be more sensitive for detecting an occult scaphoid fracture, with fewer false positives than the bone scans.29 dorsay.16 investigated the cost - effectiveness of early mri to detect occult scaphoid fractures. they noted that 75% of patients with clinical evidence of a scaphoid fracture would be immobilized unnecessarily if they were treated by the standard follow - up with repeat radiographs following immobilization. the cost differential between standard follow - up and mri was small.16 the questions that often arise are when should you prescribe mri and how early can you detect a fracture in the scaphoid ? it is possible with modern mri technology to detect a fracture within 46 h ; however, owing to most hospitals scheduling situations, obtaining an mri examination at 3648 h is a common goal. again, mri may demonstrate an occasional false - positive but rarely results in a false - negative examination. ct scanning is also a useful technology for diagnosing scaphoid fractures and can help determine nonunion or incomplete union, and it is helpful when planning for surgery to determine the intrascaphoid angle and elements of scaphoid collapse. it is not as accurate, however, as mri for diagnosing an acute, occult fracture. it is helpful to remember that most patients with a suspected scaphoid fracture have not actually injured the scaphoid and that casting for no fracture just to present a conservative treatment modality results in overtreatment at the expense of office visits, radiographs, and lost work. a technetium bone scan is sensitive but false - positive findings can occur in patients with arthrosis or prior or concurrent injury. mri tends to produce fewer false - positives, can pinpoint the location of the fracture more precisely, and can identify alternative diagnoses. bone scans rarely miss a scaphoid fracture but require a delay in performing the test ; they often do not adequately elucidate alternative diagnoses. mri is at least as sensitive and more specific, involves less radiation exposure, and may allow one to diagnose alternative problems.15 a suggested algorithm, therefore, in a patient with wrist trauma is to obtain three radiographic views of the wrist, anteroposterior (ap), lateral, and oblique. if a scaphoid fracture is identified on the radiographs, a ct scan can be then obtained for surgical planning. if the radiographs are negative or equivocal, a limited mri examination of the wrist is then obtained to determine the presence or absence of a scaphoid fracture. if needed, a ct scan can also be obtained after mri to help plan for surgical treatment. the reason to consider operative treatment is that immobilizing the patient for 12 weeks is typical for scaphoid fractures, and in more proximally located fractures it may be much longer.3,11,30,31 although few studies in the literature have documented the consequences of long - term cast immobilization, clearly it can result in significant stiffness that may require a significant rehabilitation period ; moreover, some studies have suggested a poorer outcome following prolonged immobilization. 30,22,32 in addition, union rates are higher with operative management, approaching 95% or more in most series of all types of scaphoid fracture.30,3335 multiple series have documented satisfactory outcomes and minimal complications with surgical management using percutaneous fixation of scaphoid fractures.1,30,3543 several studies have looked at surgery versus casting for acute fractures. those who underwent surgical management with percutaneous screw placement demonstrated better range of motion at the wrist, earlier time to union (7 weeks vs. 12 weeks with casting), earlier return to work, and minimal differences in the two groups with respect to outcomes and satisfaction at the final follow - up. importantly, no increased complication rate related to surgical treatment was observed.30,44,45 it appears, therefore, that percutaneous treatment of acute scaphoid fracture has a low morbidity rate and in the hands of an experienced surgeon does not result in a higher complication rate than nonsurgical treatment.1 in a meta - analysis of scaphoid nonunion that reviewed 1121 articles, 36 of which met criteria for inclusion in this review, data were obtained for 1827 scaphoid nonunions.46 when patients with avascular necrosis of the proximal pole were evaluated, union rates were 88% with vascularized bone grafting versus 47% with a standard graft.46 in addition, those who had no postoperative immobilization were demonstrated to have the same union rate as those who were immobilized for > 6 weeks postoperatively.46 this study therefore lends support for immediate screw fixation and mobilization of the wrist versus prolonged cast treatment. up to one - third of these fractures may result in nonunion even with appropriate immobilization. several studies demonstrated good results with early surgical intervention, and it appears that a careful dorsal approach does not injure the blood supply. when considering overall treatment options, it is perhaps best for all proximal pole and displaced scaphoid fractures to be treated with surgery. acute fractures may demonstrate normal or decreased t1-weighted mri intensity and increased t2 intensity.11 nonunion and impaired vascularity is often seen with low t1 and t2 marrow signal intensity, which may correlate with poor healing.11,47,48 some series, however, have failed to find a correlation with preserved vascularity of the proximal pole and successful outcome of surgery.49 despite optimal therapy, nonunion or malunion may ensue. treatment of the established scaphoid nonunion requires consideration of patient and nonunion characteristics. because the bony attachments of the dorsal intercarpal ligament and the dorsal scapholunate ligament are maintained when setting a fracture of the proximal one - third of the scaphoid, these fractures rarely demonstrate instability patterns such as dorsal intercalated segmental instability (disi). nonunion of a scaphoid fracture, however, can result in carpal malalignment and progressive radiocarpal arthrosis. 5,8,9,11 the real effect of malunion, however, is less clearly defined. in a series of 160 scaphoid nonunions treated with internal fixation and bone grafting, of which 90% healed, failure to achieve union was related to a proximal fracture location, avascularity of the proximal pole, instability of the fracture, and delay to surgery. importantly, residual flexion deformity of the scaphoid did not have an effect on the outcome. therefore, malunion was not thought to be a contributing factor to a poor result. this study, however, demonstrated that the length of immobilization negatively affects the functional outcome.50 in a smaller study of 26 patients with nonunions who underwent bone grafting for scaphoid nonunions, 13 patients ultimately had lateral intrascaphoid angles greater than 45. however, excellent function and high patient satisfaction was noted at 11 years follow - up, with clinical outcomes indistinguishable between the groups who had normal alignment and those with malunion. it was thought, therefore, that osteotomy and surgical treatment of a malunion is not indicated for most of the patients with healed scaphoid fractures.51 the nondisplaced stable nonunion without degenerative changes (mack - lichtman type i)8,52,53 may be treated with bone grafting with or without hardware. nonvascularized autogenous bone graft from the distal radius or iliac crest may be sufficient, although vascularized bone grafting should be considered in the presence of an avascular proximal pole as determined by mri or intraoperative findings. in addition, there is the caveat that if the initial bone grafting fails future surgery is less likely to be successful.3,5456 the fracture site should be freed from fibrous nonunion or interposed tissue, and hardware may or may not be placed. kirshner wires may be used, but screw fixation may provide the advantage of compression of fracture fragments. type ii nonunions, which are unstable owing to fragment displacement, require one to address the nonunion and restore normal carpal stability to prevent the downward spiral from instability to collapse and arthritis.8,52,53 a corticocancellous wedge bone graft may be harvested from the iliac crest to correct alignment. because of the instability, these fractures require fixation. scaphoid nonunion with accompanying mild arthritis are classified as mack - lichtman type iii.8,52,53 initial findings of radiocarpal arthritis include beaked changes to the radial styloid and narrowing of the joint space between the radius and scaphoid. if the scaphoid can be salvaged, open reduction and internal fixation with bone graft are required. a radial styloidectomy may be performed if isolated radial styloid arthritis is present;57 alternatively, excision of the distal scaphoid fragment may be performed if the scapholunate ligament is intact.53,58,59 however, significant arthritis of the scaphoid fossa and radial styloid is a contraindication to scaphoid salvage, and proximal row carpectomy and four - corner fusion are options.6066 mack - lichtman types iv and v nonunions are those associated with midcarpal arthritis, without and with radiolunate arthritis, respectively.8,52,53 they require partial or complete wrist arthrodesis for optimal treatment.53,67 in short, if degenerative arthritis is absent, and the carpus can be salvaged, one may consider bone grafting, either standard or vascularized, with or without internal fixation. however, if substantial degenerative arthritis is present, limited or complete wrist arthrodesis may yield a stable, painless result. alternatively, proximal row carpectomy or anterior interosseous nerve (ain) and posterior interosseous nerve (pin) denervation neurectomy may be considered. prior to surgery a trial of cast immobilization to simulate the fused wrist, or an ain or pin block may be helpful to clarify the possible effect of the desired procedure on the patient s symptoms. when the scaphoid is salvageable and bone grafting is attempted, one must consider the relative risks and benefits of nonvascularized or vascularized bone grafting. nonvascularized bone grafting is probably sufficient for most waist fracture nonunions and those with preserved vascularity of the proximal pole. as noted previously, however, should standard bone grafting fail, future surgery is likely also to be unsuccessful.3,5456 the benefits of vascularized bone grafting for scaphoid nonunion include preservation of the blood supply, primary bone healing, and maintenance of structural integrity. therefore, consideration of vascularized bone grafting is wise, particularly in the setting of osteonecrosis and proximal pole nonunions. recently the mayo clinic documented the outcomes and complications of vascularized bone grafting for scaphoid nonunion in a series of 52 nonunions in 51 patients.68 two patients (two scaphoids) were lost to follow - up, with 50 patients and 49 scaphoids available for review. in this study, the 1,2-intercompartmental supraretinacular artery (1,2-icrsa) was used as a reverse - flow vascularized bone graft for scaphoid nonunion.56,69,70 previously published union rates for vascularized bone grafts in the scaphoid have ranged from 27% to 100%, although there are presently fewer than 10 such published studies in the literature with approximately 100 total patients. in the mayo clinic study, there were 42 male patients and 9 female patients, with an average age of 23 years and a mean follow - up of 7.6 months. the nonunions were evenly divided between the proximal pole and the waist ; and evidence of avascular necrosis was present in 24 scaphoids based on intraoperative surgical findings, mri, and radiographs. overall, 72% of the scaphoid fractures achieved union with vascularized bone grafting (36/50), and healing occurred at an average of 16 weeks (range 840 weeks). factors adversely affecting the union rate included female sex (union rate : 30% vs. 82% in males) ; tobacco use (union rate : 81% in nonsmokers vs. 46% in smokers), and proximal pole avascularity [48% union rate in the presence of avascular necrosis (avn) vs. 91% in the absence of avn ]. simple k - wire fixation resulted in a 53% union rate, whereas screw fixation resulted in an 88% union rate. carpal collapse with formation of a humpbacked deformity was present in 50% of the failures versus 11% of patients who went on to union. interestingly, in this series of patients, the fracture - dislocation did not affect the union rate, with waist fractures achieving 70% union and proximal poles achieving 72% union. prior surgery resulted in a healing rate of 64% compared to 73% in those with no previous operations. complications in this series resulted in three infections, three patients with graft extrusion, two patients with graft resorption, and four patients with progressive degenerative changes that required further operative therapy. five patients underwent subsequent surgeries including ain and pin neurectomy (n = 2), repeat autologous bone grafting (n = 1), scaphoid excision with four - corner fusion (n = 1), and total wrist fusion (n = 1).68 notably, the union rate was 100% in cases of nonunion in which avn was absent and which underwent the vascularized bone grafting procedure with screw fixation.68 two studies in the literature showed relatively low healing rates of 60%54 and 27%,71 respectively. in the study by boyer.,54 10 proximal pole nonunions underwent vascularized bone grafting with a 60% union rate. the four failures occurred in patients who had previously undergone treatment with a bone grafting procedure, suggesting that prior surgery adversely affects the outcome of the 1,2-icrsa bone graft.54 in a second report by straw.,71 22 nonunions underwent the 1,2-icrsa bone graft with only a 27% union rate (n = 6). altogether, 16 of these patients had avn of the proximal pole, and the k - wires that were used for fixation were removed at 8 weeks regardless of union status. of those 16 patients with osteonecrosis of the proximal pole, only 2 had union, a 12.5% union rate, compared to 4 of 6 (67%) in the patients with a preserved vascular supply to the proximal pole.71 it could be concluded that a 1,2-icsra vascularized bone graft may not improve union rates in patients with significant avn of the proximal pole fragment.71 patients who overall are poor candidates for vascularized bone grafting are those with carpal collapse and scaphoids with a large defect or excessive comminution of the proximal fragment. likewise, patients with radiocarpal arthritis may have a more disappointing outcome.56 these series and the recent study from the mayo clinic present less optimistic results than a prior study by steinmann. in which a 100% union rate was achieved in 14 patients at a mean 11.1 weeks.56 the reasons for this increased failure rate are not entirely clear. however, it probably relates to broader acceptance of the procedure with resultant expanded indications for use of this vascularized bone grafting technique. additionally, there is enthusiasm and willingness to try everything, including a vascularized bone graft prior to performing a salvage type procedure. the limitations of this larger, more recent study relate to its retrospective nature and the multiple surgeons involved. factors overall that are thought to be associated with failure of 1,2-icsra in scaphoid nonunion include female sex, tobacco smoking, proximal pole avn, humpback deformity, comminuted proximal pole and use of a k - wire or no metallic internal fixation. when considering overall treatment options, it is perhaps best for all proximal pole and displaced scaphoid fractures to be treated with surgery. acute fractures may demonstrate normal or decreased t1-weighted mri intensity and increased t2 intensity.11 nonunion and impaired vascularity is often seen with low t1 and t2 marrow signal intensity, which may correlate with poor healing.11,47,48 some series, however, have failed to find a correlation with preserved vascularity of the proximal pole and successful outcome of surgery.49 treatment of the established scaphoid nonunion requires consideration of patient and nonunion characteristics. because the bony attachments of the dorsal intercarpal ligament and the dorsal scapholunate ligament are maintained when setting a fracture of the proximal one - third of the scaphoid, these fractures rarely demonstrate instability patterns such as dorsal intercalated segmental instability (disi). nonunion of a scaphoid fracture, however, can result in carpal malalignment and progressive radiocarpal arthrosis. 5,8,9,11 the real effect of malunion, however, is less clearly defined. in a series of 160 scaphoid nonunions treated with internal fixation and bone grafting, of which 90% healed, failure to achieve union was related to a proximal fracture location, avascularity of the proximal pole, instability of the fracture, and delay to surgery. importantly, residual flexion deformity of the scaphoid did not have an effect on the outcome. therefore, malunion was not thought to be a contributing factor to a poor result. this study, however, demonstrated that the length of immobilization negatively affects the functional outcome.50 in a smaller study of 26 patients with nonunions who underwent bone grafting for scaphoid nonunions, 13 patients ultimately had lateral intrascaphoid angles greater than 45. however, excellent function and high patient satisfaction was noted at 11 years follow - up, with clinical outcomes indistinguishable between the groups who had normal alignment and those with malunion. it was thought, therefore, that osteotomy and surgical treatment of a malunion is not indicated for most of the patients with healed scaphoid fractures.51 the nondisplaced stable nonunion without degenerative changes (mack - lichtman type i)8,52,53 may be treated with bone grafting with or without hardware. nonvascularized autogenous bone graft from the distal radius or iliac crest may be sufficient, although vascularized bone grafting should be considered in the presence of an avascular proximal pole as determined by mri or intraoperative findings. in addition, there is the caveat that if the initial bone grafting fails future surgery is less likely to be successful.3,5456 the fracture site should be freed from fibrous nonunion or interposed tissue, and hardware may or may not be placed. kirshner wires may be used, but screw fixation may provide the advantage of compression of fracture fragments. type ii nonunions, which are unstable owing to fragment displacement, require one to address the nonunion and restore normal carpal stability to prevent the downward spiral from instability to collapse and arthritis.8,52,53 a corticocancellous wedge bone graft may be harvested from the iliac crest to correct alignment. because of the instability, these fractures require fixation. scaphoid nonunion with accompanying mild arthritis are classified as mack - lichtman type iii.8,52,53 initial findings of radiocarpal arthritis include beaked changes to the radial styloid and narrowing of the joint space between the radius and scaphoid. if the scaphoid can be salvaged, open reduction and internal fixation with bone graft are required. a radial styloidectomy may be performed if isolated radial styloid arthritis is present;57 alternatively, excision of the distal scaphoid fragment may be performed if the scapholunate ligament is intact.53,58,59 however, significant arthritis of the scaphoid fossa and radial styloid is a contraindication to scaphoid salvage, and proximal row carpectomy and four - corner fusion are options.6066 mack - lichtman types iv and v nonunions are those associated with midcarpal arthritis, without and with radiolunate arthritis, respectively.8,52,53 they require partial or complete wrist arthrodesis for optimal treatment.53,67 in short, if degenerative arthritis is absent, and the carpus can be salvaged, one may consider bone grafting, either standard or vascularized, with or without internal fixation. however, if substantial degenerative arthritis is present, limited or complete wrist arthrodesis may yield a stable, painless result. alternatively, proximal row carpectomy or anterior interosseous nerve (ain) and posterior interosseous nerve (pin) denervation neurectomy may be considered. prior to surgery a trial of cast immobilization to simulate the fused wrist, or an ain or pin block may be helpful to clarify the possible effect of the desired procedure on the patient s symptoms. when the scaphoid is salvageable and bone grafting is attempted, one must consider the relative risks and benefits of nonvascularized or vascularized bone grafting. nonvascularized bone grafting is probably sufficient for most waist fracture nonunions and those with preserved vascularity of the proximal pole. as noted previously, however, should standard bone grafting fail, future surgery is likely also to be unsuccessful.3,5456 the benefits of vascularized bone grafting for scaphoid nonunion include preservation of the blood supply, primary bone healing, and maintenance of structural integrity. therefore, consideration of vascularized bone grafting is wise, particularly in the setting of osteonecrosis and proximal pole nonunions. recently the mayo clinic documented the outcomes and complications of vascularized bone grafting for scaphoid nonunion in a series of 52 nonunions in 51 patients.68 two patients (two scaphoids) were lost to follow - up, with 50 patients and 49 scaphoids available for review. in this study, the 1,2-intercompartmental supraretinacular artery (1,2-icrsa) was used as a reverse - flow vascularized bone graft for scaphoid nonunion.56,69,70 previously published union rates for vascularized bone grafts in the scaphoid have ranged from 27% to 100%, although there are presently fewer than 10 such published studies in the literature with approximately 100 total patients. in the mayo clinic study, there were 42 male patients and 9 female patients, with an average age of 23 years and a mean follow - up of 7.6 months. the nonunions were evenly divided between the proximal pole and the waist ; and evidence of avascular necrosis was present in 24 scaphoids based on intraoperative surgical findings, mri, and radiographs. overall, 72% of the scaphoid fractures achieved union with vascularized bone grafting (36/50), and healing occurred at an average of 16 weeks (range 840 weeks). factors adversely affecting the union rate included female sex (union rate : 30% vs. 82% in males) ; tobacco use (union rate : 81% in nonsmokers vs. 46% in smokers), and proximal pole avascularity [48% union rate in the presence of avascular necrosis (avn) vs. 91% in the absence of avn ]. the type of internal fixation also influenced the union rate. simple k - wire fixation resulted in a 53% union rate, whereas screw fixation resulted in an 88% union rate. carpal collapse with formation of a humpbacked deformity was present in 50% of the failures versus 11% of patients who went on to union. interestingly, in this series of patients, the fracture - dislocation did not affect the union rate, with waist fractures achieving 70% union and proximal poles achieving 72% union. prior surgery resulted in a healing rate of 64% compared to 73% in those with no previous operations. complications in this series resulted in three infections, three patients with graft extrusion, two patients with graft resorption, and four patients with progressive degenerative changes that required further operative therapy. five patients underwent subsequent surgeries including ain and pin neurectomy (n = 2), repeat autologous bone grafting (n = 1), scaphoid excision with four - corner fusion (n = 1), and total wrist fusion (n = 1).68 notably, the union rate was 100% in cases of nonunion in which avn was absent and which underwent the vascularized bone grafting procedure with screw fixation.68 two studies in the literature showed relatively low healing rates of 60%54 and 27%,71 respectively. in the study by boyer.,54 10 proximal pole nonunions underwent vascularized bone grafting with a 60% union rate. the four failures occurred in patients who had previously undergone treatment with a bone grafting procedure, suggesting that prior surgery adversely affects the outcome of the 1,2-icrsa bone graft.54 in a second report by straw.,71 22 nonunions underwent the 1,2-icrsa bone graft with only a 27% union rate (n = 6). altogether, 16 of these patients had avn of the proximal pole, and the k - wires that were used for fixation were removed at 8 weeks regardless of union status. of those 16 patients with osteonecrosis of the proximal pole, only 2 had union, a 12.5% union rate, compared to 4 of 6 (67%) in the patients with a preserved vascular supply to the proximal pole.71 it could be concluded that a 1,2-icsra vascularized bone graft may not improve union rates in patients with significant avn of the proximal pole fragment.71 patients who overall are poor candidates for vascularized bone grafting are those with carpal collapse and scaphoids with a large defect or excessive comminution of the proximal fragment. likewise, patients with radiocarpal arthritis may have a more disappointing outcome.56 these series and the recent study from the mayo clinic present less optimistic results than a prior study by steinmann. in which a 100% union rate was achieved in 14 patients at a mean 11.1 weeks.56 the reasons for this increased failure rate are not entirely clear. however, it probably relates to broader acceptance of the procedure with resultant expanded indications for use of this vascularized bone grafting technique. additionally, there is enthusiasm and willingness to try everything, including a vascularized bone graft prior to performing a salvage type procedure. the limitations of this larger, more recent study relate to its retrospective nature and the multiple surgeons involved. factors overall that are thought to be associated with failure of 1,2-icsra in scaphoid nonunion include female sex, tobacco smoking, proximal pole avn, humpback deformity, comminuted proximal pole and use of a k - wire or no metallic internal fixation. in the authors opinion, operative fixation of scaphoid fractures is the treatment of choice in cases of acute fracture in which the fracture is clearly visible on plain film radiographs, as we believe this implies that the fracture is displaced. several authors have demonstrated low morbidity and satisfactory outcomes following operative fixation.1,30,3543 the surgical time is minimal, morbidity is minimal, and complications are infrequent. when a patient with clinical symptoms suggestive of scaphoid fracture is evaluated, initial radiographs are obtained at the time of presentation. if a scaphoid fracture is readily identified on plain films, it represents displacement, and acute operative treatment is recommended, with our preference being percutaneous placement of a cannulated screw. if no fracture is seen, and clinical findings suggest a scaphoid fracture, a mri scan should be performed. if mri reveals a fracture, nonoperative treatment may be undertaken with a short arm thumb spica cast with the wrist in neutral position, immobilizing the thumb to the interphalangeal joint, unless the fracture is at the proximal pole. this is maintained for 6 weeks with a ct scan obtained at this time point. if the ct scan remains suggestive of an unhealed fracture, cast immobilization is maintained for an additional 6 weeks. the exception to this involves cases in which a fracture of the proximal pole is identified on mri. in these cases after operative fixation, the patient is placed in a volarly based thumb spica splint. if the patient is unlikely to comply with postoperative activity restrictions, activity is restricted to not lifting > 2 pounds and no repetitive use ; utilizing the hand for activities of daily living and personal hygiene only is allowed. at 6 weeks, a ct scan is obtained to evaluate for evidence of union. if evidence of healing, such as disappearance of the fracture line, spot welding between fragments, or callous formation is present, immobilization is discontinued, with gradual return to activities. the literature suggests that partial union is often present but usually progresses to full union without the need for additional immobilization.72 if no evidence of healing is noted, immobilization is continued, with another ct scan obtained 46 weeks thereafter. in the setting of an established scaphoid nonunion without significant arthritis, vascularized bone grafting with a technique such as the 1,2-icrsa graft and fixation with metallic hardware, preferably a screw, is advised. when significant degenerative changes preclude scaphoid reconstruction, salvage procedures such as proximal row carpectomy, neurectomy, or limited or complete wrist arthrodesis are indicated. in summary, this article provides an algorithm for the diagnosis, evaluation, and treatment of scaphoid fracture and possible sequelae, including nonunion. | backgroundscaphoid fractures are commonly seen in orthopedic practice. an organized and thoughtful approach to diagnosis and treatment can facilitate good outcomes. however, despite optimal treatment, complications may ensue. in the setting of nonunion or an avascular proximal pole, vascularized bone grafting may be needed.methods and resultsin this article we review the literature regarding these injuries and describe an approach to diagnosis, treatment, and management of scaphoid fractures and nonunions.conclusionscaphoid fractures and nonunions may present as challenging problems in practice, but a systematic and deliberate approach can facilitate optimal results. |
it is estimated that, worldwide, approximately 937 million adults are overweight and 396 million are obese. this rising trend continues unabated both globally and in the united states, which claims the largest population of overweight and obese adults. various etiologic factors associated with obesity have been reported, including a number of genes identified from genome - wide scans and functional genomic studies as well as some viruses and bacteria [4 - 7 ]. the current prevailing hypothesis centers on the premise that the problem of obesity is one of energy imbalance, wherein total energy intake far exceeds energy output. in addition, the global epidemic of obesity has been attributed to heuristic observations of an increase in the consumption of high - energy / high - fat content foods coupled with a sedentary lifestyle that expends little energy. for example, the increased consumption of some food types, including beverages and foods that contain high - fructose corn syrup (hfcs), is speculated to be associated with obesity. moreover, in a previous study, mice given hfcs - sweetened water gained more weight and showed increase adiposity. while the results of this animal study seem to provide experimental evidence that supports the hypothesis that consumption of hfcs causes obesity, the results from epidemiological and clinical studies in human are inconclusive, leaving the question of hfcs association with obesity unanswered. therefore, whether or not the intake of certain food types predisposes an individual to increased risk for obesity needs to be examined. quantifying the amount of food an individual consumes daily is difficult, and determining the intake of specific food types is intractable, thus posing significant challenges to the investigation of food intake and the development of obesity. it is known that the loss - adjusted food availability data from the economic research services (ers) of the united states department of agriculture (usda) constitute time series data on the national food supply of several hundred food - types targeted to the food marketing system. these data are represented as per capita food availability and are useful for studying food consumption trends, as they are an indirect measurement of actual food intake. to determine whether or not excess energy intake or the consumption specific food types contribute to the development of obesity, we surveyed loss - adjusted food availability and obesity prevalence data to investigate the correlation between total energy intake and consumption of certain food types with rising trends in obesity. we confirmed a novel association of rising obesity trends with increased corn product consumption that may be linked to the growing and ubiquitous presence of genetically modified (gm) or engineered (ge) corn in the human diet. we obtained the loss - adjusted food availability data (1970 - 2008) from the food availability (per capita) data system at the united states department of agriculture (usda) economic research services (ers). the per capita food availability data was adjusted for food spoilage, waste, and other losses, as well as converted to average daily per capita calories. the obesity prevalence and trends data spanning from 1995 and 2008, comprised of health survey data on health risk behaviors such as obesity, were obtained from the behavioral risk factor surveillance system (brfss) at the centers for disease control and prevention (cdc). trends data with body mass index (bmi) 30, classified as obese, were used in this study. the national health and nutrition examination survey (nhanes) obesity data between 1960 and 2008 were downloaded from the e - stats data bank at cdc. data for genetically modified corn varieties adopted in the u.s. were obtained from usda ers national agricultural statistics service. we analyzed the relationship between the trends in obesity prevalence and the average daily per capita calories consumed for various food types using pearson 's correlation. to validate the positive correlations, we investigated the dependence of the obesity trends on different food types by fitting a multiple linear regression using both full and reduced model functions. since the brfss obesity prevalence and trends data were only available between 1995 and 2008, we tested the correlation between each food type from 1995 through 2008 by pearson 's correlation and multiple linear regression. the food types analyzed were red meat, poultry, dairy products, grains, fats and oils, sweeteners, and average total calories consumed daily per capita. in addition, specific food types such as fish, eggs, nuts, and some dairy products exhibiting either negative or no change in the trends of consumption between 1995 and 2008 were not subjected to pearson 's analysis. we obtained the loss - adjusted food availability data (1970 - 2008) from the food availability (per capita) data system at the united states department of agriculture (usda) economic research services (ers). the per capita food availability data was adjusted for food spoilage, waste, and other losses, as well as converted to average daily per capita calories. the obesity prevalence and trends data spanning from 1995 and 2008, comprised of health survey data on health risk behaviors such as obesity, were obtained from the behavioral risk factor surveillance system (brfss) at the centers for disease control and prevention (cdc). trends data with body mass index (bmi) 30, classified as obese, were used in this study. the national health and nutrition examination survey (nhanes) obesity data between 1960 and 2008 were downloaded from the e - stats data bank at cdc. data for genetically modified corn varieties adopted in the u.s. were obtained from usda ers national agricultural statistics service. we analyzed the relationship between the trends in obesity prevalence and the average daily per capita calories consumed for various food types using pearson 's correlation. to validate the positive correlations, we investigated the dependence of the obesity trends on different food types by fitting a multiple linear regression using both full and reduced model functions. since the brfss obesity prevalence and trends data were only available between 1995 and 2008, we tested the correlation between each food type from 1995 through 2008 by pearson 's correlation and multiple linear regression. the food types analyzed were red meat, poultry, dairy products, grains, fats and oils, sweeteners, and average total calories consumed daily per capita. in addition, specific food types such as fish, eggs, nuts, and some dairy products exhibiting either negative or no change in the trends of consumption between 1995 and 2008 were not subjected to pearson 's analysis. to determine the correlation between obesity and food type intake, we first assessed the suitability of the brfss dataset for our analysis. use of the nhanes obesity prevalence data was hampered by the availability of only five data points, nhanes (1999 - 2000), (2001 - 2002), (2003 - 2004), (2005 - 2006), and (2007 - 2008), in contrast to the 14 data points in the brfss dataset between 1995 and 2008, which is statistically more favorable for our study. however, the brfss dataset, which is based on self - reported weights and heights, is generally considered inferior to the nhanes obesity prevalence data. despite the qualitative and quantitative differences between the nhanes and brfss data, our results show that the obesity trends between 1995 and 2008 derived from the two datasets were remarkably similar by regression analysis (fig. 1). therefore, we used the brfss data for our correlation study with the food trends data since it is statistically more robust than the nhanes dataset. we analyzed the usda ers loss - adjusted food availability data, which include seven major aggregated food groups including 1, meat, eggs, and nuts ; 2, dairy ; 3, fruit ; 4, vegetables ; 5, flour and cereal products ; 6, added fats and oils, and dairy fats ; and 7, caloric sweeteners. these groups are further comprised of more than 100 individual or specific food types (commodities). analysis of these food types revealed that a large number of them including fresh vegetables, fresh fruit, beverage milk, fish and shellfish, fruit juice, nuts, and others, showed either negative trends or no change in trends of consumption and did not coincide with rising trends in obesity (fig. is thought to contribute to obesity, we first analyzed the trends in calorie intake between 1995 and 2008. the food availability data indicated that the average daily per capita total calorie intake has plateaued since year 2000, whereas obesity exhibited a rising trend (fig. 3a), and pearson 's analysis showed a correlation coefficient of 0.79 (table 1). in contrast, strong positive correlations with obesity were unexpectedly found for chicken and corn products (fig. 3b and c), with pearson 's correlation coefficients of 0.96 and 0.99, respectively (table 1). 3d). however, further analysis revealed that, with the exception of cheddar and mozzarella cheese, most other cheeses, such as provolone, parmesan, swiss cheese, blue cheese, and others, showed little or no changes in consumption trends between 1995 and 2008, and pearson 's analysis of either cheddar (fig. 3f) did not show correlation with rising obesity. even though correlation with obesity was not found for " added fats and oils, and dairy fats " (fig. 3 g), with a correlation coefficient of 0.86 (table 1), analysis of salad and cooking oils (fig. 3i) revealed correlation with obesity, each with a correlation coefficient of 0.97 (table 1). these correlations subsequently did not cross - validate upon further analysis by multiple linear regression (see below). additionally, either poor or negative correlations were found for foods such as flour and cereal products, shortening, red meat, caloric sweeteners, and hfcs, with correlation coefficients of -0.03, -0.18, -0.40, -0.74, and -0.38, respectively (fig. 3j - n, and table 1). the consumption of refined cane and beet sugar (fig. the consumption of corn as a fresh vegetable constituted only a small percentage (averaging 0.01%) of the total calorie intake between 1995 and 2008. to further test these positive correlations with obesity trends, we performed a fitting by multiple linear regression analysis with food types that showed correlation coefficients > 0.95, which included chicken, corn products, dairy fats, salad and cooking oils, and total cheese, in a full model function. this analysis showed that only corn products had p - values smaller than 0.05 (table 2), suggesting that consumption of corn products had a significant effect on rising obesity trends. in the reduced model, we analyzed corn products and total cheese, which have p - values closest to 0.05 from the full model analysis, and our results confirmed a correlation between corn products, but not total cheese, and obesity trends (table 2). it is noteworthy that hfcs is classified separately as a caloric sweetener and not aggregated with other corn products. we were not able to fully analyze whether or not corn product consumption correlated with obesity trends between 1970 and 1994 because the national health and nutrition examination survey (nhanes) datasets are only available in four cross - sectional, nationally representative surveys prior to 1995, including nhanes i (1971 - 1975), ii (1976 - 1980), and iii (1988 - 1994), thus yielding only three data points for pearson 's correlation analysis of corn - rich products. nevertheless, we showed that the trends in obesity prevalence and corn product consumption between 1970 and 1994 did not align (fig. we were also aware that genetically modified (gm) corn has been planted in the u.s. since 1996. to further investigate the relationship between bioengineered corn and rising obesity, we obtained data on the adoption of gm corn from the usda, which covered the period between 2000 and 2008, for comparison with rising obesity. these data did not take into account the use of gm corn for other purposes besides as a food or animal feed. despite this limitation, our result shows that the trends of obesity and adoption of gm corn were similar (fig. we further asked whether or not the consumption of corn products might be associated with the demographic distribution of the population. using the nhanes stratified obesity prevalence data between nhanes iii (1988 - 1994), nhanes (1999 - 2000), (2001 - 2002), (2003 - 2004), (2005 - 2006), and (2007 - 2008), we examined the relationship between corn product consumption and race / ethnicity of men and women between 1995 and 2008. our results show that the trends of obesity and corn product consumption rose in parallel irrespective of gender among non - hispanic white men and women (fig. 5a and b), non - hispanic black men and women (fig. 5c and d), and mexican - american men and women (fig. 5e and f), thus suggesting that the association of rising obesity trends with increased corn product consumption is independent of race / ethnicity and gender. our analysis of obesity and food type consumption trends data in this report yielded three major findings. first, it has been long accepted that overconsumption of food coupled with a sedentary lifestyle results in a positive energy imbalance, which is a formula for obesity development. our analysis in this report, however, indicates that even though total calorie intake in the u.s. has plateaued in recent years, the incidence of obesity continues to rise, thus suggesting that rising obesity trends do not correlate with total energy intake. alternatively, it is conceivable that the total caloric intake has plateaued while the levels of physical activity have also not increased, thus explaining the intransigent obesity trends. it was shown recently that rats given hfcs along with a regular chow diet gained more weight than control rats, even when they consumed the same amount of calories. further, consumption of an hfcs - containing diet increased visceral fats and blood triglycerides over time. however, our results show a negative correlation of hfcs with rising obesity, as hfcs consumption has been on the decline since 2000. however, this negative correlation does not refute the underlying biological role of hfcs in obesity. instead, it suggests that hfcs consumption on the whole may not contribute to rising obesity trends. though we initially also observed positive correlations between increased consumption of chicken, salad and cooking oils, dairy fats, and total cheese with obesity, subsequent multiple linear regression analysis and cross - validation of these results revealed a lack of significance in these correlations. the above observations suggest that additional factors may be involved in rising obesity trends. therefore, our third finding of a correlation between increased corn product intake and rising obesity between 1995 and 2008 is intriguing, as these foods are not generally considered unhealthy. what are the underlying etiologic links between these foods and obesity ? in the ers dataset, corn products are considered an aggregate comprised of flour and meal, hominy and grits, cornstarch, and other corn products, which are widely used in the manufacture of a large variety of food products consumed by humans. recently, it was reported that approximately 85% of the corn grown in the u.s. the increased ubiquity of gm or genetically engineered corn products in human food sources is noted, but their potential impact on human health has not been investigated despite recent reports of hepatorenal toxicity in rats fed gm maize. moreover, the rising trends in obesity coincide, in part, with the introduction of gm corn in foods and animal feeds in the u.s.. these observations prompted us to hypothesize that consumption of gm corn products may contribute to rising obesity trends. the implications of our results and the new hypothesis raised here are provocative but testable, as the effects of gm corn products can be assessed in molecular and animal models of obesity. no data are currently available on how much genetically engineered food is on the market due to a lack of proper labeling and traceability. we further speculate that the bacterial antigen derived from the bacillus thuringiensis (bt) entomocidal crystalline protein protoxin, which is genetically engineered into corn to confer resistance to insect pests, may be the underlying culprit that causes anomalous adipose tissue dysregulation and obesity development. while our trends study has yielded novel insights into the potential impact that some food types may have on the development of obesity, there are some possible confounding factors that should be discussed. it is noteworthy that the loss - adjusted data do not reflect actual consumption or the quantities of food ingested. moreover, it was difficult to collect data on the actual amount of food or the specific food types consumed, as most previous clinical studies have relied on questionnaires and voluntary reporting or recollection of food consumed by study subjects, which are well known to be prone to psychosocial behavioral errors. in the absence of true food consumption data, therefore, trends in food use obtained from the usda ers food availability data served as an alternative indirect measure of whether americans are consuming more or less of various foods over time. similarly, the weight and height information collected from phone interviews for the brfss obesity trends data is also highly susceptible to erroneous self - reporting. in contrast, physical measurements for weight and height were obtained from participants in the nhanes studies. despite such shortcomings, in addition, the data for the rate of gm corn adoption in the u.s. did not take into consideration the different uses of these transgenic corns other than as foods and feeds. although it is clear that transgenic corn has penetrated into human foods and animal feeds, and the consumption of gm crops has been deemed safe, precise data regarding the amounts and types of foods containing transgenic corn products are unavailable, and the correlation with increased emergence of common human diseases including diabetes and obesity has not been investigated. taken together, our results reveal a novel association of corn product consumption with rising trends of obesity, which may be linked to the increased ubiquity of transgenic corn in the diet. | the rapid rise in the incidence of obesity has emerged as one of the most pressing global public health issues in recent years. the underlying etiological causes of obesity, whether behavioral, environmental, genetic, or a combination of several of them, have not been completely elucidated. the obesity epidemic has been attributed to the ready availability, abundance, and overconsumption of high - energy content food. we determined here by pearson 's correlation the relationship between food type consumption and rising obesity using the loss - adjusted food availability data from the united states department of agriculture (usda) economic research services (ers) as well as the obesity prevalence data from the behavioral risk factor surveillance system (brfss) and the national health and nutrition examination survey (nhanes) at the centers for disease control and prevention (cdc). our analysis showed that total calorie intake and consumption of high fructose corn syrup (hfcs) did not correlate with rising obesity trends. intake of other major food types, including chicken, dairy fats, salad and cooking oils, and cheese also did not correlate with obesity trends. however, our results surprisingly revealed that consumption of corn products correlated with rising obesity and was independent of gender and race / ethnicity among population dynamics in the u.s. therefore, we were able to demonstrate a novel link between the consumption of corn products and rising obesity trends that has not been previously attributed to the obesity epidemic. this correlation coincides with the introduction of bioengineered corns into the human food chain, thus raising a new hypothesis that should be tested in molecular and animal models of obesity. |
for the past decade, scientists, institutions, governments, and policymakers have warned the general public about serious health hazards associated with chemicals known as endocrine disruptors (eds). eds are exogenous compounds that interfere with the synthesis, secretion, transport, metabolism, and/or action of endogenous hormones that are responsible for normal homeostasis, reproduction, and development. chemicals with hormonal activity can be divided into three main groups : (i) synthetic compounds used in industry and agriculture as well as in consumer products, (ii) synthetic compounds used in pharmaceutical drugs, and (iii) natural compounds present in the food chain (i.e., phytoestrogens, compounds that are structurally similar to estrogen (e2)). only (i) synthetic compounds used in industry and agriculture and consumer products will be discussed in presented paper. within this class, compounds can be further subcategorized into those that are persistent in all elements of the environment, bio - accumulative, transportable over long distances, and capable of adversely affecting life forms that reside within short and long distances from the site of contamination. these compounds include dichlorodiphenyltrichloroethane (ddt, a pesticide), polychlorinated biphenyls (pcbs), polychlorinated naphthalenes (pcns), polychlorinated dibenzodioxins (pcdds), polychlorinated dibenzofurans (pcdfs), and polybrominated diphenyl ethers (pbdes) (figure 1). because xenobiotics can accumulate in the body for an extremely long period of time (i.e., decades) for instance, pesticides and other synthetic compounds used in the 1950s have polluted the air that we breathe, the water that we drink, and the soil that we grow our food in. in addition, many of these compounds are nonbiodegradable. studies have shown the presence of eds both in adipose tissue and other organs, in almost all humans and many animals. human exposure to environmental toxicants is mediated via food and water chains, breathing, and dermal absorption, with approximately 90% of the exposure coming from food. moreover, several mammalian organs (e.g., ovary, breast, uterus, cervix, bone, muscle, and skin) rely on sex steroids for normal function, and these organs are especially vulnerable to endocrine disruption by environmental toxicants. much of the information on the impact of eds on human health has come from in vivo studies where animals were exposed to a single compound, usually at an acute (i.e., pharmacological) dose ; however, humans are exposed to several compounds at unknown concentrations for unknown durations. for example, hospital patients receive an average of six drugs daily (i.e., aspirin, antihistamines, antibiotics, anti - cough syrup, etc.). food and water may also contain low levels of organic and inorganic compounds (e.g., pesticides and heavy metals) and solvents (e.g., benzene, toluene, and chloroform), and air is filled with hundreds of chemicals (e.g., industrial pollutants, smoke, gasoline vapors, etc.). the ability of these environmental toxicants to affect human health depends on several factors, including interactions between compounds ; absorption, metabolism, accumulation, and excretion of compounds ; and the ability of some compounds bind to cell receptors which can affect hormone action. unfortunately, little is known about how these environmental toxicants interact with each other existing in a mixture. it is known that such xenobiotic acts in different ways depending on whether their action is observed on the fetus, newborn babies, or adult individuals, depending on the period during whose body has been exposed to these factors. in many cases, the effect of their actions may be undetected until sexual maturity, especially when exposure occurs during a period of embryonic development or shortly after birth. the female reproductive cycle is a complex process comprised of gametogenesis, embryogenesis, menstruation, ovulation, possible pregnancy, endometrial, and mammary gland changes. women are born with all the oocytes they will ever have through their life, and therefore oocytes are more vulnerable to toxic chemicals than germ cells in men who continue to make more germ cells. oocyte maturation, the final step of germ cell differentiation, determines reproductive capacity. follicles are especially susceptible to the adverse effects of environmental toxicants, and developing oocytes may be damaged directly or indirectly by action on follicular cells (i.e., granulosa cells) that may also be vulnerable to endocrine disruption, thereby affecting oocyte function indirectly. a previous study illustrated that human oocytes harvested from follicles with elevated levels of polycyclic aromatic hydrocarbons (pcahs) had fewer cell divisions after in vitro fertilization. chloroorganic mixtures such as pcbs and ddt, as well as its metabolites, have also been reported to affect puberty, development, and oocyte viability adversely. some of these studies point to unpredictable changes of translational regulation within the oocyte under the influence of pcbs. polyspermia (secretion of an excessive amount of semen) has also been demonstrated in cattle exposed to an environmentally relevant mixture of more than 15 organochlorines. presently, additional research is needed to better understand the molecular mechanisms behind mammalian ovotoxicity caused by exposure to environmental toxicants. hormone - mimicking compounds can bind to cell receptors, interfere with hormone action, and affect ovarian function. how eds affect ovarian function is not yet clear, but a disruption in gonadotropin (i.e., follicle stimulating hormone (fsh) and luteinizing hormone (lh)) secretion and feedback mechanisms involving e2 and progesterone (p4) may be involved. damaged oocytes can affect overall hormone production and follicular function, resulting in an endocrinological imbalance (i.e., a decrease in e2 and p4, but an increase in fsh and lh) and ovarian failure. fertility in sexually mature women depends largely on the maintenance of healthy follicles, and their steady production ensures that an adequate number of follicles reach the antral stage. the stage of development at which the follicles are destroyed determines the influence of these factors on the fertility of women. complete depletion of the follicle reserve results in irreversible infertility ; partial depletion of the follicle reserve results in moderate effects on periodicity. damage to large follicles can also lead to reversible acyclic disorders that affect hormone production and ovulation. nevertheless, the effects of pops on the hypothalamic - pituitary - ovarian (hpo) axis are generally reversible because they do not permanently affect the follicle reserve. tcdd (2, 3, 7, 8-teterachlorodibenzo - para - dioxin) is an isomer of pcdd, and one of the most toxic man - made compounds to pollute our environment. studies on the mechanism of tcdd action during ovulation indicate a dysfunction in the hpo axis. misregulation of fsh and lh secretion before ovulation was noted in rats exposed to tcdd. moreover, gonadotropin - releasing hormone (gnrh) inhibited the surge in fsh and lh secretion, thereby restoring ovulation [5, 6 ]. for instance, gore showed methoxychlor to affect gnrh expression in hypothalamic gt1 - 7 cells. in a study from our laboratory, tcdd inhibited e2 secretion by follicular cells and p4 secretion by luteal cells dose - dependently. these adverse effects on hormone production were mediated in part by enzymes involved in steroidogenic biosynthesis. in luteal cells, tcdd action was independent of e2 receptor stimulation, instead involving the aryl hydrocarbon receptor (ahr). on a final note, other environmental toxicants (i.e., pcdfs, biphenyls, ddt, and methoxychlor) can also block the surge in lh and fsh secretion during the female reproductive cycle [10, 11 ]. polychlorinated biphenyls (pcbs) are the man - made chemicals that may disrupt follicular steroidogenesis either by mimicking natural hormones as agonist or antagonist, altering the pattern of hormone synthesis, modulating hormone receptor affinities or numbers, or by altering enzymes involved in hormone secretion. in our previous study, we showed that the orthosubstituted pcb 153 congener accumulated preferentially in the follicular wall when compared to the nonorthosubstituted pcb 126 congener. 71%, 71.4%, and 30.4% of the total exposure for pcb 153 were in small, medium, and large follicles, respectively (figure 1). interestingly, about 70% of pcb153 accumulated in early antral and antral follicles and only 30% in preovultory follicles. the consequence was a reduction in estradiol secretion by early antral and antral follicles and lack of influence on estradiol secretion by preovulatory follicles. moreover, moreover, it has been showed that action on estradiol secretion was correlated with action on aromatase activity [1214 ]. a similar dose - responsive relationship was reported after exposure of follicular cells to a mixture of organic pollutants or a mixture of pbdes. polybrominated dibenzoethers (pbdes) are persistent and ubiquitous environmental toxicants found at increasing levels in humans and animals. despite recent bans by the european union, united states, and china on the production of penta- and octa - bde as well as on the diminished use of pbde in japan, 2,2,4,4-tetra - bde (bde-47), 2,2,4,4,5-penta - bde (bde-99), and 2,2,4,4,6-penta - bde (bde-100) are the major pbde congeners present in humans and animals. the literary data related to the effects of pbde mixture are limited predominantly to commercial mixture de-71 which contains mainly penta - bdes (bde-99 and bde-100) and tetra - bde (bde-47). indicated that de-71 (a mixture of bde-99, bde-100, and tetra - bde) affected the production of thyroid and sex steroids and the development of reproductive organs [20, 21 ]. results from our laboratory have shown an increase in the p4/testosterone (t) ratio but a decrease in the t / e2 ratio, in ovarian follicles suggesting premature luteinization of antral follicles. removal of the pbde mixture from cell cultures did not reverse adverse effects. in a follow - up study, we reported changes in the levels of steroidogenic enzymes (e.g., 17-hydroxysteroid dehydrogenase (17-hsd), cytochrome p450, family 17, subfamily a, polypeptide 1 (cyp17), and aromatase (cyp19)) by pbde congeners 47, 99, and 100. last published data showed fast activation of cyp2b1/2, and late activation of comt (with a very low basal sult1a activity) in ovarian follicles by bde-47 indicates a possible action of locally produced hydroxylated metabolites prior to their detoxification. additionally, it have been showed that 5-oh - bde-47 and 6-oh - bde-47 have a different mechanism of action in ovarian follicles from their parent compound and lead to an increase in estradiol secretion. the metabolites stimulate aromatase expression and activity, while the parent compound increases androgen production and stimulation of 17-hsd protein expression and activity. polychlorinated naphthalenes (pcns) are members of a large and diverse group of compounds with several industrial applications. pcns occur as mixtures of congeners sold under various trade names (e.g., halowax, nibren wax, and seekay wax). the toxicological characteristics of pcns are similar to those caused by pcbs, pcdds, and pcdfs [27, 28 ]. presently, there are little data on the toxicity of pcns in experimental animals, and our recently published data was the first showed direct action on ovarian function. we showed an increase in basal testosterone secretion in all doses used, with the highest stimulatory action of the smallest dose, which was accompanied by a parallel decrease in basal estradiol secretion, induced by halowax 1051 suggesting androgenic properties of halowax 1051. as a mechanism we propose direct stimulatory action on 17-hsd activity and protein expression, enzyme responsible for testosterone synthesis and inhibitory action on cyp19 activity, and enzyme responsible for conversion testosterone to estradiol. it should be taken into consideration that as in the case of bde-47, the effects of exposure to pcns may be due to the side effects of pcn metabolites. examining the effects of the halowax 1051 on phase i (cyp1a1) and phase ii (sult1a and comt) enzyme activities and expression in cultured ovarian follicles we showed fast activation of enzymes involved in phase i and concurrent inhibition of enzymes involved in phase ii metabolism confirming our suggestion that the observed effects of halowax 1051 are partially result from the action of metabolites formed locally in ovarian follicles. while hcbz is toxic to humans [31, 32 ] and animals, information on the effects of hcbz in ovarian steroidogenesis is limited despite data that showed an increase in the p4 serum level in superovulated rats exposed to hcbz. treatment of cynomolgus monkeys with hcbz, on the other hand, decreased the p4 serum level and unaltered the e2 serum level during the luteal phase. in a recently published study, we determined in vitro accumulation of hexachlorobenzene (hcbz) and pentachlorobenzene (pecbz) in porcine ovarian follicles, the effect on steroidogenesis, and the expression of enzymes responsible for steroid synthesis. we showed that sixty percent of the hcbz and almost 100% of the pecbz that was added to the culture medium accumulated in ovarian tissue, and only 1% of each was found in the medium. moreover, we showed inhibitory hcbz effect and stimulatory pecbz effect on testosterone and estradiol secretion. as a conclusion we mentioned that the greater exposure to an estrogenic action of pecbz than antiestrogenic hcbz would be a consequence of the preferential accumulation of pecbz in the ovarian follicles we showed that hcbz by fast activation of i and ii phase metabolism is probably metabolized to pecbz in placental tissue (gregoraszczuk., unpublished data). toxicants usually occur in a mixture of several toxicants, making it difficult to predict adverse effects on human health. demonstrated human follicular fluid obtained from women to contain 1, 1-dichloro-2, 2-bis (p - chlorophenyl)-ethylene (p, p-dde), mirex (a pesticide), hexachloro - ethane 1, 2, 4-trichlorobenzene, and numerous pcbs (i.e., pcbs 49, 153, and 180). of these, p, p-dde was the most frequently detected ed in follicular fluid and serum. several environmental toxicants are er agonists, and they include estrogenic steroids (natural and synthetic), phyto- and mycoestrogens, and xenoestrogens (e.g., pesticides, plasticizers, and alkylphenols). in addition to simple additive effects, interactions between different chemicals in a mixture may result in either a weaker (antagonistic) or stronger (synergistic, potentiated) combined effect than would be expected from knowledge about the toxicity and mode of action of each individual compound. these interactions may occur during toxicant uptake, distribution, metabolism, and/or excretion (i.e., toxicokinetic phase), or during toxicant binding to receptors and cellular targets (i.e., toxicodynamic phase) [39, 40 ]. moreover, some estrogenic compounds exert their effects, not by binding to er but rather by binding to estrogen plasma transport proteins, resulting in an increase in free endogenous e2. nevertheless, mixtures containing both xenoestrogens and endoantiestrogens may have no adverse effects. in our previously published data, to determine which compounds within a pcb - ddt - dde mixture stimulated e2 secretion, we exposed cells to pcbs 118, 138, 153, and 180, ddt, and dde alone or in different combinations. interestingly, ddt and dde affected e2 secretion, and these results are in agreement with another series of experiments that used mixtures of pbdes. in the next experiments, using western blot analysis indicated that pcbs mixture (marine mix) is an inducer of ahr and mixed - type cyp inducer (cyp1a1 and cyp2b1) while pbdes mixture (mjosa mix) is an inducer of er and cyp2b. early puberty is a recent growing concern as there are reports of many girls reaching their first menstruation and developing breasts earlier in life than was the case 40 years ago. early puberty associates with polycystic ovarian syndrome (pcos), obesity, breast cancer, depression, and a number of social challenges such as experimentation with sex, alcohol, or drugs at a younger age. moreover, earlier menarche and thelarche ages have been reported in girls after exposure to pcbs, pbbs, ddt, and/or phthalate esters [46, 47 ], and precocious puberty has been observed following exposure to ddt metabolites. the acyclicity of the syndrome is linked with the hyperfunctioning of theca and hypofunctioning of granulosa cells. pcos also associates with other processes (e.g., neuroendocrine function and ovarian steroidogenesis) and diseases (e.g., insulin resistance, and obesity) that are regulated by hormonal and metabolic factors. for instance, women with pcos have higher levels of bisphenol a (bpa, a plastics additive) and testosterone which is consistent with the decreased clearance of bpa that is often observed. although a cause - and - effect relationship has not been established, the role of environmental toxicants in the pathogenesis of pcos is worthy of further consideration. pof, the cessation of normal ovarian function before the age of 40, occurs in approximately 1% of women of reproductive age. the underlying causes of pof are largely known in most cases, and any factor that can decrease the ovarian reserve can result in pof. for instance, disruption of germ cell migration from the genital ridge to the developing gonad results in ovarian dysgenesis and pof. in addition, adult and in utero exposure of mice to bpa resulted in oocyte damage [52, 53 ], whereas exposure of women to cigarette smoke decreased fertility, in vitro fertilization (ivf) success rates, and the ovarian reserve resulted in earlier menopause and increased miscarriage rate. in another study, exposure of rats to tcdd in utero and throughout reproductive life resulted in premature reproductive senescence. endocrine disruption caused by acute exposure to environmental toxicants such as the ahr agonist tcdd suggests that ahr - mediated apoptosis of oocytes may be involved. breast cancer is the most frequent neoplasm affecting women residing in western countries and is the second leading cause of death. the general population is exposed to several hormonally active compounds on a daily basis. the majority of these compounds are xenoestrogens (e.g., pcahs, pesticides, pcbs, pbdes, ddt, selected drugs, fungicides, phytoestrogens, mycotoxins, bpa, and phthalates), and they can possess estrogenic action, affect estrogen levels, and/or bind to ers. the role of pcbs in breast cancer has been investigated intensively. data suggest that a correlation may exist between high levels of pcbs in mammary tissues or sera and breast cancer risk, while another study reported no association. high pcb levels upregulated cyp expression, suggesting that pcb metabolites may be critical for the pathogenesis of breast cancer. likewise, pang and colleagues reported that exposure of mcf-7 human breast cancer cells to pcbs 81, 126, and 39 increased cyp1a1 and cyp1b1 mrna levels, resulting in the formation of e2 metabolites. we demonstrated pcb 3 to induce and to be a substrate for cyp1a1 in mcf-7 cells. on the other hand, others have shown pcbs 52 and 77 to induce oxidative damage (i.e., dna strand breaks) in er ()/mda breast cancer cells but not in er (+) /mcf-7 cells, suggesting that the er receptor may play a protective role in breast cancer. moreover radice. demonstrated those pcb congeners such as pcbs 101, 118, 138, 153, and 180 increased proliferations of mcf-7 cells. our published data demonstrated that from investigated congeners (118, 138, 153, and 180), pcb138 and 153 had the highest stimulatory effects on basal mcf-7 cell proliferation as well as the highest inhibitory actions on basal caspase-9 activity. moreover, we showed that pcbs 138 and 153 contribute to the action of endogenous 17-estradiol on cell proliferation and apoptosis in the breast cancer cell line mcf-7. in vivo experiments have also shown pcbs to increase metastasis by triggering the production of reactive oxygen species (ros), thereby activating the rho - associated protein kinase (rock) signaling pathway or vascular endothelial growth factor (vegf) overexpression that stimulates endothelial hyperpermeability and transendothelial migration of cancer cells. an increase in mcf-7 cell proliferation by de-71 (a mixture of bde-47, -99, -100, -153, and -154) was also reported by mercado - feliciano and bigsby. pbde-209 also induced mcf-7 cell proliferation by affecting critical steps of the cell cycle. at the molecular level, on the other hand, kwieciska. have shown no changes in mcf-7 cell proliferation following exposure to bde-47, -99, -100, and -209 ; however, apoptosis was inhibited by decreasing caspase-9 activity in these cells. resistance to apoptosis associates with tumorigenesis as it enables tumorigenic cells to expand even in a stressful environment. thus, additional studies are needed to determine if exposure to pbdes can indeed cause breast cancer. when rodents were exposed to bpa early in life, there were changes in mammary gland morphogenesis and tumor susceptibility [7274 ]. these findings are supported by in vitro data which demonstrated bpa to induce transformation of mcf-10f human breast cancer cells. these cells formed tubule - like structures when cultured in 3d collagen matrix, but spherical masses were noted after bpa treatment, leading the authors to conclude that bpa produces adducts or ros which can inadvertently introduce a variety of dna modifications and cause breast cancer. others have shown bpa to stimulate proliferation but to inhibit apoptosis in mcf-7 cells [7678 ]. bpa may also induce breast cancer cell proliferation by upregulating cell cycle genes and downregulating antiproliferative genes, especially genes that control the g1/s transition via er signaling. ovarian cancer is the most prevalent type of gynecological cancer affecting women residing in western countries. as more than 60% of tumors are diagnosed at stage iii and certain forms of cancer are very aggressive, ovarian cancers are associated with a high mortality. while most cells undergo neoplastic transformation, including germ cells, granulose, and stromal cells, approximately 90% of tumors are derived from the ovarian surface epithelium (ose). similar to breast cancer, hormonal factors such as estrogen and xenoestrogens have been linked to ovarian cancer [80, 81 ] ; however, the role of environmental toxicants in ovarian cancer requires further study. studies in adult c57bl mice showed that orally administered pbdes-47, -85, and -99 to accumulate in the liver, adrenal cortex, and ovary in adult c57bl mice suggest a possible carcinogenic activity, especially in light of research showing that exposure of cho and ovcar-3 cells to pbde-209 initiated s and g2/m phases of the cell cycle, respectively. several in vitro studies have shown bpa to induce chromosomal aberrations in cho cells [84, 85 ], a common genetic alteration in cancer. moreover, ovarian cyst formation was observed in mice treated neonatally with bpa. ovarian cancer may stem from incessant ovulation, which may be linked to the formation of cysts that are frequently found in perimenopausal women. in a study from our laboratory, we demonstrated an increase in proliferation in ovcar- 3 cells treated with bpa. specifically, we showed bpa to promote the cell cycle by upregulating the expression of cyclin d1, cdk4, e2f1, e2f3, and pcna (a mediator of g1 to s - phase progression) and cyclin a (a mediator of g2-phase progression to mitosis), but by downregulating the expression of p21waf1/cip1, weel-1, and gadd45. additionally, we demonstrated a decrease in the expression of proapoptotic genes (i.e., fas, fadd, raidd, caspase-8, -10, -3, -6, and 7, cad, bax, bak, bok, and apaf-1) but an increase in the expression of prosurvival genes (i.e., bcl - x and mcl-1). bpa also activates a caspases - independent apoptotic pathway by inducing endonuclease g gene expression. also, hwang. using microarray analysis increased mrna levels of e2-responsive genes in er - positive ovarian cancer bg-1 cells under the influence of bpa. in a subsequent study, we showed bpa to trigger phosphorylation of stat3, erk1/2, and akt in ovcar-3 cells. cited results of research indicated that bpa acting as a mitogen as well as an antiapoptotic factor may be an additional factor responsible for ovarian cancer. in recent years there has been a growing evidence that exposure to chemicals in the environment poses a serious threat to human and animals reproduction via disrupting effects on endocrine function. despite the fact that these substances are persistent, they may be metabolized into more toxic compounds than the parent molecule in endocrine organs. this endocrine disrupting chemicals (edcs) adversely affect health and reproduction even at very low concentrations and may exert their effects on the embryo and fetus. the complexity and diversity of factors belonging to edcs, its direct action on the ovary, and disorders of the reproductive function of women indicate that the impact of environmental pollution as an important determinant factor in fertility should not be minimize. current estimates of cancer risk in humans do not account properly for transplacental and environmental (including occupational) exposure to xenoestrogens. it is important to reevaluate the role of xenoestrogens in cancer development using new approaches that better reflect the complexity of carcinogenesis. testing new compounds before they are allowed to use should be expanded to determine their effect on the endocrine system, in order to assess the hormonal activity. in addition, attention should be directed towards dose - response relationships in environmental toxicology. such studies can provide useful information that might have a significant impact on the strategies for risk assessment of toxic substances. | persistent organic pollutants (pops), such as polychlorinated dibenzo - p - dioxins (pcdds) and dibenzofurans (pcdfs), polychlorinated biphenyls (pcbs), and polybrominated ethers (pbdes), chloronaftalens (pcns), and bisphenol a (bpa), are stable, lipophilic pollutants that affect fertility and cause serious reproductive problems, including ovotoxic action, lack of ovulation, premature ovarian failure (pof), or polycystic ovarian syndrome (pcos). most of the representatives of pops influence the activation of transcription factors, not only activation of aromatic hydrocarbon receptor (ahr), but also the steroid hormone receptors. this minireview will focus on a variety of pah activities in oocyte, ovary, placenta, and mammary gland. the complexity and diversity of factors belonging to pops and disorders of the reproductive function of women indicate that the impact of environmental pollution as an important determinant factor in fertility should not be minimize. |
cardiovascular diseases are the most common cause of death worldwide (1). coronary artery disease is considered the most important cause of morbidity and mortality in the united states ; it is reported to have affected over 16 million people. in iran, cardiovascular diseases are the leading cause of mortality, also 46% of all deaths being attributed to ischemic heart diseases (2). although cardiac surgery is a successful part of cardiac care, it is considered a stressful, unwanted, life - threatening experience for many patients and is associated with fear and anxiety to the extent of affecting several aspects of the patient s personal life (3). concerns about coronary artery bypass graft (cabg) surgery are known as stressors (4). believe that stressors are circumstances and events that force people to react, and reactions to stress may manifest itself in various physical, mental, or behavioral forms (5). anxiety is an outcome of stressors such as fear of death, fear of change in health condition, environmental changes and separation from the support system (6), lack of adequate information, rumors spread by other patients, false beliefs, and distrust in surgery outcomes, all of which lead to pre - surgical stress in the patient (7). hospitalization, especially in the intensive care unit (icu), leads to a series of adverse psychological complications that can manifest itself or be aggravated after the patient s discharge from hospital (8). because of the complexity of care and treatment measures, and the complicated environment, icu is a stressful setting and can negatively affect patients recovery and rehabilitation (9). post - surgical stressors in the icu have been explored in several studies and are observed within the 4 domains of physical, psychological, environmental, and procedural factors. the main physical stressors include thirst, presence of oral or nasal tubes, inability to sleep, and pain (9 - 11). the main psychological stressors include fear of death (4, 9, 11), not being in control of oneself (11), being pressurized to accept the treatment, not knowing the length of icu stay (10), financial concerns (10, 11), fear of acquiring nosocomial infections, and treatment not explained to the patient (10). the main environmental stressors comprise medical device alarms (12), and the presence of several patients in one room(4). finally, procedural stressors were reported to encompass observing the constant presence of nurses performing their tasks around the bed, and observing intravenous bags over the bed (9). anxiety is a state of uncertainty about future threats, identified by stress, concern, negative emotions, and a sense of insecurity (13). when anxiety level is too high and disrupts daily life, it becomes irreconcilable and causes serious consequences. evidence indicates that despite the variety of manifestations of anxiety, the psychological and physical responses to them are the same (14). it is also believed that high levels of anxiety can increase the need for anesthesia, the possible complications of anesthesia, and the need for post - surgical analgesics (15). one study reported that trait anxiety levels were higher in patients hospitalized for arrhythmia, congestive heart failure, and myocardial infarction during the 4-year period after their cabg surgery (16). a review of literature showed that anxiety related to surgery is a theme explored by various studies. in tol and pourreza s study, anxiety levels before and after surgery were investigated in patients undergoing cabg surgery, and the relationship between anxiety and demographic variables was assessed. results showed that 64.7% of patients had moderate levels of anxiety before surgery and 97.3% had low levels of anxiety after surgery ; that is, anxiety levels were higher before than after surgery, and were related to variables such as age, sex, number of children, and marital status (17) gallagher and mckinley examined the relationship between anxiety levels and stressors before and after surgery in patients undergoing cabg surgery. pre - surgical anxiety predictors included being a female, concerns about the waiting period before surgery, pain and discomfort, and resuming life style. moreover, post - surgical anxiety predictors included receiving anti - anxiety and anti - depressant medications, high pre - surgical anxiety levels, lack of access to personal belongings, and sleep problems (18). a review of literature revealed that most studies merely investigated the stressors of patients in icus (4, 19, 20), with some studies reporting the stressors of cabg surgery without investigating anxiety (4). according to available databases, no study has been conducted in iran to date on the relationship between stressors and anxiety levels after surgery in patients undergoing cabg surgery, with the exception of one study on patient s perception of stressors associated with cabg surgery (4) ; this study however did not explore post - surgical anxiety. given the importance of post - surgical anxiety in the recovery process of these patients, the present study was designed to investigate the relationship between stressors and anxiety after surgery in patients undergoing cabg surgery and thereby to identify stressors and anxiety levels and propose preventive care and treatment measures for this patient group. this is a descriptive - analytical study performed using the non - random accessible sampling method on patients undergoing cabg surgery at the cardiac surgery icu of fatemeh zahra cardiac center in sari, iran. the inclusion criteria comprised first time cabg surgery ; age of 18 years and older ; being oriented in time, person, place ; having the ability to communicate ; not under treatment for severe psychological disorders ; and not having severe visual, hearing and voice impairments. the exclusion criteria included patient s unwillingness to cooperate at any stage of the study and conditions requiring emergency interventions. the sample size was calculated to be 186 according to the study conducted by shahmansouri., with a prevalence of 39% moderate level of fear (p), 95% confidence coefficient (z), and a detection accuracy of 0.07 (d) (20). the data collection tool comprised 3 questionnaires, including the socio - demographic and medical data questionnaire, the post - surgical stressors questionnaire, and the spielberger state - trait anxiety inventory. the socio - demographic and medical data questionnaire solicited information regarding the participants age, gender, marital status, level of education, employment, history of surgery, and history of psychiatric diseases. a researcher - constructed, 35-item, post - surgical stressors questionnaire was based on various studies and the 50-item intensive care unit environmental stressor scale (icuess). the post - surgical stressors questionnaire was issued to patients on the second or third day after surgery. the 50-item questionnaire was first used by ballard in 1981 and then by nastasy in 1985 ; it was subsequently revised by other researchers (9). in our study, the post - surgical stressors questionnaire was tested for face and content validity. to assess the content validity ratio (cvr), the 45-item questionnaire was first distributed among 12 experts in the fields of psychiatric nursing, psychiatry, medical - surgical nursing, and behavioral sciences and 10 items were then excluded from the questionnaire. the content validity index (cvi) was found to be 0.92. to ensure the questionnaire s reliability, internal consistency was measured (using cronbach s alpha) and was calculated at 0.863 subsequent to the completion of the questionnaire by 30 cabg patients. the 35 items were rated using a 4-point likert scale, with 4 indicating severely stressful, 3 highly stressful, 2 moderately stressful, 1 slightly stressful, and 0 not stressful or not applicable. the overall score ranged from 0 to 140, with 0 - 35, 36 - 70, 71 - 105, and 106 - 140 indicating, in the respective order, slightly, moderately, highly, and severely stressful post - surgical stressors. obtaining a score of 0 indicated the lack of any experience with the stressors after surgery. the 40-item spielberger state - trait anxiety inventory was used to measure anxiety levels before and after surgery. items 1 to 20 measures the state anxiety with 4 choices (not at all, somewhat, moderately, and very much), while items 21 to 40 measures trait anxiety with 4 choices (almost never, sometimes, often, and almost always) (21). are reverse - scored from 1 to 4, while statements showing lack of anxiety are reverse - scored from 4 to 1. to find the respondents score in each of the two scales, the total score is calculated separately for each scale given that some items are reverse - scored. items 3, 4, 6, 7, 9, 12, 13, 14, 17, 18, 22, 24, 25, 28, 29, 31, 35, 37, 38, and 40 are scored from 1 to 4, while items 1, 2, 5, 8, 10, 11, 15, 16, 19, 20, 21, 23, 26, 27, 30, 32, 33, 34, 36 and 39 are reverse scored from 4 to 1 (22). a score of 0 to 40 shows the lack of anxiety, 41 to 80 mild anxiety, 81 to 120 moderate anxiety, and 121 to 160 represents severe anxiety (23). the validity and reliability of the persian version of this questionnaire have been established by the psychology department of the tarbiat modares university (24, 25). in another study, cserep. reported the questionnaire s reliability to be 0.94 (26). ethical approval was obtained from the medical research and ethical committee of the mazandaran university of medical sciences. patients were sampled from february 2013 to june 2014 using convenience sampling method and in accordance with the study s inclusion and exclusion criteria. all patients were informed about the aims of the study, their right to withdraw from the study at any time without having to give a reason and without negative consequences to patient care. the first questionnaire, socio - demographic and medical data questionnaire, was completed for patients by one of the researchers (aj). the 35-item post - surgical stressors questionnaire was completed on postoperative days 2 or 3 in the cardiac surgery icu, and anxiety levels were measured concurrently using the spielberger s state - trait anxiety inventory. data were analyzed with spss-16 software, using descriptive statistics (such as mean, standard deviation and percentage) and analytical tests (such as mann - whitney u, logistic regression model and one - sample kolmogorov - smirnov). the mann - whitney u test was used to assess the relationship between the variables. the logistic regression model was performed to determine the relationship between stressors and anxiety after surgery. if one - sample kolmogorov - smirnov test showed that the variables (anxiety and stressors) were not normally distributed, then nonparametric tests were used. of the total 186 patients participating in the study, 52.2% were male, 44.6% were over 60 years old, 86.1% had under diploma education, and 93.5% were married. the frequencies of stressors after surgery are shown in table 2. the highest frequency (79%) of post - surgical stressors was seen in the slightly stressful group ; 7.5% and 7.5% patients fell in the moderately stressful and highly stressful groups, respectively, and 5.9% in the severely stressful group. the highest mean stressfulness score was noted for being in pain (mean sd, 1.76 1.34), followed by having a chest tube (1.75 1.23) and hearing the icu staff talking about the patient (1.43 1.18). overall, the maximum and minimum mean score of the 35 items were between 1.76 1.34 and 0.47 1.06, respectively. the median, 25 percentile (p25), 75 percentile (p75), and interquartile range of post - surgical stressors were, respectively, 14, 8, 28, and 20. following surgery, the frequency of mild, moderate, and severe anxiety was 70.4%, 28.5%, and 1.1%, respectively. the highest frequency of anxiety after surgery was noted for the state of mild anxiety (table 3). median, p25, p75, and interquartile range of post - surgical anxiety were in the following order : 68, 55, 82, and 28. the mann - whitney u test was used to assess the relationship between stressors and anxiety after surgery. because of the lack of a significant relationship with anxiety, certain post - surgical factors were excluded from the logistic regression model ; these included the presence of a urinary catheter, uncomfortable bed, and not knowing the length of stay in the icu. the logistic regression test revealed the following predictors of post - surgery anxiety : insufficient sleep during hospitalization (p = 0.010), being away from family members (p = 0.006), treatment not explained to the patient by nurses (p = 0.002), presence of a chest tube (p = 0.000), and pain in any part of the body (p = 0.031 ; table 4). the present study showed that, in general, the mean stressfulness score after surgery was low, which is similar to the results of studies conducted by parvan. (4) and gallagher and mckinley (18). in parvan.s study, the overall mean standard deviation of the revised cardiac surgery stressors scale (rcsss) was 1.63 0.36, indicating the low levels of stressfulness associated with the factors before and after surgery (4). in gallagher and mckinley s study, the highest level of stressfulness was noted for the waiting period for surgery, with a mean of 2.02 (18), a result similar to that of the present study. despite low levels of stressfulness, the results of all these studies show that cabg candidates experience stress, reinstating the importance of nursing care plans for decreasing complications of stress and anxiety. in gallagher and mckinley s study that used hospital anxiety and depression scale (hads), anxiety at the clinical level (scores above 8) was 28% during hospitalization in the icu (18). low anxiety (scores 0 - 7) had the highest frequency in that study, a finding similar to the results of the present study. also in tol and pourreza s study, which used spielberger s state - trait anxiety inventory, the highest frequency of anxiety after surgery was noted for low anxiety (66%) (17). in the present study, a majority of patients were over 60 years old ; as patients in these ages usually have a history of heart disease and hospitalization, low levels of anxiety can be expected. although our study shows low levels of anxiety, it is essential to evaluate anxiety levels because anxiety may lead to adverse effects on the cardiovascular system. all relevant studies have reported that patients experience anxiety, and therefore, it is recommended to design appropriate interventions. in the present study, the logistic regression analysis showed that insufficient sleep during hospitalization (or = 5.42) was a major post - surgery stressor (table 4). based on the 50-item icuess, stressors of icus are divided into 4 categories : physical or physiological, psychological, procedural, and environmental. in our study, (2010) examined stressors using the 50-item icuess and revealed that except for fear of death (a major stressor perceived by patients and nurses), physiological stressors such as thirst, pain, sleep problems, and presence of oral and nasal tubes had a greater significance when compared with stressors such as environmental or other psychological stressors (9). furthermore, so and chan (2004) reported that physiological stressors such as thirst, pain, and sleep problems were major stressors (11), a finding similar to that of our study. in these two studies, the relationship between the aforementioned factors and anxiety was not evaluated. in gallagher and mckinley s study, insufficient sleep (or = 1.38) was predictive of anxiety after surgery. in the present study, insufficient sleep had a greater level of anxiety when compared with that shown by gallagher and mckinley s study ; this difference could be due to difference in icu facilities. treatment not explained to the patient by nurses (or = 4.83) was another important post - surgical stressor. a study revealed that treatment not explained to the patient by nurses was a major stressor, a finding in agreement with our study (10). in so and chan s study, the relationship between this factor and anxiety has not been investigated (11). koivula (2002) investigated the receipt of in - hospital social support, which is multi - dimensional and includes emotional, informational, and tangible support, and its effect on cabg patients (27). the study indicated that patients received a substantial support from nurses and that their anxiety levels were reduced because of informational support provided by nurses (25). thus, this finding indicates the significance of psychological stressors to patients and the educational role of nurses. according to the present study, a psychological predictor of anxiety was being away from family members (or = 3.88), which is consistent with the results of other studies (4, 18). in parvan.s study, this factor, with a mean sd of 2.77 1.30, was the second major extra personal stressor (4), but the relationship between this factor and anxiety was not evaluated. considering the substantial role of family as a source of social support for patients (27), it is expected that being away from family members during surgery to be stressful and cause anxiety. it is crucial that patients to be able to meet their family members after surgery and during hospitalization in the icu. our analysis also showed the presence of a chest tube (or = 3.27) as a major physical predictor of anxiety, a finding by parvan. who reported this as a major extra personal stressor for the patients (4). found the presence of oral and nasal tubes as a major post - surgery stressor (9). given that certain stressors of cabg surgery, such as the presence of chest, urinary, nasal, gastric, and tracheal tubes, are inevitable, it is crucial for patients to be well - informed about the necessity of these intubations ; in addition, certain measures should be taken to ensure patient s greater comfort during this time. furthermore, pain in any part of the body (or = 1.95) was a major post - surgical predictor of anxiety. likewise, other studies also reported body pain to be a major post - surgical stressor (4, 9, 10). in gallagher s study, this factor only existed in patients before surgery (18). given that people have different perceptions of pain, effective pain management can only be exercised through proper objective examination of the pain, using reliable behavioral and physiological indicators and choosing proper analgesics (10). we found procedural and environmental factors to be less significant, a result aligned with those of other studies. for instance, presence of tubes and immobility can cause pain, pain can cause insomnia, and insomnia can in turn affect the perception of stress by increasing stress levels (28). our study showed that physical or physiological and psychological stressors were perceived as more stressful after cabg surgery. stressors like insufficient sleep during hospitalization, being away from family members, treatment not explained to the patient by nurses, presence of a chest tube, and pain in any part of the body were predictors of anxiety after surgery. thus, when preparing their nursing care plan, nurses should consider stressors that affect anxiety levels in patients undergoing cabg surgery and those hospitalized in icus. in this study, most of the patients were over 60 years old and had no formal education ; therefore, generalization of results to other age groups should be done with caution. based on the review of literature, many stressors have been explored in patients undergoing cabg surgery, but stressors that cause anxiety have not been identified. our study showed that physical or physiological and psychological stressors were perceived as more stressful after cabg surgery. stressors like insufficient sleep during hospitalization, being away from family members, treatment not explained to the patient by nurses, presence of a chest tube, and pain in any part of the body were predictors of anxiety after surgery. thus, when preparing their nursing care plan, nurses should consider stressors that affect anxiety levels in patients undergoing cabg surgery and those hospitalized in icus. in this study, most of the patients were over 60 years old and had no formal education ; therefore, generalization of results to other age groups should be done with caution. based on the review of literature, many stressors have been explored in patients undergoing cabg surgery, but stressors that cause anxiety have not been identified. | backgroundhospitalization and surgery are crucial adverse life events that lead to considerable anxiety in patients.objectivesthe present study aimed to investigate stressors after coronary artery bypass graft surgery and identify stressors that predict anxiety.patients and methodsthis is a descriptive - analytical study that uses a non - random convenience sampling method on patients undergoing coronary artery bypass graft surgery at the cardiac surgery intensive care unit of fatemeh zahra cardiac center in sari, iran. a total of 186 patients completed the post - surgical stressors questionnaire and the spielberger state - trait anxiety inventory on postoperative days 2 or 3 in the cardiac surgery intensive care unit. data were analyzed using descriptive statistics including frequencies, means, and standard deviations. the mann whitney u test was used to determine the relationship between the observed variables, and the logistic regression model was used to identify the relationship between stressors and anxiety after-surgery.resultspost-surgical anxiety predictors included insufficient sleep during hospitalization (odds ratio [or ] : 5.42 ; 95% confidence interval [ci ] : 1.46 - 20.00 ; p = 0.010), treatment not explained to the patient by the nurse (or : 4.83 ; 95% ci : 1.82 - 12.84 ; p = 0.002), being away from family members (or : 3.88 ; 95% ci : 1.46 - 10.26 ; p = 0.006), presence of a chest tube (or : 3.27 ; 95% ci : 1.83 - 5.84 ; p = 0.000), and pain in any part of the body (or : 1.95 ; 95% ci : 1.06 - 3.58 ; p = 0.031).conclusionsphysical or physiological and psychological stressors impose greater stress and are predictors of anxiety. when preparing their nursing care plan, nurses should consider these stressors that affect anxiety levels in patients undergoing cabg surgery and those hospitalized in intensive care units. |
temperature is one of the fundamental regulators of tissue metabolism [1, 2 ]. interest in the temperature of the eye spans almost 130 years and the ability to measure the temperature of the eye, driven by prevailing technologies, has potential importance in both research and clinical situations, including the study of ocular physiology and pathology [39 ]. new infrared ocular thermographs allow a noncontact and nonintrusive characterization of the thermal profile across the ocular surface [1017 ]. applications have included dry eye, wearing contact lens, corneal sensitivity, and ophthalmic surgery [1824 ]. in addition, some studies showed a correlation between ocular surface temperature and ocular blood flow. an increase of intraocular pressure (iop) was found to be related to a contemporary decrease of ocular perfusion pressure and ocular temperature in monkeys. a recent study on humans has also reported that eyes with ischemic central venous retinal occlusion (crvo) have lower ocular surface temperatures than nonischemic ones. moreover, in carotid artery stenosis, the eye on the affected side has been found to have an impairment in retrobulbar hemodynamics along with a reduction in corneal temperature (ct). thermography has also been applied to explore the role of vascular factors in the physiopathology of glaucoma and galassi. have recently defined ocular surface temperature as a marker of impaired retrobulbar hemodynamics in patients with glaucoma. however, to date, little work has been undertaken to determine the relationship between iop and ct [3, 22, 28 ]. the variations in iop following closed eyelid test (cet) both under normal conditions and after the administration of antioxidants have recently been investigated, leading to the conclusion that cet - induced ocular hypertension could be a response to mixed stress oxidative and thermic with degenerative effects on the trabecular meshwork (tm) [22, 29 ]. moreover, given the known influence of ambient pressure and temperature on iop, an underwater mask has been proposed as a provocative test in the diagnosis of primary open angle glaucoma (poag). poag is a multifactorial and not yet well - understood pathology [31, 32 ]. iop is generated and maintained via the aqueous humor circulation system in the anterior chamber (ac) of the eye. the major factor controlling iop is the dynamic balance between aqueous humor production in the ciliary body and its draining through so - called conventional the goal of the study here reported was to map the ct in different areas of the cornea of healthy human volunteers and follow its variations after cet at room temperature or with a cooling mask (cm - cet) and correlate such variations to iop values. this pilot, prospective, cross - sectional study was conducted following the tenets of the declaration of helsinki. after signing an informed consent, each study participant underwent a complete ophthalmological examination, including a medical history review, best - corrected visual acuity measurement (bcva), slit - lamp biomicroscopy, dry eye tests, and dilated fundus examination. all subjects were free from ocular and systemic diseases with no history of previous ocular surgery. patients enrolled in the study were accepted if they had no signs or symptoms of ocular dryness : ocular protection index (opi : calculated as the ratio between but and the blinking frequency per minute) score 1 and osdi score 12. in addition, since corneal pachymetry may influence temperature fluctuations and their determination, patients ' central corneal thickness measured by ultrasonic pachymetry (pacline compact multifeature pachymeter, optikon 2000, rome, italy) had to be within normality limits, that is, 550 20 microns. further inclusion criteria were as follows : iop 21 mmhg, without any treatment.refraction values between 4 and + 4 spheric diopters.bcva for far distance equal to 10/10.normal angle structure at gonioscopy.normal c / d ratio at slit - lamp examination.normal sap (30 - 2 sita standard program). precise spatiotemporal measurements of ct were captured through the latest generation infrared thermometer (sola electro - optics, shanghai, china). since it is known that there is an uneven distribution of temperature in the cornea, the average ct was calculated based on the recordings of cts in five different areas of the cornea (nasal, temporal, superior, inferior, and central). all patients were acclimatized to the clinical environment for at least 15 min. the room temperature was specifically set and controlled at all times at 25c (77f), humidity was maintained at 42.0%, and the average indoor illumination was maintained at 300 lux, considered a standard indoor level of illumination. the measurements were performed between 9:00 and 11:00 a.m. in a seated position and under the conditions described by mori. : the subject blinked normally, then closed both eyes for 5 s, and then kept the eyes open for more than 10 s. the thermography device was set up 20 cm in front of the eye and the head was held steady with a frame. during that time, the subject was asked not to blink. three measurements were taken consecutively during a single session for each eye and the average value was recorded. the measurements were repeated after one hour of cet and after the same test following the application of a cooling mask on the volunteers ' eyelids. to avoid any iop is reported as the mean value of three consecutive readings of each eye by goldmann applanation tonometer (at-900, haag streit diagnostics, switzerland) registered between 9:00 and 11:00 a.m. to minimize the effect of daily variations. to avoid interexaminer and intertonometer variances, all iop measurements were taken by the same trained resident. to simulate the temperature distribution of the human eye, we adopted the physical model developed by karampatzakis and samaras that solves the pennes bioheat transfer equation coupled with the incompressible navier - stokes equation of fluid dynamics. according to such a physical model, the eye can be modeled by seven regions with different thermal properties the cornea, the anterior chamber, the trabecular meshwork, the iris, the lens, the vitreous humor, and the sclera. the basal metabolic heat generation, as well as the blood perfusion, mainly occurs in the iris and in the sclera. the results obtained were statistically analyzed by student 's t - test for paired samples. p values below 0.05 were considered significant and denoted by one (p < 0.05) or two (p < 0.01) asterisks (spss v.19, ibm spss statistics, usa). figure 1 shows the distribution of basal values across the cornea. in each eye, the lowest temperature was observed at the temporal side, the highest temperature was observed at the nasal side, and intermediate values were observed along the corneal longitudinal axis (superior, central, and inferior). ct mean basal values were 36.33 0.33c in the right eye (re) and 36.32 0.24c in the left eye (le). after cet, all the temperatures tended to increase with respect to the basal values (mean values : 36.89 0.20c in the re and 36.80 0.26c in the le) whereas after cm - the temperatures showed a tendency to decrease (mean values : 36.22 0.39c in the re and 36.12 0.23c in the le). on average, after cet a highly significant increase of the corneal temperature of 0.56c for the re and 0.48c for the le (p < 0.001 for both eyes) was observed and after cm - cet there was a significant decrease of 0.11c for the re and 0.20c in the le (re p = 0.04 and le p = 0.024 for both eyes) (figure 2(a)). correspondingly, we observed significant variations in iop, which increased after cet by 1.13 mmhg in the re and 1.46 mmhg in the le (basal mean values : 12.20 1.72 mmhg in the re and 12.87 3.6 mmhg in the le ; after cet mean values : 13.33 1.2 mmhg in the re and 14.33 3.8 mmhg in the le ; p < 0.001 for both eyes), whereas it significantly decreased after cm - cet by 2.19 mmhg in the re and by 1.54 mmhg in the le (basal mean values : 12.20 1.72 mmhg in the re and 12.87 3.6 mmhg in the le ; after cm - cet mean values : 10.01 1.78 mmhg in the re and 11.33 2.1 mmhg in the le ; re p < 0.001 and le p = 0.0019) (figure 2(b)). figure 3 shows that there is indeed a direct correlation between average ct and iop, with a correlation coefficient scoring 0.74 in the re and 0.94 in the le. finally, the application of the physical simulation model by karampatzakis and samaras and pennes bioheat transfer equation (figure 4) shows the following : the mean temperature of the eye is influenced by the ambient temperature : it increases by about 1.0c when the ambient temperature rises from 15c to 30c. the data that we obtained at an environmental temperature of 25c were 36.3c with open eyelids and 36.8c after cet, in agreement with the theoretical prediction.the evaporation rate of the thin tear film on the cornea decreases its temperature, similarly to sweat evaporation on the skin ; therefore, blocking the evaporation increases the overall surface temperature, as less heat is being exchanged between the cornea and the environment. mathematical simulations estimate such a temperature rise at about 0.4c, in agreement with the experimental data after cet, when the evaporation was stopped for some time, and consequently the ct increased by 0.56c (re) and 0.48c (le). the mean temperature of the eye is influenced by the ambient temperature : it increases by about 1.0c when the ambient temperature rises from 15c to 30c. the data that we obtained at an environmental temperature of 25c were 36.3c with open eyelids and 36.8c after cet, in agreement with the theoretical prediction. the evaporation rate of the thin tear film on the cornea decreases its temperature, similarly to sweat evaporation on the skin ; therefore, blocking the evaporation increases the overall surface temperature, as less heat is being exchanged between the cornea and the environment. mathematical simulations estimate such a temperature rise at about 0.4c, in agreement with the experimental data after cet, when the evaporation was stopped for some time, and consequently the ct increased by 0.56c (re) and 0.48c (le). this study shows that, in normal eyes, following cet and cm - cet, iop significantly changes in response to variations of ct. in agreement with previous studies [3437 ] in addition, our study is the first to report an overall decrease of ct after application of a cooling mask on the lid surface (cm - cet). moreover, a direct correlation was found between ct and iop, with iop values following the increase or the decrease of average surface ct values. to date, the physiological link between ct and iop still remains somewhat inconclusive. previous investigations demonstrated that eyelid closure, or the use of an underwater mask with open eyelids, can raise iop because of an increase of local temperature. further studies confirmed the relationship between local temperature, variation in iop, and anterior chamber oxidative stress following cet [29, 3941 ]. as previously reported, our data confirm that ct varies in response to tear film evaporation rate, which in turn is influenced by environmental temperature and blinking rate [19, 4244 ]. however, our results also show a peculiar pattern of ct distribution across the corneal surface, not in line with previous findings [12, 14, 15, 45 ]. in fact, in all experimental settings (basal, after cet, and cm - cet), we detected the coolest area at the corneal temporal region while the warmest area was at the nasal side. along the corneal vertical axis, the temperatures showed intermediate values. to explain and validate our data, the physical model by karampatzakis and samaras and pennes bioheat transfer equation was applied and allowed us to conclude that ah flow depends on ct distribution. numerical simulations show that the circulation of ah follows the temperature difference between the temporal and nasal side of the cornea and that an increase of ah flow from stagnation to fast vortices correlates with the increasing asymmetry between temporal and nasal temperatures, thus influencing the balance between production and outflow, and finally iop values. accordingly, ct differences between nasal and temporal sides were higher after cm - cet (0.80 and 0.96 for the re and le, resp.) than after cet (0.52 and 0.44 for the re and le, resp.). this may indeed suggest a faster flow with lower temperatures, favoring a higher discharge of ah, and therefore a lower iop. iop depends on the dynamics of ah turnover, which in turn is a balance between secretion and excretion. three mechanisms are involved in ah formation by the ciliary body : diffusion, ultrafiltration, and active secretion, the last being the major contributor to its formation. ah can be considered analogous to a blood surrogate as it provides nutrition, removes excretory products from metabolism, transports neurotransmitters, stabilizes the ocular structure, and contributes to the regulation of the homeostasis of the surrounding ocular tissues [3336 ]. ah, as part of a vascular circulatory loop, returns to the blood flow by passing through the tm, a uniquely modified vessel wall interposed between the anterior chamber and sc or through the uveoscleral pathway [34, 36 ]. our results suggest that temperature may affect ah balance through the regulation of its secretion, excretion, and flow dynamics. secretion is enhanced by increasing temperatures, because of increased blood flow in the anterior segment of the eye due to vasodilation and upregulation of metabolic processes in the ciliary body. the opposite effect is therefore expected after decreasing the temperature, since it is known that low temperatures stimulate the sympathetic system inducing vasoconstriction., there is an increase of oxidative stress and inflammatory processes due to increased metabolic activities. these events may negatively affect the draining through the tm, since it has been shown that high temperature increases the cellularity of the anterior chamber angle structures [33, 34 ]. the application of the physical model by karampatzakis and samaras confirmed the direct correlation between iop and ct and the influence of tear film evaporation and environmental temperature on ct. moreover, and more interestingly, such numerical simulation suggested that ah responds to increasing surface temperature generating vortices that might contribute to the variations in iop observed in our experiments. finally, the presence of several transient receptor potential (trp) channel isotypes that are responsive to temperature variations in the three corneal layers and in corneal nerve fibers may suggest an involvement of this receptor type also in controlling the aqueous humor dynamics, although no reports exist yet indicating such kind of interaction. iop is the only treatable risk factor in glaucoma, one of the world 's leading causes of blindness. the existing correlation between ct and iop suggests that ct may affect short - term iop control and its fluctuations. therefore, controlling iop and its fluctuations is a main therapeutic goal in glaucoma treatment. temperature and oxidative stress remain additional targets in the control of glaucoma progression [29, 31, 34, 36, 40 ]. it will be interesting to extend these findings studying the influence of temperature on iop not only in the seated position but also in the supine position and on a larger population. moreover, it could be interesting to see whether the same correlation persists in the presence of ocular hypertension. in such case, the use of a cooling mask during the night, when peaks of iop are mostly expected with negative effects on poag progression, might be advisable. | purpose. to study the geographical distribution of corneal temperature (ct) and its influence on the intraocular pressure (iop) of healthy human volunteers. materials and methods. fifteen subjects (7 m, 8 f), 33.8 17.4 years old, were enrolled in this pilot, cross - sectional study. measurements of ct were taken after one hour with closed eyelids (cet) or closed eyelids with a cooling mask (cm - cet) and compared to baseline. results. if compared to baseline, after cet, average ct significantly increased by 0.56c in the re and by 0.48c in the le (p < 0.001) and iop concomitantly significantly increased by 1.13 mmhg and 1.46 mmhg, respectively, in each eye (p < 0.001). after cm - cet, average ct significantly decreased by 0.11c and 0.20c, respectively, in the re and le (re p = 0.04 ; le p = 0.024), followed by a significant iop decrease of 2.19 mmhg and 1.54 mmhg, respectively, in each eye (re p < 0.001 ; le p = 0.0019). conclusion. significant variations of ct occurred after cet and cm - cet and were directly correlated with significant differences of iop. it can be speculated that both oxidative stress and sympathetic nerve fiber stimulation by temperature oscillations may affect the regulation of ah vortex flow and turnover, thus influencing iop values. |
death anxiety is a multifaceted construct. it was defined and described the different ways in which it is manifested [13 ]. death anxiety has been conceptualized as fear of death of oneself ; fear of death of others ; fear of dying of self ; and fear of the dying of others [47 ]. many factors influence death anxiety such as age, sex, culture, religion, physical health, and mental health. aging is a stage in developmental psychology and associated with various medical problems, loss of loved ones, and deteriorating cognitive abilities. old - aged individuals being nearer to the end of life may experience death anxiety or death fear [8, 9 ]. cultural variations in conceptualizations of death can determine death anxiety in different ages [1012 ]. many researches showed that death anxiety exists among different cultures including iranian culture [8, 1315 ]. missler. revealed that dutch elderly people who were in an assisted living facility had higher levels of fear for others and of the dying process than of fear of the unknown. the fear for important others correlated with poor somatic health ; and fear of the dying process correlated with low self - esteem, small goal in life, and poor mental well - being. for most of old - aged individuals who encounter death of their spouses, on a national cross - sectional survey in malaysian middle - aged and older couples, reported that women (52%) had significantly higher spousal death anxiety compared to men (45%). caregiving issues for older men and financial security for older women could predict increasing spousal death anxiety. death anxiety impacts behavior and life and death decisions [18, 19 ], and one study found that depression and severity of illness positive correlated with death anxiety. one study showed that death anxiety could be a determining factor of health promoting behaviors both in young and older adults. abdel - khalek and lester found that somatic symptoms were associated with death anxiety, on the arabic scale of death anxiety (asda) and the collett - lester fear of death scale (clfds) ; the kuwaiti young adults with good somatic health were anxious about death compared with american young adults. the asda has been used in various populations, for example, health professionals, drug dependents, psychiatric patients, healthy individuals, college students, and middle - aged individuals, to investigate the factors related to death anxiety. in spite of the fact that death anxiety is one of the issues related to health, well - being, and welfare of elderly persons in the societies, the little studies have focused on it among the elder population. in the present study it is hypothesized that (a) iranian old - aged women would have higher death anxiety, (b) death anxiety levels would differ between iranian young adults and older adults, and (c) and (d) the farsi form of the arabic scale of death anxiety (asda) has good psychometric properties of factors among iranian old - aged individuals. a sample of 146 volunteer iranian old - aged individuals took part in the study. the elders had come to the neighborhood parks for leisure, recreation, and the activities done for enjoyment when they were not working. the range of age was 6090 years, with mean ages 68.58 (sd = 7.10), male being 68.81 (sd = 7.44) and female being 68.28 (sd = 6.76), respectively. 80 (58%) were male and 58 (42%) female. the majority of them were married (n = 107 ; 75%) and had an education level of diploma (n = 56 ; 39%). demographic characteristics sheet of the sample includes age, gender, education status, and marital status. the arabic scale of death anxiety (asda) was developed by abdel - khalek, and it is one of the scales for measuring death anxiety. the asda has been validated in the arabic countries of egypt, kuwait, and syria [2628 ], the western countries, and the asian countries of turkey. it has 20 items, and each item is answered on a five - point intensity scale anchored by no (1) and very much (5). abdel - khalek and al - kandari reported that cronbach 's alpha of the asda was high (0.93), and the asda yielded three factors disease in kuwaiti middle - aged individuals labeled (f1) fear of dead people and tombs ; (f2) fear of postmortem events ; and (f3) fear of lethal disease. sariek aydoan. found cronbach 's alpha for turkish version of the asda was 0.86, for each item was from 0.85 (for items (11) and (12)) to 0.90 (for item (20)), the guttman coefficient was 0.86, and the asda was highly associated with the das (r = 0.68). they obtained five factors in the sample of turkish college students, accounting for 65.6% of the total variance and labeled (f1) fear provoked by visual stimuli related to death ; (f2) fear of physical and psychological pain related to death ; (f3) fear of other situations reminding of death ; (f4) fear of postmortem events ; and (f5) fear of the act of dying. the pearson correlations between the total score of the asda and age was 0.012 (no significance) ; sex.443 (p >.01) ; education.155 (no significant) ; and marital status.302 the cronbach alpha was 0.94, the split - half reliability was 0.86, the spearman - brown coefficient was 0.92, and the guttman coefficient was 0.92. the intercorrelations between the items ranged from 0.15 to 0.79, and the item - total correlations ranged from 0.60 to 0.80 in the sample. the mean score of the asda was 51.09 (sd = 20.19), for women 62.36 (sd = 17.00) and men 44.33 (sd = 18.93). old - aged women had a significantly higher mean asda total score than men (t = 5.75, p <.001). the mean scores of the asda items were from 1.67 (sd = 1.10) for item (12) i dread walking in graveyards to 3.58 (1.49) for item (6) i worry that death deprives me of someone dear to me. old - aged women had significantly higher mean scores in all of the asda items than men (table 1). the mean score of the asda for elders was 51.09 (sd = 20.19) and for college students 41.40 (sd = 12.73). old - aged individuals had a significantly higher mean asda total score than younger college students (t = 5.86 and p <.001) (table 2). the criteria for the factor analysis were evaluated using the kaiser - meyer - olkin measure of sampling adequacy (kmo) and the bartlett test of sphericity. the kmo was 0.919, indicating the adequacy of the sample of college students, and the bartlett test of sphericity was 2.203e3 (df = 190 and p <.001) indicating that the factor analysis was justified in the elders sample. three components with eigenvalues greater than one were retained in the sample of elders as reported in table 3. factor 1 (8 items) explained 49.71% of the observed variance and was labeled fear of lethal disease and death. i am afraid of looking at a corpse, witnessing the burial procedure terrifies me, i dread walking in graveyards, the sight of a dying person frightens me, and talking about death upsets me. factor 2 (8 items) explained 11.95% of the observed variance and was labeled fear of lethal disease and postmortem events and included the following items : the possibility of having a surgical operation terrifies me, i am preoccupied with thinking about what will happen after death, the pain accompanying death terrifies me, and i am afraid of getting cancer. factor 3 (4 items) explained 6.20% of the observed variance and was labeled death fear. it included the following items : i fear death whenever i because ill, i fear visiting graves, i am afraid of sleeping and not waking up again, and i fear death. the study showed that female iranian elders had a significantly higher mean asda total score compare to men. the gender and age results in this iranian sample are similar to that in some western, arabic, and indian samples. abdel - khalek using the asda declared that egyptian women in clinical and nonclinical groups had higher mean scores than men. another study showed that there were strong sex differences in the das scores of men and women. abdel - khalek and al - kandari found kuwaiti middle - aged women had a significantly higher mean asda total score also on 17 out of 20 items of the scale. on the death anxiety survey schedule from an indian perspective, madnawat and kachhawa indicated that women were significantly more anxious about the word of death. found that, on the asda, egyptian, kuwaiti, lebanese, syrian, spanish, and american women (no english women) had significant higher mean scores than men. missler. reported that women compared to men had more fear of the death of loved ones and of the consequences of their own death on these loved ones. our results were not similar to findings of fortner and neimeyer that reported gender did not predict death anxiety in elderly people but death anxiety was higher in young female adults ; wu. showed that gender does not affect death anxiety in chinese elderly people ; chuin and choo found that malaysian women had lower death anxiety ; and mimrot indicated that gender had no impact on death anxiety ; and there was no significant difference between indian old age men and women in death anxiety scores. rasmussen and johnson found female undergraduate college students had higher levels of death anxiety than males. on the death anxiety scale (das), death depression scale (dds), and death obsession scale (dos), bahrami. reported that iranian muslim female older adults had higher death distress than men but no significant difference. overall, it seems that iranian elder women were more expressive of their death anxiety, and elder men were more in denial of their death anxiety. indicated that causes of higher death anxiety in women include encouraging to pursue success and attain accomplishments for men in many cultures, more readily admitting troubling feelings in women, and different connotations and implications for men and women. chuin and choo indicated that men like women have death anxiety but suppress it or deny it ; also process of socialization, emotional evaluation of death by women, and cognitive evaluation of death by men can justify lower death anxiety in men. our findings showed that old - aged individuals had a significantly higher mean asda total score than younger college students. keller. reported that middle age and late - middle age individuals were significantly less anxious in regard to evaluation of death in general than older and younger persons.. studies of feifel and nagy ; maiden and walker ; walker and maiden, cited in chuin and choo, declared that age was not related to death fear and age did not impact death anxiety as elders were no more likely to fear death compared to younger persons. goebel and boeck, cited in mimrot, reported low death anxiety in late adulthood ; and age was not a significant factor in determining the degree of death anxiety in late adulthood. rasmussen and brems indicated that psychosocial maturity was a stronger predictor of death anxiety than age ; and both age and psychosocial maturity were significantly inversely related to death anxiety. abdel - khalek and al - kandari found that kuwaiti middle - aged individuals had a significantly lower mean asda total score than younger college students. chuin and choo revealed that death anxiety levels did not differ between malaysian young adults and older adults. goebel and boeck, cited in mimrot, found no relationship between death anxiety and age. one study found that individuals with physical and mental health problems are more sensitive to death anxiety. another study showed that indian old age people living in the family had high death anxiety. there was a difference between indian old age people living in family and old age institutionalized people in death anxiety ; and old age people living in the family had high death anxiety. one study indicated that elders living in the more dangerous situations had higher death anxiety than those living in less dangerous situations. overall, death anxiety levels change according to used tools for death anxiety, place of living, and lifestyle of old - aged persons, and are also related to experiences that elders had during their lives. mimrot concluded that ego integrity or an accomplishment and fulfillment of personal sense, founded in religion or some other related measures, is probably the greatest factor in death anxiety. our finding may reflect cultural differences in death anxiety. also our study did not support erikson 's theory because the iranian old - aged sample could not be viewed in the ego integrity stage of erikson 's theory and they experienced high death anxiety. it is probable that an old person can equilibrate for psychosocial maturity in rasmussen - brens 's theory with ego integrity erikson 's theory, so we can consider supporting erikson 's studies in iranian old - aged persons. the third hypothesis of the present study was to explore the reliability, validity, and descriptive statistics of the farsi version of abdel - khalek 's asda in the iranian samples of elders. the results indicate that the asda has high internal consistency, split - half, and spearman - brown reliabilities (ranging from 0.86 to 0.94) in the sample. the principal components analysis identified three components of the asda in iranian elders as follows : fear of dead people and tombs, fear of lethal disease and postmortem events, and death fear. these factors were highly replicable with previous high internal consistency and factors extracted from a kuwaiti college student sample and a kuwaiti middle - aged sample. abdel - khalek obtained four components in an egyptian sample, labeled fear of dead people and tombs, fear of postmortem events, fear of lethal disease, and death preoccupation. fear provoked by visual stimuli related to death, fear of physical and psychological pain related to death, fear of other situations reminding death, fear of postmortem events, and fear from the act of dying. there were some similarities in specific factors between the previous results and the present findings. in the present study, mean score of the asda was 51.09 (sd = 20.19). in study of chuin and choo mean score of the das was 7.35 (sd = 3.49) in middle adulthood and elders ; and, in study of bahrami. (2014), mean score of the das was 7.48 (sd = 3.32) in iranian elders. the asda items receiving the highest mean score in the present sample of old ages were as follows : item (5) i am afraid of suffering heart attack (3.07), item (6) i worry that death deprives me of someone dear to me (3.58), item (15) the pain accompanying death terrifies me (3.19), item (10) i fear getting a serious disease (3.32), and item (19) one study found that religiousness buffered against the death and dying anxiety in american late adulthoods. several studies investigated the relationship between religiosity and death anxiety [46, 47 ]. dadfar. found that there was no significant association between religious spiritual well - being and death anxiety total scores among iranian muslim elders. in view of pyne, our present findings showed that death anxiety has no significant correlation with age ; the sex - related differences on death anxiety are striking in the iranian samples ; and the asda has a highly replicable factor structure. these results are similar to findings of abdel - khalek and al - kandari of the volunteer kuwaiti middle - aged individuals. therefore, we conclude that the asda is useful for assessing death anxiety in iranian elders in clinical and nonclinical samples. the present results must be viewed within the limitations imposed by the data. despite the good psychometric characteristics of the asda, the recruitment of the sample was not random but rather one of convenience. the sample was related to the population who live in tehran, the capital of iran and tehran province, in the north of the country, with a population of around 9 million in the city and 16 million in the wider metropolitan area. it is known that population who live in tehran may differ from other regions in the country of iran. religious spiritual attitudes can predict death anxiety in old - aged individuals, but they were not assessed in our study. however, the asda may be useful in research on death anxiety among iranian old - aged and middle - aged persons, and it is hoped that this study will stimulate further cross - cultural research on the asda in the elders. future research should be differentiated between the concept of death and the process of dying. constructing validity of the asda with the other scales, using multidimensional fear of death scale (mfods), death attitude profile - revised (dapr), longitudinal and qualitative studies, comparing old age people living in institutions with old people living in the family, improving the quality of life and health care services for elderly, and investigating death anxiety and factorial structure of the asda on the iranian middle - aged population, physical patients, addicted patients, and the assessing of components and correlates of demographic, psychological, social, religious spiritual, and subcultural of death anxiety in future studies are recommended. | the present study is aimed at examining the level of death anxiety and the sex - related differences among old - aged iranian individuals sample to compare the old - aged persons with young college students and to explore the psychometric properties of the arabic scale of death anxiety (asda) factors in old - aged sample. a sample of 146 volunteer iranian individuals took part in the study. the mean ages were 68.58 (sd = 7.10), men 68.81 (sd = 7.44) and women 68.28 (sd = 6.76), respectively. the mean score of the asda was 51.09 (sd = 20.19). cronbach 's alpha of the asda was found to be high (0.94) ; and spearman - brown coefficient was 0.92. women had a significantly higher mean total score on the asda. old - aged individuals had a significantly higher mean asda total score than younger college students (m age = 25.77). the factor analysis of the asda items yielded three factors accounting for 67.88% of the total variance labeled (f1) fear of dead people and tombs ; (f2) fear of lethal disease and postmortem events ; and (f3) death fear. these factors were highly replicable with previous factors extracted from a middle - aged kuwaiti sample. on the basis of the present results, there are the following three general conclusions : death anxiety is not significantly correlated with age ; the sex - related differences on death anxiety are striking in the iranian samples ; and the asda has a highly replicable factor structure among two iranian and arab countries. |
osteogenesis imperfecta (oi), also known as brittle bone disease (marini., 2007), is a group of rare heritable connective tissue disorders characterized by skeletal fractures with mild trauma, secondary deformities, and extraskeletal manifestations (ben amor., 2011 ; marini., 2007) such manifestations may include blue sclerae, progressive hearing loss, dentinogenesis imperfecta, and hyperlaxity of ligaments and skin (ben amor., 2011 ; marini., 2007). the prevalence of oi ranges from 1 in 10,000 to 1 in 25,000 (byers and cole, 2002). iv, based on its clinical features and radiographs (sillence., 1979 ; table s1). type v has been associated with hyperplastic callus formation (glorieux., 2000), type vi with a mineralization defect (glorieux., 2002) and type vii with a range of severity that may be lethal (ward., 2002). oi types viii and ix are usually lethal (barnes., 2012 ; moul., oi severity ranges from intrauterine fractures and perinatal lethality to extremely mild forms without fractures (ben amor., 2011). the severity of bone fragility of oi patients is highest in types viii and ix, and declines progressively from type ii to type vii. most oi types (i vi) show autosomal dominant inheritance, but two of the rarer types (vii and viii) exhibit autosomal recessive disorders (ward., 2002 ; ben amor., 2011 ; the vast majority of fibrils are composed of two 1 and one 2 chains, but a small number are composed of three 1 chains in a triple helix structure. the translated collagen chains include propeptides at each end, one at the amino terminus end and one at the carboxyl terminus end. these propeptides help to connect and arrange the three chains which subsequently interwine in a zipper - like fashion into the triple helix configuration. most patients with types i iv oi (90%) have a mutation in one of the two collagen type i genes, col1a1 or col1a2 (ben amor., 2011 ; van dijk., 2012 ; more than 200 mutations in collagen 1 genes have been associated with oi (ben amor., 2011). these mutations occur in most exons and introns of col1a1 and col1a2 (http://www.le.ac.uk/ge/collagen/ ; dalgleish, 1997, 1998). many oi type i patients have col1a1 and col1a2 frameshift mutations, splice site mutations, and insertion mutations resulting in haploinsufficiency, or the expression of approximately half the amount of collagen type i mrna and protein, leading to weaker bones (redford - badwal., 1996 ; slayton., 2000 ; peng., 2012). other deletions, insertions, and point mutations in col1a1 or col1a2 induce conformational changes in the collagen 1 and 2 chains and lead to knot microfibril structure disorders, altered deposition of hydroxyapatite, brittleness, reduced resistance to external force, altered stability of the collagen triple helix, and fragility (pallos., 2001 ; witecka., 2008 ; ben amor., 2011). clinical diagnosis of osteogenesis imperfecta is straightforward in individuals with a positive family history and several typical oi features (van dijk., 2012). however, despite the presence of several physical manifestations, some patients are difficult to diagnose in the absence of an affected family member and without apparent association between bone frailty and obvious extraskeletal abnormalities. in these cases, analyses of type i collagen genes for mutations can provide helpful information since 90% of oi - associated mutations arise in collagen i (van dijk., 2012). here, we present the results of col1a1 and col1a2 sequencing analysis from dna samples collected from two related chinese patients with several typical features of oi. these results will help to provide a better diagnosis of mild oi type i when physical symptoms are mild. two related han chinese patients, mother and daughter, presented with blue sclerae, bone pain, and dentinogenesis imperfecta. we reviewed their clinical features, medical history, physical examination, radiologic images, and biochemical parameters, and diagnosed them with type i osteogenesis imperfecta. both affected family members, unaffected family members (maternal grandmother, father, and two sisters), and controls agreed to a genetic analysis and provided informed consent for the study approved by the endocrinology department of general hospital of pla. blood samples were obtained from the two patients, their family members, and 50 ethnically matched, unrelated controls. genomic dna was isolated from peripheral leukocytes by using a whole blood dna extraction kit (qiagen co., valencia, ca) according to the manufacturer s instructions. pcr was performed to detect sequences in all exons and exon - intron boundaries of col1a1 and col1a2. fifty - seven pairs of primers, 17 for col1a1 and 40 for col1a2 (sequences available upon request), were designed and verified with primer 5 software (softonic, shanghai, china) using the genomic sequences of col1a1 (genbank accession ng_007400.1) and col1a2 (genbank accession ng_007405.1). dna sequence analysis was performed by automated sequencing in an abi genetic analyzer (model 377 ; abi, usa) by the ying jun gene company (shanghai, china). sequencing results were compared with the genbank database and confirmed through sequencing of clones. dna was cloned into a pgem - t easy vector following the manufacturer s instructions (promega, madison, wi). the osteogenesis imperfecta and ehlers - danlos syndrome variant databases compiled of previously reported oi mutations in col1a1 and col1a2, the 1000 genomes database, and the uniprokb database were also used for comparison. pcr and sequencing of col1a1 and col1a2 genes in samples of other family members were performed with the same methods. open reading frame analysis of the mutated sequence was performed by the orf finder, part of the blast software suite. the parental protein sequence and the protein sequence corresponding to the c.700delg mutation the 1000 genomes database was also used to search for the presence of this mutation by using the alignment function of the blast software and the following search sequence : 5-ttttctctccctctcagggga agctggaaaacctggt cgtcctggtgagcg-3. two related han chinese patients, mother and daughter, presented with blue sclerae, bone pain, and dentinogenesis imperfecta. we reviewed their clinical features, medical history, physical examination, radiologic images, and biochemical parameters, and diagnosed them with type i osteogenesis imperfecta. both affected family members, unaffected family members (maternal grandmother, father, and two sisters), and controls agreed to a genetic analysis and provided informed consent for the study approved by the endocrinology department of general hospital of pla. blood samples were obtained from the two patients, their family members, and 50 ethnically matched, unrelated controls. genomic dna was isolated from peripheral leukocytes by using a whole blood dna extraction kit (qiagen co., valencia, ca) according to the manufacturer s instructions. pcr was performed to detect sequences in all exons and exon - intron boundaries of col1a1 and col1a2. fifty - seven pairs of primers, 17 for col1a1 and 40 for col1a2 (sequences available upon request), were designed and verified with primer 5 software (softonic, shanghai, china) using the genomic sequences of col1a1 (genbank accession ng_007400.1) and col1a2 (genbank accession ng_007405.1). dna sequence analysis was performed by automated sequencing in an abi genetic analyzer (model 377 ; abi, usa) by the ying jun gene company (shanghai, china). sequencing results were compared with the genbank database and confirmed through sequencing of clones. dna was cloned into a pgem - t easy vector following the manufacturer s instructions (promega, madison, wi). the osteogenesis imperfecta and ehlers - danlos syndrome variant databases compiled of previously reported oi mutations in col1a1 and col1a2, the 1000 genomes database, and the uniprokb database were also used for comparison. pcr and sequencing of col1a1 and col1a2 genes in samples of other family members were performed with the same methods. open reading frame analysis of the mutated sequence was performed by the orf finder, part of the blast software suite. the parental protein sequence and the protein sequence corresponding to the c.700delg mutation the 1000 genomes database was also used to search for the presence of this mutation by using the alignment function of the blast software and the following search sequence : 5-ttttctctccctctcagggga agctggaaaacctggt cgtcctggtgagcg-3. the proband was a 13-year - old girl of han descent from the shanxi province of china. the first fracture occurred in the right tibia / fibula after a slight injury at 16 months old and its healing was achieved with fixation of a plate. physical examination revealed short stature, blue sclerae, discrete signs of dentinogenesis imperfecta, pigeon breast, deformities of slight scoliokyphosis and joint hypermobility. x - rays showed thoracic - lumbar vertebra compression fracture and osteoporosis, acetabulum, and femoral head protrusion acetabulum into the pelvis. the mother is a 43-year - old of han chinese descent. she had two fractures in childhood : one in the right tibia / fibula that occurred at age 5 and the other in the right radius at age 8. physical examination revealed blue sclerae, discrete signs of dentinogenesis imperfecta, deformity of scoliosis, and joint hypermobility. blood samples were analyzed from the two patients and blood serum calcium, phosphorus, and parathyroid hormone levels were all within the normal range (data not shown). sequence analysis revealed no mutations in the col1a2 gene from the proband or her mother. however, sequence analysis of col1a1 identified a heterozygous frameshift mutation in exon 10 from both subjects at nucleotide 700 of the mrna, named as c.700delg, with nucleotide 1 defined as a of the initiating atg (figure 2). this mutation was not identified in other family members (two sisters, father, and maternal grandmother of the proband) or ethnically matched controls. importantly, a comparison with the compiled list of col1a1 mutations associated with oi in the osteogenesis imperfecta and ehlers - danlos syndrome variant databases (dalgleish, 1997, 1998 and http://www.uniprot.org/uniprot/p02452a) revealed that the mutation is novel and had not been previously reported. this novel, heterozygous c.700delg frameshift mutation in col1a1 was inherited in an autosomal dominant manner and was correlated with oi symptoms (figure 2). an open reading frame (orf) analysis indicated that translation of the col1a1-c.700delg mrna would initiate at the standard methionine and would be 100% homologous until amino acid 233. however, the mutation would produce distinct amino acids from p234 to p263 when compared to wild - type collagen 1 and would terminate at the uga stop codon at nucleotide 792 in exon 10 (figure 3)., the putative 263 amino acid collagen 1 700delg mutation would contain the signal peptide (p122), propeptide (p23161) and the first 102 residues of the main collagen 1 chain (p1621218 and the triple helical region from p1791192), but no c - terminal propeptide (p12191464). the proband was a 13-year - old girl of han descent from the shanxi province of china. the first fracture occurred in the right tibia / fibula after a slight injury at 16 months old and its healing was achieved with fixation of a plate. physical examination revealed short stature, blue sclerae, discrete signs of dentinogenesis imperfecta, pigeon breast, deformities of slight scoliokyphosis and joint hypermobility. x - rays showed thoracic - lumbar vertebra compression fracture and osteoporosis, acetabulum, and femoral head protrusion acetabulum into the pelvis. the mother is a 43-year - old of han chinese descent. she had two fractures in childhood : one in the right tibia / fibula that occurred at age 5 and the other in the right radius at age 8. physical examination revealed blue sclerae, discrete signs of dentinogenesis imperfecta, deformity of scoliosis, and joint hypermobility. blood samples were analyzed from the two patients and blood serum calcium, phosphorus, and parathyroid hormone levels were all within the normal range (data not shown). sequence analysis revealed no mutations in the col1a2 gene from the proband or her mother. however, sequence analysis of col1a1 identified a heterozygous frameshift mutation in exon 10 from both subjects at nucleotide 700 of the mrna, named as c.700delg, with nucleotide 1 defined as a of the initiating atg (figure 2). this mutation was not identified in other family members (two sisters, father, and maternal grandmother of the proband) or ethnically matched controls. importantly, a comparison with the compiled list of col1a1 mutations associated with oi in the osteogenesis imperfecta and ehlers - danlos syndrome variant databases (dalgleish, 1997, 1998 and http://www.uniprot.org/uniprot/p02452a) revealed that the mutation is novel and had not been previously reported. this novel, heterozygous c.700delg frameshift mutation in col1a1 was inherited in an autosomal dominant manner and was correlated with oi symptoms (figure 2). an open reading frame (orf) analysis indicated that translation of the col1a1-c.700delg mrna would initiate at the standard methionine and would be 100% homologous until amino acid 233. however, the mutation would produce distinct amino acids from p234 to p263 when compared to wild - type collagen 1 and would terminate at the uga stop codon at nucleotide 792 in exon 10 (figure 3). this col1a1 mutation would be named p.e234kfsx264. according to the orf analysis, the putative 263 amino acid collagen 1 700delg mutation would contain the signal peptide (p122), propeptide (p23161) and the first 102 residues of the main collagen 1 chain (p1621218 and the triple helical region from p1791192), but no c - terminal propeptide (p12191464). mutations in col1a1 and col1a2 account for approximately 90% of oi cases and usually occur in patients with oi types i iv. oi symptoms can also occur in patients with mutations in genes that code proteins that interact with type i collagen, including cartilage associated protein (crtap ; starman., 1989 ; barnes., 2006 ; morello., 2006 ; valli., 2012), prolyl-3-hydroxylase 1 (p3h1/lepre1), cyclophillin b (ppib ; morello., 2006 ; fratzl - zelman., 2010 ;, 2012 ; zhang., 2012), fkbp10 (barnes., 2012), wnt-1 (pyott., 2013), and serpin peptidase inhibitor clade f member 1 (serpinf1, homan., 2011). in our report, the clinical presentation of oi in the proband and her mother was mild, and the associated finding of a frameshift mutation in both patients allowed diagnosis of type i osteogenesis imperfecta. many patients diagnosed with mild oi type i have null variants in either col1a1 or col1a2 genes and a second allele with the wild - type sequence. the phenotype that results from these null variants is due to approximately 50% decrease of the wild - type production of the type 1 collagen 1 or 2 chains, known as haploinsufficiency (redford - badwal. ben amor., 2013 ; peng., 2012 ; zhang the phenotypes of patients with haploinsufficiency may be caused by increased degradation of the abnormal type 1 collagen chain (garnero., 2009), alterations of the mutant mrna, such as nonsense mediated decay of the mrna from the mutant allele, or inefficient transport of mrna to the cytoplasm (redford - badwal. nonsense mutations, frameshift mutations, and splice site mutations can induce a premature termination codon in the orf. for example, 23 of 33 chinese patients with oi type i had mutations leading to haploinsufficiency in col1a1 ; the mutations included eight frameshift mutations, eight splice site mutations, and seven nonsense mutations (zhang., 2012). mutations associated with haploinsufficiency occurred in exons 2, 3, 5, 7, 9, 11, 17, 18, 19, 24, 28, 31, 36, 42, 43, 46, 47, 49, and 52 (slayton., 2000 ; zhang., 2012) and introns 44i (peng., 2012). several of the previously described null mutations of col1a1 appear to be transcribed into (hnrna) at equivalent levels to wild - type col1a1, but the mrna levels of the null mutations are very low or undetectable in the cytoplasm (slayton., 2000). similar mutations have been observed less frequently in col1a2 (zhang., 2012). in a recent study, eighty six canadians with oi type i due to haploinsufficiency that resulted from nonsense and frameshift mutations showed a risk of 0.62 bone fractures per year which was approximately 95 times greater than that of healthy person of british decent (ben amor., 2013). the proband and mother described here had suffered only two fractures each in their lifetimes. a second class of col1a1 and col1a2 mutations are translated into procollagen chains that exhibit altered structure. these mutated procollagen chains become en - twined with normal collagen chains and the resulting triple helix has a modified configuration, reducing bone strength. the most common mutations result in replacement of one single glycine in the many essential gly - x - y triplets in the triple helical region (p.1791192). the substituted amino acid may include serine, arginine, aspartic acid, valine, or alanine at a single position (ben amor. these point mutations that replace glycine can occur throughout most of the length of col1a1 and affect collagen stability (witecka. these expressed col1a1 and col1a2 peptides usually cause more severe oi types (ii, iii, iv) and usually result from point mutations, insertions, deletions, duplications, and frameshift mutations in the last 200 nucleotides of the reading frame (xiao., 2011). table 1 is a summary of current known mutations of col1a1 that have been documented. seven mutations were described in the ninth intron that may reduce the efficiency of the 3 splice site. interestingly, three patients with the same mutation presented with oi of varying severity (oi type i or type iv). six mutations were described in exon 10 in two col1a1 databases. g > a, c.725 g > a, c.740 g > t, c.742 g > a, and c.743 g > a were diagnosed with type iv, unclassified oi, oi type i, oi type iii, and oi type iii, respectively using the osteogenesis imperfecta and ehlers - danlos syndrome variant databases (dalgleish, 1997, 1998) and the uniprokb database. taken together, these data, combined with the description of the novel mutation c.700delg described here, suggest that additional genes and possibly environmental factors such as nutritional status during development may influence the severity of oi. adapted from the osteogenesis imperfecta and ehlers - danlos syndrome variant databases (dalgleish, 1997 ; dalgleish, 1998). location of clinician who submitted the mutation and characteristics of the carrier of the database. if translated, the c.700delg allele would result in a mutant collagen 1 that would be significantly shorter than the wild - type collagen 1 (1464 residues) and would not contain two regions, helix positions p.691893 and p.910964, in which single point mutations are usually lethal ((ben amor., 2011). because of the mild oi type i symptoms and the dominant inheritance in the pedigree, the col1a1-c.700delg variant likely leads to haploinsufficiency. the col1a1-c.700delg was not detected in samples from the proband s maternal grandmother, father, two sisters and was not present in the 50 control samples from unrelated individuals who did not express the col1a1 mutation. limitations of this study include the fact that we only sequenced col1a1 and col1a2 genes. while the dominant inheritance pattern suggests involvement of col1a1 or col1a2, other known oi candidate genes which induce a recessive oi were not examined, including crtap (barnes., 2006 ; morello., 2010 ; zhang., 2012), fkbp10 (barnes., 2012), wnt-1 (pyott., 2013), and serpinf1 (homan., 2011). it is theoretically feasible that variants in the aforementioned proteins that interact with collagen may compensate for a collagen 1 or 2 variant and thereby contribute to the heterogeneity of the oi symptoms. although mutations of col1a1 and col1a2 have been associated with oi for several decades, the relationship between the clinical symptoms and location of the variants need further elucidation. additional genetic or epigenetic factors must influence the variety and severity of symptoms since clinical symptoms of patients with the same mutations can vary among family members (rauch., 2013). the effect of collagen 1 and 2 variants on proteins that interact with collagen, such as crtap, lepre1, ppib, fkbp10, serpinh1, and sp7 (garnero. the novel frameshift variant in exon 10 described here is located only 16 nucleotides upstream of another previously described exon 10 point mutation, c.716 g > a, which was detected in a patient with oi type iv (sarafova., 1998). comparison of the interacting factors for hnrna, mrna, and mrna transport of these two col1a1 variants may help elucidate the phenotypic differences. in summary, the col1a1-c.700delg variant described here in a proband and her mother, if translated, would produce a truncated form of collagen 1 chain without the two essential helical regions (691893 and 910964) found in the wild - type form. because of the mild oi type i phenotype, we propose that the novel col1a1-c.700delg variant likely leads to haploinsufficiency of the collagen 1 chain and that this frameshift variant is the molecular basis of oi type i in this han chinese family. its identification adds further insight to col1a1 mutations in the han chinese population with oi type i. | osteogenesis imperfecta (oi) is a family of genetic disorders associated with bone loss and fragility. mutations associated with oi have been found in genes encoding the type i collagen chains. people with oi type i often produce insufficient 1-chain type i collagen because of frameshift, nonsense, or splice site mutations in col1a1 or col1a2. this report is of a chinese daughter and mother who had both experienced two bone fractures. because skeletal fragility is predominantly inherited, we focused on identifying mutations in col1a1 and col1a2 genes. a novel mutation in col1a1, c.700delg, was detected by genomic dna sequencing in the mother and daughter, but not in their relatives. the identification of this mutation led to the conclusion that they were affected by mild oi type i. open reading frame analysis indicated that this frameshift mutation would truncate 1-chain type i collagen at residue p263 (p.e234kfsx264), while the wild - type protein would contain 1,464 residues. the clinical data were consistent with the patients diagnosis of mild oi type i caused by haploinsufficiency of 1-chain type i collagen. combined with previous reports, identification of the novel mutation col1a1-c.700delg in these patients suggests that additional genetic and environmental factors may influence the severity of oi. |
zebrafish have been a popular model organism in genetics and developmental research for decades and have more recently attracted the interest of scientists studying behavior as well. they present a number of advantages relative to rodent models, most notably their amenity to forward genetic screening and the relative simplicity of the system, which nonetheless shares good degree of homology with other vertebrates including humans. one emerging area of translational research for which zebrafish have been proposed as an excellent model is the study of stress. the zebrafish stress system appears to be quite comparable to the hypothalamic - pituitary - adrenal (hpa) axis in mammalian models, with homologies between corticotrophin - releasing factor (crf), adrenocorticotropic hormone (acth), and cortisol function in zebrafish and in mammals. researchers have used a wide variety of stimuli to induce stress in adult zebrafish, including restraint or confinement, social crowding and isolation, heat and cold stress, predator cue exposure, handling or netting, water or tank changes, and mild electric shock. indices of stress have included cortisol and crf measures, as well as behaviors such as diving, locomotor activity (including both hyperactivity and immobility), turn angle, shoal cohesion, and scototaxis. the majority of stress research with zebrafish has involved unpredictable stressors, administered either acutely in a single session or chronically over several days (chronic unpredictable stress : cus). indicate that acute restraint stress increases whole - body cortisol levels and behavioral measures of locomotor activity, without any changes in diving behavior. in contrast, chakravarty. found that cus led to an increased diving response and suppression of locomotor activity. also report increased diving and suppressed locomotion as a response to cus as well as reduced shoal cohesion. it therefore appears that acute and cus may induce quite different behavioral signatures, although they have not yet been compared directly. while unpredictable stressors clearly induce a variety of behavioral and physiological effects in zebrafish, it is not yet known how predictable stress may differ from unpredictable stress. on one hand, it is possible that predictability attenuates stress, leading to reduced effects of treatment. on the other hand, it could be that stimuli predictive of stress become stressors themselves, leading to an exaggerated effect of treatment. our goal with this experiment was to examine the behavioral and proteomic effects of predictable and unpredictable stress on adult zebrafish. fear conditioning, one of the most commonly used techniques for the study of learning in rodent models, is basically a predictable stress procedure. standard fear conditioning is a classical conditioning procedure in which an initially neutral light or tone (cs) is paired with an electric shock (us). although classical fear conditioning has not been widely used with adult zebrafish (but see refs (810)), electric shock appears to be an effective aversive stimulus for fish. in this study, the use of electric shock as a stressor was appealing for two primary reasons. first, the variety of stressors often used to induce chronic stress (such as crowding, heat, and predator exposure) is difficult to control temporally in relation to a predictive stimulus and almost by definition introduce a degree of unpredictability. second, using a variety of stressors is likely to produce a range of proteomic effects due directly to stimulus exposure rather than stress per se. although electric shock may also produce direct effects unrelated to the subsequent stress response, the use of a single highly controllable stressor minimizes this problem. in the current experiment the behavior and proteome of an experimental group, which received a standard fear conditioning treatment in which a light predicted shock exposure, and an unpaired group, in which the light was not predictive of shock exposure, were compared with that of a control group receiving only light exposure. following behavioral training, animals were sacrificed, and their whole proteomes were analyzed and compared across groups. subjects were 28 adult wild - type zebrafish (50:50 male / female, age 69 months) purchased from a local aquarium supply store (aquatic warehouse, san diego, ca). subjects were housed in an aquaneering table - top housing rack, with a recirculating filtration system using mechanical, biological, and chemical filtration. prior to the experiment, all animals were housed together in a single 10 l tank ; during the experiment, animals were separated into pairs in 1.8 l tanks within the same housing system. the temperature of the tanks was held at 26 c, and the room was maintained on a 14:10 h light / dark cycle. subjects were fed twice daily on a mixed diet of live brine shrimp (artemia franciscana), freeze - dried brine shrimp (san francisco bay brand, san francisco, ca), and tetra - min (tetra, melle, germany) flake food. the housing conditions and experimental protocols were approved by the university of san diego institutional animal care and use committee. the conditioning apparatus was a small rectangular tank made of opaque white acrylic (12 cm 6 cm 6 cm ; length width depth), with a black cover opaque to visible light but transparent to infrared light. the tank was filled with water from the housing system to a depth of 4 cm. there was a bank of white led lights along one of the shorter walls of the tank that served as a cs. each of the two longer walls was lined with a grid of silver wire ; the grid along one wall was grounded, and the other opposite grid was connected to a shock scrambler that administered a 7 v electric shock through the entire tank. the magnitude of electric shock was primarily selected based on pilot data, indicating this to be the lowest shock level that reliably produced a measurable behavioral response in the fish, which is consistent with other reports in the literature. an infrared video camera suspended 40 cm above the testing tanks was used in conjunction with a bank of infrared lights beneath the apparatus to monitor the location and activity of the fish. the video camera fed into a desktop computer using viewpoint videotrack v3.2 to control stimulus administration and to track the locomotor activity of the fish. circles represent the led lights used as a conditioned stimulus, and the heavy dashed lines represent electrodes used to administer the unconditioned stimulus shock. animals were divided randomly into three groups of 810 subjects each : experimental (exp), unpaired (unp), and no shock control (nos). subjects in all groups were given 16 trials, distributed across 4 days with four trials each day (figure 2). intertrial intervals (itis) were variable with a mean of 180 s (60 s). the cs was a 15 s white light, and the us was a 5 s shock train (1 shock / s). at the beginning of training, each subject was placed individually into the experimental tank and given 30 s to acclimate prior to the start of the session. subjects in group exp then received four trials per day in which the cs and us were paired ; after a variable iti, the cs light came on for 10 s, and the us was presented during the final 5 s of cs exposure, with light and shock train coterminating. subjects in group unp received four exposures per day to the 15 s cs and the 5 s us, but the two were not presented concurrently. the cs were presented according to the same schedule used in group exp, with us occurring between 30 and 175 s (varying pseudorandomly) after the cs (and at least 30 s prior to the subsequent cs). subjects in group nos received four exposures per day to the 15 s cs but did not receive any us exposures. the cs group was presented according to the same schedule used in both exp and unp. activity during the iti, the cs, and the us, was monitored using video - tracking software. following each training session, each animal was removed from the experimental tank and returned to its home tank. at the last session, four randomly selected animals from each group were anaesthetized in an ice bath and immediately decapitated within 10 min of completion of behavioral testing and processed for proteome samples immediately. a diagram of the experimental procedure is presented in figure 2. experimental design. overview of the experimental treatments for each group : experimental (exp), unpaired (unp), and no shock control (nos). subjects received paired light (conditioned stimuli ; cs) and shock (unconditioned stimuli ; us) for 4 days as previously described. squares represent presentations of the cs light, and the bolt signifies the us shock. zebrafish (4/group) were decapitated and rinsed with ice - cold pbs containing protease inhibitor cocktail tablet (roche, indianapolis, in) ; then, the whole fish was placed in 3 ml of lysis buffer containing 8 m urea, 500 mm tris (hydroxyethylamine), ph 8.5 and protease inhibitors cocktail (roche, indianapolis, in). precellys 24 tissue homogenizer (bertin technologies) was for protein extraction by adding 2.8 ceramic beads (zirconium oxide) to tubes and homogenizing at 6000 rpm for 30 s at 4 c under electrostatic condition, followed by bca protein quantification (sigma - aldrich, st. the precipitated protein pellet was resuspended in 2%sds and 0.1 m triethylammonium bicarbonate (teab) dissolution buffer, followed by reduction with 200 mm tris(2-carboxyethyl)phosphine (tcep) and alkylation with 375 mm iodoacetamide. for endopeptidase digestion, modified trypsin (promega, madison, wi) was added at 50:1 (protein / protease mass ratio) along with 1 mm cacl2 and incubated overnight in a thermoshaker at 600 rpm at 37 c. to recover short peptides that did not undergo acetone precipitation, 1.3 ml of cold methanol and 15 l of acetic acid were mixed with the supernatant and spun down briefly at 18 000 g for 15 min. delipidation was performed by adding 200 l of ethyl acetate to the sample, followed by pellet drying and resuspending in 0.1 m teab. the tandem mass tag (tmt) labeling was performed according to the manufacturer s instructions (thermo fisher scientific, rockford, il) with some modifications. the tmt reagents (0.8 mg) were dissolved in 100 l of anhydrous acetonitrile (acn). for the triplex experiment, each of the labeling reaction mixtures contained 25 l of the tmt reagent and 75 l (50 g) of the tryptic digest in teab buffer to ensure that the organic (acn) content was between 25 and 30% (v / v) for the reagent s stability. aliquots of the tryptic digest were derivatized with triplex chemical labels 126.127, 128.134, and 130.141 th (thomson). after the labeling, reaction mixtures were incubated at room temperature for 1 h, and 15 l of 5% hydroxylamine solution in water was added to quench the labeling reaction. each tmt - modified digest from triplex was then combined into one sample and vacuum - dried. the lyophilized tmt - labeled peptides were reconstituted with 500 l of buffer a (0.1% fa, 5% acn in water) centrifuged at 14 000 rpm for 30 min to remove particulates prior to loading into the multidimensional protein identification technology (mudpit) trapping column. three ms runs comprising three technical replicates were performed by loading 150 g of the tmt - modified digest into the mudpit column. mass spectrometric (ms) analysis of tmt triplex samples was performed using mudpit technology. an analytical rplc column was generated by pulling a 100 m id/360 m od capillary (polymicro technologies, phoenix, az) to 3 m i d tip. the pulled column was packed with reverse - phase particles (aqua c18, 3 m diameter, 90 pores, phenomenex, torrance, ca) until it was 12 cm long. a mudpit trapping column was prepared by creating a kasil frit at one end of an undeactivated 250 m id/360 m od capillary (agilent technologies, santa clara, ca), which was then successively packed with 2.5 cm strong cation exchange particles (partisphere scx, 5 m dia, 100 pores, phenomenex) and 2.5 cm reverse - phase particles (aqua c18, 5 m dia., 90 pores, phenomenex). the mudpit trapping column was equilibrated using buffer a prior to sample loading. after sample loading and prior to ms analysis, the resin - bound peptides were desalted with 1 ml of buffer a by letting it flow through the biphasic trap column. mudpit and analytical columns were assembled using a zero - dead volume union (upchurch scientific, oak harbor, wa). ms / ms analysis was performed on q - exactive (thermo scientific, san jose, ca) interfaced at the front end with easy - nlc ii hplc pump (thermo scientific) using an in - house built electrospray stage. electrospray was performed directly from the analytical column by applying the esi voltage at a tee (150 m i d, upchurch scientific) directly downstream of a 1:1000 split flow used to reduce the flow rate to 250 nl / min through the columns. a fully automated 11-step mudpit run was performed on the combined sample using a three mobile phase system consisting of buffer a (5% acn ; 0.1% formic acid (fa) (sigma - aldrich), buffer b (80% acn, 0.1% fa), and buffer c (500 mm ammonium acetate, 5% acn, 0.1% fa). the first step was 60 min, whereas subsequent steps were 135 min each. each mudpit run includes steps 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100% buffer c run for 4 min at the beginning of the gradient. as peptides were eluted from the microcapillary column, they were electrosprayed directly into a mass spectrometer with the application of a distal 2.4 kv spray voltage. for each cycle, survey full - scan ms spectra (m / z 4001800) were acquired in the orbitrap with a mass resolution of 30 000 at m / z 400 and an automatic gain control (agc) target of 1 10 ions with a maximal injection time of 250 ms. each full scan was followed by the selection of the most intense ions, up to 10, for higher - energy collisional dissociation (hcd)ms / ms analysis in the orbitrap. from our experience, hcd fragmentation is optimum for quantifying labeled peptides based on their reporter ion intensity. in all cases, ms / ms scans were acquired in the orbitrap with a mass resolution of 17000. the selected peptide ions were dynamically excluded from further analysis for 120 s to allow for the selection of lower - abundance ions for subsequent fragmentation and detection using the setting for repeat count = 1, repeat duration = 30 ms, and exclusion list size = 500. the minimum ms signal for triggering ms / ms was set to 5000, and an activation time of 0.1 ms was used. the m / z isolation width for ms / ms fragmentation was set to 2 th. for ms / ms, tandem mass spectra were extracted from raw files using rawextract 1.9.9 and searched with the prolucid algorithm against danio rerio uniprotkb / trembl database with reversed sequences (56 285 entries). the search space included all fully and semitryptic peptide candidates (at least 6 amino acids). carbamidomethylation of cysteine (+ 57.02146 amu) was considered as a static modification as well as n - terminal and lysine modification (+ 229.1629 amu) for triplex tmt labels analysis. the search parameter includes 10 ppm precursor mass tolerance, 0.6 da peptide mass tolerance. exported prolucid files were assembled and filtered using the dtaselect2.0, which combines xcorr and deltacn values using a quadratic discriminate function to compute a confidence score. the false discovery rate (fdr) was kept at 1% at the protein level. for quantitative analysis, census was used to extract the relative intensities of reporter ions for each peptide from the identified tandem mass spectra for normalization. statistical analysis for both behavioral and proteomic analysis werewas performed using repeated - measures analyses of variance (anova) with tukey s post hoc test. prolucid, xcorr, deltacn, and zscore values were used to generate a bayesian discriminator. outlier points in the two distributions having a mahalanobis distance greater than four were discarded. peptide expression alteration (fold changes), log values, and confidence were calculated based on reporter ion peak intensities generated from the ms analysis after extracting confident protein spectra with p 4 cm / s) in each 5 s time interval. the third was the duration traveling at high velocity (seconds) in each 5 s time interval. the final measure was the duration spent immobile (seconds) in each 5 s time interval. these measures were taken during the presentation of the cs, during the iti, and during the us. there were no significant changes across days in the total distance moved, but there was a significant difference between groups (f(2, 25) = 50.72, p 0.001), with group exp producing the highest total distance and group nos producing the lowest. there was a significant reduction across days in high - velocity distance (f(3, 75) = 3.51, p 0.05), with no main effect of group and no group day interaction. there was also a significant effect of day (f(3, 75) = 2.96, p 0.05) on duration traveling at high velocity, again with less high - velocity activity across days, and no effect of group. these results indicate that there was, in general, a reduction in high - velocity activity across days for all groups, likely reflecting habituation to the experimental context. there were no significant effects of group on any of the behavioral measures during the iti. there was a significant main effect of day on distance traveled at high velocity during the iti : overall, there was more high - velocity activity in the first 2 days than in the second 2 (f(3, 75) = 2.96, p 0.05). however, none of the other behaviors varied by day. there were no day group interactions for any behavior, so the reduction of activity across days did not differ across groups. therefore, there were no apparent differences between the three groups in baseline locomotor activity. as expected, the two groups receiving a shock us (exp and unp) exhibited a pronounced unconditioned hyperactivity response during the presentation of the shock. there was a significant main effect of group on the total distance moved (f(1, 25) = 24.41, p 0.001), on distance at high velocity (f(1, 25) = 14.38, p 0.001), on duration at high velocity (f(1, 25) = 25.98, p 0.001), and on duration immobile (f(1, 25) = 17.67, p 0.001). both distance at high velocity and duration at high velocity also exhibited a main effect of day (f(3, 75) = 3.06, p 0.05 and f(3, 75) = 4.077, p 0.01), with a reduction in high - velocity swimming across days, but there was no day group interaction. overall, these results indicate that the two shocked groups show more high - velocity swimming and less immobility than group nos (figure 3). they suggest the possibility of some long - term habituation to the shock across days, although evidence of this possibility is weakened by the lack of a significant day group interaction. the three groups produced similarly low levels of high velocity swimming during the iti, indicating that baseline locomotor activity was comparable across the three groups. the two groups receiving electrical shock (experimental, exp, unpaired, unp) produced a vigorous high - velocity swimming response when the shock was presented. y scale represents distance moved in centimeters. error bars represent standard error, and represents significance at p 0.05 (n = 810 in each group). there were no significant differences in total distance moved during the light cs. there was no main effect of group on high - velocity distance, but there was a significant effect of day (f(3, 75) = 5.66, p 0.001), with an overall reduction of activity across days, and a significant day group interaction (f(6, 75) = 2.56, p 0.05), with group exp suppressing activity the most across days, moderate suppression by group unp, and no suppression by group nos. there was no main effect of group on duration at high velocity, but there was a significant effect of day (f(3, 75) = 6.17, p 0.001), again with less activity across days, and a significant day group interaction (f(6, 75) 2.40, p 0.05). there were no significant effects of day or group on duration immobile during the cs. posthoc analysis indicates that group exp showed a significant reduction of high - velocity activity across days relative to group nos, while group unp did not differ significantly from either exp or nos. these results indicate that while absolute levels of activity during the cs did not differ between groups, group exp exhibited significant suppression of activity across days relative to the control group. conditioned response is presented in terms of the proportion of distance moved at high velocity on day 4 relative to that on day 1. a proportion of 1.0 represents no change in response to the conditioned stimuli (cs) across days of training. all three groups exhibited a similarly small reduction in activity during the intertrial intervals (iti) across days. group no shock control (nos) showed a similar pattern of response to the cs, with relatively little change across days. both groups unpaired (unp) and nos suppressed activity during the cs somewhat on day 4 relative to day 1, with the most suppression of activity by group experimental (exp). error bars represent standard error, and represents significance at p 0.05 (n = 810 in each group). to investigate the consequences of the shock exposure on the proteome profiles of the experimental groups global proteome screening identified an average of 295, 280, and 337 nonredundant protein candidates in exp, unp, and nos groups, respectively (see supplement 3 in the supporting information). as shown in figure 5a, however, 27 proteins were dysregulated (up- or down - regulated) in stressed groups (exp and unp) compared with the nos control group (data not shown). on the peptide level, a modest increase (p < 0.05) in peptide frequencies was detected when exp and unp groups were compared with the control nos group (figure 5b). (a) violin plot of normalized protein abundance showing box plot overlaid with kernel density distribution. (b) peptide ratio and frequency of matched peptides between exp / nos (black) and unp / nos (red). we further quantified the significant proteins between experimental groups based on the reporter ion intensity of the tmt - labeled tags. this precise method enables accurate protein quantification and overcome technical variability, such as lc retention, time draft, and nanospray instability. to properly view protein changes as a result of stress conditioning, we used log - transformed data (based on reported ion intensity) to obtain a guassian distribution of protein abundances and plotted against probability scores (figure 6a). among the identified candidate proteins, eight proteins passed the filtering process (at least one - fold difference and p < 0.05) and were believed to be up - regulated significantly in stressed groups compared with control nos group. these proteins are trifunctional enzyme subunit beta (hadhb), heat shock 70 kda protein 8 (hspa8) and 5 (hspa5), actin beta 1 (actb1), myosin heavy chain 4 (myhc4), calcium - transporting atpase (atp2a1), and two novel actin proteins [zgc:86709 and zgc:86725 ]. additionally, heat map and hierarchical cluster analysis were used to identify proteins with certain patterns of changes under different stresses. the differentially regulated proteins were clustered according to similarities in change profiles across all conditions, as shown in figure 6b. (a) volcano plot showing significance and magnitude of fold change of the same protein hit between exp and nos (in black dots) and unp and nos (in red dots) based on quantitative reporter ion intensity of tmt labeled peptides. (b) heat map and hierarchical display of top 30 differentially expressed proteins between experimental groups. asterisks shown represent proteins passed filtering threshold (at least one - fold difference and p < 0.05) excluding the two novel proteins. the differentially regulated proteins were clustered according to similarities in change profiles across all conditions. trifunctional enzyme subunit beta (hadhb), heat shock 70 kda protein 8 (hspa8) and 5 (hspa5), actin beta 1 (actb1), myosin heavy chain 4 (myhc4), calcium - transporting atpase (atp2a1), and two novel actin proteins [zgc:86709 and zgc:86725 ]. proteins were tmt - labeled, quantified, and listed along with their accession numbers, gene symbol, sequence length, theoretical isoelectric point (pi), and molecular weight (mw) in kilodaltons, peptide number, normalized intensity of corresponding reporter ions, fold changes of exp and unp in relation with nos control. exp, experimental ; unp, unpaired ; nos, no shock control group. to access the major biological themes perturbed by stress in zebrafish, we performed a gene ontology (go) analysis for the output proteome of the experimental groups. the go annotation was extracted using panther and searched against danio rerio reference genome database (zfin). illustration of protein molecular function, biological process, and protein classes are shown in figure 7. interestingly, compared with control nos group, both exp and unp groups showed overrepresentation of proteins related to stress response (hspa8 and hspa5), catalytic activity (hadhb), cation transport (atp2a1), and motor activity (actb1 and myhc4). in addition, two novel actin proteins (zgc:86709, and zgc:86725) were significantly up - regulated and have atpase - like activity. (a) molecular function, (b) biological processes, and (c) protein classes. p 0.05. the goal of this study was to investigate the behavioral and proteomic effects of predictable and unpredictable stress in zebrafish. overall, our results indicate that exposure to electric shock produces a behavioral hyperactivity response and induces proteomic changes related to locomotor activity and oxidative stress in experimental groups relative to control. additionally, although differences between the group exp and unp were minor, there were some indications that predictable shock produced a behavioral response to the predictive stimulus. however, this behavioral response was not reflected significantly on the proteome level, at least under the current experimental condition. all three groups showed a reduction in high - velocity swimming across the 4 days of testing. this suggests that either animals habituated to the experimental context across days and therefore exhibited less escape behavior or handling and transportation stress caused animals to sensitize to the experimental setting and suppress activity across days. although further experimentation is needed to determine whether a reduction in locomotor activity indicates habituation of exploration, escape, or sensitization of anxiety to a novel context, because this effect was observed in all three groups, it is not central to our current question. this result is, however, consistent with observations by other researchers that chronic stress may produce a global suppression of locomotor activity. our behavioral results showed a clear unconditioned response to shock in groups unp and exp, with animals exhibiting a roughly nine - fold increase in high - velocity swimming and doubling their total swim distance during the shock interval. this increase is apparent within subjects relative to activity during the iti and also between - subjects relative to the no - shock comparison group (nos) ; see figure 3. behaviorally, there were no differences in response to the shock between the two groups, indicating that the presence of a predictive cue (in group exp) did not significantly affect response to the shock itself. it is likely that these behavioral effects are reflected on the proteome profile of exp and unp groups by the increase in motor (cytoskeletal) proteins such as actb1 and myhc4. interestingly, these two proteins are homologous to mammalian fast skeletal muscle isoforms that are recruited for very short - duration high - intensity bursts of power and thought to be up - regulated to accommodate the required fast escaping behavior during shock intervals. again, both proteins have atpase - like activities that hydrolyze atp for energy production. the increase in high velocity swimming was also correlated with elevation in the sarcoplasmic / endoplasmic reticulum calcium atpase (atp2a1 ; 1.2 fold), which catalyzes the hydrolysis of atp coupled to the translocation of calcium from the cytosol to the sarcoplasmic reticulum lumen to regulate muscular excitation and contraction. as expected, we also reported a significant up - regulation of both heat shock proteins 8 and 5 (hspa8, hspa5) with 3.3 and 1.4 fold, respectively, in both exp and unp groups. the up- regulation of heat shock proteins (hsps) during cellular stress has been well - defined. these molecular chaperones were shown to function in different cellular processes such as protein folding, actin remodeling, reduction of oxidative stress, and their ability to help cells survive under stress conditions. therefore, the release of hsp detected in this study is believed to protect the cells from damage caused by oxidative stress and acts as part of the cell s internal repair mechanism to maintain homeostasis. although both groups receiving shock exhibited a comparable behavioral response to shock and similar proteomic profiles, group exp did exhibit some effects not observed in group unp. behaviorally, the two groups differed in their conditioned response to the cs light. group exp exhibited significant suppression of activity during the cs across days of training (relative to nos), suggesting a conditioned fear response analogous to that observed in rodents. the unpaired control group (unp) showed a moderate suppression of swimming during the cs light, likely due to cross - sensitization from the shock, but this suppression was not significant in comparison with group nos, which exhibited no change in response to the light at all. some unique effects of the predictable shock might also be apparent in the proteomic data. in a clear unambiguous fashion, we detected a significantly high level of trifunctional enzyme subunit beta (hadhb ; 4.4 fold) in the exp group. this enzyme catalyzes three out of the four steps in the mitochondrial beta - oxidation cycle for energy production. the cascade of mitochondrial fatty acid oxidation is believed to be a central energy generating process particularly during long fasting, infection, and stress. in addition, a positive relationship between beta - oxidation and stress tolerance has been recently defined. for instance, drosophila melanogaster over - expressing fatty acid beta - oxidation component shows substantial resistance to oxidative stress. taken all together, the prominent increase in hadhb quantified in the current study might be an attempt to overcome oxidative stress conditions resulting from elevated fear / anxiety levels during the cs light. we also observed the involvement of two up - regulated proteins in the exp group : novel actin - like protein [zgc:86709 ] and a novel protein similar to alpha actin1 [zgc:86725 ]. on the basis of their sequence homology, these proteins these two protein identifications should be viewed as putative until confirmed using alternative experimental techniques. although groups unp and exp differed somewhat, the clearest behavioral differences were between group nos and the two groups receiving shock. likewise, global go screening of the experimental group proteomes did not reveal significant differences between exp and unp groups, suggesting undetectable / minimal effect of the predictive cue on the protein levels, at least under the current experimental condition. (see figure 7.) in contrast, the significant alteration in the pattern of protein expression between both shocked groups versus group nos receiving light only indicates the rapid induction of stress response proteins, catalytic enzymes, and locomotor proteins to counteract possible stress consequences. the sample size in this study was not large enough to establish meaningful correlations between individual subjects behavioral and proteomic outcomes (see supplement 1 in the supporting information), but this would be an interesting question for future research. finally, it is noteworthy to mention that we could not detect any significant neuropeptide regulation in this study probably due to their low abundance or analysis of the whole fish instead of brains only. additional experiments targeting the zebrafish brain should be considered in the future to disclose neuropeptide changes that might exist. to our knowledge, this is the first study to directly examine the effects of stress on both the behavior and the whole - body proteome of zebrafish. the overall pattern of results is consistent with elevated fear / stress levels in both groups exp and unp relative to group nos, with some indication of additional effects based on shock predictability in group exp. | the purpose of this study is to determine the behavioral and proteomic consequences of shock - induced stress in zebrafish (danio rerio) as a vertebrate model. here we describe the behavioral effects of exposure to predictable and unpredictable electric shock, together with quantitative tandem mass tag isobaric labeling workflow to detect altered protein candidates in response to shock exposure. behavioral results demonstrate a hyperactivity response to electric shock and a suppression of activity to a stimulus predicting shock. on the basis of the quantitative changes in protein abundance following shock exposure, eight proteins were significantly up - regulated (hadhb, hspa8, hspa5, actb1, mych4, atp2a1, zgc:86709, and zgc:86725). these proteins contribute crucially in catalytic activities, stress response, cation transport, and motor activities. this behavioral proteomic driven study clearly showed that besides the rapid induction of heat shock proteins, other catalytic enzymes and cation transporters were rapidly elevated as a mechanism to counteract oxidative stress conditions resulting from elevated fear / anxiety levels. |
drug - drug interactions, which refer to modification in the pharmacokinetics or effects of a drug by the presence of another drug (1), may convincingly result in increased or decreased efficacy of the treatment, complete treatment failure, as well as in increased toxicity of the drugs prescribed (2,3). it has been demonstrated that drug - drug interactions are mainly preventable conditions which can be carefully avoided by non - prescription of multiple drugs. as a matter of fact, putative positive effects of multiple - drug treatment should be evaluated vis - - vis the risk of the occurrence of clinically significant drug - drug interactions (4). drug - drug interactions are increasingly acknowledged as an area of major concern in medical care (5), because there have been consistent reports linking them with serious health consequences and significant clinical damage (6,7). it has been reported that the incidence of potential drug - drug interactions is close to 40% in patients taking five medications, and exceeds 80% in patients taking at least seven drugs (8,9). while several studies have investigated the frequency and nature of drug - drug interactions in community settings (10 - 12), this issue requires a particular attention in hospital settings, because more medications are usually prescribed simultaneously and more complex schemes and compounds are often used (5). therefore, the risk of drug - drug interactions is logically higher among hospitalized patients who are often affected due to severe and comorbid conditions associated with chronic polypharmacy effects, which additionally undergo frequent therapeutic alterations (4,5). as previously noted, the available information on drug interactions and their health effects for kosovo is scarce, regardless of the anecdotic evidence suggesting a high prevalence of drug interactions and its resultant toxic effect (13). furthermore, the situation is said to be particularly problematic among the older population subgroup which is highly dependent on multiple drug use given the high prevalence of comorbidity (13). in this context, the aim of our study was to assess the prevalence and socioeconomic and clinical correlates of drug - drug interactions among the adult population of kosovo, an independent country in the western balkans which is currently undergoing a difficult period of political and socioeconomic transition after a long and devastating war against serbia. this was a cross - sectional study which was carried out in the regional hospital of gjilan, kosovo, covering the period 2011 - 2013. the sampling frame consisted of all the patients aged 18 years hospitalized at the departments of internal medicine, cardiology, or infectious diseases of the regional hospital in gjilan from 01 - 01 - 2011 until 31 - 12 - 2013. based on this sampling frame, calculation of the sample size was made by use of winpepi (program for epidemiologists) for several hypotheses related to the prevalence and clinical correlates of drug - drug interactions in our study population. the significance level (two - tailed) was set at 5%, and the power of the study at 80%. based on the most conservative calculations, the required minimal size for a simple random sample was about 1570 patients records. we decided to recruit 2000 records in order to account for potential incompleteness of patients files. of the 2000 targeted patients records, there were 79 incomplete files, for which there was also not possible to re - interview the respective patients regarding their socioeconomic characteristics (educational attainment, employment status, income level, or social status). overall, 1921 patients records were included in our analysis (response rate : 1921/2000=96%). all patients records were carefully checked according to a structured checklist including clinical characteristics of the patients and other relevant data related to their hospitalization. potential drug - drug - interactions were assessed employing the drug interactions checker within www.drugs.com database (14). the drug - drug interactions evident in our study sample were subsequently classified as major, moderate and minor, depending on their severity of clinical significance and crossover checked manually for the presence of identified interacting agents, according to a few recent reports (4,15). hence, in our study, drug - drug interactions were identified and classified based on the profile of medications prescribed, as suggested by the recent literature on this field (4,15). other important clinical data included type of diagnosis, length of hospitalization and presence of comorbid conditions among patients included in the study. in addition to clinical information, demographic and socioeconomic data were retrieved from all the patients records and further verified (double - checked) by re - interviewing the patients (regarding selected socioeconomic factors such as educational attainment, employment status, income level, or social status). demographic factors included sex and age of the patients, place of residence and marital status at the time of hospitalization. socioeconomic characteristics included educational attainment (years of formal schooling), employment status, self - perceived income level and self - perceived social status. binary logistic regression was used to assess the association of drug - drug - interactions (outcome variable) with the independent variables including demographic and socioeconomic factors [age (< 40 years, 40 - 59 years, 60 years), sex (men vs. women), marital status (married vs. single / divorced / widowed), place of residence (urban areas vs. rural areas), education (low, middle, high), employment status (employed, unemployed, retired), income (low, middle, high) and social status (low, middle, high) ] and clinical characteristics of the patients [length of hospitalization (1 - 6 days vs. 7 days), diagnosis (infectious diseases, cardiovascular diseases, endocrine diseases, respiratory diseases, gastrointestinal diseases, other diseases), comorbidity (yes vs. no) and number of drugs (1 - 3 vs. 4) ]. crude (unadjusted) and multivariable - adjusted odds ratios (ors), their 95% confidence intervals (95%cis) and p - values were calculated. hosmer - lemeshow test was used to assess the goodness - of - fit of the logistic regression models. statistical package for social sciences, version 17.0, chicago, illinois, was used for all the statistical analyses. the sampling frame consisted of all the patients aged 18 years hospitalized at the departments of internal medicine, cardiology, or infectious diseases of the regional hospital in gjilan from 01 - 01 - 2011 until 31 - 12 - 2013. based on this sampling frame, calculation of the sample size was made by use of winpepi (program for epidemiologists) for several hypotheses related to the prevalence and clinical correlates of drug - drug interactions in our study population. the significance level (two - tailed) based on the most conservative calculations, the required minimal size for a simple random sample was about 1570 patients records. we decided to recruit 2000 records in order to account for potential incompleteness of patients files. of the 2000 targeted patients records, there were 79 incomplete files, for which there was also not possible to re - interview the respective patients regarding their socioeconomic characteristics (educational attainment, employment status, income level, or social status). overall, 1921 patients records were included in our analysis (response rate : 1921/2000=96%). all patients records were carefully checked according to a structured checklist including clinical characteristics of the patients and other relevant data related to their hospitalization. potential drug - drug - interactions were assessed employing the drug interactions checker within www.drugs.com database (14). the drug - drug interactions evident in our study sample were subsequently classified as major, moderate and minor, depending on their severity of clinical significance and crossover checked manually for the presence of identified interacting agents, according to a few recent reports (4,15). hence, in our study, drug - drug interactions were identified and classified based on the profile of medications prescribed, as suggested by the recent literature on this field (4,15). other important clinical data included type of diagnosis, length of hospitalization and presence of comorbid conditions among patients included in the study. in addition to clinical information, demographic and socioeconomic data were retrieved from all the patients records and further verified (double - checked) by re - interviewing the patients (regarding selected socioeconomic factors such as educational attainment, employment status, income level, or social status). demographic factors included sex and age of the patients, place of residence and marital status at the time of hospitalization. socioeconomic characteristics included educational attainment (years of formal schooling), employment status, self - perceived income level and self - perceived social status. binary logistic regression was used to assess the association of drug - drug - interactions (outcome variable) with the independent variables including demographic and socioeconomic factors [age (< 40 years, 40 - 59 years, 60 years), sex (men vs. women), marital status (married vs. single / divorced / widowed), place of residence (urban areas vs. rural areas), education (low, middle, high), employment status (employed, unemployed, retired), income (low, middle, high) and social status (low, middle, high) ] and clinical characteristics of the patients [length of hospitalization (1 - 6 days vs. 7 days), diagnosis (infectious diseases, cardiovascular diseases, endocrine diseases, respiratory diseases, gastrointestinal diseases, other diseases), comorbidity (yes vs. no) and number of drugs (1 - 3 vs. 4) ]. crude (unadjusted) and multivariable - adjusted odds ratios (ors), their 95% confidence intervals (95%cis) and p - values were calculated. hosmer - lemeshow test was used to assess the goodness - of - fit of the logistic regression models. statistical package for social sciences, version 17.0, chicago, illinois, was used for all the statistical analyses. mean age of the hospitalized patients included in our study sample was 57.811.2 years (58.410.6 years in men vs. 57.19.7 years in women) [table 1 ]. overall, 50% of the patients had a low educational level (48% in men vs. 52% in women). on the whole, 35% of the patients were unemployed with a remarkable sex - difference (28% in men vs. 42% in women). about 37% and 35% of the patients perceived as low their income level or social position, respectively. clinical characteristics by drug - drug interaction status of the patients are presented in table 2. there were 1192 patients with drug - drug interactions compared with 729 patients with no evidence of drug - drug interactions. hence, the overall prevalence of drug - drug interactions in our study sample was 1192/1921=62%. overall, 45% of the patients were hospitalized for 1 - 6 days compared with 55% of individuals who were hospitalized for seven days or more. the excessive length of hospitalization (7 days) was considerably higher among patients with drug - drug interactions compared with those without drug - drug interactions (66% vs. 37%, respectively). on the whole, 14% of the patients had infectious diseases, 26% cardiovascular diseases, 14% endocrine diseases, 18% respiratory diseases, 13% gastrointestinal diseases, whereas the remaining patients (16%) had other conditions. the prevalence of comorbidity was substantially higher among patients with drug - drug interactions compared with those without drug - drug interactions (61% vs. 26%, respectively). furthermore, the proportion of individuals who were administered 4 drugs was remarkably higher among patients with drug - drug interactions compared with those without drug - drug interactions (70% vs. 24%, respectively). distribution of demographic and socioeconomic characteristics in a representative sample of hospitalized patients in gjilan region, kosovo, 2011 - 2013. mean values standard deviations. absolute numbers and column percentages (in parentheses). distribution of clinical characteristics by drug - drug interaction status in a representative sample of hospitalized patients in gjilan region, kosovo, 2011 - 2013. absolute numbers and column percentages (in parentheses). table 3 presents the crude (unadjusted) association of demographic and socioeconomic factors and clinical characteristics with drug - drug interactions. there was evidence of a positive association of drug - drug interactions with older age (or=2.6, 95%ci=1.4 - 3.3), but no relationship with sex. furthermore, drug - drug interactions were positively associated with a lower educational attainment (or=1.7, 95%ci=1.1 - 2.2), retirement (or=2.0, 95%ci=1.3 - 2.9), a lower self - perceived income level (or=1.6, 95%ci=1.1 - 2.1) and a lower social status (or=1.6, 95%ci=1.1 - 2.2). on the other hand as for the clinical correlates, drug - drug interactions were positively related to an excessive length of hospitalization (or=3.3, 95%ci=2.7 - 4.0), presence of selected diseases [infectious diseases (or=2.0, 95%ci=1.5 - 2.9), cardiovascular diseases (or=2.1, 95%ci=1.6 - 2.9), endocrine diseases (or=1.7, 95%ci=1.2 - 2.3), respiratory diseases (or=2.3, 95%ci=1.6 - 3.1), or gastrointestinal diseases (or=1.8, 95%ci=1.3 - 2.5) ], comorbidity (or=4.5, 95%ci=3.7 - 5.5) and an excessive number of drug intake (or=7.5, 95%ci=6.0 - 9.2). association of demographic and socioeconomic factors and clinical characteristics with drug - drug interactions ; crude (unadjusted) odds ratios (ors) from binary logistic regression. odds ratios (ors) : drug - drug interactions vs. no drug - drug interactions. overall p - values and degrees of freedom (in parentheses). upon multivariable adjustment for all the demographic and socioeconomic factors as well as the clinical characteristics, drug - drug interactions were positively and significantly related to older age (or=2.1, 95%ci=1.3 - 2.8), a lower educational attainment (or=1.4, 95%ci=1.1 - 1.9), a longer hospitalization period (or=2.7, 95%ci=2.1 - 3.6), presence of three groups of diseases [infectious diseases (or=1.7, 95%ci=1.3 - 2.4), cardiovascular diseases (or=1.8, 95%ci=1.4 - 2.6), or respiratory diseases (or=1.6, 95%ci=1.2 - 2.5) ], presence of comorbid conditions (or=3.2, 95%ci=2.3 - 4.4) and an intake of at least four drugs (or=5.9, 95%ci=4.6 - 7.1) [data not shown in the tables ]. the prevalence of drug - drug interactions in our study, which included a large representative sample of patients records form the regional hospital in gjilan, was high (62%). major demographic and socioeconomic correlates of drug - drug interactions in this study sample consisted of older age and a lower educational level. conversely, the most important clinical correlates included an excessive length of hospitalization, presence of comorbidity and, particularly, the excessive number of drug intake. a recent study conducted in ethiopia reported a high prevalence of potential drug - drug interactions in the internal medicine ward in the university teaching hospital of gondar (4). thus, among all patients included in this study (n=78), the prevalence of at least one potential drug - drug interaction, regardless of its severity, was 78% (4). furthermore, in kenya, about 33.5% of the patients receiving antiretroviral medications were reported to be exposed to clinically significant drug interactions with their antiretroviral medications (16). on the other hand, a study conducted in switzerland reported that about 56% of the patients were exposed to one or more major or moderate drug - drug interaction in the internal medicine ward (17). at a broader level, a literature review reported that 0.054% of emergency department visits, 0.57% of hospital admissions and 0.12% of re - hospitalizations were caused by drug - drug interactions (18). it has been suggested that patient safety may be improved by decreasing the frequency of preventable adverse drug events (5). from this point of view, computerized alerts have been suggested as a useful warning sing for drug - drug interactions (5). however, such instruments of early detection of drug - drug interactions may also lead to excessive alerts, especially when the system produces an overload of signals that are of minor clinical relevance (5,19). our study may have some limitations which include the possibility of selection bias and potential information biases. as for the selection bias, we drew a simple random sample of patients records based on a pre - defined and well - established sampling frame available from the regional hospital in gjilan, which consisted of all the patients records hospitalized during the study period (that is 2011 - 2013). therefore, the inclusion of a large and representative sample of patients files provides reassurance for the absence of selection bias in our study sample. regarding the possibility of information bias, we should point out that the assessment of socioeconomic characteristics was based on self - reported information, which bears the risk of differential reporting among categories of individuals distinguished by selected demographic and socioeconomic profiles. notwithstanding the lack of evidence for such a differential reporting among individuals with different demographic and socioeconomic characteristics, we can not fully exclude the possibility of differential reporting among these population categories. in addition, relationships observed in cross - sectional studies are not assumed to be causal and, therefore, future cohort studies in kosovo should confirm and expand findings from our cross - sectional survey. our analysis provides important evidence on the prevalence and socioeconomic and clinical correlates of drug - drug interactions among patients hospitalized in the regional hospital of gjilan, in transitional kosovo. findings from our study should raise the awareness of decision - makers and policy makers about the prevalence and determinants of drug - drug interactions in the adult population of post - war kosovo. | aim : our aim was to assess the prevalence and socioeconomic and clinical correlates of drug - drug interactions among the adult population of transitional kosovo.methods:a cross - sectional study was conducted including a representative sample of 1921 patients aged 18 years (mean age : 57.811.2 years ; 50.3% women ; overall response : 96%) from the regional hospital of gjilan, kosovo, during 2011 - 2013. potential drug - drug - interactions were assessed and clinical data as well as demographic and socioeconomic information were collected. binary logistic regression was used to assess the correlates of drug - drug interactions.results:upon multivariable adjustment for all the demographic and socioeconomic factors as well as the clinical characteristics, drug - drug interactions were positively and significantly related to older age (or=2.1, 95%ci=1.3 - 2.8), a lower educational attainment (or=1.4, 95%ci=1.1 - 1.9), a longer hospitalization period (or=2.7, 95%ci=2.1 - 3.6), presence of three groups of diseases [infectious diseases (or=1.7, 95%ci=1.3 - 2.4), cardiovascular diseases (or=1.8, 95%ci=1.4 - 2.6), respiratory diseases (or=1.6, 95%ci=1.2 - 2.5) ], presence of comorbid conditions (or=3.2, 95%ci=2.3 - 4.4) and an intake of at least four drugs (or=5.9, 95%ci=4.6 - 7.1).conclusions : our study provides important evidence on the prevalence and socioeconomic and clinical correlates of drug - drug interactions among the hospitalized patients in the regional hospital of gjilan, kosovo. findings from our study should raise the awareness of decision - makers and policy makers about the prevalence and determinants of drug - drug interactions in the adult population of post - war kosovo. |
continuous venovenous haemofiltration (cvvh) is an established treatment for patients with acute kidney injury. during cvvh, the rate at which this happens depends on the difference in their concentrations between serum and replacement fluid, and on the rate of treatment. patients presenting with acute kidney injury may have concomitant severe hyponatraemia or hypernatraemia. over - rapid correction of the serum naconcentration if cvvh is needed, the naconcentration in the replacement fluid (usually 140 mmol / l) needs to be adjusted in order to avoid rapid changes of the serum naconcentration. in the present paper if cvvh is necessary, the naconcentration of the replacement fluid should be increased by adding concentrated nacl solution (table 1). effect of adding different volumes of 30% nacl to replacement fluid effect of adding different volumes of 30% nacl (5 mmol / ml) to a 5 l bag of replacement fluid containing a naconcentration of 140 mmol / l. generally, it is not considered safe to lower the serum naconcentration by more than 8 to 10 mmol / l over 24 hours, especially in the setting of chronic hypernatraemia. usually, a stepwise correction of the patient 's serum naconcentration is planned using replacement fluid made up to successively lower naconcentrations. if the serum nadecreases by > 2 mmol / l in 6 hours, either the rate of filtration should be decreased or the fluid bags should be changed to bags with a higher naconcentration. the volumes of 30% nacl added are small and will not affect the concentration of other electrolytes in the solution significantly. if cvvh is needed, the naconcentration of the replacement fluid should be reduced by adding sterile water (table 2). generally, it is not considered safe to increase the serum naconcentration by more than 8 to 10 usually, a stepwise correction of the patient 's serum naconcentration is planned using replacement fluid made up to successively higher naconcentrations. effect of adding different volumes of water to replacement fluid effect of adding different volumes of water to a 5 l bag of replacement fluid with a naconcentration of 140 mmol / l. [na ], sodium concentration ; [hco3 ], bicarbonate concentration ; [k ], potassium concentration. if the serum naconcentration has increased by > 2 mmol / l in 6 hours, either the rate of filtration should be decreased or the fluid bags should be changed to bags with a lower naconcentration. the concentration of bicarbonate and potassium in the final solution will also be reduced, and the patient may need additional supplementation. the authors would like to thank the icu pharmacists at guy 's & st thomas ' hospital for their contribution. | in patients with acute kidney injury and concomitant severe hyponatraemia or hypernatraemia, rapid correction of the serum na+ concentration needs to be avoided. the present paper outlines the principles of how to adjust the na+ concentration in the replacement fluid during continuous renal replacement therapy to prevent rapid changes of the serum na+ concentration. |
on hematoxylin - eosin staining, many lbs in the brainstem consist of an eosinophilic core and peripheral halo, whereas they usually appear as irregular eosinophilic inclusions in other limbic and cortical areas.7 of the numerous proteins contained in lbs, -synuclein (asyn) is the most abundant, and many of the proteins are ubiquitinated.7 the central predominance of asyn and the peripheral halo of densely ubiquitinated proteins is a conspicuous feature of brainstem lb. the aggresome hypothesis holds that the failure of the aggresome to remove unwanted proteins is a primordial event.8 a lb might form from a failed aggresome, but not in a haphazard way. the pathogenesis of pd has been discussed in association with cellular machinery such as the proteasome and autophagy for the removal of unwanted proteins.9,10 direct evidence of this came from human brain, which showed structural defects in, and functional impairment of, the proteasome.1113 experimental studies recapitulated neuronal death and the formation of asyn aggregates by proteasomal inhibition, which could be considered as a forme fruste of lb.14,15 autophagy is another important system for dealing with toxic waste. interestingly, chaperone - mediated autophagy was hampered by a mutant asyn or asyn - dopa adducts, forming asyn aggregates.16 these data suggest that the formation of the lb is closely related to the impairment of major intracellular machinery. however, the ultimate upstream mechanism responsible for the active regulation of the machinery that handles toxic waste by segregating it into aggregates is still under investigation. the neuroprotection conferred by lb formation has been studied in the human brain using in - situ end - labeling for apoptosis, in which the neurons with lbs were less frequently apoptotic than those without.17 another study showed the downregulation of tyrosine hydroxylase (th) in the neurons harboring lbs.18 the presence of lbs might alert the neurons to prepare for the threat to come. as th is a key enzyme in the metabolism of dopamine in the sn, and dopamine oxidation can produce highly toxic products, such as dopamine adducts, th downregulation could be an effective compensatory way to survive. as lbp is commonly found in a background of severe neuronal loss, as in the case of pd, lbp can not be easily exempted from neuron death. a recent pathological study showed a stable proportion of lb - containing neurons in the sn, suggesting a balance between the formation and removal of lbs.19 the authors calculated the life span of neurons with lb to be 6.2 months versus 15.9 months for those with any type of asyn inclusion. the limited life span of the neurons carrying lbs argues for the detrimental role of lbs. without prospective data, it can not be determined whether lb formation signifies a tombstone for a cell death cascade or a successful salvage operation to sequester toxicants. however, recent studies have partly suggested that the lb is not necessarily a culprit in neuronal death. incidental lewy body disease (ilbd) is a pathological entity defined by the presence of asyn pathology without any clinical evidence of pd or dlb. as ilbd is found in normal persons, by definition, its pathology offers invaluable insight into the early evolution of lbd, such as pd and dlb.2022 moreover, although ilbd has no clinical implications, by definition, it is significantly associated with a clinical prodrome of pd that includes olfactory dysfunction and bowel frequency, which provide a pathological basis for the early diagnosis of pd.23,24 in ilbd, the neuronal loss is usually minimal, despite asyn pathology.2022 the dissociation between neuronal loss and asyn pathology contrasts the marked neuronal death and widespread lbp in the sn in pd, which suggests that lbp is less toxic than expected, at least in ilbd. then, if ilbd is a true prodrome of pd and its pathology recapitulates the early evolution of the lbp of pd, the transition from ilbd to pd may be made by additional provoking factors, rather than by a simple shift in the continuum of lbd. when microgliosis and astrogliosis are studied in the brainstem nuclei, including the dorsal motor nucleus of the vagus nerve (dmv) in the medulla, locus ceruleus (lc) in the pons, and sn in the midbrain, neuroinflammation is only increased slightly in ilbd, compared to normal controls, whereas inflammatory change is conspicuous in pd.25 interestingly, although the inflammatory change was similar in the three brainstem nuclei in ilbd, the sn was more severely affected by neuroinflammation than the dmv and lc in pd. the drastic change in inflammation might underlie the pattern shift of lbp from ilbd to pd. other potential factors contributing to the pattern shift are a high iron content, excessive oxidative stress, and impaired anti - oxidative mechanism.2628 the disproportionate involvement of the sn is in line with the conspicuous motor manifestations of pd, closely associated with sn pathology, while no clinical symptoms are present in ilbd. the recent hypothesis on the caudo - rostral propagation of lbp suggested by braak assumes that the appearance of asyn pathology in sn is a key feature of entering the symptomatic phase.29,30 however, there are many ilbd cases with an extranigral distribution, even involving the neocortices.22 in fact, some cases with ilbd could be classified as a dlb - like group, due to the widespread distribution of asyn pathology. ilbd could be pro - pd (brainstem predominant ilbd) or pro - dlb (diffuse ilbd).22 these findings suggest the occurrence of lbp across the brain without a significant temporal gap, not respecting the border between the brainstem and cortices, rather than in a consecutive manner from the lower brainstem to the higher cortices, without skipping intervening tissues. other neurodegenerative diseases can accompany lbp, such as alzheimer s disease (ad) and progressive supranuclear palsy.31,32 a small proportion of the patients with multiple system atrophy and corticobasal degeneration also have associated lbd.33 as lbp is found in normal subjects and patients with various neurodegenerative diseases, it is possible to say that lbd might develop in any brain, regardless of the concomitant pathologies.31 this again raises a vexing question about the functional contribution of lbp. to understand the functional role of lbp, it is necessary to have at least two sets of data : one concerning the quantitative relationship between the clinical features and the burden of lbp, and the other concerning the interaction with other concomitant pathologies, to isolate the surprisingly, although cognitive function was reported to be correlated with cortical lbs, few studies have succeeded in showing a significant relationship between parkinsonism and asyn pathology. a recent prospective study showed that parkinsonism was significantly correlated with the burden of asyn pathology. 34 facing the problem of mixed ad and lb pathology in dlb, researchers adopted the concept of probability to discriminate the functional significance of the specific pathologies.6 the cases heavily loaded with ad pathology are less likely to be diagnosed as dlb, even with diffuse lbd according to the published consensus criteria. recently, obi.35 studied -amyloid, tau protein, and lbp together and showed the central role of -amyloid in the evolution of tau pathology. lbp was not entirely dependent on -amyloid pathology.35 these attempts underscore the importance of sifting through concomitant pathologies to identify the independent contribution of lbp. apart from ad pathologies such as tau [neurofibrillary tangle (nft) ] and -amyloid [senile plaque (sp) ], other pathologies might modify the clinicopathological presentation of lbp ; these include, vascular pathologies, argyrophilic grain disease (agd) and tar dna binding protein-43 (tdp43).3639 agd and tdp43 could contribute to cognitive decline, which would be difficult to discern in the context of coexisting limbic ad pathologies or cortical lbs. vascular pathologies have a more complicated implication because they show great variability in terms of the amount and distribution of the lesions. a comprehensive approach that considers all of the coexisting pathologies together is needed to determine a full picture of the clinicopathological interactions, and to delineate the role of lbp. recent studies of fetal grafts demonstrated the propagation of the asyn pathology from the host to the graft, which unraveled the contagious nature of asyn pathology.40,41 until recently, only grafts more than 10 years after transplantation were thought to be insinuated by asyn pathologies, and earlier studies showed no asyn pathologies in grafts younger than 10 years.4245 several mechanisms were suggested to explain the human pathology.46 in a recent case, a graft was studied 14 years after transplantation.47 although the graft was expected to be localized in the putamen, it had extended from the putamen to the amygdala. asyn inclusions were found in the graft, and they were more frequent in the portion lying in the amygdala. corpora amylacea (as a marker of aging) was more common in the peripheral portion of the graft. these findings indicated that the maturation and aging of the graft might not be critical to the development of asyn pathology. as the amygdala is more severely affected than the putamen in lbd, and could frequently be affected by various abnormal protein aggregations, such as nfts, tdp-43, and argyrophilic grains, the amygdala might have a greater tendency to develop pathological inclusions than the putamen. 36,48,49 in other words, the putamen - amygdala gradient of asyn inclusions might be affected by factors constituting the aggregation - prone milieu of the amygdala, which remains speculative. recently, cell - to - cell propagation of asyn inclusions was demonstrated experimentally.50 as the pathologic burden of asyn was heavier in the amygdala, the propagation might explain the putamen - amygdala gradient. direct propagation from the neighboring tissue might produce a center - peripheral gradient of asyn inclusions in the graft, which was, however, not observed in our case. it remains controversial whether lbp spreads from limited pathological foci (the enteric nervous system or olfactory bulb according to the hypothesis of braak) or arises nearly simultaneously, but disproportionately, in multiple sites.22,30 in some cases of ad, only the amygdala is affected by lbp, sparing other brain areas involved in the classical forms of lbd, which argues for the latter scenario.51 recent data showed that the explanatory power of braak staging for lbp is variable, and is most compelling in lbd associated with progressive supranuclear palsy. 32 as lbp commonly accompanies other pathologies, its functional role should be dissected from the influence of concomitant pathologies. | lewy body pathology (lbp) is the pathological hallmark of lewy body diseases, such as parkinson s disease and lewy body dementia. recent studies have shed new light on the role of lbp, the interactions of lbp with concomitant pathologies, and the propagation of lbp from the olfactory bulb and enteric nervous system to the central nervous system. the intrinsic difficulty with identifying clinicopathological correlates could be overcome by improving our understanding of the pathological evolution of lbp. |
the tim family of genes, which consists of eight members in mice and three members in humans (6), is located on chromosome 11 in mice and 5q33 in humans tim proteins are type 1 membrane proteins with a structurally conserved immunoglobulin variable (igv) domain and mucin stalk that connects to an intracellular tail. tim proteins were initially thought to be expressed specifically on the surface of differentiated effector t cells and to directly regulate their activity, but we now know that tims are also expressed on and regulate the function of antigen - presenting cells (7). crystal structures of the tim molecules has revealed a unique, conserved structure with ligand - binding sites in the igv domain (810). tim-3 was initially identified as a molecule expressed on t helper (th)-1, but not th2, cells in mice (11). interaction between tim-3 and its ligand galectin-9 inhibits th1 responses (12) and induces peripheral tolerance (13, 14). as in mice, tim-3 is preferentially expressed on human th1 cells, but is also expressed constitutively on macrophages and dendritic cells (dcs) (7). reduction of tim-3 expression in t cells using small interfering rna or blocking antibodies, for example, increases interferon (ifn)- secretion by cd4 t cells (15), further supporting an inhibitory role of tim-3 in human t cells. patient studies are consistent with this inhibitory function, as tim-3 expression is defective on cd4 t cells from patients with autoimmune diseases (15, 16). specifically, t cell clones isolated from the cerebrospinal fluid of patients with multiple sclerosis (ms) express lower levels of tim-3 and secrete higher levels of ifn compared with clones isolated from healthy control subjects (15). cd4 t cells from ms patients also show altered and delayed kinetics of tim-3 upon activation, resulting in lower tim-3 expression on circulating t cells and are therefore refractory to the blocking effects of tim-3specific antibodies (16). tim-1, by contrast, is predominantly expressed on th2 cells and was first identified as the hepatitis a virus cellular receptor 1 (17). a unique polymorphism in the mucin domain of tim-1 regulates hepatitis a virus infection, and also affects immune responses and resistance to asthma, allergy, and atopy (18). t cell exhaustion during hiv infection is accompanied by increased hiv replication and follows a characteristic pattern. t cells first lose the ability to enter into cell cycle, to secrete il-2, and to mediate cytotoxicity (2, 3). over time, the exhausted cells also lose the ability to secrete other cytokines, such as ifn- and tnf (2, 3, 19). initial studies suggested that t cell dysfunction is mediated by sustained pd-1 expression on t cells. upon activation, t cells up - regulates several inhibitory molecules, including pd-1, which help turn off t cell responses and thus inhibit t cell expansion. a functional role for pd-1/pd - l1 signaling in t cell dysfunction was demonstrated by studies of lymphocytic choriomeningitis virus in mice, simian immune deficiency syndrome in rhesus macaques, and hiv in humans, each of which demonstrated that blocking this pathway increased t cell responses, improved viral control in vivo, and enhanced the survival and proliferation of antigen - specific cd8 t cells in vitro (2, 3, 20, 21). although pd-1 expression appeared to identify a population of t cells entering into exhaustion, not all exhausted cells expressed pd-1 (3). thus, the molecular pathways associated with t cell exhaustion, particularly in cd8 t cells, remain incompletely understood. in this issue, jones. find elevated expression of tim-3 on both cd4 and cd8 t cells from individuals with acute and progressive chronic hiv infection, but not in chronically infected individuals who had controlled the infection or in uninfected individuals (5). the frequency of tim-3expressing cells correlated positively with viral load and inversely with cd4 t cell counts. these results are in sharp contrast to data showing decreased expression of tim-3 in patients with ms. staining with mhc class i tetramers / pentamers revealed higher tim-3 expression on hiv - specific cd8 t cells than on cmv - specific cd8 t cells. levels of tim-3 expression were variable both among different individuals and among hiv - specific t cells within the same individual, suggesting that different levels of tim-3 expression may mark cells with different functional capacities. in a subset of patients, highly active antiretroviral therapy correlated with reduced tim-3 expression on t cells. sorting of tim-3 and tim-3 populations revealed that hiv - specific t cells expressing high amounts of tim-3 secreted less ifn than did tim-3 cell, suggesting that, like pd-1, tim-3 may be an important marker of exhausted t cells during chronic viral infections. blocking the interaction between tim-3 and its ligand galectin-9 enhanced cd8 t cell proliferation and cytokine production in response to hla - a0201-restricted hiv-1 peptides, thus solidifying the functional connection between tim-3 expression and t cell dysfunction (fig. 1). inhibiting the tim-3 pathway also enhanced cd4 and cd8 t cell proliferation when peripheral blood mononuclear cells from chronic progressors were stimulated with pooled gag and nef peptides. in most of the patients, tim-3 and pd-1 were expressed on distinct populations of t cells, although infrequent tim-3/pd-1 cells could be detected. these data suggest that tim-3 and pd-1 expression mark distinct subsets of exhausted t cells. the authors suggest that tim-3 may be an important therapeutic target that is distinct from pd-1 and may help reverse exhaustion in chronic viral infections. on cd4 cells, tim-3 is expressed only on terminally differentiated th1 cells. therapeutic blockade of pd-1, on the other hand, would expand all t cells, because pd-1 is up - regulated on all activated t cells. differential expression of tim-3 on the surface of t cells regulates susceptibility to viral infection or development of autoimmune injury. (a) in patients with progressive hiv infection, tim-3 is up - regulated on the surface of cd4 and cd8 t cells. up - regulation of tim-3 on hiv - specific cd8 t cells leads to impaired cell survival, proliferation, and cytokine production (1). blocking tim-3 binding to its ligand galectin-9 using soluble tim-3 or tim-3 up - regulation of pd-1 in hiv patients also leads to t cell dysfunction (2). although a small proportion of cells express both tim-3 and pd-1, in the majority of cells, tim-3 expression is distinct from pd-1, suggesting these proteins define separate populations of dysfunctional t cells in hiv patients. (b) in autoimmune diseases, autoreactive t cells upon activation and expansion down - regulate tim-3 expression, leading to progressive uncontrolled growth and expansion of autoreactive t cells. whether down - regulation of tim-3 is mediated by genetic factors or by t cell activation signals in autoimmune disease is not known. collectively, these data suggest that tim-3 is a central regulator of t cell responses. in autoimmunity, loss of tim-3 function leads to excessive expansion of autoreactive effector t cells. in chronic hiv infection, by contrast, sustained tim-3 expression on cd4 and cd8 t cells leads to t cell exhaustion (fig. it is unclear, for example, whether hiv infection itself drives tim-3 expression or whether cd4 t cells expressing high levels of tim-3 are preferentially infected with hiv or are exhausted in their attempt to clear the virus. how the engagement of tim-3 by its ligand galectin-9 leads to the inability of t cells to enter into the cell cycle and secrete cytokines is also unknown. since tim-3galectin-9 interactions normally induce the death of terminally differentiated th1 cells (12), why are n't exhausted cells eliminated ? and why, on the other hand, is tim-3 expression reduced on th1 cells in patients with autoimmune diseases such as ms ? our recent discovery that tim-3 is constitutively expressed by human dcs and monocytes revealed a potential role for tim-3 in innate immune responses (7). it will be interesting to examine the function of tim-3 on monocytes and dcs in patients with hiv infection, whether its expression changes during the course of infection, and whether tim-3 affects the responsiveness of dcs to tlr and other innate signals. the dysregulation of tim-3 in patients with autoimmune disease and in chronic viral exhaustion highlight the therapeutic potential of targeting tim-3 in the treatment of these diseases. finally, these studies highlight the utility of investigating the same regulatory pathways in patients with hiv and autoimmunity to provide unique insights into the pathogenesis of these diseases. | exhaustion of t cell responses during chronic viral infections has been observed in both mouse and man and has been attributed to up - regulation of pd-1 on the surface of exhausted t cells. in patients with chronic human hiv infection, t cell exhaustion leads to opportunistic infections associated with aids. however, not all the exhausted t cells express pd-1, suggesting that other molecules may be involved in the phenotype. a new study now demonstrates a central role for t cell immunoglobulin and mucin domain containing protein-3 (tim-3) in t cell exhaustion during chronic hiv infection and suggests that tim-3 may be a novel therapeutic target in chronic viral diseases. |
presbyopia affects an estimated one billion people worldwide.1 current surgical treatment options for presbyopia depend on the presence of a cataract at initial assessment. if present, a cataract extraction with the implantation of a multifocal or accommodative intraocular lens implantation may be chosen.2,3 if a clear lens is present, a non - lens - based treatment, eg, presbyopic laser - assisted in situ keratomileusis (lasik), monovision with a contact lens or lasik, conductive keratoplasty or femtosecond laser intrastromal presbyopia treatment (intracor ; technolas perfect vision gmbh, munich, germany) may be the preferred choice. one emerging non - lens - based treatment modality for presbyopia is the insertion of intrastromal corneal inlays that work by a variety of different mechanisms. the vue+ (revision optics, lake forest, ca, usa) inlay consists of a space - occupying lenticule that changes corneal curvature, creating a hyperprolate cornea for both near and distance vision.4 the flexivue microlens (presbia, amsterdam, netherlands) inlay is itself a bifocal inlay, with a plano central zone for distance vision and a peripheral annulus with refractive add power for near vision.5 both the vue+ and flexivue microlens inlays have received conformit europenne (ce) marking, but are not yet us food and drug administration (fda)-approved. the kamra small aperture inlay (acufocus, irvine, ca, usa), previously known as the aci 7000, has a 3.8 mm outer diameter and an inner 1.6 mm central aperture that selects for central light rays and creates a pinhole effect, thus increasing the depth of focus. this is inserted in the nondominant eye, and has been shown to improve uncorrected near visual acuity (unva), without affecting uncorrected distance visual acuity (udva).6 the kamra inlay was the first corneal inlay for presbyopia to receive ce marking in 2005, and is currently pending approval from the fda for use in the usa outside of clinical trials. presbyopia typically affects patients from about 40 years of age, and phakic patients who undergo corneal inlay implantation may subsequently develop cataracts as they grow older. as corneal inlay implantation for presbyopia is relatively new, there are not many patients who have yet undergone cataract surgery after corneal inlay implantation. potential concerns with this procedure include technical difficulty performing cataract surgery with the corneal inlay in place, accuracy of biometry readings, and visual outcome after cataract surgery. this paper presents the experiences and results of two patients who underwent phacoemulsification and intraocular lens implantation for cataracts that developed after implantation of the kamra corneal inlay for presbyopia. the two patients in this case series, aged 53 and 62 years of age, were first seen in january 2010 and may 2007, respectively, at the singapore national eye centre, singapore, requesting for correction of presbyopia without the use of corrective lenses. slit - lamp examination of the anterior and posterior segments revealed no significant abnormalities, and both patients had lenses graded as nc1 according to the lens opacities classification system iii (locs iii)7 in their nondominant left eyes. various surgical options were presented to the patients, and each opted for implantation of the kamra corneal inlay. one patient underwent implantation of the kamra corneal inlay with a microkeratome - created lamellar flap and the other had implantation via a femtosecond laser - created lamellar flap. case 1, a 53-year - old chinese male, underwent implantation of the kamra corneal inlay in the nondominant left eye on january 6, 2010. a 7.80 mm - diameter lamellar flap was created at a depth of 190 microns with a visumax femtosecond laser (carl zeiss meditec, jena, germany). the patient s pre- and 1-month postoperative visual acuity results can be found in table 1. a slit - lamp photograph of the kamra corneal inlay in place is shown in figure 1. twelve months after implantation, even though reading well, the patient noted ghosting and slight monocular diplopia in the left eye. the patient underwent surgical repositioning of the implant, resulting in resolution of his symptoms. figure 2 shows acutarget (acufocus) photographs of the kamra implant positioning before and after surgical repositioning. seven months after repositioning of the implant, the patient had an unva of j10 and was noted to have developed cataract of grade nc3p3 (locs iii) in the left eye. various options were discussed with the patient, including cataract surgery with the implant in place, removal of the implant followed by cataract surgery and subsequent re - implantation, and implant removal followed by cataract surgery with a multifocal or accommodative intraocular lens implant. centration of the kamra implant was confirmed with acutarget photographs (figure 2). due to the excellent centration (inlay vs purkinje : 95 m x, 27 m y), the patient opted for cataract surgery, leaving the kamra implant in place. corneal topography remained stable after implantation of the kamra inlay and before cataract surgery (figure 3). the patient underwent uncomplicated phacoemulsification and intraocular lens implantation of the left eye under local anesthesia. a 2.60 mm temporal clear corneal incision was made, and a + 22.0 d monofocal tecnis zcb00 intraocular lens (abbott medical optics, santa ana, ca, usa) was implanted. no modifications were made to the surgical procedure, and the surgeon reported no difference in the ease of surgery with the kamra implant in place, apart from a few extra ocular rotations during capsulorhexis and phacoemulsification to improve visualization. one month after cataract surgery, posterior capsular opacification was noted in the left eye, and the patient underwent nd : yag laser capsulotomy 6 weeks after cataract surgery (figure 1). the patient reported good near, intermediate, and distance vision without the use of spectacles, and remains spectacle - independent. case 2, a 62-year - old chinese male, underwent implantation of the kamra corneal inlay in the nondominant left eye on july 4, 2007. a 9.00 mm - diameter lamellar flap was created at a depth of 151 microns with a schwind microkeratome (schwind, kleinostheim, germany). the patient s pre- and 1-month postoperative visual acuity results can be found in table 1. four years after kamra implantation, cataract of grade nc2 (locs iii) was noted in the implanted left eye. nevertheless, vision in this eye remained good ; udva was 20/20 and unva was j2. however, 6 years after kamra implantation, the patient felt that his distance vision in the left eye had deteriorated significantly (table 1) and, on assessment, cataract of grade nc4 (locs iii) was noted. acutarget photographs confirmed that the implant was well centered (inlay vs purkinje : 146 m x, 78 m y). various treatment options were presented to the patient, who opted for cataract surgery, leaving the kamra implant in place. the patient underwent phacoemulsification and intraocular lens implantation of the left eye under local anesthesia. a 2.60 mm temporal clear corneal incision was made, and a + 20.0 d monofocal tecnis zcb00 intraocular lens was implanted. no modifications were made to the surgical technique, and the surgeon reported no difference in ease of the surgery with the kamra implant in place, except that some extra ocular rotations during capsulorhexis and phacoemulsification to optimize the surgical view were required. surgical time was 15 minutes, and there were no intraoperative or early postoperative complications. the patient reported good near, intermediate and distance vision without the use of spectacles, and remains spectacle - independent. case 1, a 53-year - old chinese male, underwent implantation of the kamra corneal inlay in the nondominant left eye on january 6, 2010. a 7.80 mm - diameter lamellar flap was created at a depth of 190 microns with a visumax femtosecond laser (carl zeiss meditec, jena, germany). the patient s pre- and 1-month postoperative visual acuity results can be found in table 1. a slit - lamp photograph of the kamra corneal inlay in place is shown in figure 1. twelve months after implantation, even though reading well, the patient noted ghosting and slight monocular diplopia in the left eye. the patient underwent surgical repositioning of the implant, resulting in resolution of his symptoms. figure 2 shows acutarget (acufocus) photographs of the kamra implant positioning before and after surgical repositioning. seven months after repositioning of the implant, the patient had an unva of j10 and was noted to have developed cataract of grade nc3p3 (locs iii) in the left eye. various options were discussed with the patient, including cataract surgery with the implant in place, removal of the implant followed by cataract surgery and subsequent re - implantation, and implant removal followed by cataract surgery with a multifocal or accommodative intraocular lens implant. centration of the kamra implant was confirmed with acutarget photographs (figure 2). due to the excellent centration (inlay vs purkinje : 95 m x, 27 m y), the patient opted for cataract surgery, leaving the kamra implant in place. corneal topography remained stable after implantation of the kamra inlay and before cataract surgery (figure 3). the patient underwent uncomplicated phacoemulsification and intraocular lens implantation of the left eye under local anesthesia. a 2.60 mm temporal clear corneal incision was made, and a + 22.0 d monofocal tecnis zcb00 intraocular lens (abbott medical optics, santa ana, ca, usa) was implanted. no modifications were made to the surgical procedure, and the surgeon reported no difference in the ease of surgery with the kamra implant in place, apart from a few extra ocular rotations during capsulorhexis and phacoemulsification to improve visualization. one month after cataract surgery, posterior capsular opacification was noted in the left eye, and the patient underwent nd : yag laser capsulotomy 6 weeks after cataract surgery (figure 1). the patient reported good near, intermediate, and distance vision without the use of spectacles, and remains spectacle - independent. case 2, a 62-year - old chinese male, underwent implantation of the kamra corneal inlay in the nondominant left eye on july 4, 2007. a 9.00 mm - diameter lamellar flap was created at a depth of 151 microns with a schwind microkeratome (schwind, kleinostheim, germany). the patient s pre- and 1-month postoperative visual acuity results can be found in table 1. four years after kamra implantation, cataract of grade nc2 (locs iii) was noted in the implanted left eye. nevertheless, vision in this eye remained good ; udva was 20/20 and unva was j2. however, 6 years after kamra implantation, the patient felt that his distance vision in the left eye had deteriorated significantly (table 1) and, on assessment, cataract of grade nc4 (locs iii) was noted. acutarget photographs confirmed that the implant was well centered (inlay vs purkinje : 146 m x, 78 m y). various treatment options were presented to the patient, who opted for cataract surgery, leaving the kamra implant in place. the patient underwent phacoemulsification and intraocular lens implantation of the left eye under local anesthesia. a 2.60 mm temporal clear corneal incision was made, and a + 20.0 d monofocal tecnis zcb00 intraocular lens was implanted. no modifications were made to the surgical technique, and the surgeon reported no difference in ease of the surgery with the kamra implant in place, except that some extra ocular rotations during capsulorhexis and phacoemulsification to optimize the surgical view were required. surgical time was 15 minutes, and there were no intraoperative or early postoperative complications. the patient reported good near, intermediate and distance vision without the use of spectacles, and remains spectacle - independent. implantation of the kamra corneal inlay is an effective nonlens - based treatment option for presbyopia in patients with a clear lens at the time of presentation.6 however, some of these patients may eventually develop cataracts requiring surgical treatment. at this point, there are a multitude of available options, including cataract surgery with the kamra implant in place, implant removal followed by cataract surgery and subsequent re - implantation, and implant removal followed by cataract surgery with implantation of a multifocal or accommodative intraocular lens. cataract surgery with the kamra implant in place poses a few potential concerns, such as technical difficulty performing cataract surgery with the implant in place, the accuracy of biometry readings with various intraocular lens power formulae, and the visual outcome following cataract surgery. regarding the technical aspects of cataract surgery, no significant modifications to the surgical technique were required in either case, and the surgeon reported no significant difference in the ease of surgery ; surgical time was 15 minutes in both cases. the only slight difference was the need for some additional ocular rotations during capsulorhexis and phacoemulsification in order to improve surgical visualization. since both patients underwent local anesthesia, this was easily performed by asking them to voluntarily rotate the eye during the surgery. the authors feel that such rotations do not significantly increase the risk of intraoperative complications, such as radial tears or opening of the posterior capsule, as the surgical view through the 1.6 mm central aperture after rotation was very good and, as such, a 5 mm capsulorhexis and phacoemulsification were performed without difficulty. however, we acknowledge the experience of the surgeon performing the cataract operations in the patients in this series. therefore, cataract surgery with the kamra implant in place was not significantly more technically difficult to perform. the unva was j2 for both patients. corrected and unaided distance visual acuities varied from 20/20 to 20/40 (table 1). both patients were satisfied with their near, intermediate, and distance vision and remained spectacle - independent. the biometry readings and calculations were satisfactory and accurately predicted the achieved power (table 2). in this series, the surgeon used only one formula, srk / t, to calculate the power of the intraocular lenses. since there were no prior publications on this topic, an attempt was made, retrospectively, to determine if this choice of formula was reasonable. a comparison was made between four commonly used formulae for the calculation of intraocular lens power, to see which would have most accurately predicted the achieved power after surgery. the biometry readings for both cases were used for this purpose (table 2). in case 1, using srk / t, a + 22.0 d monofocal tecnis zcb00 intraocular lens was implanted, aiming for a target power of 0.33 d. the achieved spherical equivalent was 0.38 d, ie, a difference of 0.05 d. the same biometry data were entered into three other commonly used formulae (haigis, holladay 1, and hoffer q), and the same + 22.0 d tecnis zcb00 lens was chosen. for haigis, the target power was 0.76 d, with a difference of 0.38 d from the achieved power of 0.38 d. for holladay 1, the target power was 0.51 d (a 0.13 d difference). hoffer q gave a target of 0.60 d, with a difference of 0.22 d from the achieved power. in case 1, it appears that srk / t was the formula that most accurately predicted the achieved spherical equivalent. for case 2, using srk / t, a + 20.0 d monofocal tecnis zcb00 intraocular lens was implanted, aiming for a target power of 0.72 d. the achieved spherical equivalent was 0.38 d, giving a difference of 0.34 d. using the same biometry parameters and the same + 20.0 d tecnis zcb00 lens, haigis gave a target of 0.56 d (a difference of 0.18 d) ; holladay 1 gave a target of 0.87 d, with a difference of 0.49 d ; and hoffer q gave a target of 1.00 d, with a difference of 0.62 d. in case 2, srk / t was the second most accurate formula. therefore, based on these retrospective calculations, srk / t appears to be a reasonable choice of formula for choice of intraocular lens. however, this case series is too small to definitively establish the validity of srk / t in kamra implant patients. biometry readings and intraocular lens power calculations were not done prior to kamra implantation. however, in order to compare the biometry calculations before and after kamra implantation, the authors retrospectively calculated biometry for both cases before kamra implantation. the assumption was made that there was no change in axial length in either case. for case 1, the pre - kamra k readings were 47.14 and 45.99 ; hence, using the same + 22.0 d lens would have given a target outcome of 0.46 d instead of 0.33 d. for case 2, the pre - kamra k readings were 44.30 and 43.50, and so using a + 20.0 d lens, as was done in case 2, would have given a target outcome of 0.33 d instead of 0.72 d. therefore, implantation of the kamra inlay does not appear to have had a major effect on the biometry or intraocular lens calculations in these two patients. cataract surgery with the kamra implant in place is technically feasible with slight modifications to surgical technique. visual outcomes for both near and distance vision were satisfactory. additionally, the biometry readings were accurate, and srk / t appears to be an accurate formula for the prediction of eventual power. however, this case series is too small for drawing definitive conclusions, and larger studies with longer follow - up will be needed to properly evaluate the safety and visual outcome of cataract surgery with the kamra implant in place, as well as to determine the appropriate formula for calculation of intraocular lens power. nevertheless, cataract surgery with the kamra implant in place appears to be an attractive option for patients presenting with a cataract after previous kamra implantation for presbyopia correction. | intrastromal corneal inlays are an emerging treatment for presbyopic patients. the kamra small aperture inlay was the first such inlay to receive conformit europenne (ce) marking in 2005. it has been shown to improve uncorrected near and intermediate visual acuity without adversely affecting uncorrected distance visual acuity. due to the age of presbyopic patients, they may eventually develop cataracts. in two such cases, we found that cataract surgery with the kamra implant left in place was not technically more difficult, and that the surgical procedure could be improved by additional ocular rotations to improve visualization. biometry readings were reliable, and it appeared that the srk / t formula was accurate for calculation of intraocular lens power. cataract surgery with the kamra implant left in situ is a viable option for patients. |
to be in step with the future, it is necessary today for academic programs to exist that teach topics related to nanomedicine, an important and relatively recent technological advance in medicine. such programs should be included in university departments for graduate studies and research, especially in the areas of pharmacology, genetic engineering, proteomics, and molecular and cellular biology. later, as nanotechnology continues to develop, other areas will certainly be affected. research in nanomedicine began with discoveries of the novel physical and chemical properties of various carbon - based materials that can only be detected in nanometer - sized structures. nanomedicine is defined as the monitoring, repair, construction, and control of human biological systems at the cellular, molecular, and atomic levels using engineered nanodevices and nanostructures. another definition is that provided by the national institute of health in its roadmap for medical research in nanomedicine, which states that nanomedicine is an offshoot of nanotechnology, [which ] refers to highly specific medical interventions at the molecular scale for curing disease or repairing damaged tissues, such as bone, muscle, or nerve.1 nanomedicine contains five main disciplines : analytical tools, nanoimaging, nanomaterials (medications, raw materials, and devices), novel therapeutic and drug delivery systems, as well as regulatory and toxicological issues related to clinical practice.1 basic nanostructured materials, engineered enzymes, and the many products of biotechnology will most probably become essential elements in some areas of medical, chemical, and biological applications. however, the full promise of nanomedicine is yet to be defined, as this will to a large extent depend on the development of new technology, such as precisely controlled or programmable medical nanomachines and nanorobots to address medical problems. moreover, the application of nanotechniques at the cellular, molecular, and atomic levels should certainly help in the understanding of the function of organisms, as well as in the development of better techniques for imaging, diagnosing, and treating disorders at the level of molecular and cellular biology. microscopic machines were first hypothesized by the nobel prize winning physicist richard p feynman, now considered the father of nanoscience, in 1959. his article there is plenty of room at the bottom is considered a classic in the establishment of the important possibilities for this field.2 the main idea of this article was the ability to reduce the size of various apparatuses and at the same time increase their effectiveness, based on the manufacture of products by reordering atoms and molecules. of particular importance in his proposal was the reference to nanorobots, to be used in the manufacture of nanodevices, nanomaterials, and nanomachines. eric drexler, considered the father of nanotechnology, wrote the popular books, engines of creation (1986) and unbounding the future (1991), in which he announced the promise and warned of the dangers of molecular and atomic manipulation. in his more recent work, nanosystems : molecular machinery, manufacturing and, computation (1992), he predicted that nanotechnology will make the conventional means of manufacture obsolete.3 he proposed the concept of machines with the capability of ordering and reordering atoms in order to create precise molecular structures, thus resulting in the desired product. once nanomachines are available, programmable and controllable microscale robots comprised of nanoscale parts manufactured to nanometer precision will open new possibilities related to curative and reconstructive procedures in the human body at the cellular, molecular, and atomic levels (perhaps at less than 100 nm). in keeping with the adage that the best treatments are those that intervene the least, the possibility of nanotechnology to greatly increase knowledge of cellular and atomic events could lead to improved treatments. additionally, nanomedicine is on the cutting edge of medical practice and research, and for this reason it is an important focus of capital investment. consequently, it is urgent that academic programs be implemented in order to teach this discipline in universities that have a medical and biological focus of study and research. an academic discipline of nanomedicine at the university level would allow students, teachers, technicians, and researchers to become familiar with : (1) nanodevices, nanostructures, nanomaterials, nanostuffs, and precise and sophisticated nanoinstruments, such as those that could be used for molecular imaging and bedside biomarker testing and monitoring (appendix 1) ; (2) the main categories of precise nanotechnologies in real time (appendix 2) ; and (3) techniques (laboratory methods and procedures) needed for furthering research (appendix 3), including detailed procedures of nanobiosensors for medical uses (appendix 4). all of these elements of nanomedicine could be applied to cancer, cardiovascular diseases, inflammation, degenerative neurological diseases, toxicity, ethics, and societal and environmental impact.4 perhaps one of the most important potentials for nano - medical research is the gathering of extensive information about the chemical and physical properties of nanoscale biological structures. as the catalog of the interactions between individual molecules, cells, tissues, and organs develops, researchers will gain a greater understanding of the intricate operations of molecular structures, processes, and networks inside living cells based on the use of new probing tools. consequently, scientists will be able to build new devices for a wide range of biomedical applications, such as detecting infectious agents or metabolic imbalances with tiny sensors, generating miniature devices that search for and destroy infectious agents, and replacing broken machinery inside cells with new nanoscale structures.4 nanomaterials : nanotechnology used for subcutaneous treatments.5nanoprobes in vivo : targeted nanoprobes reveal early time point kinetics in vivo by time - resolved magnetic resonance imaging (mri).6methods for the preparation of small interfering ribonucleic acids (sirnas) : a direct, efficient method for the preparation of sirnas containing ribo - like north pseudo - sugars,7 and drug delivery of sirna therapeutics.8physical assessment of toxicology at nanoscale : nanodose - metrics and toxicity factor.9nanomaterials for regenerative medicine : use of nanomaterials with applications for regenerative medicine.10vascular toxicity : studies of vascular endothelial toxicity.11nanocatalizers : molecular - level biological transporters capable of converting biomass into biofuel.nanoparticle devices : molecular - level biological transporters that release medication in a precise and controlled manner throughout the organism, such as in target cells, for crossing biological barriers and for providing greater stability in the face of pathogenic deterioration, as well as use of nanoparticles for the treatment of cancer.12 nanomaterials : nanotechnology used for subcutaneous treatments.5 nanoprobes in vivo : targeted nanoprobes reveal early time point kinetics in vivo by time - resolved magnetic resonance imaging (mri).6 methods for the preparation of small interfering ribonucleic acids (sirnas) : a direct, efficient method for the preparation of sirnas containing ribo - like north pseudo - sugars,7 and drug delivery of sirna therapeutics.8 physical assessment of toxicology at nanoscale : nanodose - metrics and toxicity factor.9 nanomaterials for regenerative medicine : use of nanomaterials with applications for regenerative medicine.10 vascular toxicity : studies of vascular endothelial toxicity.11 nanocatalizers : molecular - level biological transporters capable of converting biomass into biofuel. nanoparticle devices : molecular - level biological transporters that release medication in a precise and controlled manner throughout the organism, such as in target cells, for crossing biological barriers and for providing greater stability in the face of pathogenic deterioration, as well as use of nanoparticles for the treatment of cancer.12 doctors may be able to search out and destroy the very first cancer cells before they lead to a destructive tumor in the body. perhaps a broken part of a cell could be removed and replaced with a miniature biological machine. pumps the size of molecules could possibly be implanted to deliver life - saving medicines precisely when and where they are needed. these scenarios are the long - term goals of the nanomedicine roadmap.4 with the inclusion of the academic discipline of nanomedicine in their programs, universities will be able to provide the personnel capable of accelerating the development of this field. future possibilities include the following opportunities : nanoneedles : the nucleus of live cells could be penetrated by nanoneedles used in conjunction with microscopy with the aim of introducing nucleic acids, proteins or other compounds inside human cells, or even doing surgery ; modeling the structure - property relationships of nanoneedles.13environmental nanocatalizers : biological transporters capable of greatly improving catalytic converters currently used in automobiles, thus drastically reducing the pollution emitted.nanobiosensors : sensors capable of detecting the presence of anthrax and other biological weapons ; nanosensors for probing live cells.14dendrimers : artificial molecules designed to replace defective endogenous elements in the organism, induce the production or secretion of various molecules related to the immune response in case of being genetically deficient, or act as biological markers (for cancer therapy, for the detection of adherence to medication regimens, or for the evaluation of experimental results).15nanodepots of insulin : microscopic deposits injected into the bloodstream that could act as an artificial pancreas, releasing insulin to counteract the effects of diabetes ; similar technologies could also be employed to release dopamine into neurons as therapies for parkinson s or alzheimer s disease.titanium dioxide nanotubes : microscopic tubes that could clean polluted water.carbon nanotubes : microscopic tubes aimed at replacing defective or absent genes ; use of carbon nanotubes in vivo to characterize action mechanisms at the cellular level;16 carbon nanotubes could reactivate synaptic connections and regenerate neurons damaged by an accident.biological nanosensors : a set of ultramicroscopic and fine silicon threads.molecular nanopyramids : these probes could possibly be released into the human body, with the capability of detecting and destroying the first cancer cells in the brain, heart, kidney, or lungs, without damaging neighboring tissue ; they might also be able to detect viral or bacterial infections in an early stage before the immune system has begun to respond.assembler nanomachines : machines based on the rearrangement of atoms and molecules to manufacture molecular components.smart materials : these probe - like materials could potentially detect subtle changes in body chemistry.nanoparticles : microscopic tumor - destroying genes may possibly be transported to certain sites, directed with great precision to destroy the genetic information of tumors, with the aim of inactivating the target cells and impeding their reproduction ; other genes may be transported to detect tiny alterations in proteins, as well as to scan tissue to ensure that the nanodevices are neatly congregated around the target.nanorobots : these devices could be introduced into the human body to repair damage and combat infection and disease by acting on dna without being detected and destroyed by the host immune system.1722nanobiochips : this advanced technology could work with mri to supply an abundant amount of information about the human body.23nanocomputers : computers could be developed made of material stronger than steel but with only 10% of its weight. these could possibly guide nanomachines for the purpose of examining, taking apart, and rebuilding damaged cells, tissues, and organs through the reconstruction of their molecular structures ; nanoinformatics could be applied to nanomedicine;24 new computers could search for a methodology that reproduces human organs in vitro. nanoneedles : the nucleus of live cells could be penetrated by nanoneedles used in conjunction with microscopy with the aim of introducing nucleic acids, proteins or other compounds inside human cells, or even doing surgery ; modeling the structure - property relationships of nanoneedles.13 environmental nanocatalizers : biological transporters capable of greatly improving catalytic converters currently used in automobiles, thus drastically reducing the pollution emitted. nanobiosensors : sensors capable of detecting the presence of anthrax and other biological weapons ; nanosensors for probing live cells.14 dendrimers : artificial molecules designed to replace defective endogenous elements in the organism, induce the production or secretion of various molecules related to the immune response in case of being genetically deficient, or act as biological markers (for cancer therapy, for the detection of adherence to medication regimens, or for the evaluation of experimental results).15 nanodepots of insulin : microscopic deposits injected into the bloodstream that could act as an artificial pancreas, releasing insulin to counteract the effects of diabetes ; similar technologies could also be employed to release dopamine into neurons as therapies for parkinson s or alzheimer s disease. carbon nanotubes : microscopic tubes aimed at replacing defective or absent genes ; use of carbon nanotubes in vivo to characterize action mechanisms at the cellular level;16 carbon nanotubes could reactivate synaptic connections and regenerate neurons damaged by an accident. molecular nanopyramids : these probes could possibly be released into the human body, with the capability of detecting and destroying the first cancer cells in the brain, heart, kidney, or lungs, without damaging neighboring tissue ; they might also be able to detect viral or bacterial infections in an early stage before the immune system has begun to respond. assembler nanomachines : machines based on the rearrangement of atoms and molecules to manufacture molecular components. smart materials : these probe - like materials could potentially detect subtle changes in body chemistry. nanoparticles : microscopic tumor - destroying genes may possibly be transported to certain sites, directed with great precision to destroy the genetic information of tumors, with the aim of inactivating the target cells and impeding their reproduction ; other genes may be transported to detect tiny alterations in proteins, as well as to scan tissue to ensure that the nanodevices are neatly congregated around the target. nanorobots : these devices could be introduced into the human body to repair damage and combat infection and disease by acting on dna without being detected and destroyed by the host immune system.1722 nanobiochips : this advanced technology could work with mri to supply an abundant amount of information about the human body.23 nanocomputers : computers could be developed made of material stronger than steel but with only 10% of its weight. these could possibly guide nanomachines for the purpose of examining, taking apart, and rebuilding damaged cells, tissues, and organs through the reconstruction of their molecular structures ; nanoinformatics could be applied to nanomedicine;24 new computers could search for a methodology that reproduces human organs in vitro. the academic discipline of nanomedicine could be included in university curriculums for graduate students and researchers today in the fields of pharmacology, genetic engineering, proteomics, and molecular and cellular biology. such programs would train personnel in order to accelerate advances of techniques, technology, and applications of nanomedicine. as such uses increase and | nanomedicine is on the cutting edge of technology applied to medical and biological sciences. nanodevices, nanomaterials, nanoinstruments, nanotechnologies, and nanotechniques (laboratory methods and procedures) are important for the modern practice of medicine and essential for research that could stimulate the discovery of new medical advances. accordingly, there is an eminent need for implementing an academic program in universities to teach this indispensable and pragmatic discipline, especially in the departments of graduate studies and research in the areas of pharmacology, genetic engineering, proteomics, and molecular and cellular biology. |
blue rubber bleb nevus syndrome (brbns) is a rare disorder which is characterized by multiple recurrent vascular malformations, such as hemangioma, and these primarily involve the skin and gastrointestinal tract1). brbns may also involve the brain, liver, lung and skeletal muscles1, 2). the most common clinical presentation of brbns is iron deficiency anemia which is caused by bleeding from a vascular malformation in the gastrointestinal tract. there was occult bleeding in most of the reported cases, but bleeding that presented in the form of melena or hematochezia has also been noted3). although about 200 cases of brbns have been reported in the english literature, there are currently no reports of any cases of brbns reported in the korean literature, although there have been two cases reported in the english literature3, 4). we report here of a 14-year - old patient with multiple recurrent hemangiomas on her skin and gastrointestinal tract ; she was diagnosed with brbns and treated with methylprednisolone. a 14-year - old female patient visited the department of gastroenterology at our hospital as an outpatient in october, 2005 ; her chief complaint was dizziness that had initially developed 18 months ago. the patient did not have any medical history of non - steroidal anti- inflammatory drug use, peptic ulcer or chronic liver disease. since infancy, the patient had repeatedly suffered from the recurrence of soft, compressible bluish nodules in the skin, and these nodules tended to refill with blood after compression. in order to treat these lesions, the patient had undergone a total of at least eight operations at our hospital and other hospitals. however, the nodules recurred on the patient 's toe, right arm and the left hand in may, 2004 (figure 1). consequently, she was hospitalized in the department of orthopedics at our hospital, where excision and biopsy were performed, and the biopsy results revealed hemangioma. however, she had no family history of any recurrent skin lesions or gastrointestinal bleeding. at the outpatient clinic before admission, she had appeared pale with anemic conjunctivae ; she had a blood pressure of 90/60 mmhg, a heart rate of 70/min and a body temperature of 36. there were no skeletal deformities. a digital rectal examination showed negative results. according to the laboratory examinations performed at the outpatient clinic, her hemoglobin was 5.9 g / dl and her hematocrit was 22.7%. in response to these values, we administered iron replacement therapy and transfusion of packed red cell at an outpatient clinic. after hospitalization, the hemoglobin was 13.2 g / dl, the hematocrit 38.6%, the white blood cell count was 7,700/mm and the platelet count was 322,000/mm. the serum blood urea nitrogen was 8.9 mg / dl, the creatinine 0.57 mg / dl, ast 17 iu / l, alt 14 iu / l, sodium 138 meq / l, potassium 3.8 meq / l and chloride 104 meq / l. the serum iron was 7 /dl, the ferritin was 2.2 /dl and the tibc was 379 /dl. the chest x - ray and abdomen computed tomography were all negative for any abnormalities. in order to detect the cause of her iron deficiency anemia, we performed endoscopy, a small bowel series and colonoscopy. endoscopy showed seven polyp - like mass lesions with abundant vasculature at the greater curvature of the body and fundus, the posterior wall of the gastro - esophageal junction and the anterior wall of the gastric angle (figure 2). hemangioma was diagnosed via a biopsy that was performed at this time (figure 3). the small bowel series revealed several small intra - luminal nodular filling defects in the distal jejunal loops and ileum (figure 4). in addition, eight multiple polypoid mass lesions with abundant vasculature were seen from the ascending colon to the rectum during colonscopy (figure 5). we were able to diagnose the patient with brbns due to the facts that the biopsies of both the recurrent nodules on the skin and the gastrointestinal mass lesions revealed hemangiomas, there were no recurrent episodes of epistaxis, no family history of any recurrent skin lesions or gastrointestinal bleeding, and her physical examination showed no skeletal deformity. on the 7 day after hospitalization, the patient 's hemoglobin and hematocrit had decreased slightly to 12.1 mg / dl and 35.7%, respectively. methylprednisolone (2 mg / kg / day) was given orally starting at the 8 day of hospitalization, and there was no decrease in the hemoglobin and hematocrit values until the 17 day. on the other hand, a follow - up endoscopy performed on the 21 day showed no changes in size or number of the multiple polypoid mass lesions. the patient was able to maintain a hemoglobin level of 11.7 mg / dl and a hematocrit of 34.2% until the 22 day, and it was then decided to discharge her with plans for her to undergo 4 months of methylprednisolone treatment (2 mg / kg / day for 1 month and 1 mg / kg / day for an additional 3 months). in this case, the patient had suffered from recurrent hemangiomas in the skin. in the process of evaluating the cause of the patient 's iron deficiency anemia by performing endoscopy, a small bowel series and colonoscopy, multiple hemangiomas were found in the gastrointestinal tract. diseases that involve recurrent vascular malformation of the skin and gastrointestinal tract include brbns, mafucci 's syndrome, rendu - osler - weber syndrome. however, the patients with mafucci 's syndrome also have dyschondria, and rendu - osler - weber syndrome often involves recurrent episodes of epistaxis, the pathologic findings of telangiectasia and this disease is inherited in an autosomal dominant pattern. therefore, we were able to diagnose our patient with brbns5, 6). brbns (or bean 's syndrome) is a rare disorder that is characterized by multiple recurrent vascular malformations such as hemangiomas, and these primarily involve the skin and the gastrointestinal tract1). gascoyen7) reported the disease 's association with hemagniomas in the skin and gastrointestinal tract for the first time in 1860 ; bean1) was the first to call it brbns in 1958. about 200 cases of brbns has been reported in the english literature, and with the exception of two korean cases reported in the english literature, there have been no cases reported in the korean literature3, 4). the vascular malformations that appear in brbns include telangiectasia, capillary hemangioma, cavernous hemangioma, venous angioma and on rare occasion arteriovenous fistula8). they may involve the brain, liver, lung or muscle in addition to the skin and gastrointestinal tract1, 2). the most common clinical manifestation of brbns is iron deficiency anemia which is caused by bleeding of vascular malformations in the gastrointestinal tract. in most cases, the cause is occult bleeding, but there have been cases where the bleeding presented as melena or hematochezia. intussusception, hemothorax, hemopericardium, pulmonary hypertension, dementia, paraparesis, ataxia, cortical blindness. the skin lesions of brbns mainly appear in the trunk and upper extremities and these are characterized by their small size, bluish color, softness, absence of pain and bleeding, and the tendency to refill with blood after compression14, 15). the skin lesions in this case were also small, bluish, soft and compressible, and they occurred in the upper extremities ; these are the typical features of brbns. the gastrointestinal lesions of brbns are usually distributed throughout the gastrointestinal tract and mostly in the small bowel and distal colon2, 16). upon endoscopy, the mucosal nodules are either flat, polypoid or they have central bluish nipples. also, some of these nodules showed evidence of recent or active bleeding. in our case, although brbns develops sporadically in most cases, it is sometimes inherited in an autosomal dominant pattern and its association with chromosome 9p has already been established 17). in the light of the patient 's lack of a family history of brbns, this case seems to have developed sporadically. medical treatment consists of iron supplementation for anemia2), steroids, interferon -2a and octreotide to reduce the frequency and severity of bleeding episodes. however, in most cases, discontinuing administration of these medical treatments has led to recurrence of the disease18, 19). new therapeutic modalities for treating the gastrointestinal lesions have recently been attempted such as endoscopic laser photocoagulation, sclerosis, band ligation and polypectomy3, 20). in the event that any life - threatening hemorrhage occurs, either excision of the gastrointestinal lesions or segmental resection of the involved gi tract can be performed. in a recent study performed by fishman22) on ten patients with brbns, complete gastrointestinal endoscopy was conducted followed with removal of all the gastrointestinal lesions by means of wedge resection, endoscopic polypectomy, suture - ligation, segmental bowel resection and band ligation (surgical eradication), and the patients were followed up for five years on average. as a result, gastrointestinal bleeding recurred in only one patient who had received less extensive procedures. in our case, the patient 's hemoglobin level has been constantly and appropriately maintained without any additional transfusions ever since the patient was started on methylprednisolone. however, follow - up endoscopy performed on the 21 hospital day showed no interval changes in the size or number of the gastrointestinal lesions. long term follow up is required in order to evaluate the effect of treatment with methylprednisolone on patients with brbns, and also to determine if surgical eradication may be needed. | blue rubber bleb nevus syndrome is a rare disorder that is characterized by multiple recurrent vascular malformations, such as hemangioma, and these primarily involve the skin and the gastrointestinal tract. it may also involve the brain, liver, lungs, and skeletal muscles. a 14-year - old female visited our hospital with a chief complaint of dizziness ; upon examination, we found multiple recurrent hemangiomas on the skin and gastrointestinal tract. we were able to diagnose her as suffering from blue rubber bleb nevus syndrome and we treated her with methylprednisolone (2 mg / kg / day for 1 month and 1 mg / kg / day for additional 3 months). we report on this case along with a review of the literature. |
modern kohn sham density functional theory has become the most widely used electronic structure method. a crucial contribution to this success was becke s insight into the role of exact exchange in approximations to the adiabatic connection formula. this discovery led to the creation of the class of methods known as hybrid functionals that include some (usually empirically determined) weighted contribution from exact exchange, as exemplified by the widely used b3lyp functional. the use of hybrid functionals has yielded improved results for thermochemical properties, molecular geometries, and vibrational frequencies. more recently developed hybrid functionals such as m06 - 2x have yielded significant additional improvements in accuracy. the role of exact - exchange has since been further refined with the introduction of range - separated hybrid functionals, which hold particular promise for applications in the area of time - dependent density functional theory. local functionals and standard hybrid functionals suffer from incorrect long - range behavior of charge transfer excited states due to self - interaction error of the transferred electron. range - separated hybrid functionals partition the coulomb operator into short and long - range components in order to smoothly increase the weight of exact exchange toward unity in the long - range limit, thus recovering the correct asymptotic behavior. the development of systematically optimized functionals such as the b97x - d functional led to significant improvements in accuracy over first generation range - separated hybrids. unfortunately, large basis sets are required to obtain the maximal accuracy which modern hybrid and range - separated hybrid functionals can provide. many studies have demonstrated that a basis set of at least triple- quality is required to obtain results reasonably close to the basis set limit with hybrid functionals. at least triple, and if possible quadruple- basis sets are needed for accurate thermochemistry, while at least augmented triple- basis sets are required for noncovalent interactions if no counterpoise corrections are performed. in this scenario, computation of exact - exchange typically consumes the vast majority of the computation time for a hybrid dft calculation. the k matrix required for the computation of exact exchange is computed via the expression:1the number of two - electron integrals appearing in (1) scales as the fourth power of molecular size. schwarz - inequality - based integral screening algorithms can reduce this scaling to nearly o(m) for sufficiently extended systems. in fact, linear scaling algorithms can be obtained by exploiting the density matrix sparsity that occurs in extended insulating systems, as is done in the sonx and link methods. however, such linear scaling behavior is in practice seldom obtained for small - gap systems and/or with larger basis sets which can lead to overlap ill - conditioning and resulting loss of density matrix sparsity. even with the employment of screening algorithms, the scaling of the evaluation of (1) remains fourth - order with respect to increasing the average number of basis functions per atom while molecule size is fixed. high - accuracy calculations in large basis sets are therefore most severely inhibited by the computational demands associated with exact exchange. with the introduction of still - more sophisticated treatments of electron correlation such as double - hybrid functionals, the need to compute efficiently with larger basis sets has only become more pressing. a variety of approaches have been used to improve the efficiency of constructing k in large basis sets, the most common of which is the application of the resolution of the identity (ri) approximation, also frequently referred to as the density fitting or df approximation. in the ri approximation, products of orbital basis functions are further expanded in an auxiliary basis:2this reduces the dimensionality of the integral tensors that must be calculated from four to three. the use of the ri approximation to compute exact exchange was introduced by frchtl, while the first direct implementation is due to weigend. unfortunately, without further approximation the ri - k algorithm scales fourth - order with molecular size, so performance deteriorates relative to conventional integral formation for extended systems. hence, the application of ri - scf (self - consistent field) has historically been limited to compact systems with large basis sets. it has been suggested that ri methods may be better described as inner projection methods, as in the absence of a complete auxiliary basis the ri approximation does not amount to an insertion of a resolution of the identity, but rather the insertion of an inner projection operator. the cholesky decomposition (cd) of the two - electron integrals has been shown to be equivalent to the introduction of an inner projection operator onto the space spanned by the orthogonalized cholesky vectors. in this sense, the cholesky decomposition may be seen as generating an ideal auxiliary basis for the particular problem at hand. cd methods encompass many efficient algorithms. since its introduction to the electronic structure community by beebe and linderberg, cd has been used to formulate a variety of efficient electronic structure algorithms, including the construction of the fock matrix and the evaluation of the ccsd(t) and sos - mp2 energies. cd methods are of great interest because they produce an auxiliary basis that does not depend on the model chemistry for which it was optimized, and thus can deliver consistent performance across a variety of theoretical methods. additionally, cd methods allow for fine - grained control of the accuracy of the approximation simply by varying the decomposition threshold. this is in contrast to the ri method s reliance on preoptimized auxiliary basis sets, which limits its accuracy to that attainable by the largest auxiliary basis set trained for the given theoretical method. however, as currently available ri auxiliary basis sets yield fitting errors in the tens of hartree per atom for absolute energies, which is negligible compared to orbital basis set errors, the practical advantage provided by cd in terms of accuracy is often minimal. cd methods have historically suffered from the inclusion of two - center functions in the resulting auxiliary basis, and therefore required the expensive evaluation of four - center integrals. the situation improves if the cd basis is restricted to contain only one - center auxiliary functions ; examples of such methods include the one - center cholesky decomposition (1c - cd) and the atomic cholesky decomposition (acd). the acd and 1c - cd approaches have been used to obtain more compact sets of cholesky vectors, with a size 5 times that of the orbital basis set found to yield acceptable accuracy. for comparison, the ri approximation typically requires an auxiliary basis set of 35 times the size of the orbital basis set to obtain accuracy in the hartree range. rigorous efficiency comparisons of ri and cd methods are scarce ; in one such study, weigend has shown that 1c - cd is outperformed significantly by ri - j. however, more recent work on so - called method - specific cds, in which hadamard products involving the two - electron integrals are decomposed instead of the two - electron integrals themselves, has been shown to require a drastically smaller auxiliary basis and has been found to be competitive with ri for the computation of exact exchange. the unfavorable scaling of ri - scf can be mitigated through the use of several types of local approximations. the ri expansion coefficients are typically obtained in the coulomb metric by minimizing the coulomb repulsion of the density residual. the fitting coefficients thus obtained are highly nonlocal ; the expansion of a product on a given center contains many numerically significant contributions from auxiliary functions on distant atomic centers. a root cause of this is local incompleteness of the auxiliary basis set, which leads to the inclusion of off - center auxiliary functions in order to obtain a better fit. the locality of the fit coefficients can be improved, while still retaining accuracy in the fit, by minimizing the residual repulsion based on an alternative local operator, a so - called local metric. perhaps the simplest and most drastic local metric approach to density fitting is the overlap metric first introduced by baerends. the overlap metric was subsequently employed by vahtras. in scf calculations and was found to give errors an order of magnitude larger than the coulomb metric. in an attempt to combine the locality of the overlap metric with the accuracy of the coulomb metric, jung. introduced an attenuated coulomb metric with rapid decay with interelectronic distance and similar ri errors to the full coulomb metric. the attenuated coulomb metric has subsequently been employed by reine in conjunction with localized mos to implement reduced - scaling ri - scf. in an analogous development for cd methods, aquilante. have shown that acd methods yield a set of fit coefficients with spatial locality that increases as the decomposition threshold is decreased. this is a consequence of a more complete cd auxiliary basis reducing the need to include off - center auxiliary functions in order to represent on - center products. an alternative ubiquitous approach to constructing k economically is a family of ri approximations employing local fitting domains. instead of expanding each orbital basis function product using a fitting basis set spanning the entire molecule, there have been numerous applications of local fitting domains to ri - scf. in the first development in this area, polly. developed a linear scaling algorithm using local atomic fitting domains in conjunction with localized orbitals. sodt and head - gordon have developed the ari - k algorithm, wherein each pair of orbital basis functions is expanded using the auxiliary basis functions on the atoms in a certain radius around their parent atoms. very recently, meja - rodrguez. have developed the ldf - hf algorithm, which builds upon the work of polly. with localization of the molecular orbitals at each scf cycle in order to obtain very compact local fitting domains. yet another unique approach to improving the locality and scaling of exchange matrix construction is the pseudospectral approach of neese, in which one coordinate in the integrals from (1) is integrated by numerical quadrature. the locality of the grid points coupling to the ao basis is exploited to obtain a conditionally linear scaling algorithm. this approach has been shown to yield equal or superior speedups to ri - k with comparable accuracy. however, to our knowledge its performance has not been benchmarked relative to local density fitting methods. the focus of this work is the construction of k using a drastically local form of the ri approximation, which we shall designate by the name used by merlot. as the pair atomic resolution of the identity (pari) approximation. in this approach, a given pair of orbital basis functions is expanded using only auxiliary basis functions on either of the parent atoms. subsequently by several others. when used with dunlap s robust fit functional, the pari approximation has been found to give surprisingly accurate results. however, it has been demonstrated that the use of the pari approximation for the construction of j can lead to unphysical attractive electron states. merlot. have explained this behavior on the basis of the loss of a positive semidefinite integral tensor, and have shown that this problem manifests for any local ri approximation employing the dunlap functional. they attempted to correct this problem through local completion of the auxiliary basis using the cholesky decomposition, but this approach resulted in the loss of all performance improvements derived from the pari approximation. have taken a different approach by computing subsets of the two - electron integrals analytically, and employing the pari approximation for the remainder. we circumvent this variational stability issue entirely by using pari - k alone, without pari - j. we will analytically demonstrate that the use of pari - k alone is variationally stable, and empirically demonstrate that pari - k yields negligible errors compared to exact integral evaluation. the j matrix can in turn be constructed by one of several pre - existing efficient highly efficient algorithms. have used the pari approximation and the dunlap functional to create a new algorithm, pari - k, for the construction of k. these authors report an impressive speedups of between 5 and 7-fold for their pari - k algorithm relative to the link algorithm, measured on systems of up to 42 atoms in a triple- basis set. however, their algorithm involves contraction of three - center ri integrals over the full auxiliary basis with density - contracted fit coefficients:3the auxiliary index q is coupled by sparsity to the ao - basis index as follows : for a given, all q auxiliary basis functions on atoms with at least one basis function that has non - negligible overlap with is significant. the scaling of this term is thus asymptotically quadratic, just like direct evaluation of the two electron integrals, which is highly desirable. however, it suffers from the same unfavorable scaling of o(n) with respect to basis set size for fixed molecular size, but with an improved prefactor based on the number of flops required to sum over q versus the flops required to form a given two - electron integral directly. we shall demonstrate that dramatic improvements in performance can be obtained via an mo basis algorithm. in our algorithm, the two and three - center quantities evaluated separately in merlot s formulation are evaluated simultaneously in the mo basis. unlike merlot s algorithm, the scaling of our algorithm remains fourth - order ; however, we demonstrate by numerical experiments that our algorithm performs fewer operations even for very extended 1d systems. we obtain speedups of up to 19 over conventional k construction for systems of up to 3570 basis functions. we also demonstrate that auxiliary basis sets no larger than those used in conventional ri - scf are required when the pari - k approximation is used to obtain chemical accuracy for thermodynamic properties and intermolecular interactions, despite the drastically local nature of the approximation. we shall abbreviate ri quantities using a tilde as follows:4dunlap has shown that the following robust approximation of the two - electron integrals yields errors that are quadratic in the error resulting from the ri approximation on the individual products:5merlot. have shown that the use of this robust formulation in conjunction with local ri fitting domains and/or non - coulomb fitting metrics results in a two - electron integral tensor that is not positive semidefinite. however, regardless of this complication, the following equality holds for the dunlap formulation of any fitting method:6the difference of the exact and fitted exchange energies may thus be written as7transforming to the mo basis, we have8 by an early result of slater, each term in the above sum is strictly positive. the exchange energy thus can only increase by the application of robust fitting, and densities corresponding to negative eigenvalues of the two - electron integral matrix will be avoided by the scf convergence algorithm. local fit domains and non - coulomb fitting metrics can therefore be applied to the computation of k without risk of variational instability. we now prove a stronger result concerning the relationship between standard coulomb - metric ri using a global fit domain and that of any local fit approximation. denoting coefficients for the mo pair ij computed using global ri by cijg and those with local ri by cijl, we may express their difference by9 we now consider the expansion of a single exchange integral using the dunlap functional and local ri fit coefficients, using iij to denote the three - center integral vector for this mo pair and v to denote the auxiliary basis metric : the combination of the first two terms in the above expression yields the integral computed using global ri with the coulomb metric. the next term vanishes by the inverse present in the coulomb metric fit coefficients, and we thus obtain12 the error term is non - negative, as v is positive semidefinite. we therefore see that the exchange energy computed with local ri methods is greater than or equal to both the exact and global coulomb ri variants:13 it is well - known that exchange integrals are bounded by their corresponding coulomb integrals:14 this inequality holds when both integrals are expanded using a global auxiliary basis. our treatment expands the coulomb term in the global auxiliary basis, with the exchange term treated in the pair basis. our above result proving that ekl ekg therefore guarantees that the total electronic energy of the system can not become negative. merlot. have applied dunlap s robust formulation in conjunction with the pari approximation, in which orbital basis products between functions lying on the atoms a and b are expanded only using auxiliary basis functions on those two atoms:15 using these two approximations, we shall now derive an efficient algorithm for the formation of the hartree we may write the expression for the exchange matrix in the following convenient form : we then make the following definition:16to yield the resulting expression for the exchange matrix:17 the problem of efficiently computing the exchange matrix now reduces to the efficient computation of l. we now introduce the ri approximation, presuming the use of the pari fit coefficients in eq 15:18grouping common terms yieldsreverting to the mo basis and rearranging yields an intelligently grouped expression:19here the mo coefficient matrix is m. this expression is evaluated using the steps presented in table 1. the second column gives the operation cost for each step in terms of [nb2 ] (number of significant orbital - basis function pairs, which is asymptotically linear in system size), x (mean number of auxiliary basis functions per atom, independent of system size), x (number of auxiliary basis functions), o (number of occupied orbitals), and [nbx ] (number of significant orbital - basis to aux - basis function pairs, which is also asymptotically linear). our algorithm is very similar to the ldf - hf algorithm of meja - rodrguez. but without mo localization and with additional steps to account for the explicitly robust dunlap formulation additionally, when all tensors can not be held in memory, we batch over auxiliary functions rather than over molecular orbitals, allowing us to compute the three - center ao integrals only once per scf cycle. for large systems as we demonstrate for a series of linear alkanes, the onset of sparsity in the mo - contracted fit coefficients diq is sufficiently slow even for linear systems that it is generally not economical to utilize sparsity in this step, and the algorithm is thus effectively fourth order scaling, but with a significantly smaller prefactor than ri - k. the two fourth - order steps in ri - k are given by the formation of the b matrix is more expensive than its subsequent contraction by a factor of x / n. the fourth - order step in our algorithm has the same cost as the latter fourth order step of ri - k, and the asymptotic speedup for our algorithm relative to ri - k is thus 1 + x / n. it should be noted that our algorithm is capable of treating much larger systems on an economical time scale than the largest presented in this paper ; for the purposes of this study, we were limited by the feasibility of timing the integral - driven code for comparison. the dunlap formulation converts first order error in the fitted products to second - order error in the approximate integral. we therefore may further economize our fit coefficients by using much looser screening criteria in their evaluation than in the overall calculation. specifically, we may obtain the same effect as a given integral screening threshold by neglecting the fit coefficients of pairs whose integral estimates are less than the square root of said threshold. thus, if the desired integral screening threshold for the calculation is 10, this level of accuracy can be preserved while setting the fit coefficients of products with integral estimates of approximately 10 uniformly to zero. our results demonstrate that this approximation causes negligible loss of accuracy. for range - separated functionals, both a short and long - range k build for these functionals, we reuse the fit coefficients formed with the full coulomb operator, and transfer the short and long - range operator dependence exclusively to the two and three - center integrals. our expressions for each l matrix are thereforeit should be noted that a robust fit is still obtained due to the explicit use of dunlap s robust ansatz, and our results below demonstrate that the independent use of two sets of fit coefficients is unnecessary. to obtain similar accuracy with global ri - k using a single set of fit coefficients would require the explicit use of the dunlap functional, at a significant performance penalty. we exploit this advantage to compress the evaluation of both exchange contributions into a single k build as follows : as may be seen from the above equations, only steps which are nonrate determining are duplicated. in the limit of large systems, our algorithm will therefore outperform ri - k by an additional factor of 2 for range - separated hybrids, leading to a net speedup of 2(1 + x all calculations were performed with a development version of the q - chem program. hollman and merlot have evaluated the accuracy of the pari approximation for hf and b3lyp we now wish to assess its accuracy in the context of modern density functionals. the b97x - v functional was chosen due to its excellent and highly transferable performance for a variety of types of chemical interactions. our calculations were performed in the aug - cc - pvtz orbital basis set using the corresponding ri auxiliary basis for exchange. for an orbital basis set of this size, it is expected that the user will also wish to use an ri approach for the formation of the coulomb matrix, and we therefore employ the ri - j algorithm in conjunction with pari - k. in order to benchmark performance for thermochemical properties, a subset of the g3 - 05 test set was selected consisting of all compounds for which the auxiliary basis was supported. the errors for a variety of thermochemical properties are tabulated in table 2. larger than expected errors were found when employing the ri - j approximation for the calculation of electron affinities, so this approximation was not used for these table entries. an integral screening threshold of 10 was utilized, with the screening threshold for fit coefficients set to 10 as discussed above. in order to assess the errors introduced by the pari - k approximation when applied to intermolecular interactions, we have also calculated counterpoise corrected binding energies for the s66 test set, which are given in table 3. the results in tables 2 and 3 demonstrate that pari - k can be employed with the same size of auxiliary basis set used for standard ri - jk calculations with negligible loss of accuracy. it should be noted that the b97x - v functional scales the short - range exchange energy by a factor of 0.167 and the long - range by 1.0, thus reducing the error resulting from applying the pari approximation relative to full exact exchange. functionals with larger contributions from exact exchange will experience somewhat larger errors when used in conjunction with pari - k. we use ez s original classification of h hydrogen - bonded, d dispersion, and o others. an initial guess for all systems was computed using the b97-d gga functional in the same basis set. we were able to successfully converge all systems in our truncated g3 set, including the systems for which merlot. and hollman. observe convergence problems (hexafluorobenzene, chloro - pentafluorobenze, 3-butyn-2-one, and 2-butyn). as expected from our proof of variational stability the non positive - semidefinite integral tensor raises the possibility of the scf encountering densities that lead to repulsive exchange interactions, but this is much less likely to pose a problem in practice as these densities will be actively avoided by the scf optimizer. on the basis of our results, we assert that the pari - k method may be reliably used with an appropriate guess. we consider first the performance of our algorithm for a system to which it is optimally suiteda molecule of moderate spatial extent treated with a large basis set. the chosen example is a hydrogen - terminated three ring by six ring graphene lattice (shown in figure 1) using the b97x - v functional with the cc - pvqz basis set. our method is compared against the following competing approaches implemented in q - chem : an integral - driven k build using the link algorithm (optimal for very large systems), the ari - k algorithm (optimal for midsize systems in larger basis sets), and the ri - k algorithm (optimal for compact systems in very large basis sets). our method outperforms all examined alternatives, obtaining a 19 times speedup over the conventional integral - driven k build and a 2.7 times speedup over ri - k. the speedup relative to ri - k will increase with increasing system size ; however, our ri - k code is at present unable to handle the memory requirements of jobs larger than those presented here. it should be noted that while the ri - k and ari - k timings were produced by timing a single k build and scaling the result by 2, the integral - driven timings are for a single k build without scaling, as the short- and long - range k builds can be combined by transferring all of the operator dependence to the fundamental integrals prior to contraction and application of recurrence relations. hydrogen - terminated 3 6 graphene lattice upon which qz timings were performed. wall time for the two k builds in the second scf iteration for a hydrogen - terminated 3 6 graphene lattice with the range - separated b97x - v functional. (inset) speedups relative to the integral - driven k build for the various ri methods. the first iteration is approximately 15% more expensive as of this writing due to initialization costs, but only for smaller systems. ri - k and ari - k timings were calculated by timing one k build and scaling the result by 2, as the cost of the short and long - range k builds is essentially identical for these methods. we now assess the performance of our algorithm as a function of system size in a smaller (tz) basis set, where one would expect better performance from the lower - scaling integral - driven k build. we begin with acene-5 and extend the graphene sheet in two dimensions with additional chains of six rings along the y - axis. we are limited to a four by six aromatic lattice for ri - k due to the memory usage of our benchmark ri - k implementation. our pari - k implementation obtains a 6.4 times speedup relative to exact integrals for the largest lattice, a system with 2348 basis functions. this compares favorably with the speedups obtained by merlot. for various smaller acenes. the superior efficiency of our mo algorithm thus appears to effectively compensate for its asymptotically higher scaling. our algorithm outperforms ri - k by an increasing margin as the system size is increased, reaching a 3.4 times speedup for the largest lattice. wall time for the two k builds in the second scf iteration for various n 6 graphene lattices, performed in the cc - pvtz basis. ri - k and ari - k timings were calculated by timing one k build and scaling the result by 2, as the cost of the short and long - range k builds is essentially identical for these methods. our combined k build provides a dramatic advantage even for the smallest system in figure 3 : a single k build performed for the 1 6 graphene sheet requires 312 s, whereas the combined short and long - range k build takes only 371 s. it should be recalled that the performance of ri - k and ari - k will be twice as good for functions that are not range - separated hybrids. we shall now demonstrate empirically that the loss of sparsity in the ri fit coefficients caused by contracting them with dense matrices leads to severe difficulties in practically attaining the low scaling that is expected for such local fit domains. we focus on the sparsity of the most important sparse tensor appearing in our algorithm, the mo - transformed ri fit coefficients diq, as a function of system size. as can be seen from the sparsity of this tensor for a series of linear alkanes, shown in figure 4, the onset of sparsity in diq is much slower than might be expected given the immense locality of the pari fit coefficients. the tensor is approximately 83% sparse for the longest alkane chain. as a consequence of this slow onset, exploitation of sparsity note that even as the sparsity of diq increases, this gain for the sparse implementation is offset by increasing performance of the matrix - multiply routine called by the dense implementation as the matrix size grows. thus, even for these ideal systems, the advantage gained by the formally lower - scaling algorithm which exploits sparsity is minimal. we acknowledge that a higher - performance sparse linear algebra implementation would yield a more favorable comparison. however, the tensor contraction expression in this step fundamentally takes the form of either a single matrix multiply in the dense case, or a series of what are essentially vector - matrix multiplies in the sparse case. the floating point performance difference is therefore bound to reflect the (usually large) difference in the performance of these two classes of operations for a given blas implementation. it is important to recall that this unfortunate sparsity behavior is not a consequence of the mo - basis transformation per se ; the density - matrix contracted fit coefficients appearing in the analogous step of merlot s algorithm will suffer the exact same slow onset of sparsity, assuming that density - matrix sparsity can not be efficiently utilized. rather, the lack of sparsity in the contracted fit coefficients may be viewed as a fundamental nonlocal behavior of the pari approximation. assuming a fully dense mo coefficient matrix (or density matrix for the analogous ao problem), an entry diq will be nonzero if even a single orbital - basis ao function on the atom of q has non - negligible overap with. this will hold for every single function q on the given atom. the decay of diq is thus controlled exclusively by the most diffuse ao functions in the orbital basis and is slowed significantly by the fact that auxiliary functions are grouped in large atom blocks. finally, we also note that our algorithm outperforms linear - scaling integral evaluation even for very long alkane chains, thus suggesting that widely held reservations about the use of ri - scf methods due to their higher formal scaling may be somewhat misplaced. sparsity in of diq tensor for a series of linear alkanes using the cc - pvtz basis set. screening of the ao - basis ri fit coefficients was performed using a threshold of 10. wall time for a single second scf iteration k build for several linear alkanes, performed in the cc - pvtz basis. an integral threshold of 10 was used in conjunction with a fit coefficient threshold of 10. similar sparsity arguments are relevant to a discussion of the merits of an ao versus mo implementation. a reduction in the formal scaling of the algorithm can be obtained by utilizing the gaussian product sparsity present in the ao basis representation of the relevant tensors, as is done in merlot s pari - k algorithm. however, based on our data for linear alkanes, we believe that utilizing ao sparsity would actually increase the raw operation cost of this algorithm, even for very extended systems. we illustrate this point by considering the relative sizes of the mo - transformed three - center integrals and the original ao three - center integrals, accounting for sparsity. the ratio between these two quantities represents the ratio between the rate - determining step of our algorithm and an ao algorithm such as merlot s. a plot of each of these quantities with respect to alkane length for the cc - pvtz basis is shown in figure 6. the permutational symmetry of the gaussian product indices is accounted for in this figure. on the basis of the relative size of these quantities, we propose that the ao - basis algorithm, despite formally scaling lower than our method, will in fact require more operations even for very extended systems, in addition to suffering from a significant efficiency penalty due to the high performance of dense linear algebra operations. this serves as a reminder that the tremendous compactness of the occupied mo basis and the associated high efficiency of dense linear algebra libraries must be properly accounted for when assessing the efficacy of competing lower - scaling implementations. many similar points about the relative merits of lower - scaling methods in contrast to lower - prefactor, higher - scaling competitors are stated eloquently in a review by neese. on the basis of these concerns, it seems to us that the best path toward a lower - scaling pari - k implementation is the use of localized occupied molecular orbitals. comparison of varying dimensions of tensors in the rate - determining step for ao and mo algorithms. our mo - based implementation of the pari - k approximation has been shown to significantly accelerate large scale range - separated hybrid dft calculations. benchmark results for thermochemistry and intermolecular interactions indicate that impressive accuracy can be obtained with the modern b97x - v functional, while requiring an auxiliary basis set no larger than that used in conventional ri - hf. our algorithm has been shown to out - perform commonly available alternatives for extended and compact systems in tz and qz basis sets. | an efficient new molecular orbital (mo) basis algorithm is reported implementing the pair atomic resolution of the identity approximation (pari) to evaluate the exact exchange contribution (k) to self - consistent field methods, such as hybrid and range - separated hybrid density functionals. the pari approximation, in which atomic orbital (ao) basis function pairs are expanded using auxiliary basis functions centered only on their two respective atoms, was recently investigated by merlot. [j. comput. chem.2013, 34, 1486 ]. our algorithm is significantly faster than quartic scaling ri - k, with an asymptotic exchange speedup for hybrid functionals of (1 + x / n), where n and x are the ao and auxiliary basis dimensions. the asymptotic speedup is 2 + 2x / n for range separated hybrids such as cam - b3lyp, b97x - d, and b97x - v which include short- and long - range exact exchange. the observed speedup for exchange in b97x - v for a c68 graphene fragment in the cc - pvtz basis is 3.4 relative to ri - k. like conventional ri - k, our method greatly outperforms conventional integral evaluation in large basis sets ; a speedup of 19 is obtained in the cc - pvqz basis on a c54 graphene fragment. negligible loss of accuracy relative to exact integral evaluation is demonstrated on databases of bonded and nonbonded interactions. we also demonstrate both analytically and numerically that the pari - k approximation is variationally stable. |
neoadjuvant chemotherapy in breast cancer treatment is now recognized as a standard care to increase conservative surgery [1, 2 ]. its utility is also documented in inflammatory or locally advanced tumors. long - term results of neoadjuvant chemotherapy are equivalent to those obtained with adjuvant chemotherapy if locoregional treatments are fully applied [1, 2 ]. in her2-positive breast cancer, randomized studies with and without anthracyclines have demonstrated the essential role of anti - her2 therapies in obtaining increased pathological complete response rates and good long - term results. several randomized trials have evaluated, in the neoadjuvant setting, the role of trastuzumab (herceptin, roche laboratory), a recombinant humanized monoclonal antibody that targets her2 receptor. the first randomized trial in patients with operable noninflammatory disease was stopped early when the pathological complete response (pcr) rate in the trastuzumab group was more than twice as high as that of the control group (65% versus 26%) [4, 5 ]. the pcr rates in the following studies varied between 26% and 40% in the trastuzumab arms [69 ]. these differences can be explained by various inclusion criteria, different type, and different duration of the regimens. nonetheless, all the studies showed a higher pcr rate when trastuzumab was combined with chemotherapy compared to chemotherapy alone (table 1). in the abcsg-24 study, 536 patients were randomized to receive either 6 cycles of edc (epirubicin, docetaxel, and capecitabine) or 6 cycles of ed (epirubicin, docetaxel). patients with her2-positive tumors were also randomized to receive trastuzumab or not. in the 512 eligible patients for efficacy, the pcr rate was significantly higher after neoadjuvant edc (23.8% versus 15.2%, p = 0.036). in the her2-positive subgroup (n = 90), the addition of trastuzumab to the chemotherapy increased the pcr rate (40% versus 26.7%) but this result was not statistically significant (p = 0.37). authors suggested it might be due to the unexpectedly high rate of pcr achieved in the ed / edc group (the sample size had been calculated to detect a difference in pcr rate of 20% after ed or edc versus 50% after ed or edc plus trastuzumab ; power = 80%, p 20%) during neoadjuvant therapy (after 4 cycles) was a predictor of pcr in the lapatinib - treated group only (or = 11.7, 95% ci 1.03110, p = 0.031). nonetheless, the biological relevance of elevated sher2 is still unknown and other studies have reported a limited predictive utility of baseline sher2. before neoadjuvant chemotherapy with trastuzumab, circulating mir-210 levels were significantly higher in 11 patients who had residual disease than in 18 patients who achieved a pcr (p = 0.0359). some data suggested that the fragment c receptor (fcr) polymorphism has an effect on adcc, which is one of the mechanisms of action of the trastuzumab. tamura. observed in the tumors of 15 patients that the ffr2a-131 h / h polymorphism predicts the pathological response to trastuzumab - based neoadjuvant chemotherapy in early - stage breast cancer. esteva. suggested that in 45 patients with her2 + tumors who received concomitant trastuzumab and paclitaxel followed by fec, a lower expression of genes involved with cd40 signaling was associated with a greater risk of residual disease. in the noah trial, in 171 patients with available biopsies, negative progesterone receptor and c - myc amplification were associated with higher pcr rates after addition of trastuzumab compared to chemotherapy alone. overexpression of membranous igf1r was associated with higher likelihood of residual disease after trastuzumab - based chemotherapy. in the exploratory study presented by holmes. at the 2011 asco meeting, 100 patients with stage ii / iii her2 + breast cancer were randomized to trastuzumab, lapatinib, or trastuzumab plus lapatinib. before and after the anti - her2 therapy all patients had core needle biopsies for tissue microarray, stem cell analysis, and reverse - phase protein microarrays, measuring 60 different phosphoprotein / posttranslationally modified protein signaling and gene expression analysis endpoints. molecular profiles suggested that nonresponders use autophagy and stem - cell - related pathways to evade therapy, while responders have disruption of her2-her3 linkages and known downstream regulators of growth and transcription. in her2-negative and her2-positive tumors, the standard uptake value (suv) decrease, studied with positron emission tomography (pet), is a strong predictor of pcr after only one course of chemotherapy. however pet baseline characteristics and metabolic response to neoadjuvant chemotherapy are highly dependent on the histologic type of breast cancer (i.e., luminal versus her2-positive versus triple - negative tumors). by multivariate analysis, suv was found as the only independent predictive factor of pcr in her2 subtype. a decrease of suv over 75% (suv < 75%) had a high odds ratio (or) of 6.31 (95% confidence interval = 1.1039.16 ; p < 0.03). to identify an optimal threshold for the prediction of the pathological response, receiver operating characteristics (roc) analysis gave an optimal cut - off value of 70% for suv. at this cut - off value from these results, an suv decrease greater than 70% allowed for the early identification of the highly responsive tumors, which will be completely eradicated by trastuzumab - based neoadjuvant therapy, with an accuracy of 76%. 2009 - 013410 - 26, nct01142778) aiming at confirming the role of early pet to differentiate the excellent responders to her2 neoadjuvant trastuzumab - based therapy from the less responders who are then randomized to additional neoadjuvant bevacizumab. this study is the first one to adjust therapeutic choice according to initial pet results. if successful, this type of strategy could be used to lighten or to reinforce future neoadjuvant treatments. trastuzumab (herceptin) has been a breakthrough in the treatment of her2-positive breast cancer. trastuzumab combined with chemotherapy has improved response rates, pathological complete response, progression free survival and survival in the neoadjuvant setting of these cancers. with the emergence of new anti her2 therapies like lapatinib (tyverb), pertuzumab (tarjeta) and tdm1 (kacyla), dual her2 blockade, still associated with chemotherapy, has proven to be superior. in order to better individualize targeted therapies in specific tumor subgroups, different biomarkers have been studied to predict the response to anti - her2 neoadjuvant therapies but until now, except the her2 positivity, none has been validated. pet oriented strategy could be used to separate the most responsive tumors from the less responsive ones. | since 2005, major progresses have been made in the neoadjuvant treatment of her2-positive breast cancer. trastuzumab introduction associated with chemotherapy has been the first major step leading to the improvement of the complete pathological response rate and, like in the adjuvant studies, better survivals. dual her2 blockade has been the next step and trastuzumab is associated now with other anti - her2 therapies like lapatinib or pertuzumab, the latter being much more easy to use in combination with chemotherapy. additional knowledge is necessary to better define within the her2 tumor subgroup which patients could benefit more from targeted therapies. different biomarkers have been studied to predict the response after anti - her2 neoadjuvant therapies but until now none has been validated. |
cochlear implant surgery is the only way to cure severe and profound sensorineural hearing loss. so far to identify the implantation location and avoid injury of important anatomic structures, the size of open cavity in traditional cochlear implant surgery is significantly larger than that is needed for electrodes to pass through. taking advantage of medical navigations and robotic techniques, otologic surgeons and researchers in the electrical and computer engineering fields achieved submillimeter accuracy in 2005 using registration system of new fiducial markers in otologic surgeries. based on these experiments, they hypothesized that the range of mastoidectomy could be reduced to a drill path, whose diameter was slightly larger than an electrode. this drill path was from lateral mastoid cortex to the cochlea via the facial recess. therefore, this minimally invasive path was named as percutaneous cochlear implantation (pci). in pci surgery, the edge of the drill hole is adjacent to important structures, such as facial nerves (fns), chorda tympani nerves (chts), the back wall of the external canal, and the auditory ossicles. the distance between the edge of the drill hole and anatomic structures is the safety margin, which depends on three - dimensional (3d) anatomies of the patients and the choice of the drill path. the most important factor here is the width between fn and cht. according to histological statistics among most people, this width on the oval window plane if the cht can be sacrificed, the width might reach 3.14.9 mm. as a result, considering system errors and the diameter of the drill, the accuracy of cochlear pore - forming should be 1 mm, or even better, 0.5 mm. an early study showed that it was impossible to achieve high accuracy in temporal surgeries with the traditional image - guided mode. more recently, attempts of improving accuracy by locating the implanting targets through, for instance, patient - customized microstereotactic frames achieved satisfactory results. the further employment of robot - guided frames and robot - controlled implantation instrument provided higher accuracy. in 2014, the first cadaveric feasibility study of a master - slave - assisted cochlear implant procedure in the otolaryngology - head and neck surgery field using the da vinci si surgical system was reported. they not only assured a more flexible operation but also avoided errors caused by manual surgical drill instability. pci reduced the trauma caused by mastoidectomy, but it is still necessary to drill three bone holes in advance on patients skull surface both for microstereotactic frames and robot - guided drilling. moreover, the second ct scan is required to fix the fiducial frame or fiducial markers, which imperceptibly caused additional trauma or radioactive damage during the registration. in our previous study, we demonstrated a minimal invasive registration method in which the joint registration of the bone - bed and the malleus short process was adopted to successfully obtain 1 mm registration accuracy in deep targets. assisted by the self - developed bi - planar device, this study adopted a minimal invasive registration method to conduct the image - guided minimal invasive pci on eight cadaveric temporal specimens and further verified the safety of this system. bi - planar hybrid system we designed a bi - planar hybrid system based on the special body position and the operation path in the cochlear implant operation. the fixing support could be adjusted to accommodate patients with different head shapes using two adjustable threaded rods. the hybrid mechanism was composed by two manipulators in series with 7 degrees of freedom (7-dof) for each one and a parallel mechanism with 4-dof. these two manipulators in series provided high stiffness and large workspace at the same time for holding up the parallel mechanism. the parallel mechanism was used to grasp the otology drill by two sleeves. for each moving unit, two sliding blocks could be moved manually along with orthogonal directions to control the pose of the otology drill. the lead screws with self - locking were adopted to ensure high precision and safety. the robot system could readily reach the planed position and fix the otology drill solidly. optical navigation system in this study, the photoelectric navigation, polaris spectra (ndi corporation, canada), was used. the polaris optical tracking solution gave medical simulator manufacturers exceptionally accurate and reliable 3d tracking of simulated medical tools (via attached markers) over a large measurement volume. the polaris emitted infrared light to wirelessly detect and track the tool 's position and orientation. ent navigation software system (copyright registration number : 2014sr050996, beijing, china) was independently developed by the beihang university, school of mechanical engineering and automation and peking university third hospital. the software includes functions of image - import, data - management, registration, path - planning, and real - time navigation. the overview of the study system is shown in figure 1. the whole study system process. pca : principal component analysis ; icp : iterative closest point algorithm ; pci : percutaneous cochlear implantation. cadaveric specimens preparation the institutional review board approval was obtained by the peking university third hospital medical ethics committee. every specimen had an intact external auditory canal (eac), intact tympanic membrane, and intact structure of ossicles with no apparent lesion in the middle ear. before the surgery, the bones of the mastoid and temporal regions were exposed by removing the skin, subcutaneous tissue, and muscles. on each specimen, a vertical hole of 1 mm depth, which was used in the registration process later, was drilled in the transplant bed area for conventional cochlear implants in the temporal region by a 1 mm diamond drill (nsk volvere gx, japan). imaging and specimen fixation after the preparation of each cadaveric specimen as mentioned above, a high - resolution ct (hrct) scan was performed on its temporal bone (siemens / somatom definition flash, german, thickness = 0.6 mm, pitch = 0.3 mm). the scanned cadaveric specimens were put into the head - fixed frame of the operation platform (the simulated operation table) to ensure the fixation during the registration and operational process. image processing and planning data obtained by ct scans were input to the image processing software mimics 10.01 (materialise, leuven, belgium) with the digital imaging and communications in medicine (dicom) format. the related anatomic structures were extracted by this software, and the cochlear, fn, cht ; the auditory ossicles were formed as the 3d models. this reconstructed 3d model was further generated as a stereolithography (stl) file, which was imported into the ent navigation software system. in the visualization unit of the software, the 3d positions of anatomic structures were visually analyzed. proper pci access met the following requirements : the target point of the access was located in the tympanic canal of the cochlear basal turn ; the access was tangent to the cochlear basal turn ; when the access went through the fn recess region, there was sufficient safety margins from the fn and the cht ; and the access did not penetrate posterior parties of the eac or damage the ossicular chain (oc) [figure 2 ]. polaris spectra navigation was employed. a minimally invasive method combined with a drilled hole - centered bone - bed and the short process of malleus the detailed illustrations were as follows. in the 3d visual module of the ear navigation, a 3d model was reconstructed with the 2d ct data, and the temporal region of the surgery side was selectedin the space registration unit, a 3d point cloud (so - called point cloud p) was interactively collected on the characteristic surface of the reconstructive model in the selected regions by ray - casting algorithm. this process was called the digital ct space characteristic surfaceusing the collecting ball, the point clouds were collected on the bone surface area centered around the bone hole in the temporal region with certain radius (usually, 10 mm), which was also referred as the stl file of the auditory ossicle was imported ; the collecting ball was moved to enclose the short process of malleus and all - point clouds were collected in the ball. the coordinates of point clouds of the stl file was transformed to those of the ct coordinate system. the data mentioned above were merged and point cloud p in ct space coordinate system was acquired.in the process of operation, the point data collection in the actual coordinate system was obtained. by sliding the probe on the predetermined characteristic surface of the surgical location (corresponding to characteristic surface of reconstruction model), we obtained a real - time recording of the coordinate of the end of the probe in the actual space by the position indicator. this coordinate was referred as the point cloud q, and this process was referred as the digital actual space characteristic curve surfacelikewise, after the bone exposure, a disc area with a drilled hole center and 10 mm diameter was selected. when the polaris spectra navigation began to collect, the tip of the probe should contact with the bone surface and be completely well - distributed on the disc. data of around 3000 points should be collected, so as to gain the actual space point cloud. with the endoscope, the tip of the probe was penetrated into the inside of the eac and touched the short process of the malleus lightly to collect the point cloud. this step should be softly to avoid the injury of the tympanic membrane and the oc. point cloud q in the actual space coordinate system was acquired when these two - point clouds were combined.principal component analysis of point cloud p and point cloud q was applied to acquire the eigenvector of the point cloud for the primary registration. iterative closest point algorithm was applied to performing the fine registration with a registration matrix to map locations from the actual space to the image space [figure 3 ]. in the 3d visual module of the ear navigation, a 3d model was reconstructed with the 2d ct data, and the temporal region of the surgery side was selected in the space registration unit, a 3d point cloud (so - called point cloud p) was interactively collected on the characteristic surface of the reconstructive model in the selected regions by ray - casting algorithm. this process was called the digital ct space characteristic surface using the collecting ball, the point clouds were collected on the bone surface area centered around the bone hole in the temporal region with certain radius (usually, 10 mm), which was also referred as point clouds on the bone - bed. the stl file of the auditory ossicle was imported ; the collecting ball was moved to enclose the short process of malleus and all - point clouds were collected in the ball. the coordinates of point clouds of the stl file was transformed to those of the ct coordinate system. the data mentioned above were merged and point cloud p in ct space coordinate system was acquired. in the process of operation, the point data collection in the actual coordinate system was obtained. by sliding the probe on the predetermined characteristic surface of the surgical location (corresponding to characteristic surface of reconstruction model), we obtained a real - time recording of the coordinate of the end of the probe in the actual space by the position indicator. this coordinate was referred as the point cloud q, and this process was referred as the digital actual space characteristic curve surface likewise, after the bone exposure, a disc area with a drilled hole center and 10 mm diameter was selected. when the polaris spectra navigation began to collect, the tip of the probe should contact with the bone surface and be completely well - distributed on the disc. data of around 3000 points should be collected, so as to gain the actual space point cloud. with the endoscope, the tip of the probe was penetrated into the inside of the eac and touched the short process of the malleus lightly to collect the point cloud. this step should be softly to avoid the injury of the tympanic membrane and the oc. point cloud q in the actual space coordinate system was acquired when these two - point clouds were combined. principal component analysis of point cloud p and point cloud q was applied to acquire the eigenvector of the point cloud for the primary registration. iterative closest point algorithm was applied to performing the fine registration with a registration matrix to map locations from the actual space to the image space [figure 3 ]. point cloud p in image space point cloud q in actual space point cloud simulated diagram registration matrix t after registering point cloud p and point cloud q. a : point cloud collection in bone - bed ; b : point cloud collection in short process of malleus ; c : cloud of points presented by the system. image - guided drilling by bi - planar device the first step was to fix the bi - planar robot and place the drill on the bi - planar robot. four identifiable balls for the electrooptical navigation were fixed at the end of the drill, by which the corresponding position of the drill 's tip on the image was recognized. the operator held the drill and faced the navigation display which showed the planned path and the real - time location of the drill in 3d. after ascertaining the entry point, the operator adjusted the bi - planar positions to ensure that the drill trajectory coincided with the planned path. after the manual orientation, the position of the bi - planar device was locked, and the drilling was subsequently processed following the planned pathway. three intermittent processes were carried on during the drilling involving three drill bits : in the mastoid, 2.3 mm cutting drill bits (medicom, germany) ; in the facial recess, 1.8 mm twist drill bits ; and at the basal turn of the cochlear, 1.0 mm diamond drill bits. 10,000 r / min was used at the first two processes with the continuous irrigation, and 1000 r / min for the cochleostomy. in the operation, the real - time image was displayed to verify the coincidence between the actual trajectory and the planned path, the relationship between the extension cord of the actual trajectory and the basal turn of the cochlear, as well as the relationship between the actual trajectory and the fn and the cochlear. meanwhile postoperation safety analysis the cadaveric head temporal bone was scanned by hrct with the same scanning parameters as the preoperation, and the 3d reconstruction and the image analysis were made to observe and measure the relative location and the distance between the actual trajectory and the important anatomic structures. routine anatomy was conducted on the drilled specimens to specify the relationship between the drill path and the surrounding anatomic structures. postoperative ct images were projected to the coordinate system of the preoperative ct images and registered through similar methods. since it was impossible to directly obtain the axis of the actual postoperative trajectory, using the similar planned method as the preoperation (two points to determine a straight line), the target point and the entry point of the actual trajectory were expected to be at the center of the hole in the cochlear and the temporal bone surface, respectively. this study designed a self - developed pre- and post - operation comparison procedure to calculate the distance between the actual and the planned position of the target and the entry point, as well as the deviation of the distance and the angle between the actual trajectory and the planned path, to directly obtain the location relationship between the fn and the cht. the maximal, minimal, and median values were recorded and referred as the median (inter - quartile range). bi - planar hybrid system we designed a bi - planar hybrid system based on the special body position and the operation path in the cochlear implant operation. the fixing support could be adjusted to accommodate patients with different head shapes using two adjustable threaded rods. the hybrid mechanism was composed by two manipulators in series with 7 degrees of freedom (7-dof) for each one and a parallel mechanism with 4-dof. these two manipulators in series provided high stiffness and large workspace at the same time for holding up the parallel mechanism. the parallel mechanism was used to grasp the otology drill by two sleeves. for each moving unit, two sliding blocks could be moved manually along with orthogonal directions to control the pose of the otology drill. the lead screws with self - locking were adopted to ensure high precision and safety. the robot system could readily reach the planed position and fix the otology drill solidly. optical navigation system in this study, the photoelectric navigation, polaris spectra (ndi corporation, canada), was used. the polaris optical tracking solution gave medical simulator manufacturers exceptionally accurate and reliable 3d tracking of simulated medical tools (via attached markers) over a large measurement volume. the polaris emitted infrared light to wirelessly detect and track the tool 's position and orientation. ent navigation software system (copyright registration number : 2014sr050996, beijing, china) was independently developed by the beihang university, school of mechanical engineering and automation and peking university third hospital. the software includes functions of image - import, data - management, registration, path - planning, and real - time navigation. the overview of the study system is shown in figure 1. the whole study system process. pca : principal component analysis ; icp : iterative closest point algorithm ; pci : percutaneous cochlear implantation. cadaveric specimens preparation the institutional review board approval was obtained by the peking university third hospital medical ethics committee. every specimen had an intact external auditory canal (eac), intact tympanic membrane, and intact structure of ossicles with no apparent lesion in the middle ear. before the surgery, the bones of the mastoid and temporal regions were exposed by removing the skin, subcutaneous tissue, and muscles. on each specimen, a vertical hole of 1 mm depth, which was used in the registration process later, was drilled in the transplant bed area for conventional cochlear implants in the temporal region by a 1 mm diamond drill (nsk volvere gx, japan). imaging and specimen fixation after the preparation of each cadaveric specimen as mentioned above, a high - resolution ct (hrct) scan was performed on its temporal bone (siemens / somatom definition flash, german, thickness = 0.6 mm, pitch = 0.3 mm). the scanned cadaveric specimens were put into the head - fixed frame of the operation platform (the simulated operation table) to ensure the fixation during the registration and operational process. image processing and planning data obtained by ct scans were input to the image processing software mimics 10.01 (materialise, leuven, belgium) with the digital imaging and communications in medicine (dicom) format. the related anatomic structures were extracted by this software, and the cochlear, fn, cht ; the auditory ossicles were formed as the 3d models. this reconstructed 3d model was further generated as a stereolithography (stl) file, which was imported into the ent navigation software system. in the visualization unit of the software, proper pci access met the following requirements : the target point of the access was located in the tympanic canal of the cochlear basal turn ; the access was tangent to the cochlear basal turn ; when the access went through the fn recess region, there was sufficient safety margins from the fn and the cht ; and the access did not penetrate posterior parties of the eac or damage the ossicular chain (oc) [figure 2 ]. polaris spectra navigation was employed. a minimally invasive method combined with a drilled hole - centered bone - bed and the short process of malleus the detailed illustrations were as follows. in the 3d visual module of the ear navigation, a 3d model was reconstructed with the 2d ct data, and the temporal region of the surgery side was selectedin the space registration unit, a 3d point cloud (so - called point cloud p) was interactively collected on the characteristic surface of the reconstructive model in the selected regions by ray - casting algorithm. this process was called the digital ct space characteristic surfaceusing the collecting ball, the point clouds were collected on the bone surface area centered around the bone hole in the temporal region with certain radius (usually, 10 mm), which was also referred as point clouds on the bone - bed. the stl file of the auditory ossicle was imported ; the collecting ball was moved to enclose the short process of malleus and all - point clouds were collected in the ball. the coordinates of point clouds of the stl file was transformed to those of the ct coordinate system. the data mentioned above were merged and point cloud p in ct space coordinate system was acquired.in the process of operation, the point data collection in the actual coordinate system was obtained. by sliding the probe on the predetermined characteristic surface of the surgical location (corresponding to characteristic surface of reconstruction model), we obtained a real - time recording of the coordinate of the end of the probe in the actual space by the position indicator. this coordinate was referred as the point cloud q, and this process was referred as the digital actual space characteristic curve surfacelikewise, after the bone exposure, a disc area with a drilled hole center and 10 mm diameter was selected. when the polaris spectra navigation began to collect, the tip of the probe should contact with the bone surface and be completely well - distributed on the disc. data of around 3000 points should be collected, so as to gain the actual space point cloud. with the endoscope, the tip of the probe was penetrated into the inside of the eac and touched the short process of the malleus lightly to collect the point cloud. this step should be softly to avoid the injury of the tympanic membrane and the oc. point cloud q in the actual space coordinate system was acquired when these two - point clouds were combined.principal component analysis of point cloud p and point cloud q was applied to acquire the eigenvector of the point cloud for the primary registration. iterative closest point algorithm was applied to performing the fine registration with a registration matrix to map locations from the actual space to the image space [figure 3 ]. in the 3d visual module of the ear navigation, a 3d model was reconstructed with the 2d ct data, and the temporal region of the surgery side was selected in the space registration unit, a 3d point cloud (so - called point cloud p) was interactively collected on the characteristic surface of the reconstructive model in the selected regions by ray - casting algorithm. this process was called the digital ct space characteristic surface using the collecting ball, the point clouds were collected on the bone surface area centered around the bone hole in the temporal region with certain radius (usually, 10 mm), which was also referred as point clouds on the bone - bed. the stl file of the auditory ossicle was imported ; the collecting ball was moved to enclose the short process of malleus and all - point clouds were collected in the ball. the coordinates of point clouds of the stl file was transformed to those of the ct coordinate system. the data mentioned above were merged and point cloud p in ct space coordinate system was acquired. in the process of operation, the point data collection in the actual coordinate system was obtained. by sliding the probe on the predetermined characteristic surface of the surgical location (corresponding to characteristic surface of reconstruction model), we obtained a real - time recording of the coordinate of the end of the probe in the actual space by the position indicator. this coordinate was referred as the point cloud q, and this process was referred as the digital actual space characteristic curve surface likewise, after the bone exposure, a disc area with a drilled hole center and 10 mm diameter was selected. when the polaris spectra navigation began to collect, the tip of the probe should contact with the bone surface and be completely well - distributed on the disc. data of around 3000 points should be collected, so as to gain the actual space point cloud. with the endoscope, the tip of the probe was penetrated into the inside of the eac and touched the short process of the malleus lightly to collect the point cloud. this step should be softly to avoid the injury of the tympanic membrane and the oc. point cloud q in the actual space coordinate system was acquired when these two - point clouds were combined. principal component analysis of point cloud p and point cloud q was applied to acquire the eigenvector of the point cloud for the primary registration. iterative closest point algorithm was applied to performing the fine registration with a registration matrix to map locations from the actual space to the image space [figure 3 ]. point cloud p in image space point cloud q in actual space point cloud simulated diagram registration matrix t after registering point cloud p and point cloud q. a : point cloud collection in bone - bed ; b : point cloud collection in short process of malleus ; c : cloud of points presented by the system. image - guided drilling by bi - planar device the first step was to fix the bi - planar robot and place the drill on the bi - planar robot. four identifiable balls for the electrooptical navigation were fixed at the end of the drill, by which the corresponding position of the drill 's tip on the image was recognized. the operator held the drill and faced the navigation display which showed the planned path and the real - time location of the drill in 3d. after ascertaining the entry point, the operator adjusted the bi - planar positions to ensure that the drill trajectory coincided with the planned path. after the manual orientation, the position of the bi - planar device was locked, and the drilling was subsequently processed following the planned pathway. three intermittent processes were carried on during the drilling involving three drill bits : in the mastoid, 2.3 mm cutting drill bits (medicom, germany) ; in the facial recess, 1.8 mm twist drill bits ; and at the basal turn of the cochlear, 1.0 mm diamond drill bits. 10,000 r / min was used at the first two processes with the continuous irrigation, and 1000 r / min for the cochleostomy. in the operation, the real - time image was displayed to verify the coincidence between the actual trajectory and the planned path, the relationship between the extension cord of the actual trajectory and the basal turn of the cochlear, as well as the relationship between the actual trajectory and the fn and the cochlear. meanwhile, the operator who held the drill had a real - time force feedback. postoperation safety analysis the cadaveric head temporal bone was scanned by hrct with the same scanning parameters as the preoperation, and the 3d reconstruction and the image analysis were made to observe and measure the relative location and the distance between the actual trajectory and the important anatomic structures. routine anatomy was conducted on the drilled specimens to specify the relationship between the drill path and the surrounding anatomic structures. postoperative ct images were projected to the coordinate system of the preoperative ct images and registered through similar methods. since it was impossible to directly obtain the axis of the actual postoperative trajectory, using the similar planned method as the preoperation (two points to determine a straight line), the target point and the entry point of the actual trajectory were expected to be at the center of the hole in the cochlear and the temporal bone surface, respectively. this study designed a self - developed pre- and post - operation comparison procedure to calculate the distance between the actual and the planned position of the target and the entry point, as well as the deviation of the distance and the angle between the actual trajectory and the planned path, to directly obtain the location relationship between the fn and the cht. the maximal, minimal, and median values were recorded and referred as the median (inter - quartile range). the average length of the drilling trajectory from the mastoid surface to the cochlear fenestration point was 27.69 mm (26.7729.15 mm), and the average width of the fn recess was 2.80 mm (2.503.10 mm). errors at the entrance and the target points were 0.86 mm (0.681.00 mm) and 0.44 mm (0.300.96 mm), respectively. the angular error between the planed and the drilled trajectory was 1.74 (1.262.41). summary statistics from the drilling tests (n = 8) fnr : facial nerve recess ; fn : facial nerve ; eac : external auditory canal ; max : maximum ; min : minimum ; med : median ; iqr : inter - quartile range. eight specimens all maintained intact fns, with no damage in the eac, tympanic membrane, and ossicles among specimens while five had the intact cht. taking one case as example, figure 4 shows the location between the drill path between the surrounding anatomic structures with ct scans, anatomical observation, and pre- and post - operation comparison procedure. the location between the drill path and surrounding anatomic structures. postoperative hrct ; the location relationship between drill path and fn ; postoperative verification procedures ; routine temporal anatomy ; the contoured cochlear ; postoperative micro - ct. hrct : high - resolution computed tomography ; fn : facial nerve ; oc : ossicular chain ; cht : chorda tympani nerve ; cf : cochlear fenestration ; sf : stapes footplate ; ct : computed tomography. in figure 5, we show the operation durations of each step during the complete process in pci with assistance of image - guided bi - planar device. the operation durations were reduced significantly with the practice. at specimen 8, the total time was < 60 min while the registration process consumed the longest time and the segmentation of important anatomic structures such as the cochlea was the second longest. in the actual operation, it last for 2030 min to segment the cochlea and the plan path, which was finished before the operation. consequently, 3040 min was taken to register, fixed the bi - planar and drill during the process of the operation. with the development of surgical techniques, otologists tried more minimally invasive methods to reduce the damage of the cochlear implantation. the method of minimally invasive percutaneous tunnel avoided the traditional method of opening mastoid. instead, with the computer - assisted path - planning design and the framework guidance, a tunnel with a small diameter was drilled from the mastoid outer surface to the cochlear target point. because the anatomy variations of the temporal bone are large, the cochlear implant surgery typically demands the experience and the skill of the performer. we tried to find an easier and more precise manner to perform this surgery. on the other hand, because the target point of this tunnel was at the cochlear basal turn, any deviation in the location of the target point would fail the operation and even damage the cochlea. meanwhile, since the tunnel was adjacent to the fn, cht, eac, and the ossicle, any damage of these structures, especially which of the fn, would lead to severe sequela to patients. therefore, operation accuracy is an important indicator for the clinical application of this minimal invasive theory. the operation accuracy was comprehensively determined by each element of the system, such as the position error from the navigation system, instrument error from the operation mechanism (the space framework, device, drill, etc.), and operation error as well. in the study, we succeeded in drilling on eight cadaveric head specimens with image - guided bi - planar operations, and the error of the target point was 0.86 mm (0.681.00 mm). this accuracy met the accuracy demand required by the middle ear surgery but was still lower than 0.5 mm which was reported in literature. the reasons for errors and the optimization plans are as follows : the quality of the imaging data directly affects the accuracy of the image - guided surgery. with flat - panel volumetric computed tomography, the resolution at each direction reached 0.2 mm, and the accuracy reached 0.46 0.22 mm. to evaluate the feasibility of applying this method in the current clinical settings, the temporal bone was scanned with hrct (thickness = 0.6 mm, pitch = 0.3 mm) to acquire the accuracy of 0.86 mm (0.681.00 mm). with the development of the imaging technology the accuracy of the registration algorithm and the procedure directly affects that of the entire navigation system which is crucial to the image - guided surgery. given the required target accuracy and the minimal invasion, a registration method, which was the joint registration of the bone bed malleus short process, was employed in this study. this method used the full use of bone surface of the temporal implant bone bed to avoid the additional exposure and used the short process of malleus as an obvious anatomic marker. however, due to the high demand of the accuracy in the cochlear implantation surgery, the algorithm of the method requires more improvements to further reduce the artificial error. subsequent clinical experiments are needed to prove its stability. in the current drilling operation, the former had higher accuracy dispensing with real - time navigation, but it lacked the flexibility ascribing to the one - time designed path, which was hardly adjusted in the operation. however, the industrial robot with the high accuracy was too large to apply in operation settings. in recent years, robots have been specially designed for the cochlear surgery featuring the higher accuracy, smaller size, and more convenient for operation. in this study, a passive bi - planar mechanism, with 4-dof, was specially designed for holding an electric drill to move freely in the operational space. its advantages were as follows : (1) this mechanism was passively driven and was image - guided by doctors to finish locating, and it reduced operation errors and improved clinical safety ; (2) the special design of the parallel mechanism maintained the adequate intensity and rigidity but reduced the size and mass as much as possible, so that it could be easily fixed, moved, and applied in operation rooms ; (3) this bi - planar device had a fixed electric drill leading tunnel, which could restrict the drill within the planned path to get rid of artificial factors, such as hand vibrations ; (4) while the drill was moving forward, the operator could feel the change in force generated by different bone density. particularly, at the moment when the drill penetrated the wall of cochlear, the operator might have a sense of loss. the force feedback could mutually verify the trajectory on the navigation display to guarantee the safety of the trajectory. the operator 's learning curve shows that at the early stage of the experiment, more than 45 h was taken. however, when the operator became familiar with this system and took relevant trainings, only 1 h was consumed. at the beginning of the pci study, researchers optimistically hypothesized that the duration of the cochlear implant surgery could be reduced to within 50 min. this duration is comparable to the time needed for an experienced surgeon but is significantly shorter for an inexperienced surgeon. so far, in the first clinical report about pci, the average duration from the incision to the completion was 186 36 min, which was far longer than 8090 min that was needed in the traditional cochlear surgery. as there was a process from the exploration to the perfection for the clinical application of any new technology the recognition and segmentation of the important anatomic structures were manually completed, which consumed most of the surgical time and led to artificial errors. an automated robot should be developed. during the electronic drilling process, the robot 's adaptive path was manually controlled. an automated module is required to real - time monitor any path deviation, and to cease the operation automatically once it happens. in the study, there were only eight specimens, and all the specimens were from the adults. no children cadaveric temporal specimens were used in this study while most cochlear implantation was performed in children. the small sample size allowed preliminary research on feasibility of this system, rather than fully verifies the safety, which will be fulfilled later by a larger sample - sized study. above all, we established the navigation and drilling guide system in terms of the minimally invasive cochlear implant surgery. based on this system, there was a preliminary attempt in pci with target accuracy 1 mm, which met the accuracy requirements of this surgery. the later research will further improve the accuracy of the registration and the navigation system and optimize the path planning to meet the accuracy requirement of the cochlear implant surgery. this work was supported by grants from the china capital health development project (no. this work was supported by grants from the china capital health development project (no. | background : a single drilled tunnel from the lateral mastoid cortex to the cochlea via the facial recess is essential for minimally invasive cochlear implant surgery. this study aimed to explore the safety profile of this kind of new image - guided and bi - planar device - assisted surgery procedure in vitro.methods:image-guided minimally invasive cochlear implantations were performed on eight cadaveric temporal bone specimens. the main procedures were : (1) temporal bone specimens were prepared for surgery and fiducial markers were registered. (2) computed tomography (ct) scans were performed for future reference. (3) ct scan images were processed and drill path was planned to minimize cochlear damage. (4) bi - planar device - assisted drilling was performed on the specimens using the registration. (5) surgical safety was evaluated by calculating the deviation between the drill and the planned paths, and by measuring the closest distance between the drilled path and critical anatomic structures.results:eight cases were operated successfully to the basal turn of the cochlear with intact facial nerves (fns). the deviations from target points and entrance points were 0.86 mm (0.681.00 mm) and 0.44 mm (0.300.96 mm), respectively. the angular error between the planned and the drilled trajectory was 1.74 (1.262.41). the mean distance from the edge of the drilled path to the fn and to the external canal was 0.60 mm (0.350.83 mm) and 1.60 mm (1.302.05 mm), respectively. in five specimens, the chorda tympani nerves were well preserved. in all cases, no injury happened to auditory ossicles.conclusions:this exploratory study demonstrated the safety of the newly developed image - guided minimally invasive cochlear implantation assisted by the bi - planar device and established the operational procedures. further, more in vitro experiments are needed to improve the system operation and its safety. |
any state government desiring to limit its medicaid expenditures must control payments for inpatient hospital care, a service accounting for more than 50 percent of all medicaid acute care spending in 1984 (table 1). two basic sets of policy options are available : a state government can either impose direct controls on the utilization of hospital services by medicaid enrollees or alter the rates it is willing to pay for these services. although about one - half of the states have had utilization control policies (e.g., limits on days or required prior authorization in nonemergency situations) in place since the 1970 's, prior to 1982, only 13 medicaid programs had developed alternative reimbursement systems that did not rely on medicare reasonable - cost principles. despite growing evidence that retrospective cost - based reimbursement creates inefficiencies, states generally accepted the status quo because federal guidelines required reasonable - cost reimbursement unless an alternative method waiver was granted, winning approval for such a waiver was a difficult administrative process, and many states did not acknowledge the benefits of these waivers. resistance to alternative reimbursement systems was greatly reduced by the passage of the 1981 omnibus budget reconciliation act (obra). section 2173 of this act removed the reasonable - cost requirement and allowed states to pay only reasonable and adequate rates to meet the costs of efficiently and economically operated facilities. these new rules made waivers easier to obtain and therefore a more desirable policy tool for a broader range of medicaid programs. the obra - induced shifts were so dramatic that, by 1984, the number of states with a hospital reimbursement waiver had grown to 35. they set rates prior to service delivery and do not have end - of - year reconciliations. a small number (four) of alternative retrospective systems additional details on these waiver systems are presented in the following section of this article. although obra did not directly change the conditions under which utilization controls could be implemented, several states did adopt or modify policies of these types after its passage. either states were motivated to reexamine the full range of cost - control policies in light of other obra changes, or the legislation was simply passed at a time when they were already addressing concerns about medicaid costs. eight states added utilization controls and others expanded controls already in place in the 3 years following obra. to begin to explore how these policy choices may have affected medicaid inpatient hospital spending, we compare aggregate spending growth before and after obra. in table 2, total spending, total recipients, expenditures per recipient, and a hospital input price index for the years 1977 - 84 (the study period) are shown. in order to focus on variation in real spending trends across states with different hospital policies, all monetary measures were deflated by the hcfa hospital market basket index, a nationwide annual index of the cost of hospital inputs. aged inpatient recipients and their associated payments were excluded from this table and the subsequent analyses because medicare policies, rather than medicaid policies, exert the major influence over their hospital costs. prior to obra, expenditures for nonaged inpatients grew at an average annual rate of 11.1 percent. this growth rate dropped to 5.4 percent in the years immediately following the legislation (1981 - 84). second, recipient counts began to decline at an accelerating rate. finally, real spending per recipient fell off sharply in 1982 and 1983. the last two factors could certainly be associated with the policy initiatives that resulted from obra 's passage. however, the upswing in real spending per recipient from 1983 to 1984 raises questions about the long - term effectiveness of these programmatic changes. the purpose of this article is to evaluate the importance of various reimbursement and utilization control policies in a state government 's efforts to contain medicaid inpatient hospital spending. this analysis may be used to explain why the growth in inpatient spending slowed so dramatically after the passage of obra. however, its primary objective is to identify those policies that have been able to significantly influence medicaid hospital payments. the questions addressed in this study include the following : are alternative reimbursement methods more or less effective than utilization controls as a method of containing inpatient spending ? do certain features of alternative systems seem to be more or less relevant to the system 's ability to contain medicaid costs ? is required prior authorization a weaker or stronger utilization control than fixed limits on inpatient days ? previous studies of prospective reimbursement (pr) can be broken into two general categories : those that measure the impact of pr on overall hospital spending and utilization patterns and those that specifically address the impact of pr on an individual payer, such as medicaid. eby and cohodes (1985) provide an excellent review of studies falling into the former category. studies in the latter group include morrisey, sloan, and mitchell (1983), a study of medicare effects, and zuckerman (1986), a study of commercial payer effects. the primary previous studies that focused on medicaid were conducted by cromwell and hurdle (1984, 1986). they explored the impact of pr on both total and hospital medicaid spending by analyzing state - level spending patterns and by grouping state programs according to whether the system was implemented before or after obra. their 1986 study suggests that pr effects differ for aid to families with dependent children (afdc) and supplemental security income eligibles ; however, their regression analyses uncover little impact on growth in hospital spending per recipient for either eligibility group. in this study, we extend the cromwell and hurdle studies by categorizing medicaid pr systems according to a set of potentially more meaningful dimensions than their pr implementation date relative to obra. included in these dimensions are the program 's age, non - medicaid payer coverage, and a number of specific rate - setting design features. the remainder of this article is organized as follows. in the next section, the specific alternative reimbursement and utilization control policies that states had in place or adopted from 1977 to 1984 are described. next, the results of both tabular and multivariate analyses of policy effects are presented. the implications of these results for medicaid as well as other payers are reviewed in the final section. within the categories of alternative reimbursement methods and utilization control policies, state governments have a wide range of specific policy design choices. one state may choose to institute controls by limiting the annual number of covered inpatient days per recipient, and another may require that prior authorization be granted before payment for certain types of procedures can be received. still another may implement both policies simultaneously. policy variations may also arise because of differences in the actual day limits imposed or the services designated as requiring prior authorization. when one adds to these utilization options those associated with reimbursement methods, it becomes clear that no two state governments have identical approaches to the management of medicaid hospital spending. in table 3, we identify and define a range of policy choices that states have at their disposal. policies are grouped according to whether they pertain to alternative reimbursement systems or utilization controls. we describe these choices in a generic fashion and do not explore in great detail the potential variations within each element. for example, allowing higher payment rates to hospitals with a disproportionate share of uncompensated care will vary in its effect on program payments, depending on the size of the allowance and the state 's definition of disproportionate share. we focus on these because they appear to be the most widely adopted options and also because conceptually they seem most likely to alter inpatient spending levels. in tables 4 and 5, implementation dates for the specific policy features are shown by state. for reimbursement methods (table 4) we indicate when, if ever, the state adopted prospective payment. six specific design options that may accompany a prospective (or, in limited instances, a retrospective) system are also detailed. in the 2 years immediately following obra, 1982 and 1983, the number of medicaid pr programs grew from 15 to 31. second, no new waivers were implemented in the 3 years prior to obra, 1978, 1979, and 1980. third, none of the post - obra waivers were for systems designed to include any payers besides medicaid. fourth, four states (california, 1980 - 82 ; idaho, 1979 - 84 ; louisiana, 1982 - 84 ; and wisconsin, 1981 - 84) had payment systems that incorporated several features normally associated with prospective reimbursement but maintained retrospectivity in their rate process. finally, although providing an uncompensated care allowance was a feature of the majority of the prospective systems, few systems were truly alike in terms of other design elements. turning to utilization control policies (table 5), we find that only six states (delaware, massachusetts, montana, nebraska, new york, and north dakota) went through the entire study period without some policy of this type. although obra did not directly alter a state 's ability to implement utilization control policies, it seems to be associated with an era of substantial expansion in this area. possibly obra 's cut in federal matching rates or the recession of 1982 gave states the impetus to reexamine all policies that could reduce medicaid outlays. under any circumstances, 26 states either instituted or expanded inpatient hospital utilization control policies in the years following obra. clearly, the most widespread control policy was a requirement of prior authorization for certain elective procedures or specific services. despite state - to - state variations in the precise features of this policy, this requirement however, there is little agreement on the desirability of per - year versus per - stay limits. tests of the relative effectiveness of these policies are contained in the empirical section of this article. the primary data source used in this study is information reported to the federal government by the state governments through the statistical report on medical care : eligibles, recipients, payments, and services of the health care financing administration (hcfa), known as the hcfa 2082 medicaid expenditure report. this report contains information on medicaid expenditures, utilization by eligibility status (e.g., afdc, blind and disabled, aged), and type of service (e.g., inpatient hospital, physician, nursing home), as well as an unduplicated count of recipients. because aged medicaid patients are also medicare eligible, their inpatient hospital use and payments are influenced by national as opposed to state policy decisions. all data on medicaid inpatient hospital policies were derived from the report by laudicina (1985). her primary sources were descriptions of state medicaid plans, which must be filed as part of any major policy overhauls. summary characterizations of the policies were developed from laudicina 's compilations and are shown in table 3. the 2082 form is probably the most comprehensive source of medicaid program data that is consistent in structure across states, but it is not without its drawbacks. in addition, at various points during the study period, states did not correctly exclude days paid for by medicare when reporting on utilization of medicaid - medicare joint eligibles. as cromwell and hurdle (1984) point out, working around these problems with 2082 report data would require analysis of a curtailed timeframe and a reduced number of states, as well as some degree of data interpolation. therefore, we chose to omit analyses based on utilization measures and, instead, rely on the recipient and expenditure data, about which there is a greater degree of confidence. the expenditure data were verified against audited data reported in the hcfa 64 forms, which are used to determine matching payments and seem to contain the most credible information available. some inconsistencies arise in the recipient data because of states ' difficulties in consistently and accurately assigning recipients to particular eligibility classes. hcfa also corrected, at least cross - sectionally, errors or omissions in the expenditure and recipient data by drawing on comparable data from other reports or by requesting additional information directly from the states. despite our belief that the expenditure and recipient data are of sufficiently high quality to serve as the basis of this study, some implausibly large annual fluctuations can be detected. prior to conducting any empirical analyses, we investigated all observations in which a state 's real total hospital spending per recipient changed by more than 33 percent, positively or negatively, from one year to the next. real spending per recipient was used as a screen because it is an indicator of large changes in spending and/or recipients that are inconsistent with one another. we examined the remaining data for the state involved to determine if the problems arose because of seemingly inaccurately reported payments or recipient counts. after this determination was made, we imputed a new value for the appropriate variable by using a trend regression based on the remaining observations. because only 14 out of 800 values (2 variables 50 programs 8 years) were altered, it is unlikely that the findings were substantively affected. three analysis variables were defined in order to measure the relative effectiveness of medicaid hospital policies on program outcomes : annual percent change in real inpatient hospital expenditures, annual percent change in inpatient hospital recipients, and annual percent change in real inpatient hospital spending per recipient. the 8 years of data from each state allow us to compute seven annual percent change measures, so our maximum sample size for these analyses is 350 (50 states 7 annual percent changes). the variable most appropriate for evaluating rate - setting systems, in light of the fact that utilization - based measures of payment are not available, is spending per recipient. however, rate - setting effects also could emerge as slower recipient growth if rates are reduced to the point at which access is curtailed. recipient analysis is also necessary because some utilization controls are designed to keep medicaid enrollees out of hospitals when less costly services can be substituted. using these variables enables us to determine if policies are slowing growth in total medicaid hospital outlays and if this is occurring as a result of curtailing increases in recipients or curtailing increases in real payments per recipient. both tabular and multivariate analyses are employed in this article. in the tabular analyses, presented as background information, comparisons are made among states with and without the particular policies described in table 3. for the specific aspects of rate - setting designs, all comparisons are made among states with prospective reimbursement waivers in place. using average annual percent changes is a self - controlling technique to deal with cross - sectional differences in the underlying characteristics of states or their medicaid programs. however, tabular analyses do not allow us to clearly isolate policy effects because they are confounded by potential intervening variables. to measure the true impact of a particular hospital spending policy, it is necessary to control for three specific types of confounding influences : the presence of other hospital policies, changes in the size and composition of the medicaid population, and general trends in hospital use patterns and medicaid spending. our dependent (or analysis) variables are measured as changes, so it is not necessary to include variables whose values tend to be relatively constant over time but vary from state to state, e.g., proportion of care provided in public hospitals or percent of population residing in urban areas. the reimbursement or utilization control policies are measured by categorical variables equal to one if the state had the policy in place in a given year and zero otherwise. a number of alternative specifications were employed to evaluate different aspects of hospital spending policies. in particular we then divided them into two groups : states in which medicaid is the only payer regulated by the state, and states that regulate other payers in addition to medicaid. these two types of medicaid pr programs were then analyzed with respect to the number of years they have been in effect. we were unable to isolate the independent effects of the remaining design elements shown in table 4 because of the high degree of collinearity among the variables measuring these factors ; i.e., many states with medicaid pr used similar combinations of design elements. other independent variables we control for in the regressions relate to the size and composition of the medicaid population and time trends. to hold constant changes in medicaid eligibility rules that would influence the size of the potential pool of inpatient hospital recipients, we include the percent change in the annual unduplicated count of total medicaid recipients available from the 2082 data. as the rate of change in the potential recipient group grows, the rate of change in inpatient recipients should also grow. as an alternative, we could have used the percent change in average monthly enrollee counts, which can be computed from social security administration sources and the hcfa 120 form. however, these data do not allow one to account for turnover rates, so an unduplicated enrollee count that is analogous to the 2082 unduplicated recipient count can not be derived. turnover of medicaid enrollees appears to be extensive (wilensky, walden, and kasper, 1980). therefore, we have opted to use changes in the unduplicated count of total recipients as a measure of changes in overall program size. however, as long as the proportion of medicaid enrollees utilizing some service is independent of eligibility changes among the nonaged, the unduplicated number of recipients is a reasonable proxy for program size. even if utilization patterns alter the ratio of total recipients to eligibles across all services, we still feel it is defensible to use changes in unduplicated total recipients to measure changes in the pool of eligibles for any single service. although this variable can be used to account for eligibility changes affecting the size of medicaid programs, it can not be used to account for eligibility changes affecting the composition of potential recipient groups. to control for this, we use the proportion of unduplicated recipients who are afdc adults and the proportion who are afdc children. because both child and adult afdc recipients are less likely to be hospitalized than disabled beneficiaries, we expect these proportions to be inversely related to the rate of change in inpatient recipients. finally, general trends in hospital spending are captured by the inclusion of time dummies in each of the subsequent regressions. the two general types of policies considered, prospective reimbursement and utilization controls, would be expected to affect the growth in medicaid hospital spending through different mechanisms. in terms of the analysis variables discussed here, effective pr programs are likely to save medicaid money by reducing the growth in spending per recipient. the reason for this is that pr is focused on setting payment rates (per diem, per case, or per service) but is not necessarily used to reduce admissions, i.e., inpatient recipients. medicaid 's prospective rates might be set so low that its beneficiaries would be viewed as financial liabilities for hospitals, and hospitals thus might attempt to curtail medicaid admissions. this outcome could result in pr having a negative impact on inpatient recipient growth in addition to spending per recipient. a pr recipient effect seems more likely to develop in states where medicaid is the sole regulated payer than in states in which medicaid is part of a broader regulatory process. utilization controls, on the other hand, are geared toward screening out unnecessary hospital care as opposed to controlling rates. limiting inpatient days per stay could reduce spending per recipient by shortening medicaid lengths of stay. however, if the limits are severe, hospitals could selectively shun medicaid inpatients and cause a reduction in recipients. annual day limits appear less likely than per - stay limits to shorten individual lengths of stay and, in fact, unless set at very low levels, would not reduce the unduplicated recipient counts either. prior authorization policies are more clearly directed at reducing the number of recipients. either by requiring approval for nonemergency admissions or by not allowing hospitals to provide certain services without first contacting the medicaid program, if prior authorization is effective, we would expect to see its impact in the number of recipients rather than spending per recipient. the relationship between medicaid hospital payment waivers and hospital spending patterns is described in table 6. as shown in the first row, real total spending grew significantly more slowly when a prospective reimbursement system was in place. the growth rate reductions appear to be associated with both lower growth in the number of inpatient recipients (actually an average annual reduction) and lower growth in real spending per recipient. in the remainder of table 6, data are shown only for states with medicaid prospective reimbursement waivers and their experiences with specific payment system features. in general, none of the features seem to result in significantly different patterns of hospital spending across the waiver states. the only exception is alternative payment systems containing a minimum occupancy requirement (mor) in its rate calculations. states with these mor systems experienced an average annual decrease of 1.4 percent in real expenditures per recipient, compared with 1.8-percent increase for other medicaid pr systems. however, because one - third of the observations on which this result is based come from new york data, it is conceivable that some unique aspect of the new york system, rather than the occupancy requirement, may be responsible for this finding. in table 7, spending patterns among states both with and without utilization control policies are described. the results show that neither limiting inpatient days nor requiring prior authorization for nonemergency admissions has any statistically significant effect on real inpatient spending per recipient. although these policies could act to contain the amount of care delivered to medicaid inpatients, in reality they do not appear to do so. in fact, we find that only prior authorization for elective procedures or other specific services bears any relation to inpatient hospital spending. this policy is associated with an average annual reduction in the number of recipients of 1.2 percent, compared with a 1.9-percent increase for programs that do not have this policy in effect. as might be expected, these differences translate into slower real growth in overall medicaid hospital payments. although these data suggest that the policy choices made by states have some effect on inpatient spending, we must turn to multivariate analyses in order to be certain that the policy variables are not capturing other changes in the medicaid program or the state 's environment. the means and standard deviations of the variables used in the regressions are included in table 8. regression set 1 contains estimates in which all medicaid pr programs are treated as a single group. using these results, we find that pr reduces the rate of growth in real spending per recipient by 3.1 percent per year, on average, relative to the rate in states without such programs. in general, reimbursement policies do not exert a significant influence over changes in the number of recipients. as might be expected from the tabular analysis, the only utilization control policy that affects medicaid hospital spending this requirement is associated with an average 2.7-percent annual reduction in the rate of change in recipients ; no impact on real spending per recipient is observed. these results show that both alternative methods of reimbursement and some utilization controls can reduce the growth in real hospital expenditures ; one method does so by lowering spending per recipient, and one does so by containing the growth in recipients. regressions in set 2 show that a medicaid payment system 's success appears to be related to the coverage of other payers. real spending growth is not significantly lower in medicaid - only pr systems. on the other hand, states that control medicaid rates as part of a broad - based regulatory process seem to have kept changes in real spending per recipient 4.8 percent below those in states that apply medicare reasonable - cost principles. we considered variation among these broad - based systems that, as a group, appear to contain medicaid spending without adverse consequences regarding patient access. although new york and massachusetts seemed to exert greater downward pressure on real spending growth than other states with regulatory systems that go beyond medicaid, an f - test did not allow us to reject the null hypothesis that these two states have the same impact on hospital spending as the other five states in this group (maryland, new jersey, rhode island, washington, and wisconsin). one reason that medicaid - only systems are found to be inferior may be that these approaches are, on average, newer than the more comprehensive systems. prior research has shown that, in general, pr becomes effective as a means of containing hospital costs only after it has been in place for more than 2 years (sloan, 1983). to explore the relationship between payer coverage and program age, we grouped the medicaid - only systems according to the number of years each system had been operating. the programs are separated into those that are 1 year old, 2 years old, and more than 2 years old. a similar division for broad - based programs is not appropriate because we do not observe any of the systems in year 1 and only three in year 2. results of these analyses, shown in set 3 of table 9, indicate that the effectiveness of medicaid - only systems is related to program age. growth in total hospital spending is not reduced in the first year of a medicaid - only program. this may result from the fact that the first year, as our variables are defined, is a partial year for many systems. however, in year 2 (the first full year we observe), real spending growth is 7.6 percent below growth in states without waivers. beyond year 2, these systems once again return to growth rates similar to those in states without prospective controls on medicaid rates. the lower year 2 growth rates result in lower levels of spending in year 2 than would have existed in the state if no medicaid - only pr system had ever been adopted. because growth rates in these states are no different from those in non - pr states after year 2, the level of medicaid hospital spending will remain below where it otherwise would have been, and therefore some permanent savings will be attained. what happens in the second year of medicaid - only rate - setting systems to reduce the rate of growth in hospital spending ? to answer this question, we refer to the corresponding regressions on number of recipients and spending per recipient. both the change in number of recipients and the change in real spending per recipient are lowered by about 4 percent in medicaid - only pr as compared with states with retrospective payment methods. the differential in spending per recipient, however, is not statistically significant at conventional levels of confidence. these findings suggest that when medicaid rates are the only rates regulated, hospitals may find medicaid patients less desirable and may implement procedures geared toward reducing the number of beneficiaries admitted. put differently, when medicaid rates fall relative to rates of other payers, medicaid enrollees seem to experience some hospital access problems. it may well be that nonhospital services are substituted for hospital care in these instances ; however, exploration of this issue is beyond the scope of this study. our findings with respect to later years in medicaid - only pr systems indicate that states respond to developing hospital access barriers by providing more generous rates of growth and making medicaid inpatients more desirable again. the remaining variables, which relate to general eligibility, recipient composition, and general trends, yield similar results in all of the alternative regressions. increased growth in total recipients is associated with higher growth in the number of inpatient recipients and smaller increases in real spending per recipient. this latter result suggests that, as eligibility expands, the marginal people added to the program are less costly than enrollees who were already in the program when eligibility was tight, i.e., the people added tend to be healthier than the people already covered. the share of children has a weak (not statistically significant) negative effect on changes in the number of recipients and a positive effect on real spending per recipient. these findings suggest that, as more children are covered, fewer people are hospitalized, but those who are hospitalized may be sicker. as the proportion of unduplicated recipients who are adults grows, recipient growth falls and spending per recipient slows. the time variables show that, relative to 1981 (the year of obra 's implementation), significantly different patterns of expenditure growth were exhibited only in 1982. inpatient spending grew 7.0 percent more slowly from 1981 to 1982 than it had from 1980 to 1981 (set 1, table 9). this pattern is primarily caused by lower growth in real spending per recipient over this period. no significant trend in the annual rate of change in recipients can be seen when the policy and eligibility controls are introduced in the regression. there has been a general sense that increases in hospital spending fell after obra because of the proliferation of alternative methods waivers, utilization controls, and eligibility tightening. our results indicate, however, that even after controlling for these factors, a one - time downward shock to hospital payments is still associated with obra 's passage. states may have viewed obra as a window of opportunity to contain rates below the level indicated by general pr policies. under any circumstances, it seems clear that new policies alone do not explain the immediate post - obra changes in hospital spending. the 1981 obra legislation initiated many changes in medicaid policies related to inpatient hospital services. some significant changes in eligibility standards also led to a reduction in the total pool of medicaid recipients. the majority of states that had been reimbursing according to medicare reasonable - cost principles applied for and received a waiver that allowed them to develop a prospective payment system for medicaid. in addition, many states either initiated or expanded a number of utilization control policies. real expenditures per recipient fell by 1.6 percent annually from 1981 to 1983, compared with 0.7-percent growth in the 4 years immediately preceding the legislative initiative. at the same time, the number of inpatient recipients fell by 1.1 percent per annum. based on the analyses in this article, it appears that the obra - induced policy changes definitely contributed to the reduced growth in hospital spending. although the focus of obra was on making alternative reimbursement waivers easier to obtain, it appears that expanded prior authorization requirements may have a more long - lasting effect on containing spending growth. in the post - obra era, 16 states added such policies to join the 12 states that already had them. because prior authorization for specific services had virtually the same impact in the pre- and post - obra periods, it seems clear that the expansion of this type of policy contributed to the reduction in hospital spending growth. although this policy works by reducing the growth in inpatient recipients, it should not create general medicaid access problems if recipients are truly screened on the basis of medical necessity. we find that these programs have limited (not statistically significant) capability to contain real spending per recipient in the second year of their existence (first full year), but the growth rate effect does not seem to be sustained in the subsequent years. because these policies also reduce the growth in inpatient recipients, we conclude that the impact of this approach as a means of containing spending is limited by the emergence of potential hospital access problems for medicaid enrollees. however, the one - time reduction in spending growth does result in some permanent savings because the level of medicaid hospital spending in states that adopted medicaid - only pr is driven below the level at which it would have otherwise been. states that include medicaid in a broader regulatory system are able to control the growth in spending per recipient over a longer period without any apparent barriers to access. our estimates of medicaid - only effects beyond year 2, therefore, are based on a limited time series for 13 states. these systems could mature into more effective regulatory structures, but always at the risk of curtailing service availability for eligibles. general policy changes that led to a 0.7-percent annual decline in total recipients of any service from 1981 to 1984 (holahan and cohen, 1986) also affected hospital spending. not surprisingly, as the growth in total recipients fell (a proxy for the decline in the pool of potential eligibles), changes in the number of inpatient recipients were reduced as well. hence, part of the reductions in medicaid hospital spending were caused by general programmatic changes that reduced the number of eligibles. even after taking these changes and hospital policy initiatives into account, there still appears to be some unexplained tightening in hospital expenditures emanating from slower growth in real payments per recipient. the time effect is consistent with the notion that general changes in the hospital sector (e.g., declining lengths of stay) are spilling over into the medicaid program. however, the time effect is observed only for the 1981 - 82 period, so it may be capturing some measure of immediate post - obra policy intensity. for instance, more prior authorization requests may have been denied, or payment rates may have been set well below longrun pr program targets in order to get at least some shortrun savings from obra. what are the implications of these results for the design of future medicaid policies addressing the growth in inpatient hospital spending ? in the area of utilization controls, it seems clear that states should consider some form of required prior authorization for specific services. although there can be considerable variation in how these policies are implemented, our results show that the typical approach appears effective. case study analyses of selected states with prior authorization would provide greater insight into the specific features that are associated with the more successful programs. in this study, little evidence has been uncovered that would lead us to endorse inpatient day limits or prior authorization for nonemergency admissions as an effective policy tool. our results also indicate that state governments that have implemented or are considering implementing a medicaid - only prospective payment system might want to consider expanding the system to include some other payers. medicaid - only rate - setting approaches seem to have some shortrun ability to contain spending but have not yet exhibited the sustained success evident in states with broader based regulation. however, it would be an oversimplification to suggest that any form of rate setting that includes more than one payer will necessarily be successful. further review of laudicina (1985) shows that, in addition to regulating payers other than medicaid, all seven of these rate - setting programs controlled hospitals ' total revenues by adjusting the prospectively set rates in response to the actual volume of care provided. in addition, new york and massachusetts (whose systems seemed to be somewhat more successful) applied minimum occupancy requirements in calculating the average costs of treating patients. unfortunately, given the available data and the difficulties in measuring specific policies, it is not feasible to estimate the effects of these policy choices independently. although volume adjustment, as a generic policy option, did not seem to alter hospital spending patterns, when coupled with rate setting that covers other payers, it may be a central factor in regulatory success. in this type of system, volume adjustments allow authorities to control overall hospital revenues and, thus, to contain medicaid rates in absolute terms without having them fall relative to those of other payers. in medicaid - only approaches, volume adjustments are generally made in response to the number of medicaid days and, if applied stringently, could make medicaid inpatients undesirable. as an example, in 1982, illinois began adjusting its rates based on volume to keep medicaid hospital payments at a fixed level based on state appropriations. this policy lowered real spending per recipient from 1982 to 1983 by about 13 percent, but it also resulted in a nearly 6-percent reduction in the number of medicaid inpatient recipients. nationally, inpatient recipients rose by almost 3 percent over the same period. therefore, although broad - based systems may successfully use volume adjustments without creating access problems, this does not appear to be the case when medicaid is the sole regulated payer. | numerous medicaid hospital spending policies were developed following the passage of the 1981 omnibus budget reconciliation act. the impact of reimbursement and utilization control policies on medicaid hospital spending was measured using medicaid program data for 1977 - 84. medicaid prospective reimbursement was found to contain real hospital spending by controlling spending per recipient. however, sustained reductions in the growth in real medicaid spending are achieved only when medicaid is included in a broader regulatory framework, not when it is the sole regulated payer. prior authorization for specific services reduces growth in hospital spending by reducing the growth in inpatient recipients. |
following stroke, difficulties in controlling balance may be caused by several factors, such as muscle weakness, impaired proprioception, asymmetric weight bearing, spasticity, and impaired motor control1. balance disorders due to deficits in multiple mechanisms are frequently encountered in patients with stroke. balance is an important predictor of outcome in stroke rehabilitation2. due to the weakness of dorsiflexors, the shortening of calf muscles in individuals with stroke causes stiffness, worsened hypomobility, joint mobility resistance, and decreased passive joint mobility - resulting in the limitation of ankle joint motion4. abnormal muscle tensions and movement limitations due to muscle weakness at the ankle joint cause limitation of functional activities such as sit - to - stand, locomotion, and stair - climbing5. functional weakness of the lower extremity due to stroke is caused not only by muscular weakness, but also by decreases in muscular endurance and stability of the joint, and loss of proprioceptive sense6. clinically, proprioceptive sense is an important factor in the evaluation and treatment of patients with neurologic problems ; its loss leads to declines in postural control, protective reflexes, joint movement, balance ability, and gait6. therefore, improving balance and gait function is one of the most important goals in patients undergoing stroke rehabilitation. many different types of physical therapy are used to improve balance in stroke rehabilitation. the mulligan technique for posterior talar glide (ptg) with dorsiflexion of the ankle has been applied to improve range of motion, alleviate pain, and promote earlier return to function following lateral ankle sprain7. the mobilization with movement (mwm) technique is frequently used to improve talocrural dorsiflexion deficits that are often seen following lateral ankle sprain8. a key component of the mwm treatment technique postulated to improve talocrural dorsiflexion is a ptg that is applied manually to the joint7. previous studies of the mulligan technique were conducted in patients with musculoskeletal disorders8. however, the application of the mulligan technique to ptg with dorsiflexion of the ankle for treating stroke patients had not yet been assessed. thus, the purpose of the present study was to demonstrate that ptg with dorsiflexion of the ankle improves ankle range of motion, along with muscle strength and balance in stroke patients. subjects met the following inclusion criteria : history of stroke onset > 6 months prior to the study, in order to minimise the effects of natural recovery ; mini - mental state examination score of > 24 out of 30 ; motor recovery of the paretic lower limb to within brnnstrom stages 24 ; and ability to comprehend and follow simple instructions. individuals were excluded if they had a neurological condition, an orthopedic disease, or a visual impairment. the 34 subjects were randomly assigned to either the ptg with dorsiflexion group (ptg ; n=17), or the weight - bearing with placebo ptg group (control ; n=17). there were 17 subjects in the experimental group (8 males and 9 females), with a mean age of 58.00, a mean height of 162.06 7.37 cm, and a mean weight of 61.00 8.55 kg ; there were 17 subjects in the control group (13 males and 4 females), with a mean age of 51.59 13.31, a mean height of 166.53 6.85 cm, and a mean weight of 64.71 7.54 kg. there were no significant differences between the groups (table 1table 1.characteristics of the participants (n=34)ptg (n=17)control group (n=17)gender (male / female)8/913/4affected side (rt / lt)7/108/9age (years)58.0 3.151.6 3.2height (cm)162.1 7.4166.5 6.9weight (kg)58.0 12.951.6 13.3values are expressed as mean standard deviation (sd), ptg : posterior talar glide with dorsiflexion group). the present study was approved by sahmyook university institutional review board and each participant was able to follow instructions and gave informed consent by signing an approved consent form ; thus, the rights of human subjects were protected. values are expressed as mean standard deviation (sd), ptg : posterior talar glide with dorsiflexion group all subjects were assessed using passive range of motion of the ankle (rom), a manual muscle test (mmt), a functional reach test (frt), a time up and go test (tug), and a functional gait assessment (fga). ptg treatment with dorsiflexion of the ankle joint consisted of a program to improve range of motion, and to promote functional recovery and balance ability through prioceptive control of the ankle. patients in the ptg group underwent treatment with dorsiflexion of the ankle joint for 10 glides of 5 sets / day, 5 days / week, for 4 weeks. each glide consisted of a 10-s ptg with dorsiflexion and 5 s of rest between glides. the therapist applied a sustained posteroanterior glide to the tibia using a belt by leaning backwards while the talus and forefoot were fixed with the thumb and right second finger. the other hand was positioned anteriorly over the proximal tibia to direct the knee over the line of the second and third toes. then the participant was instructed to perform a slow dorsiflexion movement until either the first onset of pain or the end range of motion, without the heel lifted off the couch. to avoid pain at the contact point of the belt with the achilles tendon, the participant was instructed to perform a slow dorsiflexion movement until the first onset of pain or end range of motion without the heel lifted off the couch. the control group was trained for 10 lunges of 5 sets / day, 5 days / week, for 4 weeks. both groups also underwent standard rehabilitation physical therapy for 30 min / day, 5 days / week, for 4 weeks. the frt is based on analysing the limits of anterior - posterior stability in an upright position, in the absence of external perturbations. it assesses the maximum forward displacement (in cm) that a subjects can reach without losing balance9. it was designed as a quick measure of the basic balance and mobility skill of stroke patients. the time taken for subjects to rise from an armchair, walk 3 m, turn, and return to the chair is measure in seconds. it was initially invented for the stroke patients who had great risk of falling down. in this study, the original form of fga spss 19.0 (spss inc., chicago, il, usa) was used for statistical analysis. the kolmogorov - smirnov test was used to test the distribution of general characteristics and outcome measures of the subjects. a paired t - test was used to compare pre- and posttest measurements of ankle rom and balance within groups, and the independent t - test was used to compare the difference in ankle rom and balance before and after training between groups. a wilcoxn s signed - ranks test was used to compare pre- and posttest measurements of ankle mmt within groups, and mann - whitney u test was used to compare the difference in ankle mmt before and after training between groups. in the ptg group, passive dorsiflexion rom of the ankle, along with frt, tug and fga, was significantly improved. ankle dorsiflexor mmt in the ptg group is significantly improved (p<0.05). however, with regards either to passive plantarflexion rom of the ankle, or to plantar and dorsiflexor mmt, there were no significant difference between the groups (table 2table 2.comparison of ankle range of motion, muscle strength, and balance as measured within groups and between groups (n=34)parametersvalueschange valuesptg (n=17)control group (n=17)ptg (n=17)control group (n=17)prepostprepostpost - prepost - prebalancefrt rt. (cm)15.7 (7.6) 17.7 (7.7) 19.2 (6.1)19.8 (6.0) 2.0 (1.5) 0.6 (0.8)frt lt. (cm)15.1 (6.4)16.8 (6.4) 18.1 (6.9)18.7 (7.0) 1.7 (0.8) 0.6 (0.9)tug (sec)13 (3.2)11.3 (2.9) 12.5 (2.8)11.4 (2.7) 1.7 (0.7) 1.1 (0.8)fga (score)19.8 (5.4)21.2 (4.4)20.9 (2.5)21.5 (2.3)1.4 (1.3)0.6 (0.6)muscle strengthdorsiflexor mmt (scale)3.12 (1.83)3.53 (1.41)3.35 (1.93)3.47 (1.80) 0.23 (0.43) 0.17 (0.48)plantarflexor mmt (scale)3.29 (1.61)3.59 (1.50)3.53 (1.83)3.59 (1.83)0.29 (0.58)0.11 (0.48)ankle romdorsiflexion (angle)14.1 (9.1)17.1 (5.0)18.8 (3.8)19.4 (2.4)2.9 (4.4)0.3 (1.2)plantarflexion (angle)41.2 (9.1)43.5 (4.2)43.2 (6.1)43.2 (6.1)2.4 (5.3)0.0 (0.0) values are mean (sd), ptg : posterior talar glide with dorsiflexion group, frt : functional reach test, tug : time up and go, fga : functional gait assessment, mmt : manual muscle testing, ankle rom : ankle range of motion values are mean (sd), ptg : posterior talar glide with dorsiflexion group, frt : functional reach test, tug : time up and go, fga : functional gait assessment, mmt : manual muscle testing, ankle rom : ankle range of motion. stroke and brain injury are diagnoses in the rehabilitation setting that often lead to decreased mobility. once a person is less mobile, more time is spent in resting positions that can predispose them to muscle shortening, which can impact recovery of function12 and can result in a significant decrease in ankle dorsiflexion rom after a stroke13. in this study, the ptg group underwent ptg with dorsiflexion for 10 glides of 5 sets / day, 5 days / week, for 4 weeks. the ptg group showed a significant improvement in passive dorsiflexion rom of the ankle and dorsiflexor mmt (p<0.05) ; the control group showed no significant improvement in passive dorsiflexion rom of the ankle and dorsiflexor mmt. however, with regard to dorsiflexor mmt of the ankle, there were no significant differences between the groups. an and jo14 twenty - six participants with chronic hemiplegia were divided into 2 groups : the mwm group (n=13) and the control group (n=13). both groups attended conventional physiotherapy sessions 3 times a week for 5 weeks. additionally, the mwm group underwent talocrural mobilization with movement (mwm) 3 times a week for 5 weeks. passive dorsiflexion rom of the ankle significantly increased in the mwm group (p<0.05). posterior talar glide with dorsiflexion facilitated the revovery of accessory movement in the talocrural joint. triceps surae muscle stretch was obtained through sustained and repeated maximal ankle dorsiflexion rom during mobilization15. muscle weakness, including weakness of the affected ankle dorsiflexors and plantarflexors, is common following stroke. it could be attributed to physiological changes in motor systems, including failures in motor unit recruitment and a reduced firing frequency of agonist motor neurones or units16. spasticity in the ankle plantarflexors might impede their ability to generate appropriate muscle force as agonists during ankle plantarflexion. balance is the ability to maintain the center of gravity from a base of support with continuity18, and is a component of prognosis for a stroke patient s functional recovery19. the most important goal of rehabilitation for a stroke patient is independence of activity, thus, priority should be given to restoring equilibrium and posture stability for optimum functioning20. therefore, one of the most important aims of therapy is the improvement of equilibrium regulation and function21. in this study, the ptg group showed a significant improvement in frt, tug, and fga ; the control group demonstrated significant improvement in frt, tug, and fga. the ptg with dorsiflexion group showed significantly greater improvement in frt, tug, and fga (p<0.05) as compared to the control group, which suggests that ptg with dorsiflexion improves the balance ability of patients recovering from stroke. mckeon.22 performed balance training 60 min / day for 4 weeks in 31 subjects who had been complaining of ankle instability. their ability to move in an anteroposterior direction, their center of pressure (cop) while moving in a left or right direction with eyes open, and their total postural sway speed at the cop were considerably decreased (p<0.05). hoch and mckeon23 performed joint manipulation in 20 patients with ankle instability, and the experimental group showed considerable improvement in anteroposterior postural control (p<0.05). the improvement in static balance in these reports corresponds to the findings of this study. to reduce falls and improve equilibrium adequate range of motion is needed24 ; the loss of somatosensory information the mechanical receptors near the ankle joint decreases postural control and results in the subsequent loss of static balance25. joint manipulation stimulates the afferent pathways of mechanical receptors near the ankle joint, and this stimulation improves afferent information of the talocrural articulation and the surrounding tissue25. hoch and mckeon23 stated that it is possible to control posture by joint mobilization through stimulation of the mechanical receptors surrounding the ankle joint. improvement in postural control is due to neuromuscular function adjustment caused by mechanical receptor activity26. for these reasons, this study demonstrates that an increase in joint range of motion and continuous ptg on the distal tibia with an active weight - bearing posture can increase afferent stimulation of the ankle joint and improve static balance with enhancement of neuromuscular function. financial disclosure statements have been obtained, and no conflicts of interest have been reported by the authors or by any individuals in control of the content of this article. financial disclosure statements have been obtained, and no conflicts of interest have been reported by the authors or by any individuals in control of the content of this article. | [purpose ] the purpose of this study was to examine the effectiveness of posterior talar glide (ptg) with dorsiflexion of the ankle on stroke patients ankle mobility, muscle strength, and balance ability. [subjects and methods ] thirty - four subjects were randomly assigned to either a ptg with dorsiflexion group (ptg ; n=17), or a weight - bearing with placebo ptg group (control ; n=17). subjects in the ptg group performed ptg with dorsiflexion, designed to improve ankle mobility, muscle strength and balance ability with proprioceptive control of the ankle, for 10 glides of 5 sets / day, 5 days / week, for 4 weeks. [results ] the experimental group showed significant improvement on the ankle dorsiflexion range of motion assessment, ankle dorsiflexor manual muscle test, functional reach test, time up and go test, and functional gait assessment compared to the control group. however, regarding ankle plantarflexion range of motion assessment and the ankle plantarflexor manual muscle test, no significant differences were found between the two groups. [conclusion ] the results of this study show that ptg with dorsiflexion can improve ankle mobility, muscle strength and balance ability in patients recovering from stroke. this exercise may prove useful in clinical rehabilitation. further research on the long - term effectiveness of ptg on gait ability is suggested. |
the success of endodontic treatment depends primarily on the eradication of micro - organisms from the root - canal system and prevention of their reinfection. the root canal system is shaped with the help of stainless steel and nickel - titanium instruments. this shaping process is accomplished in conjunction with constant irrigation to remove the inflamed and necrotic tissue, microbes / biofilms, and other debris from the root - canal space. despite the advent of numerous modern techniques and instruments in canal shaping, more than 35% of the root canal 's surface can be left uninstrumented after non - surgical root canal treatment. the presence of necrotic or vital tissue remnants within the root canal space may provide a source of nutrition for the surviving bacteria. thus, microorganisms, either remaining in the root canal space after treatment or those re - colonizing the filled canal system, are the main etiological causes of endodontic failures. the role of the irrigation protocol thereby plays a key role in the disinfection of the root canal space. the ideal root canal irrigant has been described by zehnder as being systemically nontoxic, non - caustic to periodontal tissues, having little potential to cause an anaphylactic reaction, possessing a broad antimicrobial spectrum, capable of dissolving necrotic pulp tissue, inactivating endotoxins, and either preventing the formation of a smear layer or dissolving it once it has formed. although many kinds of endodontic irrigants have been investigated ; none have been able to exhibit all the above mentioned properties. as of date, sodium hypochlorite (naocl) has been able to meet most of these criteria. naocl also dissolves pulpal remnants and collagen. in spite of its unpleasant taste, toxicity, and inability to completely remove the smear layer the endodontic irrigant with anti - bacterial activity is 2% chlorhexidine (chx) because of its substantivity. ethylenediaminetetraacetic acid (edta) is a chelating agent that is employed as a root canal irrigant. the other recently introduced irrigant is mtad, a mixture of doxycycline, citric acid, and tween 80 detergent. adjuncts to irrigation such as sonic, ultrasonic, and subsonic activation have been introduced in an effort to improve the delivery and efficacy of irrigants to the apical third in order to improve the canal cleanliness. irrigant contact with the surfaces of the root canals can be enhanced by using systems such as endovac. (discus dental, culver city, ca). passive ultrasonic irrigation and endovac are more effective in delivering the irrigant to the working length than conventional endodontic needles in root canals. current evidence suggests that sonic and ultrasonic irrigation results in better removal of debris and smear layer from the apical third of the root. the purpose of this survey was to ascertain the current irrigation protocol followed by endodontic faculty and post - graduate students in indian dental colleges. this is the first comprehensive survey of this nature, which has been conducted in india. our survey was based on the survey employed by dutner. among members of american association of endodontists and published in the journal of endodontics (2012). a modified form was created, and a postal invitation to participate in this national survey was sent to the department of conservative dentistry and endodontic of 294 dental colleges present in india. a total of 2389 forms were successfully delivered by speed post - after accounting for seven undelivered post - covers containing a total of 35 survey forms. survey participants were asked about their irrigant selection, irrigant concentration, smear layer removal, and use of adjuncts to irrigation. questions consisted of numeric rankings, multiple choices, and multiple selections with options for write - in answers where appropriate. a total of 2389 survey, forms were successfully delivered by speed post - after accounting for seven undelivered post - covers containing, total of 35 survey forms. as the purpose of this survey, was to ascertain the clinical irrigation protocol being employed in dental colleges in india, the survey participants comprised of post - graduate students and endodontic teaching faculty with varying years of experience. the respondents of our survey, comprised of 47% being post - graduate students, 26.4% were teaching faculty with less than 5 years of experience, 11.8% were faculty with 5 - 10 years of experience, 9.3% were faculty with 11 - 20 years of experience, 3.8% were faculty with 21 - 30 years of experience and 1.6% respondents were faculty with a clinical experience of more than 30 years. among all experience groups, our data indicate that 92.8% of respondents are employing naocl as their primary irrigant [figure 1 ]. when asked to rank the reasons for their primary irrigant selection, antibacterial capability was most important, followed in order by tissue dissolution, biocompatibility, substantivity, and expense. the majority of respondents are also including saline (83.6%), chx (73.3%), and edta (56.3%) in their irrigation protocol [figure 2 ]. most respondents surveyed (49.3%) use 2.6 - 4.0% strength naocl [figure 3 ]. 49.4% of the respondents claim to use 5 - 10 ml of irrigants per canal. the results of this survey indicate that 73.3% of respondents also employ chx as an irrigant with 83% of the positive respondents using 2% as the preferred concentration of chx. percentage of respondents who utilize each irrigant as their primary irrigant during root canal treatment percentage of respondents who include each irrigant as any part of their irrigation protocols, whether as primary or secondary irrigants. naocl : sodium hypochlorite, chx : chlorhexidine, edta : ethylenediaminetetra - acetic acid, mtad : a mixture of doxycycline, citric acid, and tween 80 detergent percentage of respondents utilizing varying concentrations of sodium hypochlorite in their irrigation protocol 68% of respondents aim to remove the smear layer during the endodontic treatment. most respondents (78%) claim to alter their irrigant selection on the basis of pulpal or periapical diagnosis. 47% of respondents use ultrasonic activation as adjunct to irrigant activation, where as 17% claim to use other activation techniques such as manual dynamic activation (mda) and k - file activation while 12% use subsonic systems such as endoactivator (advanced endodontic, santa barbara, ca). 7% of those surveyed are also using sonic activation and only 2% of those surveyed use negative pressure irrigation with systems such as endovac. our survey was based on the survey employed by dutner. amongst members of american association of endodontists. the main objective of our survey was to ascertain the irrigation trends being followed in various dental colleges present in india. the positive response rate in our survey was 33.23% while this was 94.5% in the australian survey, 85% in the united kingdom survey, 72% in north jordan survey, 58.1% in american survey i conducted during 2001, and only 28.5% in american survey ii conducted during 2012. the comparison of similar surveys similarly carried out in different countries are given in table 2. comparison of surveys conducted on irrigation protocol in different countries in our survey, an overwhelming 97% of respondents indicated that 26 gauge needle was being employed as the primary irrigation needle for the delivery of endodontic irrigants. 91% of respondents in the american survey ii and 94% in the australian survey also chose naocl as their primary irrigant. 63% of respondents selected local anesthetic solution as primary irrigant in the united kingdom survey while 33.6% selected hydrogen peroxide as primary irrigant in the north jordon survey. when asked for the percentage of naocl employed, in our survey 49.3% of respondents claim to use 2.6 - 4.0% naocl where as 57% of the respondents claim to use > 5% naocl in the american survey ii conducted in the year 2012. 1% naocl was the most common concentration reported in the australian survey (2003) and 0.5% naocl was commonly employed among the participants of north jordan survey (2004). the american survey i (2001) revealed that 51% of practicing endodontists removed the smear layer before obturation of the root canal system, while the american survey ii (2012) revealed that 77% of endodontists removed the smear layer. in our survey, 68% of our respondents claim to remove the smear layer during endodontic procedure. according to our survey the highest ranked reasons for irrigant selection is its antibacterial and tissue - dissolving capability. it was found that 49.9% of the respondents use more than 5 - 10 ml of irrigants per canal during the shaping and cleaning procedure. 78% of respondents in our study stated that their choice of irrigant might change on the basis of pulpal and periapical diagnosis. the primary irrigant employed for teeth with the radiographic evidence of periapical lesion was chx (54.2%). although, 78% of our respondents say that they change their irrigant according to periapical diagnosis, 92.8% use naocl as their primary irrigant. incidentally, in the american survey ii, 66% of the respondents said they do not alter the irrigant based on the condition of the pulp and periapex. when asked about adjuncts used for irrigation, participants were given choices including ultrasonic activation, sonic activation, subsonic activation, and negative pressure. subsonic activation was included as a choice because devices such as the endoactivator (advanced endodontic, santa barbara, ca) are sometimes described in this manner. in our study, 47% of the respondents are using ultrasonic activation, 19% using sonic (or subsonic) activation and 17% using other activation techniques such as mda and k - file activation. the findings of this survey are that the majority of post - graduate students and teaching faculties in indian dental schools are employing naocl (2.6 - 4.0%) as the primary endodontic irrigant, with 26 gauge needle being most preferred for syringe irrigation. the concept of smear layer removal is high (68%), and there is a general trend (78%) to modify the irrigant protocol according to the status of the pulp, status of the periapex and in retreatment cases. the irrigation protocol trend is similar to the trends seen in a recent survey amongst members of american association of endodontists in 2012. | background : irrigation protocol is the most critical step during the disinfection of an infected root canal system.aim:the purpose of this study was to determine the root canal irrigation trends being practiced among the endodontic teaching faculty and post - graduate students in the dental colleges present in india.materials and methods : a postal invitation to participate in this national survey was sent to the department of conservative dentistry and endodontic of 294 dental colleges present in india. a total of 2389 forms were successfully delivered out of which 794 duly filled forms were received back. survey participants were asked about their irrigant selection, irrigant concentration, smear layer removal protocol, and use of adjuncts during irrigation.results:this survey elicited a positive response rate of 33.23%. our data indicated that 92.8% of respondents use sodium hypochlorite (naocl) as the primary endodontic irrigant, with 26 gauge needle being most preferred for syringe irrigation, with 49.3% of them using it at a concentration of 2.6 - 4.0%. 68% of our respondents aim to remove the smear layer during the endodontic treatment while 47% reported using ultrasonic activation as an adjunct during their irrigation protocol.conclusions:the findings of this survey are that the majority of teaching institutions in india are employing naocl (2.6 - 4.0%) as the primary endodontic irrigant. the concept of smear layer removal is high (68%), and there is a general trend (78%) to modify the irrigation protocol according to the status of the pulp, status of the periapex and in retreatment cases. |
in the early 20th century, after extensive studies of the ovine fetal circulation, sir joseph barcroft (1872 - 1947) postulated that the environment in which the human fetus develops would be comparable to that likely endured by an adult on the summit of mount everest. he termed this intriguing hypothesis ' everest in utero ' and proposed that to survive the hypoxic uterine environ ment the fetus must develop elaborate physiological strategies comparable to those seen in climbers ascending the great himalayan peaks. in 2007, four climbers descending from the summit of mount everest (8,848 meters) took arterial blood gases from one another at 8,400 meters above sea level. their mean arterial partial pressure of oxygen (pao2) was 3.28 kpa (24.6 mm hg) with a mean calculated arterial oxygen saturation (sao2) of 54% while they rested without supplemental oxygen. among this group, one individual had a pao2 of 2.55 kpa (19.1 mm hg), the lowest pao2 ever reported in an adult human. so how far removed from intrauterine life were these measurements, and do climbers exhibit, as does the fetus, physiological strategies that may benefit the similarly hypoxemic critically ill patient ? the overwhelming challenge for a developing fetus is its complete reliance on the maternal circulation for oxygenation via placental transfer. the fetus therefore competes with other maternal organs for a share of the systemic oxygen delivery such that increased demand from other maternal tissues could be detrimental to the fetal circulation. extrapolations from animal studies suggest that human fetal pao2 is between 2.5 and 3.5 kpa. thus, barcroft 's ' everest in utero ' was a remarkably accurate prediction given the limited data available to him at the turn of the last century. table 1 compares measurements that contribute to oxygen carriage in the fetus, the typical adult male, and climbers acclimatized to high altitude near the summit of mount everest. it seems an astonishing coincidence that the highest point on the earth 's surface, the limit of human tolerance of hypoxemia, is analogous to life in utero. despite this degree of hypoxemia in utero, there is little evidence that anaerobic metabolism is a dominant source of fetal energy, suggesting that this level of fetal oxygenation is sufficient for tissue requirements. values pertinent to arterial oxygen carriage in the term fetus, male high - altitude climbers at 8,400 meters on mount everest, and typical adult male grocott and colleagues. cao2, arterial oxygen content ; hb, hemoglobin concentration ; p50, the arterial partial pressure of oxygen at which 50% of hemoglobin is saturated with oxygen ; paco2, arterial partial pressure of carbon dioxide ; pao2, arterial partial pressure of oxygen ; sao2, arterial oxygen saturation. the fetus has negligible ability to increase systemic oxygen delivery so instead must initiate mechanisms that maximize the use of available oxygen supplies. following an acute reduction in oxygen delivery, fetal tissue oxygen extraction increases to compensate until delivery falls below approximately 50% of normal. this increase in tissue oxygen extraction acts as a buffer before cellular oxygen consumption begins to fall, and the threshold of this consumption varies widely between different organs. once tissue oxygen extraction is maximal, there follows a marked decrease in fetal movement, particularly breathing effort and forelimb activity. cessation of excessive movement results in a reduced oxygen consumption, as demonstrated by the administration of neuromuscular blocking agents in experimental models. reduced activity in response to fetal hypoxic distress is used for the biophysical profile as part of an objective antepartum assessment in high - risk obstetric cases. though of clear benefit during a hypoxic event, this strategy is by no means a lasting solution for a chronic reduction in oxygen availability. the primary energy - consuming process in the developing fetus is growth. within several hours of reduced fetal oxygen delivery, this sacrifice in the battle to maintain equilibrium of oxygen flux will cause intrauterine growth retardation so that survival will be at the cost of low birth weight and the possibility of organ pathology. of note, chronic maternal hypoxia due to residence at high altitude is associated with reduced birth weight from restricted fetal growth during the third trimester. thus, fetal survival is ensured by entering a period of hibernation - like existence in which oxygen - consuming processes are downregulated in order to avoid anaerobic respiration. clearly, there will be a threshold below which this strategy leads to irreversible organ damage. transition from uterine to atmospheric life involves the loss of mechanisms imperative for fetal survival but redundant in the relatively oxygen - rich air ex utero. to facilitate this, the lungs supervene over the placenta as the gas exchange surface, and there is anatomical redistribution of blood flow. within weeks, the hemoglobin concentration of the neonate slowly declines and production of fetal hemoglobin (hbf), which possesses considerably greater affinity for oxygen than does adult hemoglobin, ceases. yet the retention of life - sustaining fetal phenomena was first demonstrated by the french physiologist paul bert some 140 years ago. bert observed that newborn rats, when submersed in water, maintained persistent respiratory movements for up to 30 minutes, yet at 20 days of age, similar rats ceased to breathe after only 90 seconds. it is feasible that the fetus, having developed in an environment with such limited oxygen availability, retains a degree of ' acclimatization ' in neonatal life. the prolonged hypoxemia in utero may serve as a time of intense ' hypoxic preconditioning ' that provides a degree of protection from untoward obstetric catastrophes. however, as with climbers returning from the mountains, this acclimatization effect subsides with time, as shown by reduced resistance to hypoxia with increasing neonatal age. historically, life on earth the earth was formed approximately 4.6 billion years ago and the earliest life - forms appeared soon after the planet 's period of heavy bombardment from meteorites, 4 billion years ago. at that time, the atmosphere consisted of gases released from within the earth 's crust, primarily nitrogen, carbon dioxide, water vapor, and hydrogen, the last of which was light enough to escape the earth 's gravitational force. to the primitive anaerobic prokaryotes that first inhabited the earth, even traces of oxygen were highly toxic. through the photochemical destruction of water vapor, releasing oxygen and hydrogen, and then photosynthesis by cyanobacteria, the concentration of oxygen in the atmosphere rose and organisms were forced to adapt to shield themselves from its destructive power. two billion years ago, a process known as eukaryotic endosymbiosis occurred in which mitochondria, evolved from a strain of purple bacteria, were engulfed by prokaryotic cells but retained their functional capacity. this unique combination provided a hypoxic sanctuary for the anaerobic - seeking mitochondria and a source of energy for the host cell, in the form of atp, which allowed the development of eukaryotic characteristics and eventually multicellular aerobic life. our understanding of the human response to hypoxia has developed considerably since barcroft 's day. the heterodimeric protein hypoxia - inducible factor 1 (hif-1) plays a central role in maintaining oxygen homeostasis. cellular hypoxia results in reduced degradation by prolyl hydroxylase (phd) and an exponential rise of the hif-1 subunit, which combines with the constitutively expressed hif-1 subunit to form the active transcription factor. this triggers upregulation of erythropoietin, angiogenic factors, and vasoactive mediators to improve oxygen delivery while induction of glycolytic enzymes permits continued energy production when oxygen for oxidative phosphorylation is scarce. additionally, hif-1 depresses mitochondrial oxygen consumption and improves the efficiency of energy production within mitochondria. during chronic fetal hypoxia, levels of hif-1 are significantly increased while a deficiency of either the or subunits in murine models is incompatible with embryonic life. another component of an individual 's ' hypoxic response fingerprint ' is the seemingly paradoxical mitochondrial production of reactive oxygen species (ros) by enzymes of the respiratory chain when oxygen is scarce. elevated concentrations of ross have been detected in the cord blood of profoundly hypoxic neonates at birth. these highly reactive molecules, if left unchecked, can cause extensive cellular oxidative damage and apoptosis. however, mitochondrial ross are also required during adaptation to hypoxia as they promote transcription via hif-1 with concomitant negative feedback from hif-1 attenuating ros generation. ros production is also governed partly by uncoupling proteins (ucps) on the inner mitochondrial membrane, allowing a leak of protons without the generation of energy. this contends the liberal use of antioxidants in hypoxic critically ill patients and may provide an axis for future interventions aimed at manipulating hypoxia tolerance in adults. as well as being a key component in the maintenance of vascular tone, nitric oxide (no) is a fundamental cellular messenger with an important role in the regulation of fetal and adult biology. fetal levels of no metabolites are raised in the presence of intrauterine growth retardation, particularly when fetal oxygenation is compromised. not only does hypoxia stimulate increased release of no from vascular endothelium, but under conditions of cellular hypoxia, mitochondria them selves can produce no, which may represent part of the cellular hypoxia - sensing apparatus. the potential significance of this metabolite is highlighted in high - altitude resident tibetans, whose circulating concentrations of bioactive no products are 10 times higher than those of low - altitude dwellers. in fact, the authors of this study stated that ' the plasma nitrite and nitrate concentrations of tibetans were unprecedented for healthy people and that average concentrations exceeded those typically seen in septic shock patients '. high circulating no metabolites appear to be beneficial, whereas in critically ill patients, it may represent a maladaptive phenomenon that leads to circulatory catastrophe. interestingly, hbf displays an no - scavenging effect that is greater than that of adult hemoglobin, which may account for the apparent circulatory stability within the fetus in the face of severe hypoxemia. the ability of the fetus to withstand profound and sustained hypoxia in utero may serve as a model from which to develop strategies to improve survival in hypoxemic critically ill adults (figure 1). with a better understanding of the relationship between hypoxemia and tissue hypoxia, ' permissive hypoxemia '. simply adding more oxygen at the top of the oxygen cascade may not be the solution to disequilibrium at a tissue level. furthermore, the toxic potential of oxygen is frequently understated despite mounting evidence in animal models and human studies [30 - 33 ]. a recent multicenter cohort study of over 6,000 patients who had sustained a nontraumatic cardiac arrest demonstrated that those patients who were hyperoxic (defined as a pao2 of greater than 40 kpa) had a significantly higher in - hospital mortality rate when compared with normoxic or hypoxic patients. in the current clinical climate of goal - directed oxygen delivery, the degree to which chronic hypoxemia should be corrected remains unknown. with accumulating evidence that normoxia may not be a prerequisite for survival or successful recovery, the possibility of permitting selected patients to remain hypoxic consequent upon their disease process may not be as absurd as it initially appears. the damage caused by high concentrations of inspired oxygen and aggressive mechanical ventilation protocols has promoted this philosophy mong pediatric critical care physicians. with more reliable monitors of specific tissue oxygenation, such as near - infrared spectroscopy, we might begin to relinquish our deep - rooted need to fully correct hypoxemia. therapy for hypoxemic critically ill adults : potential therapeutic targets derived from translational work in fetal and high - altitude research. co, carbon monoxide ; h2s, hydrogen sulphide ; hif-1, hypoxia - inducible factor 1 ; phd, prolyl hydroxylase. one group of authors has elegantly highlighted how rethinking our perceptions of oxygenation has the potential to revolutionize patient care. in two meta - analyses, they questioned the evidence base for the universal practice of supplemental oxygen administration during acute myocardial infarction, reporting a striking association between high inspired oxygen concentrations and reduced coronary blood flow, greater infarct size, and a potentially increased risk of mortality conversely, intracoronary transfer of autologous bone marrow cells has already been shown to promote improvement of left ventricular systolic function after acute myocardial infarction. the use of stem cells in targeted regenerative medicine is likely to play a major part in future interventions. reprogramming of adult cell lines by transduction of transcription factors can generate induced pluripotent stem cells (ipscs). hypoxia not only enhances the generation of ipscs but also promotes the renewal of stem cells via hif - mediated pathways. is this a window into the body 's own attempts at regeneration and repair in response to hypoxia, and can we mimic such repair mechanisms ? owing to an oxygen - carrying capacity greater than that of adult hemoglobin, hbf enhances the transport of oxygen across the placenta from the maternal circulation. consisting of 22 subunits rather than 22 possessed by most healthy adults, hbf has a lower affinity for 2,3-diphosphoglycerate than adult hemoglobin does, therefore causing a leftwards - shifted oxygen - hemoglobin dissociation curve. this accounts for the greater measured sao2 for any given pao2 in fetal, as compared with maternal, blood. in those born with sickle cell disease so significant is the persistence of this fetal mechanism that a low level of hbf in sickle cell patients is an independent predictor of early mortality and pharmacologically increasing hbf concentration with hydroxyurea improves long - term outcome in this patient cohort, possibly via an no - mediated pathway. humans homozygous for the genetic disorder ' hereditary persistence of fetal hemoglobin ' express -globin only and thus all of their circulating hemoglobin is of the fetal species. this benign condition also exists in variety of heterozygous forms and is more common in those populations in which -hemoglobinopathies are prevalent. as the presence of high circulating quantities of hbf appears to be without detrimental effect, pharmacological reactivation of the -globin and hbf in adult erythroid cells during chronic hypoxemia seems feasible. the growth - modifying cytokine, stem cell factor, in combination with erythropoietin, leads to increased production of hbf in animal models. genetic manipulation of the switching mechanism between - and -globin at dna transcription or via alteration of -globin mrna transcription, translation, and post - translational events might allow adults to revert to a fetal oxygen - carrying modality. it has been proposed that the multiple organ failure witnessed in severe critical illness may represent a rudimentary reduction in mitochondrial oxygen consumption in order to preserve organs when faced with a life - threatening challenge. this is analogous to the strategy adopted by the fetus in times of distress and the concept may hold the key to an alternative approach to managing severe hypoxemia. hydrogen sulphide (h2s) is an endogenously produced signaling molecule that belongs to a trio of biological mediators, including no and carbon monoxide (co), referred to as gasotransmitters. h2s is known to influence cellular metabolism via cytochrome oxidase and carbonic anhydrase and interaction with reactive oxygen and nitrogen species (leading to free radical scavenging). it also causes activation of atp - sensitive potassium (katp) channels and modification of gene transduction. low concentrations of h2s exert a cytoprotective effect, whereas higher concentrations have the more commonly appreciated cytotoxic properties, primarily via mitochondrial failure. the relevance of h2s to critical care medicine is that, when used in carefully titrated doses, it has the ability to induce a state of hibernation in animal models [50 - 52 ]. in such a state of torpor, the organism undergoes a reversible reduction in energy - consuming processes that facilitates a reduction in both oxygen uptake and delivery, therefore protecting tissues from oxygen deprivation. when mice are exposed to h2s gas, they undergo a dose - related reduction in metabolic, heart, and respiratory rates for a period of several hours. basal h2s production has been demonstrated in fetal membranes, and there is a significant upregulation when the tissues are exposed to low levels of oxygen in vitro. this hypoxia - induced elevation in h2s may represent a primitive protective mechanism that serves to downregulate metabolism during times of stress, a step beyond the simple reduction of fetal movements observed following acute oxygen deprivation in utero. co can also protect against hypoxic damage by interacting with hif-1-induced pathways to induce a state of ' suspended animation '. furthermore, hypoxia promotes the endogenous production of co via haem oxygenase-1, a process that prevents hypoxic damage through a variety of anti - inflammatory and anti - apoptotic mechanisms. sporadic case reports provide circumstantial evidence that prolonged hypothermia and hypoxemia can be tolerated without detrimental effects. induced suspended animation has also been proposed as a viable strategy following cardiac arrest and during severe sepsis. the possibility of pharmacologically induced hibernation in the critically ill would be as revolutionary as the iron lung, diverting the focus of oxygen supply - demand coupling to the latter rather than the former. the pivotal role of hif in oxygen sensing and hypoxic adaptation also makes it a prime target for interventional studies. already, work on specific inhibitors of phd has been shown to induce hypoxic adaption. in combination with other components of the cellular hypoxic response, such as regulators of the no pathway and ucps, these alternative strategies may avoid therapeutic interventions that resort to the administration of toxic concentrations of oxygen. crosstalk between the hif-1 transcription pathway and the ros cascade should provide a wealth of targets for future pharmacological interventions. mastery of human cellular adaptation to hypoxia may be feasible in the paradigm of scientifically robust high - altitude research. the inter - individual variation in human performance at high altitude mirrors that expressed in the hypoxemic environment experienced during critical illness. this alternative methodological approach to hypoxia research could lead to novel translational studies in critically ill patients. exposure of healthy individuals to graded ' doses ' of hypoxia provides a platform from which physiological mechanisms of adaptation and effects of their perturbation can be observed in whole - body systems. it holds further advantage in controlling for the unfavorable ' signal - to - noise ratio ' that is often encountered in the study of patient populations with multiple pathologies. our current under standing of the process of acclimatization to high altitude focuses on restoration of normal sea level systemic oxygen delivery via increases in minute ventilation, cardiac output, and red blood cell mass. however, as in survival in utero, the mechanisms that separate survivors from non - survivors may lie in the cellular control of oxygen consumption via the signaling pathways discussed. indigenous populations living at high altitudes have the advantage of lasting biological changes as a result of the evolutional pressure exerted by hypobaric hypoxia. their capacity for systemic oxygen carriage is no greater than that of an acclimatized ' lowlander ', thus suggesting more intricate changes with thousands of years of evolution, making their physiology more akin to that of the fetus. this brief develop mental era is easily forgotten when we consider adaptation to hypoxemia yet it may well hold the key to our most sophisticated compensatory mechanisms, with a potential for reawakening in disease states. the complex network of cellular signaling pathways that act to detect hypoxia and preserve cellular functioning in utero may be the answer to the failure of the systemic delivery of oxygen to tissues. manipulation of gasotransmitters to promote adaptation to hypoxemia may avoid the detrimental effects of excessive oxygen administration. all of us are triumphant mountaineers of barcroft 's everest in utero and the sequeale of that expedition may return to save us in our hour of need. co : carbon monoxide ; h2s : hydrogen sulphide ; hbf : fetal hemoglobin ; hif-1 : hypoxia - inducible factor 1 ; ipsc : induced pluripotent stem cell ; no : nitric oxide ; pao2 : partial pressure of oxygen ; phd : prolyl hydroxylase ; ros : reactive oxygen species ; sao2 : arterial oxygen saturation ; ucp : uncoupling protein. some of this work was undertaken at the university college london hospital - university college london comprehensive biomedical research centre which received a portion of funding from the united kingdom department of health 's national institute of health research biomedical research centres funding scheme. | the human fetus develops in a profoundly hypoxic environment. thus, the foundations of our physiology are built in the most hypoxic conditions that we are ever likely to experience : the womb. this magnitude of exposure to hypoxia in utero is rarely experienced in adult life, with few exceptions, including severe pathophysiology in critical illness and environmental hypobaric hypoxia at high altitude. indeed, the lowest recorded levels of arterial oxygen in adult humans are similar to those of a fetus and were recorded just below the highest attainable elevation on the earth 's surface : the summit of mount everest. we propose that the hypoxic intrauterine environment exerts a profound effect on human tolerance to hypoxia. cellular mechanisms that facilitate fetal well - being may be amenable to manipulation in adults to promote survival advantage in severe hypoxemic stress. many of these mechanisms act to modify the process of oxygen consumption rather than oxygen delivery in order to maintain adequate tissue oxygenation. the successful activation of such processes may provide a new chapter in the clinical management of hypoxemia. thus, strategies employed to endure the relative hypoxia in utero may provide insights for the management of severe hypoxemia in adult life and ventures to high altitude may yield clues to the means by which to investigate those strategies. |
bangladesh is divided into 64 administrative districts and has a population of 149.7 million, area of 147,570 km, and population density of approximately 1015 people / km2. the country has a total fertility rate of 2.3/capita gdp of $ 772, life expectancy of 69.5 years. the government 's expenditure on health is the third largest in the country, after education and defense. the paradox of bangladesh lies in its gdp and its achievements in the health, education, and socio - economic sectors. the latest international diabetes federation (idf) atlas estimated the incidence of type 1 dm (t1 dm) in bangladesh at 4.2 new cases of t1dm/100,000 children (014 years)/year, in 2013. the highest incidence of t1 dm (children 014 years) is estimated to be in europe and north america, with south - east asia closely following the trend at third position. a series of studies have reported a constant global increase in the incidence of t1 dm, and multifactorial process might be involved. at the bangladesh institute of research and rehabilitation in diabetes, endocrine and metabolic disorders (birdem), there has been an upward trend in the number of newly diagnosed children, from 112 cases in 2008 to 319 cases in 2013, as documented by the changing diabetes in children (cdic) program at birdem. the question, however, remains whether this upward trend is because of a real increase in the incidence of childhood dm or increased awareness and a decrease in prevalence of communicable diseases, brought about by hygienic practices, immunization against common infectious diseases, with a concomitant rise in non - communicable diseases (ncd). recent studies show that it is likely that multifactorial reasons are responsible for the increasing incidence e.g., hygienic practices, feeding regimens (autoimmunity to cow 's milk). badas has contributed massively to educating the public about diabetes, such that the hitherto unaccepted notion that children could get and die from dm, has been translated into a relatively low threshold for testing for dm in children with polyuria, loss of weight, unconsciousness etc. they are often considered a burden on the family, especially girls ; they have little prospect of getting married or being employed. dm is likely to be hidden from teacher, prospective spouse and employer, often with far - reaching consequences. lack of motivation, inability to manage common complications e.g., hypoglycaemia, sick day management, drop out from the clinic (which may be due to lack of motivation or extra cost involved in travel), psychological issues, are other common problems. the diabetic association of bangladesh (badas), which was established in 1956, is the largest of its kind in the world, with 68 affiliated associations in 64 districts of the country. it has more than 400,000 diabetics registered at its tertiary centre, birdem in dhaka. prior to badas, there was no care available for patients with dm needing insulin under the government health services, so poor patients faced certain death ! episodic care was the only kind available, and patients with chronic diseases, faced an uncertain and grim future. the motto was that no diabetic shall die untreated, unfed or unemployed, even if poor. starting of as an opd, badas has grown into a network of 64 affiliated associations, one in as many districts, and a chain of small hospitals and opds, as well as medical and nursing schools. all patients, irrespective of socio - economic status, are entitled to free consultation and certain tests. treatment, including insulin, is available to patients free of cost if entitled, or according to income. badas operates on a cross - financing model, such that services for the poor are subsidized from the income earned from the well - to - do, who come to seek care or be investigated. badas is a member of the idf and was a who collaborative centre until recently. children with diabetes are still managed by adult physicians or occasionally by adult diabetologists, except in institutions like birdem, where paediatricians took over the care of children, and adolescents with diabetes in 1997, as well as in dhaka shishu hospital. children and adolescents have special needs at different stages e.g., nutrition, schooling, growth, puberty etc. initially, when the department of paediatrics started, the majority of patients were classed as mrdm, and classical t1 dm were a minority. however, the situation is reversed now, with an increase across all socio - economic groups of t1 dm. the burden of providing for the care of diabetic patients places a huge demand on the stretched resources of badas. t1 two programs need special mention, the changing diabetes in children (cdic) program, and the life for a child (lfac) program, the former sponsored by novo nordisk and the latter by the international diabetes federation (idf). the central one is located in dhaka and the peripheral ones are located in 2 district headquarters ; chittagong and faridpur. the number of patients enrolled in dhaka is 1477, in faridpur 216 and 200 in chittagong. age of the children range from 0 - 18yrs. under the lfac program, there are 2200 registered (1yr-23yrs). there is a slight preponderance of females over males. with the launch of these 2 programs, the intensity of motivation and education increased and as a result, glycaemic control has improved over time from a hba1c of more than 9% to a gradual fall to < 7.5% amongst the cdic patients. both the programs are providing support by providing free consultation, free insulin, education, machines for measurement of hba1c, microalbumin on spot urine sample, and retinal camera. some blood tests, annual check - up and screening for complications, growth and pubertal monitoring are done free of cost. cdic has constructed the clinic within the premises of the women 's and children hospital (birdem2) and has been providing funds for running the clinic including doctors and supporting staff salary. under its umbrella, cdic supports some health care staff, including a psychologist. and runs an enviable education programme. the education program was set up with the help of cdic, and employs diabetes educators, a cadre specially trained in a structured diabetology course, training patients and caregivers in injection and testing techniques, and advising patients about any issues related to diabetes. however, now diabetes educators are being paid for by patients using the cross financing model. also certain components of the chec -up are available on payment by the well - to - do. the lfac program is carrying out a multi - centred epidemiological study of t1 dm, which will give us an insight into the risk factors, trends etc. it also brought to the world 's notice that dm is increasing alarmingly and every nation has a responsibility to prevent the onward march of this threat, which culminates in 14 november being declared the un world diabetes day. diabetes in children and adolescents is increasing in bangladesh, and comprehensive diabetes care is essential to achieve good glycaemic control. diabetes education combined with appropriate motivation of the patients and caregivers is the cornerstone of dm management. sustainability of the programs will ensure that the child with diabetes can lead a normal life. | diabetes mellitus (dm) is a common endocrine disorder among children and adolescents in bangladesh. the latest international diabetes federation atlas estimated the incidence of type 1 dm (t1 dm) in bangladesh as 4.2 new cases of t1dm/100,000 children (014 years)/year, in 2013. diabetes, being a lifelong disease, places a huge burden on the economy of the most densely populated, and resource - poor country of the world. the diabetic association of bangladesh (badas), the largest of its kind in the world, provides comprehensive care to the biggest number of diabetics at any one centre and is engaged in advocacy. although sounding grandiose, it 's aims that no diabetic shall die untreated, unfed or unemployed, even if poor is pursued with a passion. recently badas has been supported in its endeavor for children and adolescents by two programmes ; viz the changing diabetes in children program (a joint initiative of badas, the world diabetes foundation and novo nordisk), and the life for a child programme (lfac) supported by the idf. numerous studies from the prosperous countries have demonstrated the incidence of t1 dm is increasing. data from the cdic clinic at birdem shows a rising trend in patients presenting with classical t1 dm. in addition, the pattern of dm is changing. |
prostate cancer (pca) is the second - leading cause of cancer death in men. furthermore, imaging of localized pca still remains limited. because the disease often develops without any symptoms, utilizing additional examinations (such as biochemistry or imaging) for early detection and definition of tumor aggressiveness is of great importance. it has been estimated that approximately 20% of men will be diagnosed with pca in their lifetime. since the advent of prostate specific antigen (psa) screening, most of them are diagnosed with localized disease. while prostatectomy or radiation treatment is the standard therapy for early - stage pca, 3040% of patients will develop recurrent and/or metastatic disease. on the other hand, many non - advanced tumors have low potential for dissemination and progression. these patients probably do not benefit from radical treatment and observation only or conservative treatment should be considered ; therefore, there is a need for new pretreatment pca staging models. the diagnosis and localization of pca are based on a digital rectal examination (dre) and assessment of serum (psa) followed by a trans - rectal ultrasound (trus). magnetic resonance imaging (mri) however, the contraindications for mri (claustrophobia, metallic endoprosthesis, clips, and stents) must be considered. new functional imaging methods have come into use in recent years, using neovascularization of malignant tumors. angiogenesis, the formation of new blood vessels from the pre - existing vascular bed, is an integral part of benign prostate hyperplasia ; it is associated with prostatic intraepithelial neoplasia (pin) and is a key factor in the growth and metastasis of pca. increased neovascularity within the prostate is the marker of prostate malignancy and it is associated with an increased likelihood of metastasis, higher stage of the disease, and reduced survival. multidetector computed tomography (mdct) is already widely used as a general imaging method for evaluating cancer angiogenesis. perfusion computed tomography (pct), or dynamic contrast - enhanced ct (dce - ct), is the acquisition of serial images through the same volume over time after the administration of a bolus of iodinated contrast media. thanks to the excellent linearity between tissue attenuation and iodine concentration, dce - ct allows the analysis of blood flow and blood volume estimates in tumors. this technique was first used in the evaluation of patients with acute stroke, but has also been reported to be useful in the detection of tumor angiogenesis [712 ]. the observed tissue enhancement is related to blood flow (bf), blood volume (bv), mean transit time (mtt), and capillary permeability and surface area (ps). pct has the advantage of high spatial resolution, involves little risk to patients, and data acquisition can be incorporated into routine patient studies. our previous study proved the high sensitivity of pct in pca imaging (luczynska.). perfusion ct is a valuable method for diagnosis of prostate cancer (see our previous prospective study in 94 patients, research grant no. nn403240837 of the polish ministry of science and higher education, submitted for publication). therefore, the question is whether pct is more efficient, safe, and cost - effective than the standard methods. the aim of this study was to estimate the correlation between perfusion parameters and commonly used prognostic factors psa level, gleason score, and ptnm stage in pca patients. the study protocol was approved by the polish ministry of science and higher education, research grant no. nn403240837 titled ct perfusion and biological markers in local staging and risk assessment in surgically treated prostate cancer patients. the study was approved by the ethics committee of the center of oncology, m. sklodowska - curie memorial institute, cracow branch, and prior written informed consent was obtained from all patients. the study included 3 tasks, based on similar group of patients : 1) imaging study : pct in the visualization of pca, 2) pathological study : correlation between pct parameters and clinico - pathological features of pca, and 3) molecular study : correlation between pct parameters, immune - histological findings, and immune - histochemical expression of angiogenesis - related markers in non - advanced pca. the following report presents the data from study 2, and data analysis of studies 1 and 3 will be published separately. we prospectively enrolled patients meeting these inclusion criteria : serum psa level > 4 ng / ml and suspicious imaging results at trus suggesting prostate malignancy, scheduled for prostate biopsy, and with serum creatinine level 1 mg / dl. we excluded patients with previous cancer at other sites, androgen deprivation prior to pct, allergy to iodinated contrast media, serum creatinine 1 mg / dl, disqualification from surgery, or who refused to participate. in brief, the study design included the prostate pct, followed by prostate biopsy (performed 24 weeks after pct). if prostate malignancy was proven, radical prostatectomy was performed in the following 2 weeks. study procedures included : prospective registration of the eligible patients and signing of informed consent. evaluation of pct color maps and computation of the following perfusion parameters : blood flow (bf), blood volume (bv), permeability - surface area (ps), and mean transit time (mtt). analysis of correlation between bf, bv, ps, and mtt, and clinic - pathological findings. clinical staging was carried out according to the american joint committee on cancer (ajcc) and the international union against cancer staging system. the slides were made by sectioning the gland every 5 mm, from apex to base. an experienced pathologist, blinded to the pct results, evaluated the whole set of slides to assess the presence of malignancy and its grading according to gleason score. all diagnostic and treatment procedures were performed at the study center, which was the m. sklodowska - curie memorial institute, cracow branch, poland. ct examinations were performed using a 16-section multidetector (mdct) ct scanner (lightspeed 16 : ge healthcare, milwaukee, wi, usa). preliminary non - contrast ct of the pelvis (5-mm thickness) was performed to localize the prostate. the scanning range for the pct was chosen to include 2 levels : upper level (2 cm above the center of the prostate gland) and lower level (2 cm below the center), which together covered an area of 4 cm (top - bottom dimension). a total of 100 ml (250 ml) of nonionic iodinated contrast material was injected at 1.5 ml / kg at an injection rate 2 ml / s (ultravist 370 mg i / ml : bayer schering pharma, leverkusen, germany). pct scanning started 5 s after contrast administration ; 80 kvp and 120 ma images were acquired every 1 s for 50 s. immediately after completion of pct scanning, diagnostic ct of the abdomen and pelvis was performed with 5-mm slices. the images obtained were transferred to an image - processing workstation (advantage windows 4.2 : ge healthcare) and analyzed with commercially available software (ct perfusion 4 : ge healthcare). the arterial input was obtained from a standardized region in the external iliac artery (eia), with selection of the section that allowed best visualization to avoid partial - volume artifacts. a time - attenuation curve, expressed in hounsfield units (hu) per s, was generated for the arterial input (figure 1). the analysis of dynamic ct images was performed by means of the cine - loop tool used with perfusion ct 4.0 software. for quantitative analysis, a region of interest (roi) was manually drawn by 2 experienced radiology consultants (el and std) along the visible margins of the suspicious pca (identified as the fastest and strongest enhancement area in the gland) and for normal prostate tissue using an electronic cursor. rois were chosen so that on all images, they were drawn over regions of tumor throughout the image series, irrespective of motion (figure 2). if excessive motion artifacts precluded drawing a region of interest that stayed within the tumour margins in all the images of the pct scan, the patient was excluded from the study. functional maps of bf, bv, mtt, and ps were generated according to the deconvolution model, described in previous literature. tumor perfusion parameters were averaged over all sections of the rois drawn for each tumor and for normal gland. for display purposes, the functional maps were presented using a color scale with : pixel values of bf measured in ml/100 g wet tissue per min ; bv in ml/100 g of wet tissue ; mtt in s ; and ps in ml per 100 g of wet tissue (figure 3). the pct evaluation was performed by 2 local radiology consultants with 10 years experience in ct oncology imaging (el and std) and then reviewed by experts in the pct field. statistical analysis was performed with dedicated software (statistica ver.. 9). the correlation between separated perfusion parameter values with psa levels the correlation between values of perfusion parameters, gleason score, and pathological classification of pca were examined using anova, and duncan s test and the t - test were used for individual mean values. the spearman rank order correlations were determined and its significance was studied using the t - test. we prospectively enrolled patients meeting these inclusion criteria : serum psa level > 4 ng / ml and suspicious imaging results at trus suggesting prostate malignancy, scheduled for prostate biopsy, and with serum creatinine level 1 mg / dl. we excluded patients with previous cancer at other sites, androgen deprivation prior to pct, allergy to iodinated contrast media, serum creatinine 1 mg / dl, disqualification from surgery, or who refused to participate. in brief, the study design included the prostate pct, followed by prostate biopsy (performed 24 weeks after pct). if prostate malignancy was proven, radical prostatectomy was performed in the following 2 weeks. study procedures included : prospective registration of the eligible patients and signing of informed consent. evaluation of pct color maps and computation of the following perfusion parameters : blood flow (bf), blood volume (bv), permeability - surface area (ps), and mean transit time (mtt). analysis of correlation between bf, bv, ps, and mtt, and clinic - pathological findings. clinical staging was carried out according to the american joint committee on cancer (ajcc) and the international union against cancer staging system. the slides were made by sectioning the gland every 5 mm, from apex to base. an experienced pathologist, blinded to the pct results, evaluated the whole set of slides to assess the presence of malignancy and its grading according to gleason score. all diagnostic and treatment procedures were performed at the study center, which was the m. sklodowska - curie memorial institute, cracow branch, poland. ct examinations were performed using a 16-section multidetector (mdct) ct scanner (lightspeed 16 : ge healthcare, milwaukee, wi, usa). preliminary non - contrast ct of the pelvis (5-mm thickness) was performed to localize the prostate. the scanning range for the pct was chosen to include 2 levels : upper level (2 cm above the center of the prostate gland) and lower level (2 cm below the center), which together covered an area of 4 cm (top - bottom dimension). a total of 100 ml (250 ml) of nonionic iodinated contrast material was injected at 1.5 ml / kg at an injection rate 2 ml / s (ultravist 370 mg i / ml : bayer schering pharma, leverkusen, germany). pct scanning started 5 s after contrast administration ; 80 kvp and 120 ma images were acquired every 1 s for 50 s. immediately after completion of pct scanning, diagnostic ct of the abdomen and pelvis was performed with 5-mm slices. the images obtained were transferred to an image - processing workstation (advantage windows 4.2 : ge healthcare) and analyzed with commercially available software (ct perfusion 4 : ge healthcare). the arterial input was obtained from a standardized region in the external iliac artery (eia), with selection of the section that allowed best visualization to avoid partial - volume artifacts. a time - attenuation curve, expressed in hounsfield units (hu) per s, was generated for the arterial input (figure 1). the analysis of dynamic ct images was performed by means of the cine - loop tool used with perfusion ct 4.0 software. for quantitative analysis, a region of interest (roi) was manually drawn by 2 experienced radiology consultants (el and std) along the visible margins of the suspicious pca (identified as the fastest and strongest enhancement area in the gland) and for normal prostate tissue using an electronic cursor. rois were chosen so that on all images, they were drawn over regions of tumor throughout the image series, irrespective of motion (figure 2). if excessive motion artifacts precluded drawing a region of interest that stayed within the tumour margins in all the images of the pct scan, the patient was excluded from the study. functional maps of bf, bv, mtt, and ps were generated according to the deconvolution model, described in previous literature. tumor perfusion parameters were averaged over all sections of the rois drawn for each tumor and for normal gland. for display purposes, the functional maps were presented using a color scale with : pixel values of bf measured in ml/100 g wet tissue per min ; bv in ml/100 g of wet tissue ; mtt in s ; and ps in ml per 100 g of wet tissue (figure 3). the pct evaluation was performed by 2 local radiology consultants with 10 years experience in ct oncology imaging (el and std) and then reviewed by experts in the pct field. statistical analysis was performed with dedicated software (statistica ver.. 9). the correlation between separated perfusion parameter values with psa levels was also analyzed. the correlation between values of perfusion parameters, gleason score, and pathological classification of pca were examined using anova, and duncan s test and the t - test were used for individual mean values. the spearman rank order correlations were determined and its significance was studied using the t - test. between 2007 and 2012, abdominal and pelvic ct and prostate gland perfusion ct were performed in 161 patients who were eligible for the study protocol and signed informed consent. a total of 125 patients with pca (median age, 64 years ; range, 4977) met the selection criteria and were enrolled in study number 2 in the years 20072012 (figure 4). typical contrast enhancement of malignant lesions was visible in 119 out of the 125 patients (sensitivity 95.2%). a statistically significant correlation between bv, mtt, and ps values and psa level was found : correlation coefficients (r) were equal to 0.18 ml/100 g, 0.2 s and 0.24 ml / min/100 g (p0.05), respectively (table 2). these findings suggest that with an increase of psa values, an increase of bv, mtt, and ps can be expected (figure 5). mean values of perfusion parameters in malignant tissue were : bv 5.080.2 ml/100 g, bf 43.791.15 ml / min/100 g, mtt 8.240.15 s, and ps 31.471.2 ml / min/100 g. the mean values and range interval of perfusion parameters based on histological malignancy category of the tumors are presented in table 3. figure 6 illustrates differences between mean perfusion values (bv, bf, and ps) for poorly, moderately, and well - differentiated tumors according to gleason score. a statistically significant difference (p=0.02) was observed between bv values in well - differentiated tumors (4.85 ml/100 g) compared to poorly differentiated (6.37 ml/100 g) tumors. g in well - differentiated tumors to 53.36 ml / min/100 g in poorly differentiated tumors (p=0.01), whereas mean value of ps increased from 29.76 ml / min/100 g for well - differentiated tumors to 39.77 ml / min/100 g for poorly differentiated tumors and the difference between those values was also significant (p=0.02). mean values of bv, bf, and ps were higher in poorly than in moderately differentiated tumors, but statistical significance was achieved only for bf values (p=0.03). on the basis of variance analysis we found statistical correlation between mean perfusion values bv, mtt, ps and cancer stage, according to ptnm classification (p<0.03) (table 4). the analysis of individual tests comparing various averages revealed a significant difference between mean values of perfusion parameters (bv, mtt, and ps) for category iib and other stages (figure 7). we analyzed whether mean values of all the parameters were higher in stage iii than in iib. mean value of bv in stage iib (41.70 ml/100 g) was lower than in stage iii (46.63 ml/100 g) (p=0.002). additionally, mean value of bv in stage iii (5.67 ml/100 g) was higher than in stage iib (4.62 ml/100 g) (p=0.028). similarly, mtt and ps were significantly higher in stage iii than in iib (p=0.014 and 0.003, respectively). mean mtt ranged from 8.62 s to 7.93 s and ps from 35.45 ml / min/100 g to 28.89 ml / min/100 g in class iii and iib, respectively (table 4). to conclude, perfusion values are higher in higher cancer stages according to ajcc. between 2007 and 2012, abdominal and pelvic ct and prostate gland perfusion ct were performed in 161 patients who were eligible for the study protocol and signed informed consent. a total of 125 patients with pca (median age, 64 years ; range, 4977) met the selection criteria and were enrolled in study number 2 in the years 20072012 (figure 4). typical contrast enhancement of malignant lesions was visible in 119 out of the 125 patients (sensitivity 95.2%). a statistically significant correlation between bv, mtt, and ps values and psa level was found : correlation coefficients (r) were equal to 0.18 ml/100 g, 0.2 s and 0.24 ml / min/100 g (p0.05), respectively (table 2). these findings suggest that with an increase of psa values, an increase of bv, mtt, and ps can be expected (figure 5). mean values of perfusion parameters in malignant tissue were : bv 5.080.2 ml/100 g, bf 43.791.15 ml / min/100 g, mtt 8.240.15 s, and ps 31.471.2 ml / min/100 g. the mean values and range interval of perfusion parameters based on histological malignancy category of the tumors are presented in table 3. figure 6 illustrates differences between mean perfusion values (bv, bf, and ps) for poorly, moderately, and well - differentiated tumors according to gleason score. a statistically significant difference (p=0.02) was observed between bv values in well - differentiated tumors (4.85 ml/100 g) compared to poorly differentiated (6.37 ml/100 g) tumors. g in poorly differentiated tumors (p=0.01), whereas mean value of ps increased from 29.76 ml / min/100 g for well - differentiated tumors to 39.77 ml / min/100 g for poorly differentiated tumors and the difference between those values was also significant (p=0.02). mean values of bv, bf, and ps were higher in poorly than in moderately differentiated tumors, but statistical significance was achieved only for bf values (p=0.03). on the basis of variance analysis we found statistical correlation between mean perfusion values bv, mtt, ps and cancer stage, according to ptnm classification (p<0.03) (table 4). the analysis of individual tests comparing various averages revealed a significant difference between mean values of perfusion parameters (bv, mtt, and ps) for category iib and other stages (figure 7). we analyzed whether mean values of all the parameters were higher in stage iii than in iib. mean value of bv in stage iib (41.70 ml/100 g) was lower than in stage iii (46.63 ml/100 g) (p=0.002). additionally, mean value of bv in stage iii (5.67 ml/100 g) was higher than in stage iib (4.62 ml/100 g) (p=0.028). similarly, mtt and ps were significantly higher in stage iii than in iib (p=0.014 and 0.003, respectively). mean mtt ranged from 8.62 s to 7.93 s and ps from 35.45 ml / min/100 g to 28.89 ml / min/100 g in class iii and iib, respectively (table 4). to conclude, perfusion values are higher in higher cancer stages according to ajcc. the aim of our study was to assess the correlation between ct perfusion parameters and psa levels, gleason score, ptnm, and ajcc stage in 125 patients with pca. to the best of our knowledge, this is the largest published series of pca patients examined with pct. results of our analysis confirm high sensitivity of the method in visualization of malignant tissue in the peripheral prostate zone (luczynska.). perfusion ct is a valuable method for diagnosis of prostate cancer (see our previous prospective study in 94 patients, research grant no. nn403240837 of the polish ministry of science and higher education, submitted for publication), which suggests that pct parameters correlate with psa values, gleason score, and ptnm stage and can be useful for initial tumor staging and the definition of tumor aggressiveness. a significant correlation between bv, bf, and ps values and psa level was found, as well as a significant difference between bv, bf, and ps in poorly and moderately differentiated tumors in comparison with highly differentiated pca (p<0.08). the analysis also revealed a correlation between mean perfusion values bv, mtt, and ps and ptnm cancer stage (p<0.04). all perfusion parameters were higher in stage iii than in stage iib tumors. digital rectal examinations, psa levels with associated parameters (psa density, psa adjusted to age, excess psa, and free - psa transitional zone), and color doppler trus - guided biopsies are the main diagnostic tools for evaluation of men at risk for carcinoma of the prostate. trus - guided biopsy is the best technique for detecting cancer in patients with elevated psa or a positive digital rectal examination, but this procedure has some limitations (3). only 2040% of repeated biopsies are associated with positive findings in men with persistently elevated psa serum levels. the method provides the best depiction of the contours of the prostate as well as its internal zonal anatomy. mri also allows functional assessment with techniques such as diffusion - weighted mri (dwi), mr spectroscopy, and dynamic contrast - enhanced mri (dce - mri). although mri currently seems to be the most valuable method of pca imaging, it is necessary to keep in mind contraindications to this test as well as limitations in access to mri devices in low - income countries. therefore, other imaging methods, like pct, should be considered in pca imaging. the development of pca is a multi - step process, advancing consecutively from high - grade prostatic intraepithelial neoplasia (pin) to focal carcinoma, invasive carcinoma, and finally to metastatic disease. it is therefore important to target the molecular events that accompany the progression of each step. we were able to evaluate pca angiogenesis with the latest multidetector tomography and acquisition of high - quality, color - coded perfusion maps, which correspond with the classic tomography imaging. with the development of new multidetector ct scanners, quantitative ct imaging of angiogenesis various algorithms have been developed to quantify perfusion based on contrast - enhanced ct imaging. therefore, angiogenesis within soft - tissue neoplasms is related to contrast enhancement on ct. initial reports show that functional imaging in ct, such as pct, is important for pca staging before radical therapy. pct makes it possible to reveal excessive pathological vessel density within the prostate gland due to the increase of bv and bf values and also because pathological increase of blood vessel permeability (ps) mtt is shortened, which confirms vascular abnormality in the area. we previously proved the high sensitivity of pct in visualization of pca and confirmed this in the current study. correlations between pct parameters and clinico - pathological findings also remains in accordance with previous published reports ; for example, prando. state that ct of the prostate may be useful in the following selected groups : patients with a rising psa level and negative biopsies ; patients with elevating psa level after radical prostatectomy (suspicion of recurrence) ; and patients with routine helical ct pelvic examinations during which abnormal focal contrast enhancement in the peripheral zone is observed. the correlation between serum psa level and perfusion parameters was also described elsewhere. in the present series, the difference between mean perfusion parameters in patients with low and high gleason scores is clear. ives. demonstrated that the positive correlation between pct and malignancy was strongest in patients with high gleason scores (8). our findings suggest that quantitative perfusion measurements based on ct correlate with tumor values and may be useful in poorly differentiated tumors. according to the analyzed material, perfusion values clinical stage of pca division is based on 3 values : ptnm, psa, and gleason score. our findings suggest that higher tnm, psa, and gleason score are associated with higher perfusion parameters (bv, ps, and mtt). it has been demonstrated, however, that pathological vessel density is significantly increased compared with normal prostate gland tissue. maximal vascular density was observed in poorly differentiated pca ; moreover, reports show that pathological density of vessels within pca is one of the factors that can determine whether the neoplastic process is confined to the gland or extends beyond its capsule. it was previously determined that pathological vessel density is greater in patients with neoplastic process extending beyond the prostate gland. however, no published data are available on the correlation between ptnm stage and pct parameters in pca. consequently, our findings are also unclear, because pct parameters were higher in stage iii tumors than in stage iib, but not higher than in iia tumors, because of the relatively small number of iia tumors. also report the significance of blood vessel density within the tumor (in case of cancer confined to the gland) for pca staging. for example, in calculating the probability of cancer infiltration beyond the gland for a patient with psa serum level 8 ng / ml, gleason score after core needle biopsy and high vessel density in histopathological examination is about 93%. these are the lack of a control group, which did not allow calculating the specificity of the method, and poor visibility of the anatomy of the prostatic capsule with conventional ct or additional exposure of patients to radiation, which, in our study was equal to 16.15 msv. the dose is higher than that from a conventional ct scan but seems acceptable for whole - body examination. in conclusion, our study confirms previous findings of the high sensitivity of pct for differentiation of pca from normal prostate tissue, and suggests that pct parameters correlate with psa values, gleason score, and ptnm stage and can be useful for initial tumour staging. a significant correlation between bv, bf, and ps values and psa level was found, as well as the trend for difference between bv, bf, and ps in poorly and moderately differentiated tumors in comparison with highly differentiated ones. the analysis also revealed a correlation between mean perfusion values (bv, mtt, and ps) and ptnm cancer stage. our results may impact daily clinical practice, particularly for pretreatment staging of pca in situations when routine access to mri is not available. | backgroundthe aim of the study was to assess the correlation between computed tomography perfusion (pct) parameters and psa levels, gleason score, and ptnm stage in patients with prostate cancer (pca).material / methodsone hundred twenty - five patients with localized pca were prospectively enrolled in the study. all patients were diagnosed due to suspicious prostate findings and elevated psa serum levels and underwent pct followed by core biopsy and radical prostatectomy. blood flow (bf), blood volume (bv), mean transit time (mtt), and permeability - surface (ps) area product were computed in the suspected pca area and normal prostatic tissue. core biopsy followed by prostatectomy was performed 24 weeks after pct. correlation between pct findings and psa levels, gleason score, and ptnm stage were analyzed.resultsthe mean age of patients was 64 years. all patients had elevated psa levels (mean value 6.2 ng / ml). nineteen patients (15.9%) were at low risk of recurrence, 91 (76.5%) were at moderate risk, and 9 (7.6%) were at high risk according to national comprehensive cancer network criteria. pca was visible on pct as focal peripheral ct enhancement in 119 out of 125 patients (sensitivity 95.2%). significant correlations between bv, bf, and ps values and psa level were found (p<0.05), as well as a trend for difference between bv, bf, and ps in poorly and moderately differentiated tumors (according to gleason score) in comparison with highly differentiated pca (p<0.08). the analysis also revealed a correlation between mean perfusion values and bv, mtt, ps, and ptnm cancer stage (p<0.04).conclusionsour study suggests that in low- and intermediate- risk patients, pct parameters correlate with psa values, gleason score, and ptnm stage and can be useful for initial tumor staging. |
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