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the main reasons for the widespread use of thyroid sonography are availability, low cost, limited discomfort to the patient, and absence of ionizing radiation. sonography has many favourable features, such as detection of nonpalpable nodules, estimation of nodule size / goiter volume, and guidance for fine needle biopsy (fnb). high - resolution ultrasound is very sensitive in detection of thyroid nodules, enabling differentiation of solid and liquid lesions. however, with introduction of sonography it became evident that thyroid nodules are very common, with prevalence ranging from 17% to as much as 67% in some cohorts. nodular goiter does not include thyroid cancer, but one of the main aims of the clinical evaluation is to exclude the risk of overlooking thyroid cancer which is much less prevalent than benign nodules. sonographic characteristics such as hypoechogenicity, microcalcifications, and increased nodular flow visualized by doppler are all to some extent predictive of malignancy. microcalcifications visible on ultrasound examination are considered to be a specific feature of thyroid cancer (85,895%) ; however, the sensitivity of this sign is relatively low (2959%). presence of calcifications doubles the risk of malignancy, whereas microcalcifications increase the risk of thyroid cancer three - fold. however, in pathologic examinations of benign thyroid nodules - inspissated colloid, fibrosis, and microcalcifications often coexist. palpation the earliest and most common form of tissue hardness estimation was practiced by egyptian physicians as early as 2600 bc. a more recent and sophisticated method of imaging of tissue hardness is the technique known as elastography. the term elastography was coined by ophir. to refer to an ultrasound - based imaging technique, where local axial strains were estimated by computing the gradient of axial shifts in echo arrival times along the ultrasound beam direction following quasistatic tissue deformation. elastography, however, has now been used as a more general term to identify methods that image tissue stiffness, using different imaging modalities for example, ultrasound, magnetic resonance imaging, optical coherence tomography, different perturbation techniques to deform tissue, based on the elasticity parameter being measured or imaged. roughly 20 years have elapsed since the first images depicting the local elastic properties of tissues were obtained. the first decade of development produced a remarkable proliferation of techniques and optimization strategies. in the second decade this trend continued, but with an important extension to dedicated platforms for conducting clinical trials in the hands of radiologists and skilled clinicians. there are 3 main types of ultrasound elasticity imaging : elastography that tracks tissue movement during compression to obtain an estimate of strain (quasi - static elastography), sonoelastography that uses color doppler to generate an image of tissue movement in response to external vibrations (harmonic elastography), and a technique tracking shear wave propagation through tissue to obtain the elastic modulus (transient elastography). the first commercially available, clinical ultrasound scanners with option of tissue elasticity evaluation were equipped with strain elastography. application of strain elastography to thyroid nodules examination resulted in different results of evaluation of usefulness of this technique in regard to differential diagnosis of benign and malignant thyroid nodules [914 ]. diagnostic value of strain elastography is limited in evaluation of small nodules, large nodules (with diameter approaching or exceeding the length of the probe), nodules with calcifications, and nodules with liquid content. different results are obtained in different anatomical planes (e.g., axial versus sagittal), and the reproducibility is poor. finally, multinodular goiters with scarce or no normal thyroid tissue for reference are difficult to evaluate [9, 1116 ]. the new generation of elasticity imaging called supersonic shear wave elastography (sswe) has been introduced since 2006 to imaging of superficial organs as breast and thyroid with high - frequency linear probes [1721 ]. this type of transient elastography does not require the compression of the tissues during their elasticity examination. the obtained information is based on calculated elastic modulus (described in kpa) of the examined tissues. based on multinational large - scale studies in the field of breast cancer detection and characterization, sswe proved to be highly reproducible, and it increased specificity without loss of sensitivity [20, 21 ]. in the field of thyroid sswe, it was proved that autoimmune thyroiditis does not hinder the evaluation of elasticity of thyroid nodules. the aim of this study was to compare the usefulness of the new supersonic shear wave imaging elastography with strain elastography in evaluation of thyroid nodules. during a few weeks trial time in 2010, four consecutive patients with single thyroid nodule (n = 1) and nodular goiter (n = 3) were evaluated. approval for this study was obtained from the ethics committee of the medical university of warsaw, and all patients provided informed consent. the b mode and power doppler ultrasound of whole thyroid and neck lymph nodes was performed. six dominant thyroid nodules (in regard to b - mode and power doppler ultrasound features) were evaluated with shear wave and strain elastography qualitatively and quantitatively as well as some with contrast - enhanced ultrasound (sonovue (bracco)). france)sswe, aplio xg (toshiba, japan)strain elastography, technos (esaote, italy)contrast - enhanced ultrasound, with linear high - resolution transducers : 154 mhz, 187 mhz, and, 83 mhz respectively. for strain elastography, we adopted qualitative scale of rubaltelli. with threshold score of 2/3 and quantitative scale of cantisani. with threshold thyroid tissue / nodule strain ratio of 2 measured with elasto - q (toshiba). for shear wave elastography, we adopted quantitative scale of sebag. with the threshold stiffness (mean elastic modulus) of thyroid nodule of 65 kpa. the final diagnosis was based on clinical evaluation, multiple fnb, 1 year followup, or surgery. final diagnosis (pathology examination after surgery in 5 nodules, double fnb, and 1 year followup in 1 nodule) was established : 1 papillary carcinoma (figure 1), 4 colloid nodules, and 1 benign nodule (figure 2). shear weave elastography revealed 1 true positive and 5 true negative diagnoses in regard of thyroid cancer. false positive diagnoses with strain elastography were found in nodules with liquid (not evident on b - mode ultrasound) or degenerative content of the nodules visible on contrast - enhanced ultrasound and/or pathology examination. a novel finding were the punctate increased stiffness foci in microcalcifications seen in 4 nodules, some not visible on b - mode ultrasound as opposed to soft inspissated colloid foci visible on b - mode ultrasound in 2 nodules (figure 3). supersonic shear weave elastography consists of the generation of remote radiation force by focused ultrasonic beams, the so - called pushing beams, a patented technology called sonic touch. using sonic touch, the source is moved at a speed that is higher than the speed of the shear waves that are generated. in this supersonic regime, shape, which increases their amplitude and improves their propagation distance. for a fixed acoustic power at a given location, sonic touch increases shear wave generation efficiency by a factor of 4 to 8 compared to a nonsupersonic source. after generation of this shear wave, an ultrafast echographic imaging sequence is performed to acquire successive raw radiofrequency dots at a very high - frame rate (up to 20000 frames per second). based on young 's modulus formula, a color - coded image is displayed, which shows softer tissue in blue and stiffer tissue in red. quantitative information is delivered ; elasticity is expressed in kilo - pascal (kpa). this preliminary paper based on small number of cases indicates that sswe indicated correctly thyroid nodules suspicious for cancer in contrast to strain elastography. false positives on strain elastography could be due to liquid or degenerative content of nodules. however, imaging with sswe, as a sensitive method of evaluation of stiffness of human tissue, the operator should be aware of physiological processes influencing the elasticity and easily apply a few rules to avoid artifacts (figures 4, 5 and 6) (supplementary material 1-cine loop video). among well - known artifacts on sswe that should be mentioned is the one that can be encountered in any region when the sswe can be applied : the increased stiffness of the structures under externally applied pressure (figures 4 and 5) (supplementary material 1-cine loop video) that can be due to nonlinear elastic effects, well explained by theory. another artifact that can be encountered in thyroid sswe is one of increased stiffness in the isthmus of the thyroid due to trachea (figure 6). it can be avoided with imaging in paracoronal plane of the nodule that does not incorporate the trachea. however, it is important to state that these artifacts when properly interpreted do not hinder the accurate diagnosis. supersonic shear wave elastography may add a new dimension to ultrasound evaluation of thyroid nodules in several ways, for example : improve general performance in elasticity differentiation of thyroid nodules over strain elastography due to its high reproducibility, independence of examiners skill and numeral scale of elasticity measurement in kpa ; overcome the limitations of strain elastography : nodules with liquid components or with degenerative changes;small nodules (very good spatial resolution of the technique);large nodules (possibility of subsequent determination of stiff regions even of large nodules, without the need of visualizing the whole nodule on one image);multinodular goiter with no or scarce normal thyroid tissue as a reference ; nodules with liquid components or with degenerative changes ; small nodules (very good spatial resolution of the technique) ; large nodules (possibility of subsequent determination of stiff regions even of large nodules, without the need of visualizing the whole nodule on one image) ; multinodular goiter with no or scarce normal thyroid tissue as a reference ; differentiation between soft - inspissated colloid and stiff microcalcifications ; visualization of microcalcifications, even not visualized on b - mode imaging (may increase sensitivity and decrease specificity of thyroid cancer diagnosis) ; introduction of three - dimensional elastographic images to routine clinical practice and to national thyroid cancer databases, as this technique is already available and enables rapid acquisition of 3d ultrasound and elastographic data. this would devoid diagnostic process and data archiving of image selection bias attributable to 2d ultrasound examination. further multicenter large scale studies of thyroid nodules evaluating different elastographic methods are warranted, including (a) investigation of developmental models of diseases that link biomechanical properties (elastography findings) with genetic, cellular, biochemical, and gross pathological changes ; (b) comparison of accuracy of different elastographic methods ; (c) establishment of optimal diagnostic elastographic criteria ; (d) establishment of limitations of different elastographic methods in relation to evaluation of thyroid pathology. | although elastography can enhance the differential diagnosis of thyroid nodules, its diagnostic performance is not ideal at present. further improvements in the technique and creation of robust diagnostic criteria are necessary. the purpose of this study was to compare the usefulness of strain elastography and a new generation of elasticity imaging called supersonic shear wave elastography (sswe) in differential evaluation of thyroid nodules. six thyroid nodules in 4 patients were studied. sswe yielded 1 true - positive and 5 true - negative results. strain elastography yielded 5 false - positive results and 1 false - negative result. a novel finding appreciated with sswe, were punctate foci of increased stiffness corresponding to microcalcifications in 4 nodules, some not visible on b - mode ultrasound, as opposed to soft, colloid - inspissated areas visible on b - mode ultrasound in 2 nodules. this preliminary paper indicates that sswe may outperform strain elastography in differentiation of thyroid nodules with regard to their stiffness. sswe showed the possibility of differentiation of high echogenic foci into microcalcifications and inspissated colloid, adding a new dimension to thyroid elastography. further multicenter large - scale studies of thyroid nodules evaluating different elastographic methods are warranted. |
dental caries is a multifactorial disease caused by interactions between acidogenic bacteria, biofilm and individual caries risk factors (e.g., saliva composition, fluoride exposure and dietary components). in a global context, its prevalence is still high, particularly in children. because dental caries is preventable, caries risk assessment (cra) is an important tool assisting the dentist to better understand the cariogenic profile of a patient. the process includes collecting relevant historical data of the patient, such as the medical and dental history. the complex nature of caries risk has led to the development of different protocols that are not all validated. cariogram, a software program from sweden, stands out as it has been clinically proven to be effective in evaluating caries risk. the outcomes are presented graphically to the patient, indicating the probability of avoiding new carious lesions. cariogram is based on a set of pathological and protective factors - caries experience, systemic diseases, diet contents and frequency, amount of plaque, mutans streptococci, fluoride sources, saliva secretion and buffer capacity - in addition to the professional clinical judgment. as some factors are considered more relevant than others regarding caries development, most information is easily collected at the dental practice, but saliva and bacteria assessment require specific tests which are not frequently performed by a dentist. the use of cariogram is limited to places where a computer system is available and where those tests can be performed. for that reason, alternatives ways of performing cra have been proposed ; it is suggested that the best way of implementing cra in clinical practice would be by the use a form, ensuring that all patients could be assessed systematically in the same manner. the main differences between the cariogram software and the new form include the following : filling in the form does not require a computer ; the variables " mutans streptococci ", " salivary buffer capacity " and " clinical judgement " are excluded and the form assesses risk based on the sum of values attributed to each etiological factor. finally, it classifes the patient as having a low, moderate or high caries risk and provides treatment guidelines for each category. this study aimed to determine whether the newly developed cra form could classify schoolchildren according to their caries risk and to evaluate relationships between caries risk and the variables in the form. this study is part of a clinical trial that aims to compare the performance of different sealant materials in the prevention of dental caries. therefore, children considered at moderate and high caries risk needed to be identified. with that purpose, a survey was carried out in an elementary school located in parano, which is a deprived area of brazil 's federal district. all 298 children aged 5 to 7 years old and enrolled at school were invited to participate. children whose parents did not sign the informed consent form and those who needed special care were excluded (figure 1). the study was approved by the ethics committee of the school of medicine of the university of braslia (reference no. information about the prevalence of dental caries was obtained according to international caries detection and assessment system (icdas) ii. the dentist was trained in using icdas, having completed its e - learning program. additionally, a pilot study was also conducted at the braslia university hospital for the examiner calibration covering other oral conditions that were assessed : toothache, gingival bleeding and visible plaque. the oral examinations took place in a dental office located inside the school, with the children sitting on a conventional dental chair under optimal lighting. a plane buccal mirror, who - cpi probe and air syringe were used in the clinical examination. then, the examiner cleaned all tooth surfaces with only a toothbrush, after which dental caries was recorded in primary and permanent teeth. caries risk was assessed according to a form based on cariogram, in which seven variables were included, as shown in figure 2. the form is able to convert into scores the information that was collected about each patient. according to the cariogram built - in algorithm, each variable in the form was weighted at a relevant ratio and the scores were assigned. variables assessed, their description, the instrument used and the scores of each variable according to severity dmft = decayed, missing, filled teeth the variables " caries experience ", " oral hygiene " and " salivary flow " were obtained clinically. for all other variables, the information was retrieved from questionnaires previously sent to parents. for calculating caries experience, data were converted into dmft / dmft scores according to amorim,. all children were classified as having a normal salivary flow during the clinical examination. after completion of the forms and summing up the scores for all variables, each child was classified as belonging to a low (033), moderate (3466) or high caries risk (> 67) group. first, linear regressions were performed, considering caries risk as a dependent variable and the variables presented in figure 2 as the independent ones. this study is part of a clinical trial that aims to compare the performance of different sealant materials in the prevention of dental caries. therefore, children considered at moderate and high caries risk needed to be identified. with that purpose, a survey was carried out in an elementary school located in parano, which is a deprived area of brazil 's federal district. all 298 children aged 5 to 7 years old and enrolled at school were invited to participate. children whose parents did not sign the informed consent form and those who needed special care were excluded (figure 1). the study was approved by the ethics committee of the school of medicine of the university of braslia (reference no. information about the prevalence of dental caries was obtained according to international caries detection and assessment system (icdas) ii. the dentist was trained in using icdas, having completed its e - learning program. additionally, a pilot study was also conducted at the braslia university hospital for the examiner calibration covering other oral conditions that were assessed : toothache, gingival bleeding and visible plaque. the oral examinations took place in a dental office located inside the school, with the children sitting on a conventional dental chair under optimal lighting. a plane buccal mirror, who - cpi probe and air syringe were used in the clinical examination. then, the examiner cleaned all tooth surfaces with only a toothbrush, after which dental caries was recorded in primary and permanent teeth. caries risk was assessed according to a form based on cariogram, in which seven variables were included, as shown in figure 2. the form is able to convert into scores the information that was collected about each patient. according to the cariogram built - in algorithm, each variable in the form was weighted at a relevant ratio and the scores were assigned. variables assessed, their description, the instrument used and the scores of each variable according to severity dmft = decayed, missing, filled teeth the variables " caries experience ", " oral hygiene " and " salivary flow " were obtained clinically. for all other variables, the information was retrieved from questionnaires previously sent to parents. for calculating caries experience, data were converted into dmft / dmft scores according to amorim,. all children were classified as having a normal salivary flow during the clinical examination. after completion of the forms and summing up the scores for all variables, each child was classified as belonging to a low (033), moderate (3466) or high caries risk (> 67) group. first, linear regressions were performed, considering caries risk as a dependent variable and the variables presented in figure 2 as the independent ones. the sample population comprised 150 children (81 girls and 69 boys) with a mean age and standard deviation of 6.80.62. the majority of them were from low - income families (less than one brazilian minimum wage). caries prevalence in primary dentition, including enamel and dentine carious lesions, was 98.6%, and 77.3% when only dentine lesions were considered. the mean dmft score was predominantly influenced by the d - component, as only 18.6% children presented fillings or extracted teeth. the permanent dentition caries prevalence, including enamel and dentine lesions, was 34% and when only dentine lesions were considered, 12.6%. intra - examiner reliability in diagnosing carious lesions was determined by re - examining 9.3% of the children. the kappa - coefficient value was o.924, showing a high level of reliability. the mean dmft scores of the children classified as being at low, moderate or high risk, according to the form based on cariogram, are presented in table 1. distribution of variables scores according to assessed caries risk is presented in table 2. children distribution and mean dmft according to caries risk dmft = decayed, missing, filled teeth. sd= standard deviation distribution of variables scores occurrence according to caries risk assessed in order to establish the variables that influenced caries risk the most, a univariate model was applied, including all variables presented in figure 2. results are presented in table 3 and show that the variable " related diseases " was the only one that did not have a statistically significant effect on the caries risk profile. variables included in the univariated model the significant factors were then included in a multivariate forward model, showing that the most relevant variable in caries risk prediction was caries experience (table 4). the sample population comprised 150 children (81 girls and 69 boys) with a mean age and standard deviation of 6.80.62. the majority of them were from low - income families (less than one brazilian minimum wage). caries prevalence in primary dentition, including enamel and dentine carious lesions, was 98.6%, and 77.3% when only dentine lesions were considered. the mean dmft score was predominantly influenced by the d - component, as only 18.6% children presented fillings or extracted teeth. the permanent dentition caries prevalence, including enamel and dentine lesions, was 34% and when only dentine lesions were considered, 12.6%. intra - examiner reliability in diagnosing carious lesions was determined by re - examining 9.3% of the children. the kappa - coefficient value was o.924, showing a high level of reliability. the mean dmft scores of the children classified as being at low, moderate or high risk, according to the form based on cariogram, are presented in table 1. distribution of variables scores according to assessed caries risk is presented in table 2. children distribution and mean dmft according to caries risk dmft = decayed, missing, filled teeth. sd= standard deviation distribution of variables scores occurrence according to caries risk assessed in order to establish the variables that influenced caries risk the most, a univariate model was applied, including all variables presented in figure 2. results are presented in table 3 and show that the variable " related diseases " was the only one that did not have a statistically significant effect on the caries risk profile. variables included in the univariated model the significant factors were then included in a multivariate forward model, showing that the most relevant variable in caries risk prediction was caries experience (table 4). caries risk assessment is an important tool that contributes to identification of risk factors and guides the dental professional in the decision - making process involved in effective prevention and management of dental caries. the common practice is to assess these factors individually but, as dental caries is a multifactorial disease, more than one factor can predict future caries. therefore, the use of validated instruments for guiding the professional in establishing the risk profile of a patient is essential. for that reason, the form based on the cariogram software, which has been validated in schoolchildren, was chosen for use in this investigation. it includes factors involved in the caries process which can be easily assessed, clinically or with questionnaires, without requiring a computer system, thus allowing, in our case, its application in a school located in a low - income community. the fact that neither salivary buffer capacity nor microbial tests were included in the form can be justified by the low predictive values of both tests in relation to dental caries. it is known that in the presence of fluoride, a high number of mutans streptococci and/or lactobacilli may be tolerated in the oral fora without causing any harm to the teeth. for the univariate model used in the present study, all variables that are included in the form based on cariogram were analyzed, except " salivary flow ", as none of the children presented clinical aspects of hyposalivation. for the forward stepwise multiple regression model, five variables were included in the analysis and, together, could explain 90.4% of the caries risk observed in the sample. these results are corroborated by previous studies using the cariogram software, which showed high correlation between caries experience and caries risk profiles. furthermore, caries experience is considered the strongest predictor for future caries, even when other forms of cra are used. results show that the form based on cariogramwas able to classify the schoolchildren according to the three caries risk categories : low, moderate and high. however, few children were allocated to the low and high categories. regarding the low risk group, this finding can be ascribed to the high caries prevalence among the children included in the sample. this outcome is in line with a previous study conducted in the same area, where caries prevalence was 67% for children in the same age group. however, how sure are we that children with a mean dmft of 3.38 (86% of whom were classified as belonging to the moderate risk group) do not belong to the high caries group ? these children had a low proportion of missing and filled teeth and a high proportion of decayed teeth. the variable " caries experience ", even though it was the most relevant for caries prediction in the analyzed form, might still not show enough influence on the overall results, since children with an average high dmf score were mostly considered as being at only moderate risk of developing caries, particularly those living in areas with fluoridated water and access to fluoridated toothpaste. it is important to discuss the weighting given to the " fluoride sources " variable in the form. among all factors included in the form, the complete absence of a fluoride source is the one that counts the most for the fnal outcome (50 points). this makes sense as the effectiveness of fluoride in preventing and controlling caries progression has been highly documented. however, in the present study, all children received the lowest score (zero) for the variable " fluoride source ", as they live in an area which has fluoridated water (0.07 ppm), and brush their teeth with fluoridated toothpaste. another variable that may be responsible for the low differentiation of the children between caries risk groups was " salivary flow ", since the absolute majority of the examined children presented normal salivary flow. firstly, it is common to observe an increase in salivary flow rate with an increase in childhood age. furthermore, children who suffer from dental caries generally do not present clinical aspects of decreased salivary secretion and, if present, these are often related to systemic diseases. the long - term use of sugary medications, as well as the presence of systemic diseases, is related to an increment in caries risk. however, the form was effective in classifying the children with the highest dmft scores (> 4) as having a high risk of developing carious lesions. it was observed that, although these children received the lowest scores for " fluoride sources " and " salivary flow ", they got maximum scores for all other factors related to diet and hygiene included on the form, showing the multifactorial etiology of dental caries. these patients would normally score 68 points, just 1 point above the borderline between moderate and high caries risk. any minimal control of diet or hygiene would already lead them into the moderate risk group. further discussion of the proper weighting of the variables in the simplified form is encouraged. also, it is important to note that the outcome of the cariogram software is the chance (%) of avoiding new carious lesions, while the simplified form classifes children according to three risk groups categories. transformation of cariogram results into risk groups has already been performed, using five subgroups according to the chance of avoiding new lesions [very low (0 - 20%), low (21 - 40%), medium (41 - 60%), high (61 - 80%) and very high (81 - 100%) ]. the school in which the survey was carried out is located in an unprivileged community with difficulties in access to health care. this can explain, in part, the high number of children presenting dental treatment needs. also, this fact reinforces the " feeling " of the authors that the number of children identified as high - risk should be higher. in the cariogram software a professional clinical judgement variable allows the examiner to increase the risk for patients that are believed to have a higher chance to develop compared to the data results only. maybe, in the simplified form, in which this variable is absent, a modification in the caries risk group threshold values could act as the clinical judgment variable of the original software. the fact that a substantial number of parents did not sign the informed consent form might suggest that their children were caries - free and the parents did not feel that exposing them to a dental examination was a necessity. if this assumption is correct, the absence of these children could help to explain the very high prevalence of caries in the studied population and, therefore, some of the results found in the analysis of the form usage. it is suggested that the form based on cariogramshould be applied in a population with different oral health needs, in order to confirm the ability of the instrument to properly identify subjects who are at low risk. the form based on the cariogram software was able to classify the schoolchildren according to low, moderate and high caries risk, with a large concentration in the moderate risk group, despite the high average dmft. caries experience, oral hygiene, frequency of food consumption, sugar consumption and fluoride sources are the variables that were shown to be highly correlated with caries risk. | identifying caries risk factors is an important measure which contributes to best understanding of the cariogenic profile of the patient. the cariogram software provides this analysis, and protocols simplifying the method were suggested.objectivesthe aim of this study was to determine whether a newly developed caries risk assessment (cra) form based on the cariogram software could classify schoolchildren according to their caries risk and to evaluate relationships between caries risk and the variables in the form.material and methods150 schoolchildren aged 5 to 7 years old were included in this survey. caries prevalence was obtained according to international caries detection and assessment system (icdas) ii. information for filling in the form based on cariogram was collected clinically and from questionnaires sent to parents. linear regression and a forward stepwise multiple regression model were applied to correlate the variables included in the form with the caries risk.resultscaries prevalence, in primary dentition, including enamel and dentine carious lesions was 98.6%, and 77.3% when only dentine lesions were considered. eighty - six percent of the children were classified as at moderate caries risk. the forward stepwise multiple regression model result was significant (r2=0.904 ; p<0.00001), showing that the most significant factors influencing caries risk were caries experience, oral hygiene, frequency of food consumption, sugar consumption and fluoride sources.conclusionthe use of the form based on the cariogram software enabled classification of the schoolchildren at low, moderate and high caries risk. caries experience, oral hygiene, frequency of food consumption, sugar consumption and fluoride sources are the variables that were shown to be highly correlated with caries risk. |
ubb, a loss - of - function variant of ubiquitin b (ubb), accumulates in neurofibrillary tangles, a pathological hallmark in alzheimer s disease (ad) (van leeuwen., 1998). ubb is translated from an aberrant mrna encoding a + 1 frameshift protein in which the c - terminal glycine residue required for ubiquitylation is replaced by an extension of 20 amino acids (dennissen., 2010). the detrimental impact of ubb has been studied in neuronal cell cultures, transgenic mice, and yeast (de vrij., 2001 ; ubb is a substrate for truncation, ubiquitylation, and proteasomal degradation (dennissen., 2011 ; lindsten., 2002 ; whereas the ubiquitin - proteasome system (ups) can assure the degradation of low levels of ubb, higher levels impair the ups and subvert the homeostatic mechanisms allowing for its elimination (fischer., 2009 ; lindsten., 2002 ; at high levels, ubb affects mitochondrial dynamics and triggers neuronal cell death (de vrij., 2001 ; tan., 2007) through as - yet elusive mechanisms. yeast is an established model for studying programmed cell death mechanisms that are often shared with animal cells, including the contribution of caspases and mitochondrion - associated cell death proteins, such as cytochrome c (carmona - gutierrez., 2010). yeast models have been used to explore cell killing by neurotoxic proteins, such as parkinson - disease - associated -synuclein, and the outcome could be successfully translated to fly, worm, and murine disease models, as well as to human disease (braun., 2010 ; bttner., 2013). driven by these premises, we established a yeast cell death model for ubb - triggered neurotoxicity. our findings revealed that ubb interfered with the ups and triggered the perturbation of the mitochondrion - associated basic amino acid synthesis executing cell death. the mitochondrion - associated ups subroutine, depending on the aaa - atpase cdc48 and its co - factor vms1, strongly antagonized ubb cytotoxicity. since vms1, the human homolog of yeast vms1, co - exists with ubb in neurofibrillary tangles, these data imply a potential pivotal role of the ups at mitochondria in ad. to investigate whether the introduction of ubb into yeast recapitulates hallmarks of ubb accumulation in neurons, we expressed monomeric ubiquitin b (ubb), ubb, as well as an ubb variant lacking two lysine residues (k29,48r) that are important for its ubiquitylation. when expressing ubb, we detected a discrete immunoreactive band at the size of monomeric ubiquitin (9 kda), and an immunoreactive smear across a wide range of the immunoblot that corresponds to ubiquitylated proteins (figure 1a). this smear was not detectable upon transformation with ubb or ubb - k29,48r, reflecting their loss of function. instead, ubb or ubb - k29,48r were detectable as 12 and 9 kda protein species (full - length and truncated ubb ; fl - ubb and tubb) that accumulated over time (figures 1a, s1a, and s1b). in cells expressing ubb, a faint higher molecular weight species corresponding to the size of monoubiquitylated fl - ubb (21 kda) appeared (figure 1a, flag long exposure, asterisks). consistent with a role of lysines 29 and/or 48 in the ubiquitylation of ubb, this band was absent in cells expressing ubb - k29,48r. these results suggest that in yeast human ubb (but not ubb) can serve as a substrate for ubiquitin ligases and that, like in neurons, ubb is ubiquitylated and truncated. next, we investigated whether ubb expression results in ups impairment by means of three complementary assays : (1) the measurement of polyubiquitylated endogenous proteins by immunoblot ; (2) the assessment of the abundance of transgenic ubiquitin - g76v - gfp, which is a substrate of the ubiquitin - fusion degradation pathway ; and (3) an enzymatic assay designed to quantify the chymotrypsin - like proteasomal activity. cells expressing ubb or ubb - k29,48r contained a higher level of polyubiquitylated proteins than cells transformed with vector controls (figures 1b and 1c), suggesting decreased ups - dependent protein turnover. the steady - state levels of ubiquitin - g76v - gfp were significantly increased upon expression of ubb or ubb - k29,48r (figure 1d). in contrast, ubb or ubb - k29,48r expression did not reduce chymotrypsin - like proteasomal activities (figure s1c). these data suggest that, in yeast like in neurons, ubb expression impairs the ups. however, in yeast ubb does neither directly affect the enzymatic activity of proteasomes, nor is its ubiquitylation essential for ups dysfunction. to assess its effects on the fitness of proliferating cells, we performed growth assays on agar plates and in liquid cultures. as a positive control of cytotoxicity, tdp-43, a causal factor for motor neuron degeneration, was expressed. in sharp contrast with tdp-43, ubb and ubb - k29,48r failed to compromise the growth of cells on agar plates (figure 2a), and in liquid cultures (figure 2b), suggesting that ubb is unable to kill proliferating cells. next, we studied the effects of ubb or ubb - k29,48r on chronologically aged cultures. for this, the proportion of viable cells capable of forming a colony (clonogenicity) on nutrient - containing solid medium was studied at different time points following ubb or ubb - k29,48r expression. consistent with the growth assays, 16 hr (day 1) after ubb or ubb - k29,48r expression cells exhibited a similar clonogenic potential as did cells expressing vector controls (figure 2c). in contrast, we observed a 10% and 25% decrease in clonogenic cell survival when expressing ubb for 2 and 3 days, respectively. exogenously applied stressors, including acetate and hydrogen peroxide, further enhanced the cytotoxicity of prolonged ubb expression (figures s2a and s2b, left). upon both chronological aging and stress experiments, ubb - k29,48r turned out to be slightly less cytotoxic as compared to ubb (figures 2c, s2a, and s2b, left), suggesting that ubiquitylated ubb is slightly more cytotoxic than ubb. we next examined whether the ubb - induced loss of clonogenicity correlated with the manifestation of oxidative stress, which can be detected by the intracellular conversion of the reactive oxygen species (ros)-sensitive stain dihydroethidium (dhe) to fluorescent ethidium. we observed indistinguishable low levels of oxidative stress after expressing ubb or ubb - k29,48r for 16 hr (day 1) (figure 2d). at later time points, the levels of oxidative stress progressively increased in all cultures with chronological aging, and ubb - expressing cells exhibited a mild but significant increase in oxidative stress as compared to vector controls. thus, upon ubb expression increased markers of oxidative stress coincided with decreased clonogenic cell survival (cf. figures 2c, 2d, and s2c). when combined with protracted ubb expression, acetate or hydrogen peroxide exacerbated the signs of oxidative stress (figures s2a and s2b, right). as shown for clonogenic survival, ubb - k29,48r demonstrated slightly decreased levels of oxidative stress upon chronological aging or exogenously applied stress as compared to ubb (figures 2d, s2a, and s2b). to determine the mode of cell death triggered by the expression of ubb or ubb - k29,48r, we performed double staining with annexin v - fitc and propidium iodide (pi). annexin v - fitc labels externalized phosphatidylserine that appears on the surface of apoptotic cells, whereas pi is a vital dye that stains cells that have lost plasma membrane integrity during necrosis. two days after ubb expression the frequencies of early apoptotic (annexin v - fitc pi), late apoptotic or secondary necrotic (annexin v - fitc pi), and necrotic cells (annexin v - fitc pi) were increased, as compared with vector controls (figures 2e and s2d). apoptosis induction by ubb could be confirmed by the terminal deoxynucleotidyl transferase dutp nick - end labeling (tunel) that detects fragmentation of nuclear dna (figures 2f and s2e). consistent with the results obtained from the clonogenic survival and oxidative stress experiments, ubb - k29,48r triggered cell death in a lower number of cells as compared to ubb (figures 2e and 2f). altogether, these results indicate that the protracted expression of ubb can induce apoptotic and necrotic killing of yeast cells, and that ubiquitylated ubb is a slightly better killer than ubb. to investigate the putative contribution of dysfunctional ups to ubb - triggered cytotoxicity, we measured the cytotoxic potential of ubb in the context of enhanced or suppressed ups. since full knockout of genes coding for proteasomal subunits is lethal, yeast strains bearing point mutations in one or two proteasomal genes were employed (heinemeyer., 1993). the chymotrypsin - like proteasomal activity was reduced in strains carrying mutant alleles in the proteasomal subunits pre1 and pre2 by > 88% (figure 3a). in these conditions of close - to - complete proteasomal inactivation, significantly reduced clonogenic cell survival was only observed in the pre1 - 1 and the pre1 - 1/pre2 - 2 strains upon ubb expression for day 1, and in the pre1 - 1/pre2 - 2 strain upon ubb expression for day 2 (figures 3b and s3a), as compared to wild - type strain. one explanation for the increased ubb - triggered cytotoxicity would be that ubb accumulates in these strains due to impaired ubb degradation. however, we could not observe increased steady - state levels of ubb in these strains (neither fl - ubb, nor tubb, nor ubiquitylated fl - ubb) (figures s3d s3f ; data not shown). thus, although severe proteasomal inactivation can increase ubb - triggered cell death, there is no strict correlation between the loss of proteasomal capacity on the one hand, and the increase in ubb - triggered cytotoxicity or the increase in the steady - state levels of ubb on the other hand. next, we measured ubb - induced cytotoxicity in knockout strains lacking selective ups genes, including (1) ubi4 encoding ubiquitin (finley., 1987), (2) rpn4 encoding a major transcriptional ups activator (mannhaupt., 1999), (3) ubr2 encoding the e3 ligase responsible for rpn4 degradation (kruegel., 2011), (4) yuh1 encoding the ubiquitin protease that cleaves fl - ubb into tubb (dennissen., 2011), and (5) ubp6 encoding a deubiquitinase, which can be inhibited by extended ubiquitin proteins (krutauz., 2014). only rpn4 deletion significantly impaired the chymotrypsin - like proteasomal capacity of the cells (figure 3c). notably, upon comparable fl - ubb steady - state levels (figures s3 g and s3i), ubb - triggered cytotoxicity was significantly increased in ubi4 as compared to rpn4 upon both stressed und unstressed conditions (figures 3d and s3b), although the proteasomal capacity was lower in rpn4 as compared to ubi4 (figure 3c). these data propose that the ratio of mutant ubiquitin (ubb) to wild - type ubiquitin (encoded by ubi4) is more relevant for determining ubb - triggered cytotoxicity than the proteasomal capacity. upon stressed conditions, ubb - triggered cytotoxicity was markedly increased in yuh1 as compared to wild - type cells upon comparable fl - ubb steady - state levels (figures 3d, s3b, s3h, and s3i). these data suggest that ubb truncation is a putative protective event, for instance, as part of a mechanism to degrade excessive ubb. in ubp6 as compared to wild - type cells ubb - triggered cytotoxicity was unaltered upon comparable fl - ubb steady - state levels (figures 3d, s3b, s3 g, and s3i), suggesting that ubp6 activity is not protective against the accumulation of the extended ubiquitin ubb. ubb - triggered cytotoxicity was significantly relieved in ubr2 cells upon stressed conditions (figures 3d and s3b), in which rpn4 is stabilized and consequently the ups activity is increased (kruegel., 2011). consistently, rpn4 expression, which also leads to increased ups activities (figure 3e), was protective for ubb - expressing wild - type cells (figures 3f and s3c) but not for cells lacking ubi4 (figure s3c). in both cases, the protective effect can not be explained by decreased steady - state levels of ubb (figures s3 g and s3i s3k). these data show that increasing ups capacity is protective for ubb - expressing cells, but not by affecting the turnover of ubb itself but rather by interrupting the lethal signaling cascade triggered by ubb. oxidative stress and mitochondrial impairment are hallmarks of neurotoxin - elicited death in yeast and neurons (braun, 2012 ; debattisti and scorrano, 2013). therefore, we analyzed whether oxidative stress, which occurred starting by day 2 of ubb expression (figure 4a), correlated with mitochondrial impairment. two days after inducing ubb expression, the mitochondrial network was fragmented in both ubb - expressing cells, as well as in cells carrying vector controls (data not shown), which is typical for stationary phase cultures. however, after shifting these cultures to fresh growth medium (which represses ubb expression) the recovery of the mitochondrial network was significantly compromised in cultures transformed with ubb - encoding constructs as compared with vector controls (figures 4b and s4a). we further tested for mitochondrial impairment by measuring the cellular oxygen consumption, the mitochondrial membrane potential, and the atp levels in cells expressing ubb for days 2 and 3. whereas the cellular oxygen consumption and mitochondrial membrane potential were significantly increased by day 3 in (surviving) cells expressing ubb (figures 4c and 4d), the cellular atp levels were significantly decreased by days 2 and 3 (figure 4e). these data hint at hyperactive mitochondria, which are incapable to prevent a metabolic crisis in ubb - expressing cells. in yeast, alterations in the cytochrome bc1 complex of the mitochondrial respiratory chain may contribute to the loss of respiratory capacity and the production of lethal ros (diaz. for instance, loss of the rieske iron - sulfur protein rip1, a key component of the cytochrome bc1 complex, results in increased ros generation and mitochondrial dysfunction (diaz., 2012). indeed, the cellular level of rip1 was markedly decreased by days 2 and 3 upon ubb expression as compared with vector controls (figures s4b and s4c). consistently, rip1 and cytochrome c were depleted in the mitochondrial fraction of ubb - expressing cells (figures 4f and 4 g). these data further hint at a major ubb - induced mitochondrial dysfunction, in which the respiratory chain is impaired (depletion of rip1 and cytochrome c), leading to the production of ros (for which cellular oxygen is needed), and the decline of cellular atp levels. hyperpolarization of mitochondria may precede mitochondrion - dependent yeast death (eisenberg., 2007) ; therefore, we expressed ubb in strains deleted for genes encoding a range of mitochondrial cell death proteins, including the yeast bh3-only protein (ybh3) that translocates to mitochondria to mediate their permeabilization, and several potentially cytotoxic proteins that can be released from mitochondria such as apoptosis - inducing factor 1 (aif1), endonuclease g (nuc1), and the two cytochrome c isoforms (cyc1, cyc7). deletion of nuc1 resulted in a paradoxical increase in ubb - triggered cytotoxicity, and loss of ybh3 did not have any effect upon both stressed and unstressed conditions (figures 4h and s4d). in contrast, ubb - mediated cytotoxicity was significantly decreased in strains depleted from isoform 2 of cytochrome c (cyc7) upon stressed conditions (figures 4h and s4d). the steady - state levels of ubb were not decreased in the cyc7 as compared to wild - type strain (figure s4f), and this strain maintained a normal state of respiratory competence (presumably due to the presence of the cytochrome c isoform 1 cyc1) (figure s4i), excluding trivial explanations for the cytoprotective action of cyc7. thus, our data suggest the implication of mitochondria in ubb - triggered cell death. next, we tested for a possible role of the unfolded protein response (upr) and the er in ubb - triggered cytotoxicity and expressed ubb for 2 days in cells lacking the upr kinase ire1 and its downstream target hac1, as well as in cells lacking the er cell death protease kex1 (which executes cell death in which mitochondria play a pivotal role [hauptmann and lehle, 2008 ]). upon stress, ubb - triggered cytotoxicity was relieved in ire1 and kex1 but not in hac1 cells (figures 4i and s4e), under conditions where the steady - state levels of ubb were comparable (figures s4 g and s4h). these data suggest for an implication of the er in ubb - triggered cytotoxicity, but, due to the lack of rescue in the hac1 cells, a critical involvement of the upr is unlikely. next, we performed quantitative proteomic analyses of crude mitochondria after stable isotope labeling by amino acids in cell culture (silac). this approach led to the identification of 16 proteins whose abundance was significantly altered (increased for ten or decreased for six proteins) upon ubb expression (figure 5a ; table s2). among the proteins with established mitochondrial localization, three were enzymes participating in amino acid metabolism, namely, put1 (involved in proline degradation), arg5,6, and arg8 (involved in arginine and ornithine biosynthesis). in addition, ubb induced the accumulation of the cytosolic enzyme lys1 (involved in lysine biosynthesis), an increase in the motor protein myo3 and the (putative) peroxisomal proteins gpd1 and str3, in crude mitochondria. upon acetate stress, deletion of the arg5,6, arg8, and the lys1 genes restored the clonogenic potential of ubb - expressing cells, whereas the deletion of all other genes had no effect (figures 5b and s5a). these data point to a hitherto unexpected involvement of the biosynthesis of basic amino acids (arginine, ornithine, and lysine) in ubb - triggered cytotoxicity. to challenge this hypothesis, we measured the cellular steady - state levels of arginine, ornithine, and lysine in cultures expressing ubb (figure 5c). indeed, we observed a marked increase in the cellular levels of all three basic amino acids, in particular, ornithine, upon ubb accumulation. to weigh the contribution of arginine and ornithine (as opposed to their metabolic intermediates) to ubb cytotoxicity, we measured ros production upon ubb expression in strains depleted from the arginine and ornithine biosynthetic enzymes (figure 5d). depletion of all enzymes operating upstream of cytosolic ornithine (arg2, arg5,6, arg7, and ort1) significantly relieved ubb - triggered cytotoxicity both in unstressed and acetate - stressed conditions (figures 5e and s5b). in contrast, none of the enzymes downstream of cytosolic ornithine (arg3, arg1, and arg4, which are needed for the conversion of ornithine into arginine) were required for the cytotoxic action of ubb. notably, all tested enzymes operating upstream of cytosolic ornithine are mitochondrion - associated (ljungdahl and daignan - fornier, 2012). therefore, we concluded that ubb triggers the mitochondrion - associated biosynthesis of ornithine, leading to increased cytosolic levels of ornithine (and its product arginine). if this model is true, increasing cytosolic levels of either ornithine or arginine (which can easily be interconverted into each other) should recover the cytotoxic effect of ubb in strains with interrupted mitochondrion - associated biosynthesis of ornithine. therefore, we measured ubb - triggered cell death in the strain depleted for the mitochondrial protein ort1 in growth media with increasing concentrations of arginine and ornithine, respectively. it turned out that ort1 cells were not able to efficiently uptake ornithine from the growth media, because the severe growth deficit of the ort1 strain in growth media lacking arginine could not be relieved by increasing concentrations of ornithine in the growth media (data not shown). in contrast, ort1 cells grew well in the presence of arginine in growth media lacking ornithine (data not shown), demonstrating the efficient cellular uptake of arginine. as expected, yeast cells lacking ort1 were protected from ubb - triggered cell death upon moderate concentrations of arginine (30 and 50 mg / l) in the growth media (figure 5f). in contrast, elevated concentrations of arginine in the growth media (150 and 300 mg / l) recovered the cytotoxic effect of ubb (figure 5f), substantiating the decisive role of increased cellular levels of arginine (and cytosolic ornithine) in executing ubb - triggered cell death. in order to address the role of cellular levels of lysine, we measured ubb - triggered cell death in the strain depleted from lys1 in growth media with increasing concentrations of lysine. whereas deletion of lys1 relieved ubb - triggered cytotoxicity as compared to wild - type strain (figure 5b), increasing the lysine concentrations did not promote cytotoxicity in the lys1 strain (figure 5 g). thus, in contrast to arginine / ornithine the cellular lysine level appears to be negligible in accelerating ubb - triggered cell death. the aforementioned data incriminate mitochondria and the ups in the execution of ubb - triggered cytotoxicity, notably because of the protective impact of the removal of mitochondrial enzymes involved in basic amino acid synthesis and the overexpression of the transcriptional ups activator rpn4. among the known rpn4 targets are the conserved aaa - atpase cdc48 and its cofactor npl4 (bosis., 2010). cdc48 and npl4 are involved in the ups, and determined by their cofactor vms1, regulate mitochondrion - associated protein degradation (heo., driven by these premises, we evaluated the involvement of the cdc48/vms1/npl4-dependent ups pathway to ubb - triggered cytotoxicity. for this, we measured ubb - triggered cytotoxicity in normal and acetate - stressed conditions in strains expressing increased levels of wild - type cdc48 or the pro - apoptotic cdc48-s565 g variant (madeo., 1997), which is characterized by decreased vms1 binding and mitochondrion - associated degradation (heo., 2010). we also determined the cytotoxicity of ubb in strains depleted from the cdc48 cofactors npl4 and vms1, or overexpressing vms1. ubb - triggered cytotoxicity was markedly attenuated in cultures expressing increased levels of wild - type cdc48, as compared to cells expressing cdc48-s565 g or controls with endogenous cdc48 only (figures 6a and s6a). ubb - triggered cytotoxicity was significantly increased in cultures depleted from npl4 under non - stressed conditions (figures 6b and s6b). depletion of vms1 resulted in a marked elevation in cytotoxicity upon stress (figures 6c and s6c), while overexpression of vms1 significantly protected against ubb upon acetate stress, as measured by the clonogenic approach (figures 6d and s6d). high levels of vms1 also protected from cell death and oxidative stress induced by ubb expression (figures 6e and 6f). notably, high amounts of cdc48 and cdc48-s565 g resulted in markedly decreased steady - state levels of ubb (figures s6e and s6f), whereas neither the deletion of vms1, nor its overexpression had an effect on the cellular ubb amounts (figures s6g s6j). these data point to a protective role of vms1, which is independent from ubb degradation, potentially by improving the quality control at mitochondria. in contrast, the beneficial role of high amounts of cdc48 could be due to both increased vms1-independent ubb degradation and improved vms1-dependent mitochondrial quality control. in order to address whether elevated vms1 levels prevent from ubb - triggered mitochondrial impairment, we measured the cellular oxygen consumption, the mitochondrial membrane potential, and the cellular atp levels in cells expressing ubb upon endogenous or elevated amounts of vms1 (figures 6g6i). whereas the cellular oxygen consumption and the mitochondrial membrane potential were significantly decreased by day 3 and days 2 and 3, respectively, cellular atp levels were significantly increased by day 2 upon high amounts of vms1. in other words, high amounts of vms1 reverted the mitochondrial damage induced by high levels of ubb (see figures 4c4e). in a next step, we used silac technology to comparatively assess alterations of the mitochondrial proteome between ubb - expressing cells with endogenous and high levels of vms1 (figure 6j ; table s3). we observed that among the 16 proteins whose abundance levels were altered by ubb as compared with the vector control (figure 5a ; table s2), ten were no more altered upon expression of both ubb and vms1 (figure 6j, blue - labeled proteins). among these ten proteins, which were particularly stringently associated with the cytopathic activity of ubb, the basic amino acid synthesis enzymes arg5,6, arg8, and lys1 were significantly decreased in ubb - expressing cells upon high levels of vms1, as compared to endogenous vms1 levels. consistently, vms1 overexpression blunted the ubb - mediated increase in the steady - state levels of arginine, ornithine, and lysine (figure 6k, see figure 5c). these data point to a pivotal role of the vms1-dependent mitochondrial ups activity in avoiding the ubb - triggered lethal overproduction of basic amino acids. pathological hallmarks include intracellular neurofibrillary tangles comprising aberrant forms of the microtubule - associated protein tau, ubb, and the mitochondrial outer membrane voltage - dependent anion channel 1 (vdac1) (reddy, 2013 ; van leeuwen., 1998). immunohistochemistry revealed expression of vms1, the human homolog of yeast vms1, in pyramidal cells within the hippocampi from ad patients and aged non - demented controls (figure 7a, all arrows ; tables s5 and s6). vms1 stained structures reminiscent of tau pathology, including tangle - like (yellow arrows) and neuropil thread - like structures (blue arrows), as well as other cellular staining patterns (green arrows), were observed in samples from ad patients, and aged non - demented controls with tau pathology. we also observed these tangle - like and thread - like staining patterns when analyzing the sections for aberrant tau, ubb, and vdac1 (figure s7). immunohistochemistry of consecutive paraffin sections from the hippocampi of ad patients (figure 7b) confirmed the identification of pyramidal cells with intracellular tangle - like structures, which co - stained for aberrant tau and vms1 (figure 7c, violet arrows), and for ubb, vms1, and vdac1 (figure 7d, orange arrows). these data suggest that vms1 is a component of neurofibrillary tangles comprising aberrant tau, ubb, and vdac1, underscoring a potential role of the cdc48/vms1 complex in ubb - mediated ad progression. we established a yeast model for dissecting cell death mechanisms triggered by ubb, in which mitochondria play a pivotal role in the execution of cell death (see the supplemental discussion). ubb triggers neuronal apoptosis accompanied by reduced mitochondrial movement (tan., 2007), and mitochondrial impairment likely contributes to ad (rodolfo., 2010). thus, our yeast model corroborates features of cell - death - relevant mitochondrion dysfunctions found in ad neurons. yeast strains expressing ubb accumulated the basic amino acids arginine, ornithine, and lysine. deletion of mitochondrion - associated enzymes involved in their synthesis abolished ubb - triggered cell killing, which could be recovered by increasing concentrations of arginine in the growth media. the accumulation of basic amino acids may trigger mitochondrial damage and cell death in mammalian cells and in yeast (almeida., 2007 ; bicz., for instance, increased production of nitric oxide from arginine executes yeast apoptosis (almeida., 2007) and increased levels of polyamines, which are produced from ornithine, may impair neuronal ion channel activities (inoue., 2013). the levels of arginine, ornithine, and/or their polyamine products were altered in aged human brains and in brains from ad patients (inoue., 2013 ; liu., 2014 ; rushaidhi., the results of these studies are controversial, and it remains not yet clear whether the observed alterations are cause or consequence of ad. despite that, our data suggest that perturbed basic amino acid synthesis is a decisive event triggering mitochondrion - dependent cell death upon ubb accumulation in yeast. further studies aiming at analyzing the role of arginine / ornithine metabolism during aging or ad progression should consider a potential pivotal contribution of ups and mitochondrial dysfunctions. we observed that ubb accumulation impaired the ups, and that the ups activity, in turn, determined ubb cytotoxicity. yeast cultures that were depleted from ubiquitin (ubi4) were highly vulnerable to ubb. in contrast, yeast cultures in which the ups was stimulated by the transcriptional activator rpn4 were insensitive to ubb, but not in cells lacking the ubiquitin gene ubi4. extended ubiquitin variants have been proposed to be specific inhibitors of the deubiquitinase ubp6 in yeast (krutauz., 2014). since the ubb - triggered cytotoxicity was unaltered in a strain deleted for ubp6 as compared to wild - type strain, our data suggest that the lethal effect of the extended ubiquitin ubb does not essentially depend on ubp6. it is tempting to speculate that the ratio of mutant (ubb) to wild - type ubiquitin determines ubb - triggered cytotoxicity with ubb as a competitive inhibitor of wild - type ubiquitin, affecting numerous ubiquitin - regulated cellular processes. we established that elevated amounts of cdc48 or its cofactor vms1 conferred tolerance against ubb expression. more specifically, vms1 overexpression relieved the ubb - triggered mitochondrial damage and accumulation of the basic amino acids arginine, ornithine, and lysine. the cdc48/vms1 complex enables the degradation of mitochondrion - associated proteins (heo., 2010). whereas under normal conditions, this complex is predominantly cytosolic, vms1 recruits cdc48 to the mitochondrial outer membrane upon stress, presumably with the scope of improving the local quality of proteins. our data suggest that cdc48/vms1-mediated processes can prevent the ubb - triggered lethal derangement of mitochondria. in one possible scenario, cdc48/vms1 might remove protein junk from the mitochondrial outer membrane. alternatively, cdc48/vms1 might specifically prevent the accumulation of arginine, ornithine, and lysine, through regulation of the turnover of the enzymes arg5,6, arg8, and lys1, which are pivotal for their synthesis. whereas the activity of the cytoplasmic enzyme lys1 could be regulated by its degradation, the activities of the mitochondrion - associated arg5,6 and arg8 could be controlled by preventing their import into mitochondria via ubiquitylation and proteasomal degradation. lys1, arg5,6, and arg8 are known targets for ubiquitylation (xu., 2009), and the ups regulates the import of mitochondrial intermembrane space proteins (bragoszewski., 2013 ; harbauer., it is tempting to speculate for a ups - dependent regulation of the import of the mitochondrial matrix proteins arg5,6 and arg8. further studies are needed to address the influence of ups (dys)function on the turnover of these and other mitochondrial proteins. this is important because recent studies demonstrated that ups dysfunction can lead to mitochondrial dysfunction and vice versa (livnat - levanon., 2014 ; maharjan., 2014 ; segref., 2014), and our data revealed the unexpected link between ubb - triggered ups dysfunction and the accumulation of functional enzymes in the mitochondrial matrix leading to potentially cytotoxic accumulation of basic amino acids. human vms1 and mitochondrial vdac1 co - existed with ubb in neurofibrillary tangles of ad patients and aged non - demented controls with tau pathology. ubb accumulates and the number of neurofibrillary tangles and damaged mitochondria markedly increase during ad progression (dennissen., 2010 ; rodolfo., 2010 we propose that vms1-dependent mitochondrial quality control might retard the ad - associated neuronal dysfunction, which is elicited by the accumulation of both aberrant tau and ubb. yeast expression constructs, strains, and growth conditions were described in the supplemental experimental procedures., cultures were treated for 4 hr with acetate. for stable isotope labeling (silac), cells expressing vector controls, ubb, or ubb and vms1 were grown in media supplemented either with lys0 and arg0 (normal isotopes), or with lys4 and arg6, or with lys8 and arg10 (heavy isotopes, silantes). briefly, growth deficits upon expression of proteins of interest on solid or liquid media, as compared to vector controls, suggest for cytotoxic effects of these proteins on (growing) yeast cells. for clonogenic assays, 500 cells from liquid yeast cultures expressing proteins of interest or vector controls, respectively, were plated on agar plates, on which expression is repressed. the number of colonies (colony forming units [cfus ]) formed after 2 days of incubation correlates with the fitness of the culture. oxidative stress was determined by measuring the conversion of dihydroethidium (dhe, sigma - aldrich) to the red fluorescent ethidium applying a fluorescence plate reader or a flow cytometer. propidium iodide (pi, sigma - aldrich) in cells that have lost their plasma membrane integrity using a flow cytometer. annexin v / pi co - staining (annexin v - fluos staining kit, roche applied science) for discriminating early and late apoptosis, as well as necrosis, and terminal deoxynucleotidyl transferase dutp nick end labeling (tunel) for measuring apoptosis (in situ cell death detection kit, roche applied science) were performed by flow cytometry. oxygen consumption of stationary yeast cultures was analyzed using the firesting optical oxygen sensor system (pyro science). mitochondrial membrane potential was assessed with flow cytometry after staining cells with tetramethylrhodamine methyl ester (tmrm, molecular probes, life technologies), a fluorescent dye that accumulates within mitochondria dependent on their membrane potential. to determine the atp level of yeast cultures atp was measured using the atp determination kit (molecular probes, life technologies). this assay is based on an atp - dependent reaction of recombinant firefly luciferase, which induces bioluminescence of its substrate d - luciferin and is directly correlated with the atp content. see the supplemental experimental procedures for details. for determining the level of polyubiquitylated proteins in cellular extracts, immunoblots of cellular extracts were incubated with an ubiquitin - specific antibody and immunosignals were quantified with imagej 1.47 m. for measuring the turnover of ups substrates, the ubiquitin - fusion protein ubiquitin - g76v - gfp was co - expressed with ubb or vector controls. gfp fluorescence (relative fluorescence units [rfus ]) and optical densities (od600) were determined using the fluostar omega plate reader. rfu was normalized to od600, in order to determine the level of ubiquitin - gfp fusion proteins per culture. measurement of chymotrypsin - like proteasomal activities were performed using the fluostar omega plate reader, applying the luminescence - based proteasome - glo cell - based assay (promega). yeast cultures were incubated in expression media (scgal) for the indicated time points. cell extracts were generated by pre - treating yeast pellets in naoh followed by heating in sds lysis buffer. protein extracts were separated on tricine - sds polyacrylamide gels, transferred on pvdf membranes, and incubated with primary and secondary antibodies coupled to horseradish peroxidase. immunoblot quantification was done with the gel analysis method in imagej 1.47 m. see the supplemental experimental procedures for details. crude mitochondrial extracts were taken up in sds lysis buffer, thawed, reduced with dtt, and alkylated using iodoacetamide (sigma - aldrich). protein mixtures were separated by sds - page using bis - tris gels (nupage, invitrogen). the gel lanes were cut into slices, which were in - gel digested with trypsin (promega), and the resulting peptide mixtures were processed on stage tips. mass spectrometry was performed on a ltq orbitrap xl mass spectrometer (thermo fisher scientific) coupled to an eksigent nanolc - ultra. electro spray high - resolution mass spectrometry (ip - rp - lc / hrms). lc / ms measurements were normalized to the total number of cells of each sample. see the supplemental experimental procedures for details. experiments with human materials were in accordance with the local ethical committees at the universities of bayreuth (germany) and maastricht (the netherlands). postmortem tissues of hippocampi from ad patients and non - demented controls were obtained from the netherlands brain bank (table s6) as paraffin sections. for immunohistochemistry, sections were deparaffinated, incubated with primary antibodies against the indicated proteins, and with biotin - coupled secondary antibodies followed by the avidin - biotin - peroxidase complex. | summaryneuronal accumulation of ubb+1, a frameshift variant of ubiquitin b, is a hallmark of alzheimer s disease (ad). how ubb+1 contributes to neuronal dysfunction remains elusive. here, we show that in brain regions of ad patients with neurofibrillary tangles ubb+1 co - exists with vms1, the mitochondrion - specific component of the ubiquitin - proteasome system (ups). expression of ubb+1 in yeast disturbs the ups, leading to mitochondrial stress and apoptosis. inhibiting ups activity exacerbates while stimulating ups by the transcription activator rpn4 reduces ubb+1-triggered cytotoxicity. high levels of the rpn4 target protein cdc48 and its cofactor vms1 are sufficient to relieve programmed cell death. we identified the ubb+1-induced enhancement of the basic amino acids arginine, ornithine, and lysine at mitochondria as a decisive toxic event, which can be reversed by cdc48/vms1-mediated proteolysis. the fact that ad - induced cellular dysfunctions can be avoided by ups activity at mitochondria has potentially far - reaching pathophysiological implications. |
pseudomonas aeruginosa (p. aeruginosa) is an aerobic gram - negative bacterium ; that is ubiquitous, exist in aqueous habitats, and take advantage of humid environments. it is also saprophytic and naturally resistant to antibiotics (beta - lactams, hydrophilic), and may become an opportunistic pathogen responsible for serious infections when favorable circumstances exist. because it is environmentally present in soils, plants (including fruits and vegetables), aqueous habitats and humid environments, it has an ability to acquire resistance to antibiotics, and has multiplicity of virulence factors (diffusible or constituent) that baffle the host s defenses and allow the development of infections in susceptible patients, such as malnourished patients ; burn victims, trauma patients, patients suffering from diabetes, cystic fibrosis, cancer, human immunodeficiency virus (hiv), and blood disorders, patients undergoing mechanical ventilation or long - term corticosteroid therapy ; patients in whom carrier surveyed peripheral and central catheters are being used. aeruginosa infections are most often acquired in a hospital, but are sometimes community acquired ; factors limiting p. aeruginosa proliferation on the skin are dryness of the skin s surface and normal flora (gram + cocci). the bacterium p. aeruginosa can grow or become permanent on the skin in case of the traumatic rupture of the skin barrier ; chronic wounds such as leg ulcers, extensive burns, diabetes include ulcers, oozing dermatitis, and wet or macerated lesions the disappearance of gram + cocci normal flora caused by the use of systemic or topical antimicrobials. the first signs of infection of a chronic wound or ulcer are the production of greenish pus and the appearance of a characteristic aromatic odor. these are vesicular or bullous lesions with a content of serum are hemorrhagic based on an erythematous and edematous base and can initially have the clinical appearance of multiform erythema. these lesions can change very quickly in a few days to ulceration, and they then take the appearance of a so - called ecthyma gangrenosum [3 - 6 ]. one can also have abscesses or single or multiple subcutaneous nodules that can change very quickly to necrosis and ulceration. biofilm formation seems to facilitate the survival of p. aeruginosa in the environment and in their hosts. bacterial biofilms are structured masses of bacterial cells coated with a hydrated polymeric matrix of their own synthesis. the biofilm bacteria can withstand the immune response of the host and are much more resistant to antibiotics and disinfectants than planktonic bacterial cells. the ability to form a biofilm the presence of biofilms during infections therefore requires new methods of prevention, diagnosis and treatment. the existence of biofilms and antibiotic resistance has emerged as one of the greatest threats to global health and, brings an additional level of complexity to the control of chronic ulcers. the black beast of burn centers, pseudomonas, is responsible for 10% of bloodstream infections and 28% of deaths. therefore, alternative antimicrobial strategies are urgently needed, and this situation has led to a re - evaluation of the therapeutic use of ancient remedies, such as herbal extracts and herbal - based products. these include proteins, lipids and carbohydrates that are used for subsistence and reproduction ; in not only the plant itself but also the animals that feed on them. in addition, plants synthesize an extraordinary range of other compounds, called secondary metabolites, whose functions are far from being unanimously accepted. antibiotics as they protect plants against fungi, bacteria, animals and even other plants. phenol compound are involved in plant - plant interactions (allelopathy, and inhibitions of both germination and growth). these compounds include lignin, flavonoids, phenylpropanoids, anthocyanins and nitrogen compounds including alkaloids and glycosides. recently, phenol compounds have drawn much attention due to their antioxidants properties and their potential beneficial implications for human health, such as the treatment and the prevention of cancer, cardiovascular disease and other inflammatory diseases. polyphenol are composed of various phenol compounds such as phenolic acid, catechins, flavonoids, and tannins. they usually have antiseptic, antibacterial actions. the simplest is phenol (an antimicrobial) present in thyme oil. other phenols have analgesics, anti - inflammatory, cholesterol - lowering, hypotensive, anticoagulant, anti - allergenic, hypotensive, and hepato - protective effects these substances can be yellow (origin of the word flav), red, blue or purple. the main properties of flavonoids are their veinotonic, protective (vessels), anti - cholesterol or antioxidants effects [11, 12 ].. these complex compounds may be soluble in water or alcohol, are the flavonoid family. they are common both in gymnosperms and angiosperms. within angiosperms, tannins are more common in dicotyledons than they are in monocotyledons. tannins are mainly used externally, particularly to treat patients with injuries, wounds or hemorrhoids. catechin is a molecule of the flavonoid family ; the name comes from the fact that this molecular compounds found in the fruits of acacia catechu. catechins are astringent juices from various sources and involve decoction of the fruit of areca catechu l. (palms), and the wood of the acacia catechu. the fermentation of the leaves being stopped soon after harvest, they are barely oxidized, which is the reason that green tea retains most of its original catechins, which gives them their slightly bitter and astringent taste. interesting amounts of catechins also exist in chocolate, red wine, apples and grapes. five catechins molecules are : catechins, epicatechin, epigallocatechin, epicatechin gallate and epigallocatechin gallate molecules. the latter is the most abundant and most active of all the catechins. according to numerous studies, catechins have, in addition to their antioxidant properties, a protective role in the prevention of certain chronic diseases such as diabetes and osteoporosis. several studies that have been conducted in recent years have manifested the antibiotic activities of some phenol compounds in natural plants extracts, including acacia catechu (l.f.) honey, maple saps, phoenix dactylifera l. (dates) extract and pomegranate which are well known for their high levels of antioxidants and polyphenols have also shown promise as novel antimicrobial agents. phenol compounds have recently attracted much interest due to their antioxidants properties and their potential beneficial implications for human health, such as their use in the treatment and the prevention of cancer, cardiovascular diseases and inflammatory diseases. the effectiveness of these above substances ; has been demonstrated in several studies [16 - 19 ]. however, no effort has been made to evaluate the synergic effects of combined natural sweeteners on enhancing the antibiotic activities of natural plants extracts. the topical formulations that on certain vitamins and nutrients, are rich in phenol compounds, and are applied directly to wounds are more effective for reducing the risk of infection and for stimulating healing. meanwhile, phenol - rich compounds ; provides a number of essential minerals such as zinc, potassium, iron, magnesium and calcium (table 1). in order to obtain a natural antibiotic for the dressing and the treatment of chronic wounds, we conducted this study to find an innovative strategy for using natural medicine derived from phenol - rich compounds that have been tested on bacterial strains of a reference such p. aeruginosa. this research was conducted through 2015 at the laboratories of dubai pharmacy college united arab emirates (uae). the processing of the plants performed in this study was the same as the traditional method used by the people in the iranian bakhtiari tribe, as mentioned in ref. the phenol - rich compound sweet gel was prepared by blending four natural herbal extract acacia catechu (l.f.) willd, castanea sativa, ephedra sinica stapf, and momia in to combination of sweet gel medium, including honey, maple saps, date syrup, pomegranate extract and azadirachta indica gum as a stabilizer. the combinations were screened by using a well - diffusion assay with cloxacillin as a control. suspension assays were used to determine the antimicrobial activities of the medium gel alone and of the medium gel in combination with the four natural herbal extract. the test organism was p. aeruginosa. in this study, eight plant species were used as shown in (table 2) and 3. the ingredients of the sweet gel compound are presented in (table 4), bacterial strains and growth conditions. the inhibition of bacterial growth was studied by using the well - diffusion method with nutrient agar, as commonly practiced in medical bacteriology, and was purchased from himedia laboratories, india. plates were inoculated with 100 l of each pathogenic microorganism adjusted to standardized inoculum (1.5 10 cfu / ml) in triplicates and were spread with sterile swabs. eight mm wells were drilled into the agar by using a sterile stainless steel borer. for the preparation of phenol - rich compound herbal extracts, one gram of aslan crude extract (table 3) was added to 10 ml of distilled water to form 10% aslan (w / v), which was heated and stirred until all ingredients had dissolved. it was then mixed with an equal amount of sweet gel, which included honey, date, maple syrup and pomegranate in specific percentages, as shown in (table 3), after which neem gum as much as 5 percent of the total weight, was added as stabilizer. one drop of each sample solution (50 ul) was applied to each well on the plate by using a pasteur pipette. the petri dishes thus prepared were left at room temperature for ten minutes to allow diffusion of the extract into the agar. anti - bacterial activity was indicated by an inhibition zone surrounding the well (including the well diameter) containing the plant extract. the diameters of the zones of inhibition were measured in millimeters and interpreted based on published standards. anti - bacterial activity was recorded if the zone of inhibition was greater than 8 mm. the interpretation of the anti - bacterial activity results was done according to the diameter of the zone of inhibition as follows : zones with diameters 18 mm were considered very active. the means and standard deviations of the diameters of the inhibition zones were calculated. data were evaluated using the ibm spss software program (version 19 ; ibm spss inc. were compared at the 95% confidence interval, and the results were expressed as means standard deviations. differences between the control group and the herbal extract groups were the criteria for the anti - bacterial activities. the t - test (paired tests) was used to detect differences between the treatment groups and the control group. the table 5 shows the zones of inhibition for the individual ingredients in the phenol - rich compound that were used against p. aeruginosa and that produced a greater inhibition of pseudomonas spp. table 6 shows that the phenol - rich compound sweet gel produced a greater inhibition of pseudomonas spp. than cloxacillin did. also, the table shows that phenol - rich compound sweet gel had a greater inhabitation had a greater inhabitation on pseudomonas spp. based on the results for the antibacterial effects, we can state that phenol - rich compound sweet gel is a good candidate for the prevention and the treatment of chronic ulcers. our findings showed that the phenol - rich compound sweet gel had a significant antiseptic effective against pseudomonas spp. and were in agreement with those of previous studies [15 - 19 ] that found that phenol compound exhibited fairly good antimicrobial activities against both gram - negative and gram - positive bacteria and that remarkable activity was exhibited by p. aeruginosa. also, the greater inhibition due to the compound compared to the inhabitation due to the individual ingredients on pseudomonas spp. indicates significant synergic effects. however, no effort has been made so far to evaluate the synergic effects of combined natural sweeteners on enhancing the antibiotic activities of natural plants extracts. phenol compounds rich in minerals and trace elements are involved in many biological processes in wound healing : several studies revealed a higher postoperative morbidity, delayed healing, and more frequent secondary infections in malnourished patients with amputations than in non - malnourished patients. zinc as a trace element is quantitatively the most important one and is involved in the construction of over 200 enzymes with effects as follow : improving cell growth and differentiation, fibroblast proliferation, collagen synthesis, strengthening the immune system and increasing steroid receptors [21, 22 ].. it also promotes collagen synthesis, improves oxygen delivery to tissue, and is a component of many enzyme systems. chronic iron deficiency or anemia is associated with an extended period of healing and other painful complications. copper is considered one of the most effective nutrients for improving the wound healing rate. some studies shown that copper can destroy several bacterial strains ; hence, it is interesting for use in dressings and bandages devices. globally, copper strengthens bones, organs and connective tissues, and provides better overall immune response. this is because copper, iron and zinc use the same routes to cross the intestinal barrier and reach the bloodstream. therefore excessive inputs of a given mineral salt can interfere with the absorption of others. no significant benefit for wound healing is seen with nutritional supplements such as vitamins c, a, e, and zinc in a non - deficient individuals. [25 - 28 ] in contrast to oral administration, topical administration of zinc appears to be superior due to its action in reducing super infections and necrotic material via enhanced local defense systems and collagenolytic activity ; and to the sustained release of zinc ions, which stimulates epithelialization of wounds in normozincemic individuals. zinc oxide in paste bandages (unna boot) protects and soothes inflamed peri - wound skin. zinc from these formulations is transported through the skin although the systemic effects seem insignificant. the topical administration formula that focuses on certain vitamins and nutrients applied directly on the wounds is more effective as a supplements to reduce the risk of infection and to stimulate healing. furthermore, phenolrich compounds, provides a number of essential minerals, such as zinc, potassium, iron, magnesium and calcium (table 1). in previous studies [29 - 31 ] on phenol compounds, no contraindications and side effects have been reported to date for any of the ingredient used in our study, even though all of the benefits seem to have been reported. the topical use of phenol - rich compound sweet gel should be safer than that of the other formulations, but to be on the safe side, more clinical studies and toxicology studies need to be conducted. the antibiotic effect of phenol- rich compounds is due to the presence of hydrophilic components, such as polyphenols, polysaccharides, flavonoids and tannins in one or more parts of the plant. thus is an innovative approach to fighting the antibiotic - resistant pseudomonas. our results and those of previous studies provide pharmacology and microbiology information to explain the advantages of the phenol - rich compound sweet gel and its mechanisms of action as a bioactive dressing material in treating chronic ulcer : sterilization of wounds rapid autolytic debridement inhibition of potential pathogens of wounds and enzymes that destroy tissue, stimulations of tissues growth to speed healing, protection against cross - contamination, reduction of scars, anti - inflammatory effect : reductions of infections, deodorization of wounds, reducing edema and against maceration due reduced of exudate, provision of moist healing environment without risk of surrounding skin maceration and preventions of adhesion of the dressings to the wound, thus preventing pain and tissue damage when dressing is changed. the increase in the number of bacterial infections resistant to current antibiotics is an extremely worrying phenomenon, especially in the hospital setting (nosocomial infections). therefore new strategies and innovative antibiotics for use against these particularly virulent microorganisms need to be developed if a therapeutic impasse is to be avoided. this work evaluated the synergic effects of combined natural sweeteners on enhancing the antibiotic sensitivities of natural plants extracts and found the phenol - rich compounds sweet gel to be an alternative medicine and bioactive dressing material, for the treatment of patients with various types of wounds, including burns, venous leg ulcers, ulcers of various etiologies, diabetes induced ulcers on the feet, unhealed sampling sites grafts, abscesses, boils, surgical wounds, necrosis process, post - operative and neonatal wound infection. based on these results, that compound should be considered an alternative to the usual methods of cure. on burns its antibacterial and anti - inflammatory properties allow a moist healing environment that protects the wounds from deterioration and fibrosis to be maintained. united states department of agriculture, national nutrient database for standard reference, the national agricultural library, release 28, slightly revised may, 2016, software v.2.5.4. | objectives : the purpose of this study was to obtain a natural antibiotic from phenol - rich compounds ; for the dressing and the treatment of chronic wounds.methods:the phenol - rich compound sweet gel was prepared by blending four natural herbal extracts, acacia catechu (l.f.), momia (shilajit), castanea sativa, and ephedra sinica stapf, with combination of a sweet gel medium, including honey, maple saps, phoenix dactylifera l. (date), pomegranate extract and azadirachta indica gum as a stabilizer. the combinations were screened by using a well - diffusion assay with cloxacillin as a control. pseudomonas spp. was tested with our novel antimicrobial compound. the zones of inhibition in agar culture were measured for each individual component and for the compound, and the results were compared with those of the control group which had been treated with cloxacillin. data were expressed as means standard deviations. quantitative analyses were performed using the paired t-test.results:the antibiotic effect of the phenol - rich compound sweet gel was statistically shown to be more significant than that of cloxacillin against pseudomonas aeruginosa (p < 0.05).conclusion : our novel approach to fighting the antibiotic resistance of pseudomonas proved to be successful. the phenol - rich compound sweet gel was found to be suitable for use as an alternative medicine and bioactive dressing material, for the treatment of patients with various types of wounds, including burns, venous leg ulcers, ulcers of various etiologies, leg ulcers on the feet of diabetic, unhealed graft sampling sites, abscesses, boils, surgical wounds, necrotic process, post - operative and neonatal wound infection, and should be considered as an alternative to the usual methods of cure. |
oral cancer is considered as an international health disease that affects an individual 's health, psychology, lifestyle, and loads the whole family with its effect. on the other hand, these kinds of diseases load the governments as well with economical overheads including the need for developmental programs and advanced research groups and centers. the total number of cases of oral cancer is around 263,900 and the mortality rate is 40%. but when the disease is detected early, the percentage of recurrence is reduced to 1020%. unfortunately, only 35% of the malignant lesions are detected in the early stages due to the fact that these lesions start at the basal cell layer and can not be seen by the naked eye. potentially malignant oral lesions are those lesions that exhibit a change in shape or color on clinical or histological appearance. these kinds of changes are due to the different types of biopsies, which, in most cases, are surgical in nature and may cause trauma for the patient. so, it is important to have another technique to detect such lesions, especially in their early stages, such as using fluorescent light. risk factors associated with oral cancer : smoking : the risk of oral cancer is about 510 times greater among smokers compared to people who never smoked. this risk further multiplies among smokers who also drink alcohol. on the other hand, several studies relate this habit with the consumption of alcohol, as well as the number of cigarettes smoked and the period of smokingalcohol consumption : many studies showed that more than 7580% of the oral cancer patients drink alcohol continuously. it is thought that the effect of alcohol on the liver, especially liver cirrhosis, might be one of the main reasons for oral cancerhuman papillomavirus (hpv) : this virus plays an important role in the epithelial cancer of the tonsils and oropharynx, wherein more than 10% of these cancers have hpv dna integrated in their genomesyphilis : the percentage of cancerous ability of the leukoplakia originating from syphilis is 30100%. but the disease is rare even in the third worldage : more than 98% of oral cancers occur at more than 40 years of agemalnutrition : there is epidemic evidence to the relation of deficiency of vitamin a and oral epithelial cancerintraoral infectionsdirect sunlight : direct exposure to ultraviolet light for long periods of time is considered as an important risk factor for oral cancers, and it incidence is higher in whites than othersprecancerous lesions : the transformation of white lesions in the oral cavity into malignant ones is low ; but, on the other hand, it is high when they are red lesions and traumatic ulcers which is one of the categories. smoking : the risk of oral cancer is about 510 times greater among smokers compared to people who never smoked. this risk further multiplies among smokers who also drink alcohol. on the other hand, several studies relate this habit with the consumption of alcohol, as well as the number of cigarettes smoked and the period of smoking alcohol consumption : many studies showed that more than 7580% of the oral cancer patients drink alcohol continuously. it is thought that the effect of alcohol on the liver, especially liver cirrhosis, might be one of the main reasons for oral cancer human papillomavirus (hpv) : this virus plays an important role in the epithelial cancer of the tonsils and oropharynx, wherein more than 10% of these cancers have hpv dna integrated in their genome syphilis : the percentage of cancerous ability of the leukoplakia originating from syphilis is 30100%. but the disease is rare even in the third world age : more than 98% of oral cancers occur at more than 40 years of age malnutrition : there is epidemic evidence to the relation of deficiency of vitamin a and oral epithelial cancer intraoral infections direct sunlight : direct exposure to ultraviolet light for long periods of time is considered as an important risk factor for oral cancers, and it incidence is higher in whites than others precancerous lesions : the transformation of white lesions in the oral cavity into malignant ones is low ; but, on the other hand, it is high when they are red lesions and traumatic ulcers which is one of the categories. clinical objective analysis remains the most important method for the early detection of oral cancers so far. it was noticed that only 13% of the american population performed routine check - up in 2010 and it is hoped that this number will increase to 20% in 2020. nevertheless, the use of other instruments and machines is highly important in conjunction with visual examination, such as the brush biopsy, oralcdx, toluidine coloration, vizilite machine, and velscope. clinical objective analysis remains the most important method for the early detection of oral cancers so far. it was noticed that only 13% of the american population performed routine check - up in 2010 and it is hoped that this number will increase to 20% in 2020. nevertheless, the use of other instruments and machines is highly important in conjunction with visual examination, such as the brush biopsy, oralcdx, toluidine coloration, vizilite machine, and velscope. these patients were attending the college of dentistry, al - moasat hospital, al - mojtahed hospital, and al - karama nursing home, all in damascus. the basic forms were used to record the personal information, social information, and medical history and habits of the patients. many questions were asked about the patient : 1. the duration of the lesion since its occurrence ; 2. the change in the size of the lesion ; 3. did the characteristics of the lesion change ? 4. what are the signs and symptoms associated with the lesion, such as pain, sensation alteration, anesthesia, paresthesia, swellings, bad taste or odor, and enlargement or pain of nearby lymph nodes ? 5. any general symptoms associated with the lesion, such as fever, vomiting, nausea, etc., 6. was there any related disorder with the lesion like continuous trauma to the site or dental diseases at the site ? it is one of the machines used for the detection of oral cancers that uses blue light with a special wavelength of 400460 nm as the source that penetrates the epithelial layer and the basal layer reaching the stroma (led dental, burnaby, canada, 2007). this ability is called autofluorescence in which invisible fluorescent light filtered from the blue light gives fluorescent color to the normal tissues and different dark green colors reaching black color to the abnormal tissues [figure 1 ]. the velscope machine and intraoral camera (led dental, canada, 2007) the machine is portable, easily stored as it does not need much space in the clinic, painless for the patient, and it needs 1 cm) or is located at a critical place or has high susceptibility for malignancyexcisional biopsy carried out in small lesions (1 cm) or is located at a critical place or has high susceptibility for malignancy excisional biopsy carried out in small lesions (< 1 cm) and has normal readings. the biopsies were sent to the histopathology department in damascus university college of dentistry. after obtaining the histopathological results, potentially malignant lesions observed in each patient were examined twice : the first examination consisted of a biopsy while the second one was with velscope instrument. the malignancy of each detected lesion was determined ; 26 cases were high - risk lesions. sensitivity, specificity, accuracy, positive predictive value, and negative predictive value were calculated using the following formulas : sensitivity = [true positives/(true positive + false negative) ] 100 specificity = [true negatives/(true negative + false positives) ] 100 accuracy = sensitivity + specificity predictive value for a positive result (pv+) = [true positive/(true positive + false positive) ] 100 predictive value for a negative result (pv) = [true negatives/(true negatives + false negatives) ] 100. potentially malignant lesions observed in each patient were examined twice : the first examination consisted of a biopsy while the second one was with velscope instrument. the malignancy of each detected lesion was determined ; 26 cases were high - risk lesions. sensitivity, specificity, accuracy, positive predictive value, and negative predictive value were calculated using the following formulas : sensitivity = [true positives/(true positive + false negative) ] 100 specificity = [true negatives/(true negative + false positives) ] 100 accuracy = sensitivity + specificity predictive value for a positive result (pv+) = [true positive/(true positive + false positive) ] 100 predictive value for a negative result (pv) = [true negatives/(true negatives + false negatives) ] 100. the sample consisted of 414 males and 334 females with a mean age of 37 years [figure 2 ]. the study sample according to gender the results presented in figure 3 show the occurrence and gender wise distribution of potential malignant lesions. clinically undifferentiated lesions were higher in females (92.2%) compared to males (89.1%), while clinically differentiated lesions were higher in males than females (10.9%). data distribution according to gender there were no statistically significant differences between male and female patients with potentially malignant lesions, as shown in figure 3 and table 1. potential malignant lesions occurrence and gender wise distribution subjects distribution according to the types of lesions is shown in figure 4. traumatic ulcer had the highest incidence (26.8%) and ossifying fibroma or pemphigus vulgaris (1.4%) was the least common. distribution according to the types of lesions tables 24 show that the percentage of benign lesions was 61.5% according to their histological analysis, while the efficiency of velscope in detecting lesions was higher (81.7%). lesions malignancy according to their histological analysis velscope 's efficiency in detecting lesions malignity chi - square test of benign and malignant in biopsy group table 4 shows that p value was much lower than 0.05, so we can conclude that at 95% confidence level, there were significant differences in velscope results between benign in biopsy group and malignant in biopsy group subjects ; percentage of benign subjects in benign in the biopsy group was greater than the percentage of benign subjects in malignant in the biopsy group. also, the percentage of malignant subjects in benign in the biopsy group was lower than the percent of malignant subjects in malignant in the biopsy group. agreement between velscope results and biopsy results to know if there were significant differences in velscope results between benign in biopsy group and malignant in biopsy group subjects, a chi - square test was applied as shown in table 4. kappa coefficient value was calculated between velscope results and biopsy results, as shown in table 6. velscope results in statistical calculations table 6 shows that p value was much lower than 0.05, so we can conclude at 95% confidence level, there was significant agreement between velscope results and biopsy results. kappa coefficient value was approximately 0.5, which means that the strength of the agreement was medium. table 8 shows the sensitivity, specificity, accuracy, positive predictive value, and negative predictive value, while table 9 shows that accordance between velscope and biopsy failure was 21.1% and between velscope and biopsy success was about 78.9%. true-/false - positive and -negative status of the results sensitivity, specificity, accuracy, positive predictive value, and negative predictive value of the calculated results accordance between velscope results and biopsy results after applying fluorescent light to the patients tissues, the response of the tissues reveals the nature of the tissues as shown in figure 5 ; in case of tissues with malignant or premalignant lesions, the color changes into dark colors as shown in figure 6. normal fluorescent color of the tongue using velscope, as seen in our study fluorescent discontinuity and dark color showing abnormal tissues. by biopsy the sample consisted of 414 males and 334 females with a mean age of 37 years [figure 2 ]. the results presented in figure 3 show the occurrence and gender wise distribution of potential malignant lesions. clinically undifferentiated lesions were higher in females (92.2%) compared to males (89.1%), while clinically differentiated lesions were higher in males than females (10.9%). data distribution according to gender there were no statistically significant differences between male and female patients with potentially malignant lesions, as shown in figure 3 and table 1. potential malignant lesions occurrence and gender wise distribution subjects distribution according to the types of lesions is shown in figure 4. traumatic ulcer had the highest incidence (26.8%) and ossifying fibroma or pemphigus vulgaris (1.4%) was the least common. distribution according to the types of lesions tables 24 show that the percentage of benign lesions was 61.5% according to their histological analysis, while the efficiency of velscope in detecting lesions was higher (81.7%). lesions malignancy according to their histological analysis velscope 's efficiency in detecting lesions malignity chi - square test of benign and malignant in biopsy group table 4 shows that p value was much lower than 0.05, so we can conclude that at 95% confidence level, there were significant differences in velscope results between benign in biopsy group and malignant in biopsy group subjects ; percentage of benign subjects in benign in the biopsy group was greater than the percentage of benign subjects in malignant in the biopsy group. also, the percentage of malignant subjects in benign in the biopsy group was lower than the percent of malignant subjects in malignant in the biopsy group agreement between velscope results and biopsy results to know if there were significant differences in velscope results between benign in biopsy group and malignant in biopsy group subjects, a chi - square test was applied as shown in table 4. kappa coefficient value was calculated between velscope results and biopsy results, as shown in table 6. velscope results in statistical calculations table 6 shows that p value was much lower than 0.05, so we can conclude at 95% confidence level, there was significant agreement between velscope results and biopsy results. kappa coefficient value was approximately 0.5, which means that the strength of the agreement was medium. table 8 shows the sensitivity, specificity, accuracy, positive predictive value, and negative predictive value, while table 9 shows that accordance between velscope and biopsy failure was 21.1% and between velscope and biopsy success was about 78.9%. true-/false - positive and -negative status of the results sensitivity, specificity, accuracy, positive predictive value, and negative predictive value of the calculated results accordance between velscope results and biopsy results after applying fluorescent light to the patients tissues, the response of the tissues reveals the nature of the tissues as shown in figure 5 ; in case of tissues with malignant or premalignant lesions, the color changes into dark colors as shown in figure 6. normal fluorescent color of the tongue using velscope, as seen in our study fluorescent discontinuity and dark color showing abnormal tissues. by biopsy the survival rate for the patients suffering from oral cancer depends on three main factors : early detection of the lesiona good differential diagnosis of the lesionthe way of treatment. early detection of the lesion a good differential diagnosis of the lesion the way of treatment. elimination of the risk factors should be taken into consideration, such as smoking and alcohol drinking. clinical intraoral examination and proper radiographic study are important for the detection of the lesion, but unfortunately, they are not enough for detecting malignant and premalignant lesions. final diagnosis should be based on surgical biopsy. the velscope machine is an efficient diagnostic instrument for the differentiation of premalignant and malignant lesions. several techniques have been introduced to aid in the early detection of malignant or premalignant lesions, such as oralcdx, toluidine coloring, vizilite machine, identafi machine, and velscope. this explains the big number of articles explaining the importance of these techniques as non - traumatic procedures in comparison with surgical biopsy. therefore, we had this study done to evaluate the role of velscope in such detection. this machine works based on fluorescence activity [figure 2 ] as it uses blue light with a wavelength of 400460 nm inside the oral cavity, penetrating the epithelial tissues and reaching the stroma through the basal layer. this will allow the practitioner to observe the suspicious lesions in different colors [figure 7 ]. as and when the light comes across abnormal tissue, it will discontinue its fluorescence activity that was seen on the normal tissues by changing from the green fluorescent light to a dark green color. the mode of action of fluorescent light the machine 's efficiency to act as a diagnostic instrument for the differentiation of malignant and the premalignant lesions, depends on the final decision made on the basis of the golden standard surgical biopsy. the results of our study are in accordance with the results of laneh. who found in 2006 that in the differentiation between normal and defective mucosa, the sensitivity value was 98% and the specificity value was 92%. poh. in 2006 stated they had used the machine inside the surgical chamber while performing an enucleation. we obtained results similar to that of trolneck and erikson (2009) whom did a comparative study between different ways of early detection of intraoral lesions using vizilite and biopsy from oralcdx and velscope ; they obtained a sensitivity of 98100% and a specificity of 94100%. in 2011, matsomoto. they noticed that the machine was a good aid in the detection of changes in tissues. the study emphasized that the machine has a good sensitivity as the clinical examination, and it can be used as an additional device for diagnosis in patients with risk factors. the reason for the lesser value of sensitivity was that the study included all the intraoral lesions in the high - risk patients and not only the lesions that were diagnosed histopathologically as malignant or hyperplastic in nature. but we agree with them that we can use the machine as an aiding device with the clinical examination. our study results agreed with the study of lee and wong who reported in their study that the sensitivity and specificity of the machine is high and almost absolute when it comes to the diagnosis of malignant lesions ; they performed their study on lesions that were prediagnosed as saethre our results are similar to the results of farah. who studied 44 cases histologically and by the machine clinically, and obtained a sensitivity of 100% and a specificity of 96%. but our results are not in agreement with that of camily and ledegy as their study showed that the machine is not effective in the detection of hyperplasia as the sensitivity was 30% and the specificity was 63%. the difference in results could be because the machine was depended as a single definitive diagnostic tool in their study without clinical examination. as their study did not support using the machine in suspected lesion, as the sensitivity was 87.1% and the specificity was 20%. this drop might be due to not combining the clinical examination with the use of the machine. they noticed loss of fluorescent effect on benign lesions like varicose of the tongue and pigmentation. so, based on the results it can be concluded that we can depend on the machine as a reassuring device for the clinical examination, especially in the examination of the high - risk population, and the big advantage is that it is a non - traumatic technique used by dentists for the detection of premalignant and malignant lesions in early stages. the use of velscope is effective in the detection of malignant and premalignant lesionsvelscope aids in the detection of the site of biopsyvelscope is used an aiding tool inside the surgical theater for the detection of the borders on enucleation. the use of velscope is effective in the detection of malignant and premalignant lesions velscope aids in the detection of the site of biopsy velscope is used an aiding tool inside the surgical theater for the detection of the borders on enucleation. | aim : the purpose of this study was the early detection of premalignant and malignant oral soft lesions by fluorescent light (velscope).materials and methods : a total of 748 patients were evaluated through clinical and fluorescent light analysis of the entire oral cavity. any lesion that was detected underwent a surgical excision biopsy as the golden standard for the detection of the lesion 's histology ; then a comparison was made between the results to assure the efficacy of the fluorescent light analysis outcome.results:about 9.4% of the lesions detected were abnormal lesions and 83.09% had loss of fluorescent light effect. based on the use of surgical biopsy, the machine had a sensitivity of 74.1% and a specificity of 96.3%. according to the statistical analysis, the p value was much lower than 0.05, so we can conclude that at 95% confidence level, there was significant agreement between velscope results and biopsy results. kappa coefficient value was approximately 0.5, which means that the strength of the agreement was medium.conclusion:velscope can be used as a clinical diagnostic aid in the detection of premalignant and malignant lesions of the oral cavity. in addition, it helps in the detection of the borders in both surgical biopsy and surgical excision. |
frontal fibrosing alopecia (ffa) is increasingly considered as a distinct variant of lichen planopilaris (lpp), with scarring alopecia of frontotemporal hairline and nonscarring alopecia of the eyebrows. ffa and graham little piccardi syndrome (glps) can be considered as spectrum of a disease as per the current literature due to the presence of overlapping features. a 49-year - old female presented with hair loss from both center and sides of the scalp for the last 1 year which did not improve following minoxidil therapy. her family history was unremarkable, and she did not suffer from any chronic disease. no history of chronic medication could be elicited. personal and family history of photosensitivity was absent. on examination, we found scarring alopecia with follicular papules and mild scaling over the vertex [figure 1a and b ]. she had definite recession of frontotemporal hairline with bilaterally symmetrical band - like scarring alopecia of the same region [figure 2 ]. there were numerous violaceous follicular spinous papules distributed over the abdomen, back, arms, and legs [figure 3a c ]. (b) close - up view recession of frontotemporal hairline with symmetrical band - like scarring alopecia (a) violaceous follicular spinous papules over the abdomen. (c) violaceous follicular spinous papules over the leg nonscarring alopecia over the axilla routine investigations with complete hemogram, blood sugar, liver function test including hepatitis profile and thyroid screening were within normal limit. there was definite basal cell degeneration with band - like chronic inflammatory infiltrate around perifollicular region. all these features were consistent with lpp [figure 5a and b ]. (a) follicular plugging with band - like chronic inflammatory infiltrate around perifollicular region, occasional loss of hair follicles, and perifollicular fibrosis (h and e, 10). (b) follicular plugging with definite basal cell degeneration with band - like chronic inflammatory infiltrate around perifollicular region (h and e, 40) based on above findings, a diagnosis of glps with ffa was made, and the patient was put on oral prednisolone (1 mg / kg / day) and oral retinoid (0.5 mg / kg / day) for initial 2 weeks. dose of the steroid was tapered to 20 mg / day and continued along with the same dose of oral isotretinoin (20 mg) for another 2 weeks with strict monitoring of blood parameters without any alteration. at the end of 1 month, the patient had significant improvement of pruritus and hair loss with flattening of the follicular papules. doses of steroid and isotretinoin were further reduced to 10 mg / day and 15 mg / day, respectively, for another 1 month with further flattening of follicular papules and improvement of ffa patch. now, the patient is on only 15 mg oral retinoid every alternate day and with regular follow - up on a monthly basis. the entity, glps was born with a lot of controversies as far as proper clinical definition is concerned. our observation of simultaneous presence of two variants may further add fire to the unsolved issues of this strange entity. glps is an unusual variant of lpp characterized by multifocal cicatricial alopecia of the scalp, noncicatricial alopecia of the axillae, and/or pubis and follicular lichen planus (lp) involving the trunk and extremities. on the other hand, this progressive form of fibrosing alopecia has failed to show any significant difference from other forms of lpp as far as histopathology and immunophenotyping are concerned. if we look into the pathogenesis, glps is generally considered of having an autoimmune origin. in ffa, the lymphocytic infiltrate and fibrosis affect selectively the intermediate and the vellus - like follicles of the frontal margin and eyebrows. although the reason for this selective involvement is still an enigma, it is hypothesized that it may represent a variety of lpp with selective involvement of certain androgen - dependent areas. the affected follicles may have typical biologic markers that could explain typical characteristic feature of ffa. there was several existing literatures where ffa was found to be associated with different forms of lp. in one report, authors concluded ffa as a phenotypic variation of glps where additional features are nonscarring alopecia of the eyebrow and axillae and follicular lp over the face. other authors described ffa in the association of cutaneous lp and oral mucosal lp. to the best of our knowledge, like our case, no such case has been reported till date where ffa is found along with full blown glps with all features. there are several treatment options available for lpp such as oral steroid, oral retinoid, cyclosporine, mycophenolate mofetil, hydroxychloroquine, and psoralen plus ultraviolet a therapy. surprisingly, our case responded very well in terms of symptomatic improvement, clearing of lesions, and halt of progressive alopecia. the classical and extensive presentation of glps along with concomitant ffa in the index case portrays a rare and unique presentation of follicular lp. such a coexistence of these two variants of lpp may be a mere overlap, a phenotypic variation of the same entity, or it may help in future to frame a newer nosology for lpp. the authors certify that they have obtained all appropriate patient consent forms. in the form the patient(s) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal. the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity can not be guaranteed. the authors certify that they have obtained all appropriate patient consent forms. in the form the patient(s) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal. the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity can not be guaranteed. | graham little piccardi syndrome (glps) is a rare form of follicular lichen planus and comprises cicatricial alopecia of the scalp, noncicatricial alopecia of the axillae, and/or pubis and spinous follicular papules involving the trunk and extremities. we herein report a classic case of glps in a 49-year - old female. uniqueness of our case is due to its additional features of frontal fibrosing alopecia (ffa). although overlap between glps and ffa has been mentioned in literature, such numbers are scanty. therapeutic response with oral steroid and retinoid is also far better in contrast to what is described in the existing literature. |
iron insufficiency has been proposed to play a central role in restless legs syndrome (rls) pathophysiology indicated primarily by the secondary causes of rls such as pure iron deficiency anemia and end stage renal disease or pregnancy which are both associated with altered iron metabolism. it has also been shown that frequent blood donors have an increased risk of rls.1 overall, it has gained wide clinical acceptance that rls is commonly occurring with iron deficiency anemia, although there have been few studies documenting this relationship.24 in addition, it has been shown that rls severity negatively correlates with serum ferritin.3,5 correcting iron status in rls patients with iron deficiency has been found to reduce rls symptoms.3,6 complete remission of rls symptoms has also been reported after intravenous substitution of iron in rls patients with and without clear anemia.710 thus pharmacological data as well as data from secondary rls support a relation between iron deficiency and rls. for evaluating the iron status of rls patients, serum and cerebrospinal fluid (csf) measures involved in iron metabolism,11 autopsy evaluation of brain iron status,12,13 transcranial parenchymal ultrasound,14,15 and magnetic resonance imaging (mri) measurements16,17 have been performed. all of these data supported the view that there is a measurable brain iron deficiency particularly in the substantia nigra in rls patients.18 t2-hypointensity is thought to represent iron deposition as iron deposits shorten t2 relaxation times on t2-weighted mris.19 using a cranial mri scan with a standard t2 -sequence, differences of regional mean t2-values can be measured throughout the brain as a consequence of different brain iron concentrations. up to now, significant differences in brain iron concentrations were found in the substantia nigra in rls patients as compared to controls in two studies using t2 -weighted mri.16,17 in this study we investigated regional brain iron concentrations using a standard t2 -sequence but failed to replicate earlier findings of reduced brain iron in the substantia nigra in patients with rls compared to healthy controls. twelve patients (nine women, three men, mean age 58.5 8.7 years, range 4346 years) with idiopathic rls for 12.1 9.1 years (range 230 years) were included. five patients had early - onset rls with symptom onset before the age of 45 years. all patients met the standardized diagnostic criteria of the international restless legs syndrome study group,8 and neurological examination revealed no evidence of additional neurological or psychiatric disease. eight patients were treated with dopaminergic agents for rls (levodopa, n = 5 ; pramipexole, n = 3). the mean overall duration of treatment with dopaminergics (including previous medication) in these patients was 3.4 2.4 years (range 19 years). rls severity was assessed considering the overall status when not taking the current medication (if applicable) by means of the clinical global impression (cgi ; 1 = normal to 6 = very severe). cgi ratings revealed that six patients suffered from moderate rls (cgi = 4) and six patients from severe rls (cgi = 5). controls consisted of 12 gender- and age - matched subjects (mean age 56.8 10.6 years, range 4174 years) in whom rls was clinically excluded. mol / l (range 12.4 to 21.1) ; ferritin, 111.9 63.4 g / l (range 22 to 193) ; transferrin, 2.5 0.4 g / l (range 1.9 to 3.2) ; transferrin saturation, 26.7% 8.9% (range 20 to 44) ; soluble transferrin receptor, 3.3 1.0 mg / all values were within the normal range and did not significantly differ from control subjects who had the following mean values : iron, 18.7 6.6 mol / l (range 8.5 to 27.1) ; ferritin, 124.7 171.8 g / l (range 7 to 614) ; transferrin, 2.4 0.4 g / l (range 2 to 3.2) ; transferrin saturation, 31.1% 11.3% (range 12 to 46) ; soluble transferrin receptor, 3.1 0.8 mg / all subjects were scanned using a whole head t2 weighted gradient echo sequence (tr 800 ms, te 26 ms, fa : 20, fov 230 230 mm, matrix 256 192, slice thickness 5 mm) on a siemens 1.5 t sonata scanner (siemens medical solutions, erlangen, germany). average t2 signal intensity (si) values were determined in selected regions of interest. regions of interest (rois) were placed in the following areas in each hemisphere : the substantia nigra, the pallidum, the caudate head the thalamus as well as the occipital white matter and the frontal white matter bilaterally. all rois were placed using a standardized placement procedure utilizing atlas - based rules with morphological landmarks in each individual participant s volumes. all rois were placed by the same rater who was blinded to the subjects status (patient or control) and were created as a sphere with a diameter of 2 mm (totalling a number of 100 isotropic voxels) to avoid arbitrary sizes of rois across participants, and to avoid inclusion of multiple white matter bundles in regional measurements. the average t2 si value and its standard deviation were determined for each roi and for two reference regions of interest. one of these reference rois was placed in the middle of the ventricles, measuring csf, the other was placed at the scull measuring the t2 si value of the bone. si was measured as a surrogate marker for t2-values. in order to provide objective measurements the t2-change ratio (tcr) was calculated as followed : t2cr = (si [roi ] - si reference)/si reference. differences in tcr between patients and control subjects were analyzed using the mann - whitney u test. for correlation analysis, spearman rank correlation coefficients were calculated. the significance level was set at = 5% with adjustment for multiple testing by the bonferroni method. the study was performed in accordance with the declaration of helsinki and approved by the local ethics committee. the t2cr values did not significantly differ between rls patients and controls for any of the investigated brain regions both for the csf and bone ratios (see tables 1 and 2). in rls patients there was no significant correlation between brain iron values and rls severity or cgi scores. in both groups we did not find a significant correlation between t2cr values (csf and bone ratios) and age or sex. in addition, serum iron, ferritin, transferrin, transferrin saturation and the soluble transferrin receptor concentration did not correlate with brain t2cr values (csf and bone ratios) in both groups. subanalysis of untreated and treated patients did not reveal significant differences in t2cr values (csf and bone ratios) and we found no significant differences between early - onset and late - onset rls. in this study we did not find significant abnormalities in brain iron content of rls patients in any of the investigated brain regions such as the substantia nigra, pallidum, caudate head, or thalamus. serum measures involved in iron metabolism such as serum iron, ferritin, transferrin, transferrin saturation, and soluble transferrin receptor did not correlate with t2cr - values. in an earlier study allen and colleagues16 investigated five rls patients with a similar mri method and reported decreased iron concentration in the substantia nigra and the putamen compared to five age- and sex - matched controls. in a subsequent study they performed mri in a considerable larger sample including 22 early - onset rls patients, 19 late - onset rls patients, and 39 controls. in that study they found a significant lower iron index in the substantia nigra only in early - onset rls but not in late - onset compared to controls.17 these results are consistent with csf studies of the same group showing iron insufficiency in csf predominantly in early - onset rls.11 in agreement, autopsy evaluations of brain iron status which were all performed in early - onset rls showed a brain iron deficit in the substantia nigra.12,13 in addition, transcranial ultrasound studies revealed a significantly reduced hyperechogenicity (hypoechogenicity) in the substantia nigra in rls patients when compared with normal control subjects which is supposed to indicate reduced regional iron concentration.14,15 all of these data supported the view that there is a measurable brain iron deficiency particularly in the substantia nigra in rls patients. in contrast, a recent mri study investigating patients with rls with significant hypoechogenicity in the substantia nigra in the transcranial ultrasound found no significant differences in t2 values reflecting iron concentration in the substantia nigra,20 a finding that is also supported by the present study. however, godau and colleauges20 found mr signal changes in the caudate nucleus, the thalamus, and the red nucleus, which could not be confirmed in the present and earlier studies.21 in all of the studies describing an iron deficiency in the substantia nigra, patients were either on dopaminergic medication or pretreated with dopaminergics and taken off medication for the investigation. therefore it was discussed that dopaminergic medication could possibly have affected brain iron concentration16 and thus the results of above mentioned studies. using a similar technique,19 we could not replicate the mri findings of earlier studies16,17 and did not find a regional brain iron deficit neither in early- or in late - onset rls. since we also investigated some patients who were on dopaminergic medication it is unlikely that the rls - specific medication accounts for the different results. discrepancies might have arisen as t2-values are not only affected by iron concentration but also by water content of the brain. therefore, correlation with volumetric measures should be performed in future. since iron increases in the brain with aging,22 age differences one possible reason for the different results might be differences in serum iron variables which were either not assessed in the previous mri studies16 or only used for exclusion of patients with iron deficiency (serum ferritin < 18 g / l, iron saturation < 16%).17 therefore it is possible that patients in the previous mri studies had lower iron values than our patients or lower compared to their control group resulting in significant differences. however, we did not find a significant correlation between t2-values and serum iron parameters. another possible explanation for the different mr findings concerning the iron concentration in the substantia nigra and various other brain regions might be the different mr sequences used in the different studies which may be influenced by different aspects of tissue iron composition20 und thus lead to different results. further studies with larger sample sizes, assessment of iron variables, all patients off rls medication and consistent mr sequences are necessary to better clarify the role of brain iron in rls. in addition, studies using more sophisticated methods which can be applied in vivo to study untreated patients and which are able to detect also small quantitative differences in brain iron, eg, moving to higher field strengths or the use of new imaging methods such as three - dimensional (3d) gradient echo imaging or susceptibility - weighted imaging19 should be performed. despite the limitations in the mri measurements of brain iron, our results do not support the view of substantially impaired regional brain iron in rls. | using a t2 gradient echo magnetic resonance imaging (mri) sequence, regional t2 signal intensity (si) values, a surrogate marker for t2 values, were determined in 12 regions of interest (substantia nigra, pallidum, caudate head, thalamus, occipital white matter, and frontal white matter bilaterally) and in two reference regions (cerebrospinal fluid and bone) in 12 patients suffering from moderate to severe idiopathic restless legs syndrome (rls ; mean age 58.5 8.7 years) for 12.1 9.1 years and in 12 healthy control subjects (mean age 56.8 10.6 years). iron deposits shorten t2 relaxation times on t2-weighted mri. we used regional t2 si to estimate regional t2-values. a t2-change ratio was calculated for each region of interest relative to the reference regions. we did not find significant differences in any of the investigated brain regions. in addition, serum measures involved in iron metabolism did not correlate with t2 si values. we could not replicate earlier findings describing reduced regional brain iron concentrations in patients with rls. our results do not support the view of substantially impaired regional brain iron in rls. |
percutaneous radiofrequency (rf) ablation is a minimally invasive and nonsurgical technique used to treat various solid tumors. many studies have shown that this technique is safe and effective in treating hepatic and renal tumors [13 ]. recent investigations of rf ablation s practicability and outcome as a successful method for extricating primary and metastatic lung tumors have been promising [48 ]. studies have shown that rf ablation is evolving into a suitable alternative and adjuvant method to open thoracic surgery of primary and metastatic lung cancer, which is commonly associated with high morbidity and mortality [9, 10 ]. however, there are many issues concerning rf ablation that need to be addressed. these questions mainly involve patient selection, location and type of lung malignancy, accessibility of the lesion, possible risks for acute and chronic complications and management, and finally, pain management, both during and after the rf ablation. in this article, we present a case that utilized a technique to decrease the pain associated with rf ablation of a malignancy located within the subpleural lung. under ct guidance, we created an artificial pneumothorax prior to the rf ablation, which resulted in minimizing the pain usually experienced during and after the procedure. the patient s written informed consent was obtained after the risk, benefits, and alternatives of the procedure were fully explained. a waiver of informed consent was obtained from the irb for this report. a 43-year - old woman with a history of metastatic colorectal cancer to the liver, originally diagnosed in 1999, presented with newly rising carcinoembryogenic antigen (cea), and ct scan of the chest confirmed the development of three new subcentimeter nodules within both lungs. these nodules were consistent with pulmonary parenchymal metastases : one within the right middle lobe and, notably, two nodules within the anterior segment of the left upper lobe, one within the subpleural aspect of the lobe (fig. due to slow progression of the tumor within the lung, complications of hypercoagulability related to the patient s current systemic chemotherapy and her prior familiarity with rf ablation for at least three hepatic metastases, rf ablation was preferred over surgical metastasectomy.fig. a a 22-gauge chiba needle was placed adjacent to the peripherally located metastasis (white arrowhead : subpleural tumor). b an intentional and iatrogenic artificial pneumothorax was created displacing the visceral pleura (white arrows : artificial pneumothorax). c enlargement of the artificially created pneumothorax further separated the ablation site from the parietal pleura. d following completion of the ablation, the tines were retracted and a localized pneumothorax was decompressed by active aspiration using a syringe sequential ct images of radiofrequency ablation of subpleural tumor. a a 22-gauge chiba needle was placed adjacent to the peripherally located metastasis (white arrowhead : subpleural tumor). b an intentional and iatrogenic artificial pneumothorax was created displacing the visceral pleura (white arrows : artificial pneumothorax). c enlargement of the artificially created pneumothorax further separated the ablation site from the parietal pleura. d following completion of the ablation, the tines were retracted and a localized pneumothorax was decompressed by active aspiration using a syringe the patient was prepped and draped in sterile fashion, and midazolam hydrochlorate (1 mg of versed ; roche laboratories, nutley, nj, usa), fentanyl citrate (25 g of sublimaze ; akorn, buffalo grove, il, usa), and diphenhydramine hydrochloride (25 mg of benadryl ; pfizer inc., new london, ct, usa) were administered intravenously to induce moderate sedation. utilizing the ge hispeed advantage ct scanner (ge medical systems, milwaukee, wi, usa) with the patient in the supine position, a 22-gauge chiba needle (cook, bloomington, in, usa) was placed adjacent to the peripherally located metastasis within the anterior segment of the left upper lobe via anterior intercostal approach (fig. following ct confirmation of the needle tip, a rita 6.4-fr starburst - xl multiarray electrode (rita medical systems, mountain view, ca, usa) was placed so that its leading tip was situated to enter proximally into the parietal and visceral pleura and the underlying lung. based on results by others using iatrogenic pneumothorax for easy access to a tumor [1214 ], we deduced that iatrogenic pneumothorax may decrease pain by separating the two pleural layers or the visceral from the somatically innervated parietal pleura. with slow and deliberate tine deployment to 3 cm, an intentional and iatrogenic artificial pneumothorax was created, the tines of the electrode piercing the lung and surrounding the targeted nodule, while displacing the visceral pleura away from the parietal pleura (fig. in order to further enlarge the potential pneumothorax, the deployed electrode was then advanced, ensuring that its 1-cm active tip was entirely within the pleural space and pneumothorax (fig. subsequently, a single rf ablation was performed according to the manufacturer s guide (fig. 1c). by separating the visceral and somatically innervated parietal pleura with a potential space, we observed very minimal patient discomfort during the ablation session. in addition, we were still able to achieve effective capture of the targeted nodule within the expanded array, without negatively affecting the patient s oxygen saturation and vital signs. following completion of the ablation, the tines were retracted with the electrode tip still residing within the pleural space and pneumothorax. with a 20-ml luer lock syringe attached to the side port of the electrode, the rf electrode was withdrawn with negative suction applied to the syringe, thereby manually decompressing the associated localized pneumothorax and actively aspirating any potential air leak (fig. the second nodule was then localized with the chiba needle, and via tandem needle technique, the starburst electrode was driven to the nodule, its tines deployed, and the metastasis ablated using standard technique. due to the second nodule s more central location, repeat creation of an artificial pneumothorax was not performed. before rf ablation, as the chiba needle was removed, intraprocedural ct scans of the thorax showed no air within the pleural cavity following the first ablation and a minimally sized residual pneumothorax following the second ablation, which required no subsequent intervention or further treatment. postablation ct images demonstrated progressive ground - glass attenuation envelopment of each targeted nodule, suggestive of a successful rf ablation. throughout the rf ablation procedure and postprocedure observation period, the patient s vital signs, including oxygen saturation, remained stable. following the procedure the patient was discharged on the same day with minimal oral nsaid for pain control. nine - month follow - up ct images demonstrated dense bands of scarring with signs of shrinkage of ablation - induced areas of necrosis, as well as complete resolution of the pneumothorax. rf ablation is a minimally invasive and effective method used to treat various tumors by inducing complete coagulation necrosis and cell death of a targeted tumor, with generally few complications. the physiological heat insulation created by air surrounding the tumor produces more effective thermal energy for complete cell necrosis ; therefore, rf ablation is thought to be a suitable treatment option for pulmonary malignancy. however, the literature on the percutaneous ablation of thoracic tumors, although expanding, is still incomplete, and multiple technical issues require address and optimization. one issue that has been a constant challenge for rf operators has been patient pain management. for the most part, rf ablation has been performed as an outpatient procedure, usually under moderate sedation. operators have at times favored deep sedation and even general anesthesia, particularly in patients with targeted lesions involving the pleura and/or chest wall, and especially in those seeking palliation for pain. rf ablation - induced pain causes discomfort and agitation for patients during and following the procedure. it can cause a wide spectrum of pain ranging from tolerably mild pain to severe pain requiring high doses of narcotics or sedatives, or even general anesthesia, which necessitates anesthesiology service one study has shown that rf ablation - induced pain is the second most frequently cited complication of the rf ablation procedure, requiring general anesthesia at their institution. in particular, rf ablation involving the pleural margin, as in our case, has been known to cause more severe pain, usually requiring multiple intercostal nerve blocks or general anesthesia to proceed with the procedure [11, 17 ]. the consequences of rf ablation - induced pain can present additional problematic issues. with severe intra - ablational pain and its management, subsequent electrode repositioning for overlapping ablations required in larger tumor therapy may be jeopardized, in that a higher and deeper level of moderate sedation and pain control during the procedure will diminish lung aeration and expansion and patient cooperation. the technical difficulties encountered in repeated electrode repositioning and deployment may overly lengthen the procedure and induce additional complications, especially in those patients with advanced lung diseases and poor pulmonary reserve, leading to difficult and prolonged recovery. with prolonged postprocedural pain, patients may splint and limit their respiratory excursion. with decreased lung ventilation and clearance function,, rf ablation - induced pain reduces the benefits of the noninvasiveness of rf ablation and may cause relatively extended care and potential hospitalization for post - rf ablation pain control. therefore, the procedure may become less cost - effective and less attractive as an alternative therapeutic method to surgery. as previously mentioned, rf ablation of tumors involving the pleural margin is known to incite more pain sensors than others. however, our case presentation has suggested that an artificially created pneumothorax may mitigate the pain for those patients undergoing rf ablation of those types of tumors. in our patient, the amount of sedation (25 g of fentanyl and 1 mg of midazolam) used was substantially less than what is usually required (200 g of fentanyl and 3 mg of midazolam) in our experience. despite creation of a pneumothorax, the overall procedure time was not significantly increased, since the pneumothorax was established as the electrode was inserted and not beforehand. overall, complications related to intraprocedural pain and deep sedation are minimized, potentially with better outcomes than with the other conventional methods of treating subpleural tumors. although our case may suggest benefits of using iatrogenic pneumothorax to reduce the pain, possible risks and limitation must be evaluated. no major unpredicted complications are expected other than the usual potential complications that rf ablation of the lung may cause. however, a larger than planned pneumothorax or a pneumothorax related to an actual air leak may be anticipated. this may cause respiratory distress, but immediate awareness and treatment should minimize further deleterious respiratory deterioration. there are reports of utilizing artificially created pneumothorax to reach tumors in other target organs, such as the upper pole of the kidney, the hepatic dome, and the mediastinum / pulmonary hilum to prevent traversal of aerated lung parenchyma. however, this is one of the first observations of utilizing an artificial pneumothorax to reduce rf ablation - induced pain in ablation of an intrathoracic intrapulmonary subpleural tumor. future investigation focused on followings will be useful to potentiate the pain - reducing effect of iatrogenic pneumothorax in lung rf ablation : (i) evaluating a cohort of patients utilizing a subjective and objective pain scale, both during and after the procedure ; and (ii) investigating how reproducible and feasible this technique is in different clinical settings such as patients with a history of lung / pleural surgery with scarring or patients with emphysematous disease. | one of the main issues with radiofrequency (rf) ablation of the subpleural lung malignancy is pain management during and after rf ablation. in this article, we present a case that utilized a technique to decrease the pain associated with rf ablation of a malignancy located within the subpleural lung. under ct guidance, we created an artificial pneumothorax prior to the rf ablation, which resulted in minimizing the pain usually experienced during and after the procedure. it also decreased the amount of pain medications usually used in patients undergoing rf ablation of a subpleural lung lesion. |
paratuberculosis (map), is a disease affecting mainly domestic but also wild ruminants, including mouflons (ovis musimon ; wild sheep) [13 ]. in wild ruminants, paratuberculosis often manifests subclinically without overt symptoms which makes the disease more concerning from an epidemiological point of view [3, 4 ]. during the subclinical as well as clinical phase of the infection, the animals spread the agent into the environment by faecal shedding, where it can act as a source of infection for other animals., animals intended for transport across different regions or countries must undergo quarantine lasting at least 30 days with respect to major epizootic and notifiable diseases, such as bluetongue, bovine tuberculosis, brucellosis, and foot and mouth disease. paratuberculosis does not belong to the above mentioned group and the examination of imported ruminants therefore depends on the preference of the owner of the receiving farm. in addition, due to economic constraints, the belief that paratuberculosis does not represent a problem, or because of its low priority compared to other animal diseases, paratuberculosis control programmes in europe are mostly based on voluntary participation. in some countries, paratuberculosis numbers among the notifiable diseases, but there is no obligation to test animals with clinical signs. thus, animals are not examined due to concerns regarding their obligatory elimination or problems with selling animal remains (herds with paratuberculosis status). animals can be negatively influenced either by physical (hunger, thirst, injury, etc.) or psychological / ethological stress (handling, restraint, new environment, etc.). compared to domesticated, calm animals, wild species with excitable temperaments react to stressors more sensitively. while physical stress is connected with pain, psychological stress is characterised by fear which is one of the most important stressors. various stressors can detrimentally affect the immune system of animals, causing disruption of commensal microflora [10, 11 ], enhancing susceptibility to diseases, or activating latent infections. in the best case scenario, animals are examined for the presence of map before their import into a new farm. healthy animals diagnosed as map - negative are then released into the area of a new farm with other animals. the goal of this work was to assess the influence of stress connected with transport to a new farm in animals that were negative for map but which had originated from a herd in which map was previously detected. although pcr is not able to distinguish between live and dead organisms, a key main advantage lies in its higher sensitivity compared to cultivation. in the case of mouflons (sheep) in which cultivation is complicated and the presence of nongrowing isolates was described, real time quantitative pcr (qpcr) represents a sensitive tool for detection of map, based on copy number of is900 in map chromosome. the imported group of mouflons consisted of six individuals of different ages ; two mouflon ewes with their offspring were included (table 1). all animals came from a mouflon farm free of clinical paratuberculosis, although map dna was found in the faeces of a few individuals (18.2% positivity ; low concentrations between 10 and 10 map cells ; data not shown) based on copy number of is900 in map chromosome. in the six animals selected, map dna was repeatedly (twice) not detected in their faeces (is900 qpcr) four and two weeks before the transportation. all animals were transported from the farm of origin to the receiving farm in boxes used for animal transport (one hour journey). the experiment was conducted on an eleven hectare farm which consisted of about 70 heads of mouflons and 25 heads of fallow deer. the farm has for many years been subjected to monitoring for the presence of paratuberculosis. in the mouflons, the presence of map however, at the time of the experiment, neither culture nor qpcr revealed map in the mouflons inhabiting the receiving farm. after arrival, the mouflons were placed into a quarantine enclosure (circle ground plan, 50 m) which consisted of an open space connected with a sheltered sleeve used for capturing animals and the collection of samples (tunnel connected with the enclosure and with reclosable partitions on both ends). the ground in the quarantine was covered with sand (not originating from the farm ; 10 cm layer) and the mouflons were fed with map - free hay. the experiment lasted 17 days which included 12 days in the quarantine enclosure and subsequent five days when the mouflons were released into the area of the farm with other mouflons. during the quarantine enclosure period seven times during quarantine, samples of blood were collected from each animal into a vial with edta (amresco, solon, oh, usa), and samples of milk were collected (on the same days as blood samples) from mother ewes (animals number 2 and 3 ; table 1). dna isolation from faeces was performed using a modified qiaamp dna stool kit (qiagen, hilden, germany) protocol. according to the procedure described by slana. blood was processed according to the instructions of a commercial blood and tissue kit (qiagen). the absence of map in hay used for feeding was confirmed using is900 qpcr on dna extracted using the powerfood microbial dna isolation kit (mobio, carlsbad, ca, usa). each qpcr run included a calibration curve to allow calculation of the absolute number of map cells per gram or ml of the sample. all is900 qpcr analyses were performed on a lightcycler 480 instrument (roche diagnostics). statistical analysis was performed using the graphpad prism 5.04 (graphpad inc.) statistical software. the frequencies of occurrence of positive results in the quarantine and postquarantine periods were compared using fisher 's exact test. none of the animals showed overt clinical signs of disease or the presence of map in faeces before the transport. in spite of this, map dna was found in the faeces of all animals either on the first or second day after their transport. during quarantine, map dna was continuously found in the faeces, although the shedding for individual animals was irregular. the highest number of positive results (in days) was found for lamb number 5 and the second highest rate in its mother (number 3). after the release of animals, map was detected for a further two days, after which it was not detected for the subsequent three days, except for one positive case (lamb number 5 ; table 1). statistically significant differences were found between frequencies of map - positive animals (young and adult) in quarantine and after it and between adult animals in the quarantine and postquarantine periods (table 2 ; p > 0.05). odds ratios (odds of is900-positive result in animals in quarantine compared to animals out of quarantine) were 3.3 and 3.1 for the group of adults and all animals (young and adults together), respectively. examination of blood was negative in all cases (table 1). on two occasions, a very low map positivity was found in the milk of both ewes (table 3). the hay used for feeding was demonstrated to be map - negative (data not shown). various stress factors can significantly influence the microbial diversity of gut microflora [10, 11 ]. in addition, disturbing the balance and diversity of gut microflora can affect levels of pathogens in the gastrointestinal tract and lead to their shedding from subclinically infected animals. in our case, handling, transport, a novel environment, and dietary changes could have induced the shedding of map in the mouflons (tables 1 and 2), which were previously demonstrated to be clinically healthy and negative by pcr examination. these mouflons, which originated from a herd in which map was previously detected in a few animals using qpcr, must have been therefore challenged by map infection before the transport. these animals then harboured the latent form of paratuberculosis, which is characterised by an absence of map shedding in faeces and clinical symptoms. a similar effect was observed in clinically healthy cattle and wild deer in which stress probably activated latent malignant catarrhal fever [19, 20 ]. described the reactivation of herpes simplex virus infection in mice after their exposure to restraint stress. generally, paratuberculosis characterised by a long incubation period is mostly induced by stress factors occurring during the life of animals, such as late pregnancy or early lactation. lamb number 5 was the highest map shedder in this study. in lamb number 6, the frequency of map shedding was comparable with other animals (table 1). however, due to the low age of the lambs (both 1/4 of year) and slow character of the disease, the detection of map in these lambs was probably caused by thus, the demonstration of map in the lambs would most likely reflect the presence of map in other animals, particularly in their mothers. the shedding in faeces was not regular in individual animals but was intermittent, which is characteristic for paratuberculosis. map shedding in milk is also known to be irregular and its likelihood is higher in symptomatic compared to asymptomatic animals [23, 24 ]. the proportion of map shed in faeces is generally higher compared to milk, which could explain why map was observed only twice in the milk of the two ewes over the examined period (table 3). blood samples were always negative which could be explained by the short - term influence of stress. this stress manifested as map shedding through faeces but not as systemic map bacteraemia, which occurs especially in animals in late stages of infection with accompanying clinical symptoms. previously, map was detected in the blood of one bull which shed a high concentration of map in its faeces and showed clinical signs of the disease. map was also detected in the blood of subclinical cows ; however, the number of positive cases was significantly lower compared to animals in the clinical phase. after the release of the animals from quarantine, map was still detected in faeces for two days, but then the excretion was undetectable. except for one case (lamb number 5), no positive finding was made on the subsequent three days (tables 1 and 2). releasing the animals from quarantine together with their grazing on pasture instead of hay feeding could be probably the most important factors which decreased map shedding in the imported animals. therefore, novelty, transport, novel environment, or change of feeding, together with the enclosure of animals in a confined and cramped space could be considered as the factors which induced the map shedding in this study. in this regard, the placement of imported animals into quarantine can help to distinguish truly healthy animals from those with latent map infection. the capture of animals for sample collection did not seem to impose a significant stress as almost all samples collected from animals released out of quarantine were negative (tables 1 and 3). in conclusion, as paratuberculosis does not belong to the group of major epizootic and notifiable diseases according to european legislation, animals transported to a new farm do not need to be examined for the presence of map or to undergo quarantine. however, in this study we observed map shedding from healthy mouflons with no clinical symptoms and previously determined as map - negative after their transport to a new farm. the results of this study call into question a sole reliance on examination of animals in their environment before transport and also underline the risk of admitting such animals onto new farms without previous placement in quarantine and further examination. obviously, therefore, clinically healthy and diagnostically negative animals coming from exporting farms can potentially represent a risk of paratuberculosis for other animals in a receiving farm and its environment. for this reason, we recommend that animals be examined not only before the transport to a new farm but also after the transport. we suggest keeping animals in a quarantine enclosure until the results of the examination are known. due to the intermittent shedding of map, | there is no european legislation concerning paratuberculosis that requires that imported animals be kept in quarantine and commonly they are directly released into areas with other animals. in this study, detection of latent infection of paratuberculosis in healthy mouflons previously diagnosed as paratuberculosis - free, but originating from a real time quantitative pcr- (qpcr-) positive herd, occurred after their transport to a new farm. during a twelve - day quarantine period, all mouflons irregularly shed mycobacterium avium subsp. paratuberculosis (map) in faeces, and in a small number of cases also in milk. after the animals were released from quarantine, map was detected for a further two days, after which, testing was negative, except in one case. therefore, the stress connected with transport, novel environment, dietary change, or limited area with high density of animals might have contributed to the induction of paratuberculosis and the shedding of map from the animals, previously diagnosed as map - negative. according to these results, the keeping of imported animals in quarantine and their examination for map presence not only before the transport but also afterwards should be recommended. the designation of a particular area of a farm as a quarantine enclosure could help to mitigate the impact of stress caused by a confined space with a high density of animals. |
the host colonization by microbiota is a gradual and complex process initiated by the exposure of newborns to the maternal vagina or skin microbiota, according to the mode of delivery, which fluctuates throughout life due to external and internal factors, including dietary habits, antibiotic treatments and host diseases. the adult human intestine contains approximately 4 10 microorganisms including viruses, fungi and 500 to 1000 bacterial species. the bacterial density increases along the gastrointestinal tract to reach 3.8 10 bacteria per gram of fecal matter in the colon, and the composition of this microbiota also varies according to spatial localization., escherichia coli, whereas the colon is enriched in dietary fiber - consuming bacteria, such as clostridium sp. and bacteroides species. depending on the origin of their vendor, the small intestine of mice can also be colonized by segmented filamentous bacteria (sfb). the gut microbiota and microbiota - derived metabolites are essential for healthy host functions, influencing the immune system, the nervous system (the gut brain axis), the endocrine system and the metabolism. despite its key role within the organism, the microbiota encompasses millions of proteins and putative antigens to which the host must be tolerant. physical and chemical protective barriers, such as the mucus layer, production of antimicrobial peptides, the secretion of immunoglobulin a (iga) and the epithelium per se, allow microbiota sequestration within the lumen and prevent damage associated with bacterial dissemination. when the gut barrier integrity is impaired, the immune system can respond to antigens of the microbiota, inducing chronic and noxious inflammation, as well as a disequilibrium in bacterial species composition with direct consequences on the different key physiological systems influenced by the microbiota. the role of the intestinal microbiota in shaping the immune system was clearly demonstrated by the use of axenic mice. these animals, born and raised under germ - free conditions, display a strong decrease in the expression of most cytokines (except type 2 cytokines), antimicrobial peptides, iga, cd4 t cells and b cells. the immune system can also influence the equilibrium of the microbiota in favor of certain species. one well - described example of this reciprocal interaction is the il-23/il-22 network. in response to microbiota - triggered toll - like receptor (tlr) activation, cx3cr1 myeloid cells overexpress the pro - inflammatory cytokines il-23 and il-1, which in turn induces il-22 expression by cd4 t cells and innate lymphoid cell 3 (ilc3). il-22 contributes to the gut barrier integrity and can influence the composition of the microbiota by inducing the expression of antimicrobial peptides within intestinal epithelial cells (iecs), as well as the restoration of mucus production after inflammation or the maintenance of iec fucosylation. although the role of pro - inflammatory cytokines, including il-23, il-1, il-17a and il-6 has largely been described in shaping both the immune system and the intestinal microbiota, renewed investigations suggested that certain anti - inflammatory cytokines, in particular transforming growth factor - beta (tgf-), a highly conserved cytokine, are also key modulators of the microbiota and the host immune cell cross talk. in lower life forms, including in the soil - inhabiting, bacterial - feeding nematode, caenorhabibditis elegans, and in insects such as drosophila melanogaster, two tgf- homologs, namely dbl-1 and dpp, found in these organisms, respectively, increase in response to bacterial entry in the gut and largely contribute to the gut immune response by enhancing antimicrobial peptides production, thus controlling bacterial homeostasis. the role of tgf- in the interplay between gut bacteria and the immune system is even more evident in mammals with a more complex gut flora and immune system, implying a primacy of this cytokine in the evolution of the relationship between the host organism and the microbiota. in this review, we highlight recent findings and provide an insight into the cross talk between the tgf-, the intestinal microbiota and the lymphoid cells of the lamina propria of the mammalian gut. tgf- is a potent immunosuppressive cytokine involved in the development and functions of numerous immune cells, including t and b cells, but also dendritic cells (dcs) and natural killer (nk) cells. tgf-2 and tgf-3 mainly have a role in muscle and bone development, whereas tgf-1 (tgf-) expression predominates in immune cells. all three isoforms signal through a common serine threonine kinase receptor complex composed of tgf-ri and tgf-rii subunits. signaling is initiated by binding to tgf-rii, which leads to its auto - phosphorylation, and in turn phosphorylates the tgf-ri subunit (figure 1). the latter then phosphorylates the receptor - associated r - smads, smad2 and/or smad3, which bind to smad4 (cosmad) and translocate into the nucleus to regulate gene transcription. among the tgf--activated genes, smad7 encodes a protein that binds to the cytoplasmic domain of tgf-ri and negatively regulates tgf- signaling. interestingly, smad2/3-dependent and smad4-independent signaling pathways have been described, including the smad2/3 interaction with trim33 (tif-1) and ikks, which can act either in combination or in competition with smad4 during the differentiation steps of a given cell. moreover, tgf-ri can also activate other pathways, such as the mapk / mek, jnk / p38 and akt / pi3k, which could phosphorylate the variable linker regions of smad23. in the gut, iecs as well as selective immune cells, including dcs are an important source of bioactive tgf-. tgf- is a polypeptide secreted as an inactive form bound to the latency - associated peptide, which masks its binding site to tgf-rii (figure 1). to be activated, the latent complex needs to undergo conformational changes or protein cleavage. several molecules can mediate this activation, including furin, metalloproteinases and the integrins (itg) v6 and v8. the tgf-r complex is widely expressed by immune cells, as well as other cells. the importance of tgf- signaling in gut immune cell homeostasis has been highlighted by studies that used either genetically altered tgf- signaling pathways, the deletion of factors important for tgf- activation or administration of anti - tgf-. in mice, germ - line ablation of smad3 is associated with gut inflammation and abscesses, which are totally absent when animals are crossed on a rag - ko background. dc - specific deletion of itg 8 chain causes inflammatory bowel diseases (ibd) associated with unchecked t - lymphocyte activation. interestingly, under inflammatory conditions, cd4 foxp3 regulatory t cells (tregs) constitute a unique v8-expressing t - cell subset, and are thus capable of counter - regulating gut inflammation through the provision of bioactive tgf-. tgf-1-deficient mice spontaneously develop multi - organ autoimmune lesions by the age of 3 weeks. t lymphocytes are key effectors of this autoimmunity, as mice specifically lacking tgf-r in t cells develop a similar inflammation to that observed in tgf-1-deficient animals. it is worth noting that the intestine is not severely affected in these models, probably due to the early onset of the autoimmune disease. indeed, an incomplete deprivation of tgf- signaling in t cells, due to the expression of a dominant negative receptor of tgf-r, is associated with a slower disease progression with intestinal inflammation. in patients with crohn 's disease and ulcerative colitis, the two major forms of ibd known to be associated with an excessive immune response directed against components of commensal bacteria, an impairment of tgf- signaling has largely been reported and seems likely associated with an increase in smad7 expression in t cells. in addition to t cells, gut pathologies were also associated with impairment of tgf- signaling in b cells, dcs and nk cells. the smad3/4 pathway enhances iga class switching and secretion of iga within the lumen, which contribute to maintaining gut barrier integrity against microbiota, and the deletion of tgf-rii in cd11c cells, affecting both dcs and nk cells, leads to spontaneous colitis. compared with specific pathogen - free animals, the levels of tgf- expression are largely impaired in the gut of germ - free mice. iecs are most likely an important source of bioactive tgf- when considering the huge number of cells they represent in the gut. epithelial cell injury and gut inflammation have both been demonstrated to enhance tgf- production by iecs, and there is growing evidence to suggest that iec - derived tgf- is also regulated by the microbiota. several clostridium species were shown to produce short - chain fatty acids, such as butyrate, acetate and propionate, capable of exacerbating tgf- production by colonic ecs. though the exact mechanisms by which clostridium - derived short - chain fatty acids increase tgf- in iecs remain to be unraveled, it is noteworthy that not only do the clostridium species increase tgf- secretion by iecs in the colon, but also the expression of metalloproteinases at the surface of iecs, providing a large source of bioactive tgf- within the colon. microbiota - derived products were also reported to influence the tgf- production by lamina propria dcs, including atp (adenosine 5-triphosphate), which increases the expression of tgf- in a cd70 subset of dcs of the small intestine. similarly to the effects of short - chain fatty acids on iecs, microbiota - derived atp can both directly increase iec tgf- production and indirectly increase active tgf- levels by promoting integrin v8 expression at the surface of dcs. after dextran sodium sulfate treatment, clostridium butyricum was recently reported to enhance tgf- production by colonic lamina propria dcs mainly in a tlr2, ap1erk pathway - dependent manner. furthermore, microbiota - mediated tlr signaling in intestinal dcs also promotes the expression of the integrin v8 essential for tgf- activation. the long co - evolution of commensal bacteria and the mammalian gut has resulted in multiple mechanisms to increase tgf- levels, which include microbiota - induced tgf- production as well as enhancement of active and hence functional tgf- levels. although direct and indirect mechanisms may be used by iecs and dcs in response to different commensal bacteria and bacteria - derived products, future investigations should address the specific contributions of each mechanism during homeostasis, healing, inflammation initiation and inflammation resolution more precisely. tregs are characterized by constitutively high levels of the forkhead family transcription factor foxp3, which is deemed to provide them with their regulatory activity. although tregs constitute approximately 10% of the cd4 t cells in most organs, they are more abundant within the lamina propria, often exceeding values of 20% and 30% in the small intestine and in the colon, respectively. in the gut, tregs can regulate the different branches of the mucosal immune response through several mechanisms. they inhibit t - cell responses to maintain immune tolerance to dietary and microbiota - derived antigens. tregs express high levels of ctla-4, icos, il-10, il-35 and to some extent tgf-. however, foxp3-cre ; tgf1 mice, with tregs unable to produce tgf-, remain healthy without any signs of ibd, implying that production of tgf- by tregs is not essential for their regulatory functions in the gut. however, a cellular source of bioactive tgf-, such as dcs, is essential to sustain the high levels of foxp3 in tregs and their regulatory functions. tregs differentiate either in the thymus (ttreg) or in the periphery of naive t cells (ptreg). the ttreg cells and ptreg cells appear to have non - redundant immunoregulatory functions in the gut. the deprivation of tgf- signaling in thymocytes was associated with a delay in the ttreg cell development potentially due to their apoptosis. within the foxp3 locus, conserved noncoding dna sequences (cns1 - 3) promote stability, size and composition of the foxp3 treg populations. in the gut, the binding of smad2/3/4 to the cns1 induces foxp3 expression in peripheral naive t cells. cns1-deficient mice display unaffected ttreg cell populations, but a defective induction of foxp3 in the gut - associated lymphoid tissue. unlike foxp3-deficient mice the relative proportion of ttreg cells and ptreg cells in the intestine remains a matter of debate. although dietary antigens induce the development of ptreg cells in the small intestine, independently of microbiota, commensal bacteria and their derived products have an instrumental role in the differentiation of ptregs within the colon. clostridium butyricum enhances tgf- production by lamina propria dcs of colon, which was associated with ptreg differentiation. among bacterial products, butyrate, a clostridium - derived short - chain fatty acid, not only induces tgf- expression by iecs and potentially helps enforce ptreg - cell differentiation, but also acts directly on ptreg - cell differentiation by inhibiting histone deacetylase, resulting in histone 3 lysine 27 acetylation (h3k27ac) on cns1 (and cns2 at high concentrations of butyrate), opening the chromatin and leading to the transcription of foxp3. it is noteworthy that in the colon of patients with ulcerative colitis, the levels of butyrate and the capacity of treg cells to suppress effector t cells are reduced, whereas the levels of tgf- are largely reported to be increased. this could either be due to the increased expression of smad7 in t cells (including tregs) from patients with ibd or to different but complementary roles of tgf- and microbiota - products in ptreg - cell differentiation. retinoic acid (ra), a vitamin a - derived metabolite largely produced in the gut by cd103 dcs, and iecs not only contributes to mucosal homing but also has a role in tolerance and inflammation. cd103 dcs exhibit an exacerbated ability to induce ptreg - cell differentiation in vitro, which is dependent on their ra production and tgf- signaling in t cells. on stimulation, in the presence of tgf- tgf--induced high expression levels of il-6r at the surface of t cells, which constrains treg differentiation, can be reversed by the addition of ra into the culture medium. of note, vitamin a - deficient mice were reported to have a comparable proportion and number of total intestinal tregs implying that ttregs can replenish the intestinal treg compartment in the absence of ra - induced ptreg. interestingly, the gut microbiota was recently reported to repress ra synthesis. though this repressive role was observed in the context of colitis - associated cancer and the contribution of specific bacteria or bacterial - derived products repressing ra remains unclear, these recent works suggest that microbiota could finely tune the differentiation of colonic ptreg cells by either promoting tgf- or repressing ra synthesis. within the gut, these rort foxp3 cells were shown to be very stable and to strongly express immunosuppressive molecules, such as ctla-4, lag-3, tim3 and icos. mice lacking rort expression, specifically in foxp3-expressing cells, do not develop spontaneous colitis but are more susceptible to chemically induced colitis and t - cell - driven colitis. the development of rort - expressing tregs is also dependent on the wide - range of commensal bacterial species, including the butyrate - expressing clostridia species, but also sfb and b. thetaiotaomicron, as well as on the levels of vitamin a, il-6 and il-23. although not shown in these studies, we could hypothesize that tgf-, described to induce the co - expression of foxp3 and rort in cd4 t cells is also one of the key cytokines involved in the development of rort - expressing tregs. in conclusion, bacteria and bacteria - derived products from the gut microbiota can influence tgf- levels both under healthy and pathological conditions. whether the imbalance in gut bacteria composition (dysbiosis), observed in patients with ibd, similarly affects tgf- production and its corresponding alterations on the immune cell tolerance remains to be addressed. the type 17 helper (th17) cells constitute a subset of cd4 t cells that express the transcription factor rort and produce cytokines, such as il-17a / f, il-22, often in association with other cytokines, including il-10, gm - csf and ifn, depending on local inflammatory conditions. th17 cells are commonly associated with the development of chronic inflammatory diseases, particularly autoimmunity and tumorigenesis. however, tissue - protective roles have also been attributed to th17 cells in the gut. neutralization of il-17a with antibodies increases tissue damage in patients with crohn 's disease, and il-17a injection into mice maintains the tight junctions in the epithelium and therefore the intestinal barrier integrity. th17-cell differentiation from naive cd4 t cells requires antigen stimulation and was largely reported to be influenced by tgf-. th17 cells generated with tgf- and il-6 produce il-17a but fail to be pathogenic without further exposure to il-23. moreover, th17 cells can be generated in the absence of tgf- signaling but in the presence of il-23, il-6 and il-1. these tgf--independently generated th17 cells were described as strongly pathogenic, reinforcing the importance of tgf- in the control of pathogenic features of th17 cells. in cd4 t cells, in the presence of il-6, tgf- signaling promotes the expression of both rorc and ror encoding for rort and ror, respectively, two transcriptional factors essential for th17 differentiation. interestingly, the induction of rorc and ror by tgf- is independent of smad4, and an opposite role for smad2 and smad3 in the regulation of rort transcriptional activity was described. using mice with specific ablation of tgf- in cd4 t cells, it has been proposed that the autocrine production of tgf- by cd4 t cells is essential for th17-cell differentiation. however, a recent study revealed that effector cd4 t cells are unable to convert tgf- into its bioactive form suggesting that some other cells, able to activate the tgf-, are mandatory for th17-cell differentiation. thus, within the gut, it is likely that iecs and dcs, which express large amounts of tgf- and can lead to its activation, contribute to th17-cell differentiation. alongside its ability to enhance ptreg - cell differentiation, ra was shown to inhibit il-6r and il-23r upregulation induced by tgf- and il-6, respectively. however, ra was also proposed to work in concert with microbial - driven signals through tlr-5 on dcs of the lamina propria to enhance t - cell differentiation into th17 cells. these observations suggest that depending on the accessibility of microbial - derived products for immune cell actors and thus on the gut barrier integrity of the host, ra can differently influence th17-cell differentiation. specific bacteria, such as atp - producing bacteria, or iec - adhering bacteria like sfb, were described to induce intestinal th17 cells. although most intestinal th17 cells bear a tcr specific for sfb antigens, sfb antigens alone do not drive th17 differentiation and tlr signaling is not required for th17 cells, as myd88-deficient mice display a normal number of intestinal th17 cells compared with control littermates. the binding of sfb to iecs directly induces the expression of the serum amyloid a1 and 2 (saa1/2) and reactive oxygen species, which in turn promote th17-cell differentiation. whether sfb promote the expression of tgf- by iecs and/or dcs has so far not been addressed. however, a source of bioactive tgf- is required to induce th17-cell differentiation in response to the commensal bacteria. thus, within the gut, both microbiota and tgf- signaling in cd4 t cells promote the differentiation of th17 cells. the contribution of these two key actors is largely influenced by the inflammatory conditions, which in turn influence the profile of cytokines secreted by th17 cells and their functions. ilcs are the most recently described immune cells to be associated with the regulation of gut homeostasis. ilcs share common features with cd4 t cells but lack t - cell lineage markers and have been categorized into at least three subsets ilc1, ilc2 and ilc3, based on differential transcription factor expression patterns. ilc1 express the transcription factor t - bet, the surface marker cd127, nkp44 (or nkp46 in mice) and produce cytokines, such as tnf- and ifn-. in the human gut, ilc1s have a strong tropism for the epithelium where they produce ifn- in response to il-12 and il-15. a recent study, targeting tgfbr2 in ilc1, showed that tgf-, in a smad4-independent manner, is required for ilc1 development in the salivary gland. so far, the role of tgf- signaling on intestinal ilc1 development and function has not been tested and needs further investigations. ilc2s are identified as lineage negative gata-3 st2 crth2. in response to the il-33, an iec - produced cytokine, gut ilc2s produce amphiregulin (areg), involved in tissue repair, and areg - deficient mice are more susceptible to dextran sodium sulfate, a microbiota - dependent colitis model. in vitro, tgf- was shown to be a potent inducer of areg in human lung adenocarcinoma. whether tgf-, a microbiota - derived cytokines expressed by iecs, could be involved in areg production by intestinal ilc2s remains to be elucidated. ilc3s express the transcription factor rort and secrete cytokines, such as il-17a, and il-22, in association with ifn-, gm - csf or alone. ilc3s have been divided into three subsets : lti (lymphoid tissue inducer), nkp46 ilc3 and nkp46 ilc3. in the adult colon, the role of the microbiota on both the development and functions of ilc3s remains controversial. through their production of il-22, both nkp46 and nkp46 ilc3s have a redundant function with an important role in microbiota segregation, pathogen clearance, maintenance of the intestinal barrier integrity, mucosal healing. interestingly, tgf- signaling was recently proposed to control the balance between nkp46 and nkp46 ilc3 ; however, the source of tgf- and the actors of the tgf- signaling pathway involved remain unknown. thus several recent studies have established a link between the ilc biology, tgf- and microbiota. regarding the role of ilcs in gut homeostasis, and the protective role of tgf- in the gut, future investigations should address whether tgf- induced by the microbiota could allow ilcs to regulate the intestinal barrier integrity and in turn protect against bacterial and parasitic entry in the deeper layers of the gut. immunoglobulin a (iga) can uniquely pass through the epithelium and additionally modulate luminal bacterial composition. in the gut, iga secreted from b cells can bind to polymeric ig receptors on the surface of iecs and subsequently translocate into the lumen. iga can be categorized into two subsets : high - affinity igas, which neutralize pathogens, and low - affinity igas, which prevent bacterial adhesion by aggregating them together. it has been proposed that specific commensal bacteria have a crucial role in iga production. sfb, but not escherichia coli, are capable of inducing iga secretion in peyer 's patches and in isolated lymphoid follicles. so far, the mechanisms linking sfb and iga production remain unclear and require further investigations. tgf- is a master regulator of iga production among all of the other iga - modulating immune cell factors and cytokines, with b - cell - specific deletion of tgfr resulting in the loss of iga - producing b cells. tgf--induced iga production is dependent on canonical pathways, as evidenced by the fact that smad2, smad3 and smad4 deficiency results in the decrease in iga levels. interestingly, tgf- can directly and indirectly control iga production. indeed, in addition to its effect on b cells, tgf- also regulates t - follicular - helper - cell development, a cd4 t - cell subset involved in the control of isotype - switched antibody production. alcaligenes species, gut - associated lymphoid tissue - resident bacteria, as well as lactobacillus gasseri sbt2055, a probiotic bacterium, were shown to induce tgf- production by small intestinal dcs in a tlr2-dependent manner and in turn induce iga production by b cells. thus by influencing tgf- production in the gut, the microbiota regulates iga secretion and thus the strength of the intestinal barrier. largely overlooked until the last decade, the interplay between the microbiota and the mammalian host organism is now widely regarded as essential for host homeostasis and health. in this review, we have highlighted the complexity of this interplay within the gut and the central role of tgf- in regulating these interactions. tgf- levels in the gut are directly and indirectly modulated by the gut microbiota, which impacts the development and functions of immune cell subsets, which in turn regulates microbiota sequestration within the mammalian lumen (table 1 and figure 2).. this area of research should contribute to fully understanding the effects of tgf- on microbiota homeostasis and the risks of utilizing either neutralizing tgf- antibodies or tgf- analogs in human patients. | the relationship between host organisms and their microbiota has co - evolved towards an inter - dependent network of mutualistic interactions. this interplay is particularly well studied in the gastrointestinal tract, where microbiota and host immune cells can modulate each other directly, as well as indirectly, through the production and release of chemical molecules and signals. in this review, we define the functional impact of transforming growth factor - beta (tgf-) on this complex interplay, especially through its modulation of the activity of local regulatory t cells (tregs), type 17 helper (th17) cells, innate lymphoid cells (ilcs) and b cells. |
insulin resistance syndromes are a heterogeneous group of disorders with variable clinical phenotypes, associated with increased blood glucose and insulin levels. hyperglycemia did not respond to age appropriate insulin dosage ; therefore, insulin dosage was increased, but did not lead to appropriate glycemic control. twenty two exons of insulin receptor gene (insr), on short arm of chromosome 19, were sequenced, but no identifiable disease causing mutation was detected. although a rare mutation within the intronic or promoter region has not been excluded in this case, further molecular studies on patients with insulin resistance syndromes associated with certain features are needed. insulin resistance syndromes are a group of disorders with variable clinical spectrum, including extreme high insulin levels, glucose intolerance, and diabetes mellitus. various endocrine and metabolic conditions syndromes associated with insulin resistant state, are leprechaunism or donohue 's syndrome (abnormal facial appearance, early life growth retardation), rabson - mendenhall syndrome (dental and nail abnormalities, skin lesions), werner syndrome (features of premature aging), and alstrom syndrome (childhood blindness, impaired hearing). abnormal facial appearance and dentition in association with insulin resistance resembles rabson - mendenhall syndrome (rms) (omim # 262190), an autosomal recessive disorder caused by mutation in the insulin receptor gene (insr, omim147670). growth and developmental delay, coarse facies, prognathism, gingival hypoplasia, premature and dysplastic dentition, enlarged genitalia, acanthosis nigricans, lichenified skin, onychauxis, and hypertrichosis are some of the clinical manifestations observed in these patients. herein, we present a patient with insulin resistance syndrome with the clinical diagnosis of rms ; however, molecular analysis of the insr gene did not show any mutation. a 10-year old girl was referred to the children 's medical center, the pediatrics center of excellence in iran, with history of poor - controlled diabetes mellitus since six years of age. the parents of this patient were first consanguine ; the father is a 42-year old man with height of 175 cm. her mother is a 35-year old female with height of 152 cm. the patient had a history of low birth weight (2200 g) ; developed irritability at 22 days of age ; the work - up performed at that time, showed hyperammonemia. she developed progressive abdominal protrusion ; hepatomegaly and bilateral renal microlithiasis were revealed at one year of age, based on clinical and radiological evaluation, respectively. hyperammonemia resolved, while benzoate therapy was stopped at six years of age. at that time all of her deciduous teeth were extracted after showing severe dental caries before the school age. fasting blood sugar (fbs) was more than 350 mg / dl in repeated tests. the diagnosis of diabetes mellitus was made ; consequently insulin treatment was started, but glycemic response was poor ; so, insulin dosage was increased gradually, without salient effect on blood glucose level. she has been under insulin therapy (105 - 110 units) and metformin (1000 milligram per day) since 6 years, and 8 years old, respectively. in spite of such treatment, the results of recent laboratory tests were as follow : insulin > 500 mu / l (normal range : 7 - 24 mu / l), ca in 24 hrs urine : 92 mg (normal > 4 mg / kg), oxalate : 54 mg (normal up to 35 mg / kg), hba1c : 10.5 (normal up to 5.5). the height was 115.5 cm (g, c.1230 g > t and c.1650 g > a), but no disease causing mutation was detected. indeed being suspect to pigmented hypertrichosis with insulin dependent diabetes (phid), the slc29a3 [solute carrier family 29 (nucleoside transporter), member 3 ] gene rabson - mendenhall syndrome is a rare insulin resistance disorder, which was first reported by rabson and mendenhall in 1956 in three siblings, who presented with cutaneous and skin abnormalities as well as phallic enlargement. diabetes refractory to large doses of insulin, acanthosis nigricans and abnormal dentition are some other features of this syndrome. as rms is the only insulin resistance syndrome with dental manifestations, this clinical diagnosis was most likely for our patient. other insulin resistance syndromes, including donohue 's syndrome, werner syndrome, and alstrom syndrome, have different clinical phenotypes which were not compatible with this presented case. she had coarse facial appearance from birth and dysplastic, late erupting teeth, extracted before school age. she did not respond to age appropriate insulin dosage and therefore the diagnosis of an insulin resistance syndrome was confirmed. however, she did not have clitoromegaly, hypertrichosis, and further molecular studies did not show insr exons mutations, therefore not further substantiating the diagnosis of rms. it should be noted that mutations in the insr gene are not limited to the rms ; and mutations within this gene are usually associated with a spectrum of inherited insulin - resistance syndromes ranging from severe leprechaunism (donohue syndrome) to type a insulin resistance. analysis of 22 exons of insr gene in our patient did not show any disease causing mutation and only 3 homozygous snps (c.5c > g, c.1230 > t and c.1650 g > a) were identified. although it might be possible that a very rare mutation is located within the intronic or promoter region, a new syndromic insulin resistance disorder could also be considered. indeed, it seems that such dysmorphic phenotype is not always a valid parameter for predicting insr mutations in insulin resistance condition. although rms seems to be the most likely clinical diagnosis for this case, it could be considered as an unusual and possibly even hitherto not yet described entity, or with rms but not caused by insr mutations. | backgroundinsulin resistance syndromes are a heterogeneous group of disorders with variable clinical phenotypes, associated with increased blood glucose and insulin levels.case presentationherein, a 10-year old girl with abnormal face and dentition is presented. she has suffered from diabetes mellitus type i since she was 6 years old. hyperglycemia did not respond to age appropriate insulin dosage ; therefore, insulin dosage was increased, but did not lead to appropriate glycemic control. twenty two exons of insulin receptor gene (insr), on short arm of chromosome 19, were sequenced, but no identifiable disease causing mutation was detected.conclusionalthough a rare mutation within the intronic or promoter region has not been excluded in this case, further molecular studies on patients with insulin resistance syndromes associated with certain features are needed. |
low back pain (lbp) is an extremely prevalent and costly condition, affecting over 90% of adults in the united states at some point during their lifetime.1 the prevalence of chronic lbp in the us has increased from 3.9% in 1992 to 10.2% in 2006.2 claims associated with lbp among medicare beneficiaries increased by 131.7% from 1991 to 2002 in the us, and the associated charges to medicare during this time period increased by 387.2%.3 overall, the total cost burden associated with lbp in the us, including lost productivity and decreased wages, is estimated to range between $ 60 billion and $ 200 billion annually.4,5 the sacroiliac (si) joint is a common cause of chronic lbp.6 the prevalence of si joint pain has been reported to range between 15%30% of patients with chronic lbp, recognizing that not all of these patients require surgery.6,7 therefore, it is reasonable to expect that the disability burden associated with si joint pain will parallel that of lbp. a study of treatment of spinal disorders among medicare beneficiaries found that costs for epidural injections increased from $ 396.7 million in 2002 to $ 743.8 million in 2006, an increase of 121.1%.8 total medicare costs for inpatient lumbar spinal surgery totaled more than $ 1 billion in 2003.9 determining that the si joint is the pain generator is based on three or more positive provocation tests that indicate that the si joint is the source of pain, followed by a confirmatory image - guided diagnostic injection of the si joint ; a 50% or greater reduction in pain immediately after the injection of a local anesthetic is used to confirm that the si joint is the pain generator.10,11 the traditional si joint pain treatment options have included either nonoperative care, such as physical therapy, epidural injections, si joint injections, radiofrequency ablation, and pain medications, or traditional open si joint arthrodesis surgery. less invasive, nonoperative therapies often have limited benefit, as they only address the symptoms of the condition and do not treat the underlying cause. the only option for patients with si joint pain that is refractory to nonoperative therapies has been open si joint arthrodesis. this invasive, open surgery requires large incisions, bone harvesting, joint destabilization, and lengthy inpatient hospital stays. moreover, patients may be required to abstain from weight bearing for several months.12 to address the unmet need for improved surgical treatment of these patients, several minimally invasive surgery (mis) arthrodesis systems have been developed to minimize tissue destruction, shorten the length of hospital stays, and facilitate faster recovery. minimally invasive surgical procedures involve placing implants across the target si joint in order to achieve arthrodesis through a permanent linkage across the joint. the clinical safety and effectiveness of mis has been demonstrated based on a safety surveillance database of 5,319 patients treated with mis over a 4-year period, a retrospective study of 50 patients at 40 months postimplant, and a retrospective study of 40 patients with 1-year follow - up.1315 however, the economic implications of si joint fusion with mis have not been fully explored. this study evaluates the cost of si joint fusion with mis compared to nonoperative care in patients who suffer from lbp due to si joint disruption and degenerative sacroiliitis in the hospital inpatient setting among the us medicare population. this research was performed according to guidelines good publication practices (gpp2) established to minimize the conflict of interest when conducting pharmacoeconomic studies.16,17 a multispecialty panel comprised of clinicians and methodologists (the coauthors) provided the framework for the economic analysis, and the panel made all of the decisions regarding the data analysis and interpretation of the results. an economic model was developed to simulate the lifetime cost savings (2012 us dollars [usd ]) associated with treating medicare patients with mis si joint fusion in the hospital inpatient setting compared to the cost of treating the same patients with nonoperative care. the savings in lifetime costs was estimated by subtracting the cost of treating medicare patients with mis si joint fusion from the cost of nonoperative care. the economic model incorporated data from multiple sources, including published literature, medicare claims data, and clinical expert judgment. the costs included in the analysis pertained to medical treatments, follow - up care, diagnostic tests, and retail pharmacy pain medication. a multispecialty clinical panel of six physicians (three orthopedic surgeons, one neurosurgeon, and two physiatrists) provided the clinical framework for the economic evaluation, and this panel made the final decisions on parameter values. several assumptions were made during the development of the economic model : 1) this analysis applies only to medicare patients in the us who suffer from chronic lbp due to si joint disruption and degenerative sacroiliitis, and who are eligible for mis ; 2) this analysis applies only to mis patients treated in the hospital inpatient setting ; 3) mis patients who were classified as clinical failures underwent additional treatment, as described in table 1, where mis treatment failure was defined as having one or more of the following : implant failure, loosening, and/or malpositioning ; failure to relieve pain requiring repeat intervention ; and infection requiring reoperation ; 4) mis patients who were classified as clinical successes incurred minimal additional medical resources, such as a reduced class or a reduced dose of pain medications ; 5) late complications of mis, such as infection or loosening, requiring revision were reflected in the 1-year treatment failure rate for mis ; 6) the quality of life effects of mis and nonoperative care have not been included in the present analysis ; 7) the indirect costs associated with lost productivity and intangible costs of pain and suffering related to treatment morbidity have not been included in the present analysis ; and 8) the analysis assumed that a single cohort of patients was followed over several years with no new patients entering the cohort in subsequent years. not all patients with si joint pain and dysfunction necessarily have chronic pain and dysfunction, despite medical intervention. however, it remains unknown how many patients truly seek care, as little data exist on the effectiveness of nonoperative treatment. we assumed the percentage of si joint disruption in patients who experience chronic pain despite medical treatment strategies is 75%, recognizing that progression is substantial. further, all patients with si joint pain and dysfunction are not necessarily surgical candidates for mis. we have assumed that 90% of patients are eligible for mis and the remaining 10% are too ill for general anesthesia. the costs associated with degenerative sacroiliitis / si joint disruption patients managed with nonoperative care were estimated using the medicare 5% standard analytic file (saf) for the years ranging between 20052010.18 patients with a primary international classification of diseases, ninth revision, clinical modification (icd-9-cm) diagnosis code for degenerative sacroiliitis / si joint disruption (icd-9-cm diagnosis codes 720.2, 724.6, 739.4, 846.9, or 847.3) with continuous enrollment for at least 1 year before and 5 years after diagnosis were included in the analysis. spine - related health care claims attributable to degenerative sacroiliitis / si joint disruption were identified using icd-9-cm diagnosis codes (claims with a primary or secondary icd-9-cm diagnosis code of 71x.xx, 72x.xx, 73x.xx, or 84x.xx), and the 5-year direct medical costs were totaled across practice settings, including hospital inpatient settings, hospital outpatient settings, physicians offices, and emergency departments. a subgroup analysis was performed among patients who underwent lumbar spinal fusion. among medicare patients with degenerative sacroiliitis or si joint disruption (n=14,552), the mean 5-year direct medical costs attributable to degenerative sacroiliitis / si joint disruption was $ 18,527 (standard deviation [sd ] $ 28,285) per patient.18 among patients with lumbar spinal fusion (n=538 [3.7% ]), the 5-year cost was $ 63,913 (sd $ 46,870) per patient. among patients without lumbar spinal fusion (n=14,014 [96.3% ]), the 5-year cost was $ 16,769 (sd $ 25,753) per patient. to estimate the total number of medicare beneficiaries with degenerative sacroiliitis or si joint disruption annually, the 14,552 patients identified from the medicare 5% saf is multiplied by 20, which yields an estimated 291,040 medicare beneficiaries annually ; this figure includes 10,760 patients who underwent a lumbar spinal fusion procedure. while pain medications, such as nonsteroidal antiinflammatory drugs, are used by many patients treated with nonoperative care,19,20 pharmacy claims data are not available in the medicare saf. therefore, outpatient pharmacy costs associated with pain medications were estimated among privately insured patients with degenerative sacroiliitis / si joint disruption using truven health marketscan (truven health analytics inc., ann arbor, mi, usa) data from january 1, 2004 through december 31, 2010. marketscan is a large, nationally representative longitudinal database of medical and pharmacy claims from over 150 million individuals. the population was identified using the same primary icd-9-cm diagnosis codes that were used in the medicare saf analysis. pain medication costs were estimated as the costs of pharmacy claims for the following drug categories : salicylate analgesics / antipyretics ; antiinflammatory analgesics / antipyretics ; opiate agonists ; antidepressants ; benzodiazepines ; anxiolytics ; sedatives ; and hypnotics. the cumulative mean costs for outpatient pharmacy pain medications at 1 year, 2 years, and 3 years were $ 1,003, $ 1,809, and $ 2,567, respectively (2012 usd). parameter estimates for mis were derived from the published literature, medicare claims data, and the expert clinical opinion of the multispecialty clinical panel, and are further described in table 1. in a retrospective study14 of 50 consecutive patients treated with a mis device (ifuse implant system ; si - bone, inc.),21 early and sustained clinically significant improvements were reported in seven out of nine quality of life domains, with 82% of patients reaching minimal clinically important difference (mcid) (> 2 point change)22 at 40 months postimplant.11 in another retrospective study of 40 consecutive patients treated with the same device,15 a clinically significant improvement (> 2 point change from baseline) was observed in all but one patient at 1-year follow - up. therefore, the mis 1-year treatment success rate was assumed to be 82% in the economic model. complications were reported among 3.8% of 5,319 patients treated with the new mis system (ifuse implant system) over a 4-year period (204 of 5,319 patients),13 which included clinical, device - related, and procedure - related events. clinical events included pain due to nerve impingement, recurrent si joint pain, hematoma / excessive bleeding, iliac fracture, superficial wound infection, deep venous thrombosis, and deep wound infection. device - related events included pin bending / breakage and device migration, whereas procedure - related events included improper device placement or improper device size. mis revisions were performed in 1.8% of patients (n=96) at a median follow - up of 4 months, and were typically performed in the early postoperative period for the treatment of symptomatic malpositioned implants (n=46), or in the late postoperative period due to symptom recurrence (n=34).13 therefore, based on miller,13 complications were assumed to occur in 3.8% of patients undergoing si joint fusion with mis, and revisions were assumed to occur in 1.8% of patients in the economic model. the reason why this procedure is conducted in this manner is that it offers postoperative pain control, it helps to ensure avoidance of urinary retention, it allows provision of physical therapy education on toe touch weight bearing, and ensures safety in ambulation. the cost of mis hospitalization was based on the national average adjusted drg payments of $ 46,700 for drg 459 (spinal fusion except cervical with major complication or comorbidity) and $ 27,800 for drg 460 (spinal fusion except cervical without major complication or comorbidity). a weighted cost was calculated using the percentage of patients (3.8%) with clinical, device - related, or procedure - related events based on 5,319 patients treated with mis fusion (ifuse implant system).13 the mis device cost is bundled into the drg payment. the costs associated with major adverse events that occur during the mis hospitalization (such as implant malpositioning requiring revision, as well as medical complications including hematoma and deep vein thrombosis) are reflected in the medicare payment for drg 459 (with major complications). the professional fee of $ 1,033.38 for the mis procedure was based on the 2012 payment for current procedural terminology (cpt) code 27280 (arthrodesis, si joint [including obtaining graft]).23 the medicare policy has designated cpt code 27280 as an inpatient only service.22,24 medical resource use for mis si joint fusion follow - up care (including pain medications) was determined by three surgeons (dwp, th, and jc) based on their experience treating over 360 patients with mis (table 1). it was assumed that two of the office visits in year 1 would fall under the postsurgical global period and would not incur additional costs, per cms regulations and guidance.25,26 cpt codes and reimbursement amounts from standard physician fee schedules23 were used to enumerate costs for professional services for mis patients (table 2). retail pharmacy pain medication costs for mis patients were enumerated using the thomson reuters redbook online (table 2).27 the 5-year costs for nonoperative care and mis were extrapolated to an overall lifetime cost impact to the medicare population. for this extrapolation, it was assumed that medicare patients are 70 years old in year 1 (the mean age of the medicare saf sample), and that patients have a life expectancy of 84 years (the sex - weighted average life expectancy of americans who reach the age of 65 years, per the social security administration);28 as such, cost savings after year 5 are extrapolated over an additional 10 years. the costs in each of the additional years beyond 5 years were estimated by adding the treatment - specific average annual difference over the first 5 years to the cost totals at the end of year 5. the net present value was discounted at 3% per annum, based on the standards used in economic analyses and the approach employed by the congressional budget office.29 because there is significant overlap of si joint pathology and lbp requiring spinal fusion, a subgroup analysis was performed on degenerative sacroiliitis / si joint disruption patients who underwent lumbar spinal fusion. it is unclear how often lumbar fusion is performed on patients who truly have si pathology ; however, sembrano and polly6 previously suggested at least 5% of the time. in a recent study of the medicare population of the 538 patients in a lumbar spinal fusion subgroup, 7% underwent lumbar spinal fusion within 1 year prior to receiving a diagnosis of si joint disruption and/or degenerative sacroiliitis,18 which may represent patients with concomitant disease, new si joint disease, or misdiagnosis. in addition, lumbar spinal fusion patients with si diagnoses represent a group requiring greater medical resource utilization for treatment than patients with the same diagnosis, but who have not had lumbar spinal fusion.18 sensitivity analyses were performed to determine the consequences of making alternative assumptions for the following model parameter inputs : the durability of the mis treatment success rate ; the percentage of mis index hospitalizations that fall under drg 459 (with major complications) ; the distribution of subsequent treatments for mis failures ; the exclusion of retail pharmacy costs for pain medications ; the inclusion of icd-9-cm code 721.3 (lumbosacral spondylosis) ; and the discount rate for extrapolation. of note, for the base case analysis, we adjusted the medicare population size to reflect patients who suffer from chronic lbp due to si joint disruption and degenerative sacroiliitis who are eligible for mis. as such, sensitivity analyses were also performed for the percent of patients with chronic pain and the percent of patients who are eligible for mis surgery. several assumptions were made during the development of the economic model : 1) this analysis applies only to medicare patients in the us who suffer from chronic lbp due to si joint disruption and degenerative sacroiliitis, and who are eligible for mis ; 2) this analysis applies only to mis patients treated in the hospital inpatient setting ; 3) mis patients who were classified as clinical failures underwent additional treatment, as described in table 1, where mis treatment failure was defined as having one or more of the following : implant failure, loosening, and/or malpositioning ; failure to relieve pain requiring repeat intervention ; and infection requiring reoperation ; 4) mis patients who were classified as clinical successes incurred minimal additional medical resources, such as a reduced class or a reduced dose of pain medications ; 5) late complications of mis, such as infection or loosening, requiring revision were reflected in the 1-year treatment failure rate for mis ; 6) the quality of life effects of mis and nonoperative care have not been included in the present analysis ; 7) the indirect costs associated with lost productivity and intangible costs of pain and suffering related to treatment morbidity have not been included in the present analysis ; and 8) the analysis assumed that a single cohort of patients was followed over several years with no new patients entering the cohort in subsequent years. not all patients with si joint pain and dysfunction necessarily have chronic pain and dysfunction, despite medical intervention. however, it remains unknown how many patients truly seek care, as little data exist on the effectiveness of nonoperative treatment. we assumed the percentage of si joint disruption in patients who experience chronic pain despite medical treatment strategies is 75%, recognizing that progression is substantial. further, all patients with si joint pain and dysfunction are not necessarily surgical candidates for mis. we have assumed that 90% of patients are eligible for mis and the remaining 10% are too ill for general anesthesia. the costs associated with degenerative sacroiliitis / si joint disruption patients managed with nonoperative care were estimated using the medicare 5% standard analytic file (saf) for the years ranging between 20052010.18 patients with a primary international classification of diseases, ninth revision, clinical modification (icd-9-cm) diagnosis code for degenerative sacroiliitis / si joint disruption (icd-9-cm diagnosis codes 720.2, 724.6, 739.4, 846.9, or 847.3) with continuous enrollment for at least 1 year before and 5 years after diagnosis were included in the analysis. spine - related health care claims attributable to degenerative sacroiliitis / si joint disruption were identified using icd-9-cm diagnosis codes (claims with a primary or secondary icd-9-cm diagnosis code of 71x.xx, 72x.xx, 73x.xx, or 84x.xx), and the 5-year direct medical costs were totaled across practice settings, including hospital inpatient settings, hospital outpatient settings, physicians offices, and emergency departments. a subgroup analysis was performed among patients who underwent lumbar spinal fusion. among medicare patients with degenerative sacroiliitis or si joint disruption (n=14,552), the mean 5-year direct medical costs attributable to degenerative sacroiliitis / si joint disruption was $ 18,527 (standard deviation [sd ] $ 28,285) per patient.18 among patients with lumbar spinal fusion (n=538 [3.7% ]), the 5-year cost was $ 63,913 (sd $ 46,870) per patient. among patients without lumbar spinal fusion (n=14,014 [96.3% ]), the 5-year cost was $ 16,769 (sd $ 25,753) per patient. to estimate the total number of medicare beneficiaries with degenerative sacroiliitis or si joint disruption annually, the 14,552 patients identified from the medicare 5% saf is multiplied by 20, which yields an estimated 291,040 medicare beneficiaries annually ; this figure includes 10,760 patients who underwent a lumbar spinal fusion procedure. while pain medications, such as nonsteroidal antiinflammatory drugs, are used by many patients treated with nonoperative care,19,20 pharmacy claims data are not available in the medicare saf therefore, outpatient pharmacy costs associated with pain medications were estimated among privately insured patients with degenerative sacroiliitis / si joint disruption using truven health marketscan (truven health analytics inc., ann arbor, mi, usa) data from january 1, 2004 through december 31, 2010. marketscan is a large, nationally representative longitudinal database of medical and pharmacy claims from over 150 million individuals. the population was identified using the same primary icd-9-cm diagnosis codes that were used in the medicare saf analysis. pain medication costs were estimated as the costs of pharmacy claims for the following drug categories : salicylate analgesics / antipyretics ; antiinflammatory analgesics / antipyretics ; opiate agonists ; antidepressants ; benzodiazepines ; anxiolytics ; sedatives ; and hypnotics. the cumulative mean costs for outpatient pharmacy pain medications at 1 year, 2 years, and 3 years were $ 1,003, $ 1,809, and $ 2,567, respectively (2012 usd). parameter estimates for mis were derived from the published literature, medicare claims data, and the expert clinical opinion of the multispecialty clinical panel, and are further described in table 1. in a retrospective study14 of 50 consecutive patients treated with a mis device (ifuse implant system ; si - bone, inc.),21 early and sustained clinically significant improvements were reported in seven out of nine quality of life domains, with 82% of patients reaching minimal clinically important difference (mcid) (> 2 point change)22 at 40 months postimplant.11 in another retrospective study of 40 consecutive patients treated with the same device,15 a clinically significant improvement (> 2 point change from baseline) was observed in all but one patient at 1-year follow - up. therefore, the mis 1-year treatment success rate was assumed to be 82% in the economic model. complications were reported among 3.8% of 5,319 patients treated with the new mis system (ifuse implant system) over a 4-year period (204 of 5,319 patients),13 which included clinical, device - related, and procedure - related events. clinical events included pain due to nerve impingement, recurrent si joint pain, hematoma / excessive bleeding, iliac fracture, superficial wound infection, deep venous thrombosis, and deep wound infection. device - related events included pin bending / breakage and device migration, whereas procedure - related events included improper device placement or improper device size. mis revisions were performed in 1.8% of patients (n=96) at a median follow - up of 4 months, and were typically performed in the early postoperative period for the treatment of symptomatic malpositioned implants (n=46), or in the late postoperative period due to symptom recurrence (n=34).13 therefore, based on miller,13 complications were assumed to occur in 3.8% of patients undergoing si joint fusion with mis, and revisions were assumed to occur in 1.8% of patients in the economic model. the reason why this procedure is conducted in this manner is that it offers postoperative pain control, it helps to ensure avoidance of urinary retention, it allows provision of physical therapy education on toe touch weight bearing, and ensures safety in ambulation. the cost of mis hospitalization was based on the national average adjusted drg payments of $ 46,700 for drg 459 (spinal fusion except cervical with major complication or comorbidity) and $ 27,800 for drg 460 (spinal fusion except cervical without major complication or comorbidity). a weighted cost was calculated using the percentage of patients (3.8%) with clinical, device - related, or procedure - related events based on 5,319 patients treated with mis fusion (ifuse implant system).13 the mis device cost is bundled into the drg payment. the costs associated with major adverse events that occur during the mis hospitalization (such as implant malpositioning requiring revision, as well as medical complications including hematoma and deep vein thrombosis) are reflected in the medicare payment for drg 459 (with major complications). the professional fee of $ 1,033.38 for the mis procedure was based on the 2012 payment for current procedural terminology (cpt) code 27280 (arthrodesis, si joint [including obtaining graft]).23 the medicare policy has designated cpt code 27280 as an inpatient only service.22,24 medical resource use for mis si joint fusion follow - up care (including pain medications) was determined by three surgeons (dwp, th, and jc) based on their experience treating over 360 patients with mis (table 1). it was assumed that two of the office visits in year 1 would fall under the postsurgical global period and would not incur additional costs, per cms regulations and guidance.25,26 cpt codes and reimbursement amounts from standard physician fee schedules23 were used to enumerate costs for professional services for mis patients (table 2). retail pharmacy pain medication costs for mis patients were enumerated using the thomson reuters redbook online (table 2).27 the 5-year costs for nonoperative care and mis were extrapolated to an overall lifetime cost impact to the medicare population. for this extrapolation, it was assumed that medicare patients are 70 years old in year 1 (the mean age of the medicare saf sample), and that patients have a life expectancy of 84 years (the sex - weighted average life expectancy of americans who reach the age of 65 years, per the social security administration);28 as such, cost savings after year 5 are extrapolated over an additional 10 years. the costs in each of the additional years beyond 5 years were estimated by adding the treatment - specific average annual difference over the first 5 years to the cost totals at the end of year 5. the net present value was discounted at 3% per annum, based on the standards used in economic analyses and the approach employed by the congressional budget office.29 because there is significant overlap of si joint pathology and lbp requiring spinal fusion, a subgroup analysis was performed on degenerative sacroiliitis / si joint disruption patients who underwent lumbar spinal fusion. it is unclear how often lumbar fusion is performed on patients who truly have si pathology ; however, sembrano and polly6 previously suggested at least 5% of the time. in a recent study of the medicare population of the 538 patients in a lumbar spinal fusion subgroup, 7% underwent lumbar spinal fusion within 1 year prior to receiving a diagnosis of si joint disruption and/or degenerative sacroiliitis,18 which may represent patients with concomitant disease, new si joint disease, or misdiagnosis. in addition, lumbar spinal fusion patients with si diagnoses represent a group requiring greater medical resource utilization for treatment than patients with the same diagnosis, but who have not had lumbar spinal fusion.18 sensitivity analyses were performed to determine the consequences of making alternative assumptions for the following model parameter inputs : the durability of the mis treatment success rate ; the percentage of mis index hospitalizations that fall under drg 459 (with major complications) ; the distribution of subsequent treatments for mis failures ; the exclusion of retail pharmacy costs for pain medications ; the inclusion of icd-9-cm code 721.3 (lumbosacral spondylosis) ; and the discount rate for extrapolation. of note, for the base case analysis, we adjusted the medicare population size to reflect patients who suffer from chronic lbp due to si joint disruption and degenerative sacroiliitis who are eligible for mis. as such, sensitivity analyses were also performed for the percent of patients with chronic pain and the percent of patients who are eligible for mis surgery. the extrapolated lifetime cost of treating medicare patients with mis fusion in the inpatient setting was $ 48,185 per patient compared to the cost of treating them with nonoperative care of $ 51,543 per patient, resulting in a savings of $ 660 million to the medicare program ($ 3,358 in savings per patient for 196,452 patients) due to reductions in spine - related health care costs over medicare patients lifetimes (table 3). this occurs because direct medical costs of nonoperative care accumulate steadily over the patients lifetimes, as opposed to the costs associated with mis, which are higher in the first year. the per patient cost differential for mis compared to nonoperative care was similar for the overall group ($ 3,358) and for patients without lumbar spinal fusion ($ 1,033). per patient cost differential for patients with lumbar spinal fusion was much higher ($ 63,705). of note, patients managed with lumbar spinal fusion surgery (3.7% of patients) represent a disproportionate share of the cost savings in that approximately 70% of the potential cost savings ($ 463 million) would be realized from this subgroup, whereas the remaining 30% of the potential cost savings (about $ 195 million) would be realized from the 96% of patients without lumbar spinal fusion. sensitivity analyses were used to test the robustness of the results and to determine which variables have a substantial effect on the results. the results generated by the economic model are generally considered robust because the costs fall within a narrow range (figure 1) when key model assumptions and parameters are varied. as anticipated, the results were most sensitive to the mis treatment success rate, followed by the exclusion of retail pharmacy costs for pain medications, and the inclusion of the icd-9-cm code 721.3 (table 4 and figure 1). in the current study, the 1-year mis treatment success rate of 82% was estimated from two studies based on the best evidence currently available : one retrospective study of 50 consecutive patients treated with mis;14 and a second retrospective study of 40 consecutive patients.15 to address uncertainty in the durability of mis, a sensitivity analysis was performed by varying the mis treatment success rate from 72% to 92% to evaluate model robustness. overall, cost - neutrality was achieved at a 1-year mis treatment success rate of approximately 78.7% ; lower values result in mis being cost - additive over a patient s lifetime, whereas higher values result in cost savings (figure 2). in the base case, given that icd-9-cm code 721.3 may also include patients with si joint disruption and degeneration, we conducted a sensitivity analysis to estimate the costs to medicare when it was included. with the inclusion of icd-9-cm code 721.3, 35,464 patients were identified in the medicare 5% saf analysis between 2005 and 2010, resulting in up to 478,764 medicare beneficiaries with degenerative sacroiliitis or si joint disruption annually. when icd-9-cm code 721.3 is included, overall estimated lifetime per - patient costs for patients treated with nonoperative care were $ 60,867 compared to $ 52,175 for patients treated with mis ; therefore, the per patient cost differential of treatment with mis instead of nonoperative care is estimated to be $ 8,692 (table 5). when icd-9-cm code 721.3 was included in the analysis, the potential lifetime cost savings to the medicare program by treating this entire population with mis increases up to $ 4.16 billion (478,764 beneficiaries at $ 8,692 in savings per patient). for patients who underwent lumbar spinal fusion surgery (5.4% of the population with the inclusion of icd-9-cm code 721.3), lifetime costs for nonoperative care were estimated at $ 142,994 per patient compared to $ 87,503 for mis ; the cost differential of $ 55,491 per patient would result in an estimated savings to medicare of up to $ 1.4 billion over patients lifetimes. the lifetime cost savings for patients without lumbar spinal fusion were estimated at up to $ 2.7 billion with the inclusion of icd-9-cm code 721.3. due to the absence of retail pharmacy data in the medicare saf, a sensitivity analysis was performed by excluding retail pharmacy costs for pain medications (from both the mis and nonoperative care groups). as expected, because pain medications represent a standard treatment option for nonoperative care, when retail pharmacy costs were excluded from the analysis, mis became cost - additive overall and in patients without lumbar spinal fusion (table 4). the economic analysis was less sensitive to assumptions about the distribution of subsequent treatments for mis failures, the percentage of mis index hospitalizations that fall under drg 459 (with major complications), and the discount rate (table 4 and figure 1). cost savings to the medicare program over patients lifetimes (table 4) were adjusted by varying the percentage of si joint disruption patients with chronic pain (from 25% to 100%) and the percentage of patients eligible for mis si joint fusion (from 25% to 100%). sensitivity analyses were used to test the robustness of the results and to determine which variables have a substantial effect on the results. the results generated by the economic model are generally considered robust because the costs fall within a narrow range (figure 1) when key model assumptions and parameters are varied. as anticipated, the results were most sensitive to the mis treatment success rate, followed by the exclusion of retail pharmacy costs for pain medications, and the inclusion of the icd-9-cm code 721.3 (table 4 and figure 1). in the current study, the 1-year mis treatment success rate of 82% was estimated from two studies based on the best evidence currently available : one retrospective study of 50 consecutive patients treated with mis;14 and a second retrospective study of 40 consecutive patients.15 to address uncertainty in the durability of mis, a sensitivity analysis was performed by varying the mis treatment success rate from 72% to 92% to evaluate model robustness. overall, cost - neutrality was achieved at a 1-year mis treatment success rate of approximately 78.7% ; lower values result in mis being cost - additive over a patient s lifetime, whereas higher values result in cost savings (figure 2). in the base case, icd-9-cm code 721.3 given that icd-9-cm code 721.3 may also include patients with si joint disruption and degeneration, we conducted a sensitivity analysis to estimate the costs to medicare when it was included. with the inclusion of icd-9-cm code 721.3, 35,464 patients were identified in the medicare 5% saf analysis between 2005 and 2010, resulting in up to 478,764 medicare beneficiaries with degenerative sacroiliitis or si joint disruption annually. when icd-9-cm code 721.3 is included, overall estimated lifetime per - patient costs for patients treated with nonoperative care were $ 60,867 compared to $ 52,175 for patients treated with mis ; therefore, the per patient cost differential of treatment with mis instead of nonoperative care is estimated to be $ 8,692 (table 5). when icd-9-cm code 721.3 was included in the analysis, the potential lifetime cost savings to the medicare program by treating this entire population with mis increases up to $ 4.16 billion (478,764 beneficiaries at $ 8,692 in savings per patient). for patients who underwent lumbar spinal fusion surgery (5.4% of the population with the inclusion of icd-9-cm code 721.3), lifetime costs for nonoperative care were estimated at $ 142,994 per patient compared to $ 87,503 for mis ; the cost differential of $ 55,491 per patient would result in an estimated savings to medicare of up to $ 1.4 billion over patients lifetimes. the lifetime cost savings for patients without lumbar spinal fusion were estimated at up to $ 2.7 billion with the inclusion of icd-9-cm code 721.3. due to the absence of retail pharmacy data in the medicare saf, a sensitivity analysis was performed by excluding retail pharmacy costs for pain medications (from both the mis and nonoperative care groups). as expected, because pain medications represent a standard treatment option for nonoperative care, when retail pharmacy costs were excluded from the analysis, mis became cost - additive overall and in patients without lumbar spinal fusion (table 4). the economic analysis was less sensitive to assumptions about the distribution of subsequent treatments for mis failures, the percentage of mis index hospitalizations that fall under drg 459 (with major complications), and the discount rate (table 4 and figure 1). cost savings to the medicare program over patients lifetimes (table 4) were adjusted by varying the percentage of si joint disruption patients with chronic pain (from 25% to 100%) and the percentage of patients eligible for mis si joint fusion (from 25% to 100%). this study demonstrates that projected lifetime costs associated with nonoperative care were higher than with mis in the medicare population, presuming a 1-year treatment success rate of at least 78.7% for mis si joint fusion. the $ 660 million potential savings to the medicare program associated with the use of mis instead of nonoperative care would occur because direct medical costs of nonoperative care accumulate steadily over the patients lifetimes, as opposed to the costs associated with mis, which are higher in the first year. it is helpful to consider the costs associated with new spinal technologies like mis in light of other common orthopedic technologies. as a point of reference, the estimated cost savings of the mis procedure compares favorably with that of other common orthopedic procedures. for instance, mis device placement performed in the hospital inpatient setting compared with nonoperative care (per patient differential cost of $ 3,358, favoring mis over a patient s lifetime) creates cost savings far greater than artificial cervical disc replacement versus anterior cervical fusion surgery for the treatment of single - level radiculopathy or myelopathy (per patient differential cost of $ 255 [2012 usd ] favoring artificial disc from a hospital perspective).24 furthermore, mis device placement performed in the hospital inpatient setting is cost - saving relative to nonoperative care among the subgroup of patients who have undergone lumbar spinal fusion (per patient differential of $ 63,705 favoring mis over a patient s lifetime), and it is substantially more cost - saving than bone morphogenetic protein (bmp) versus autogenous iliac crest bone graft in single - level anterior lumbar fusion for treatment of degenerative disk disease (per patient differential cost of $ 14 [2012 usd ] favoring bmp over a 2-year period).30 current evidence suggests that when bmp results in a higher fusion rate, it is cost - saving.30 these findings broadly suggest that, from an economic perspective, mis is a reasonable treatment alternative along the continuum of care between continued nonoperative care and open arthodesis surgery. when nonoperative treatment fails, patients are either left to suffer, or they may be offered si joint fusion. traditionally, open si joint arthrodesis was the only si joint fusion surgery option, but recently si joint fusion has been performed with a mis technique. a recent study explored the prevalence of si joint fusion and the frequency with which it is being performed using a mis technique in the us.31 the total number of estimated si joint fusion procedures (all payers) increased from 189 in 2001 to 3,900 in 2012. mis si joint fusions accounted for an increasing percentage of the total, ranging from 0% in 2008 to 76% in 2011, with an estimate of 85% for 2012. mis si joint fusion has been broadly adopted in the us and is a viable treatment alternative for patients who have failed nonoperative treatment. presumably, this is due to improvements in diagnosis, as well as improvements of the risk benefit ratio with the use of mis. first, the mis treatment success rate of 82% at 1 year is based on two studies a retrospective study of 50 consecutive patients treated with a specific mis system,14 and a second retrospective study of 40 consecutive patients.15 despite the small sample size, clinically and statistically significant improvements were reported by rudolf14 in seven out of nine quality of life domains, with 82% of patients reaching the mcid (> 2-point change in pain score) at 40 months postimplant. similarly, in a recent retrospective study with the same mis system, sachs and capobianco15 reported a clinically significant improvement (> 2-point change from baseline) in 39 out of 40 patients at 1-year follow - up. the rate of perioperative complications (20%) reported by rudolf14 was similar to other reports in the literature ; half of these events were minor (mild hematoma at the incision site and superficial cellulitis), requiring little to no intervention, whereas the remaining five patients experienced major events. specifically, three patients were brought back to the operating room for retraction of a misplaced implant (likely due to the learning curve early on during experience), one patient experienced a deep soft tissue wound infection that resolved after 6 weeks of intravenous antibiotics, and the final patient had a nondisplaced fracture that healed without intervention. beyond the perioperative complications reported by rudolf,14 only one additional complication occurred in months 1340 (implant loosening), which resulted in the placement of two additional implants. the revision rate in the 50-patient cohort was 8% after 3 years,14 which compares favorably to that reported in the literature for other types of mis approaches (8%15%).3237 more recently, a complication rate of 3.8% (204 of 5,319 patients) has been reported for a new mis system over a 4-year period.13 revision surgeries were performed in 1.8% of patients (n=94) at a median follow - up of 4 months ; early revisions primarily for symptomatic malposition were performed at a median of 19 days postoperatively, whereas late revisions primarily for symptom recurrence or continued pain of undetermined etiology were performed at a median of 279 days postoperatively.13 these more recent findings from miller and sachs and capobianco15 further suggest that the 1-year mis treatment success rate of 82% is a reasonable base case value for the economic model (where treatment failure has been defined as having one or more of the following : implant failure, loosening, and/or malposition ; failure to relieve pain requiring repeat intervention ; and infection requiring reoperations). nevertheless, because long - term durability data for mis are limited to up to 4 years, we can only estimate the lasting effects of mis treatment on symptoms of si joint disruption / degeneration.14,15 nonetheless, based on clinical experience to date, mis failures have occurred within the first 12 months, which has been reflected in the 18% mis treatment failure rate incorporated into the economic model. in light of the small sample sizes in the rudolf14 and sachs and capobianco15 studies (50 and 40 patients, respectively), and given the question surrounding longer - term durability of mis, a sensitivity analysis was performed by varying the 1-year mis treatment success rate from 72% to 92%, which resulted in cost neutrality at a ~79% success rate at 1 year (figure 2). of note, the mis treatment success rate where cost neutrality is achieved is dependent on the medicare reimbursement for the mis procedure (that is, drg payments for drgs 459 and 460) ; for instance, if the drg payment were to increase, then the mis treatment success rate where cost neutrality is achieved would also increase. second, because the medicare saf does not include pharmacy data, the economic model estimated retail pharmacy pain medication costs for nonoperative care using data from younger, privately insured patients, which is notably imperfect. to address this limitation while the exclusion of all retail pharmacy pain medication use is not realistic clinically, the results do provide insights into the economic burden of retail pharmacy pain medications as a component of nonoperative care in patients with degenerative sacroiliitis and si joint disruption. third, the percentage of mis index hospitalizations that fall under drg 459 (with major complications) was based on complaint reporting under a us food and drug administration - mandated postmarket product surveillance program,13 which may have underestimated the true incidence of events. as such, it is possible that the true rate of complications is higher than the 3.8% reported by miller.13 nevertheless, an analysis of drg 459 (with major complications) and drg 460 (without major complications) using the medicare provider analysis and review (medpar) 2011 data (a medicare database reflecting 100% of hospital inpatient stays for medicare beneficiaries) estimated the percent of patients with drg 459 at 5% and drg 460 at 95%. these drgs include lumbar spinal fusion in addition to mis si joint fusion. to address this limitation, a sensitivity analysis was conducted by increasing the complication rate from 3.8% to 15%. among medicare patients with degenerative sacroiliitis or si joint disruption, 3.7% underwent lumbar spinal fusion 1 year prior to or 5 years following diagnosis.18 other investigators have reported that between 18% and 48% of their patients treated with mis si joint fusion underwent lumbar spinal fusion,14,15 which suggests that the 6-year window may not have been long enough to fully capture those patients who previously or subsequently underwent lumbar spinal fusion. as the percentage of patients who underwent lumbar spinal fusion increases, the overall per - patient cost differential (cost of nonoperative care as noted earlier, the health - related quality of life (hrqol) effects of mis and nonoperative care were not included in the present analysis. nonetheless, among mis patients, rudolf14 reported early and sustained clinical improvement through 12 months of follow - up among multiple hrqol domains, including pain, activities (light, moderate, and vigorous), sleep, overall happiness, and pain effects on social interest. on the other hand, nonoperative care often requires continued therapy over time,18 which suggests that mis may lower lifetime costs and result in greater improvement in hrqol, as compared to nonoperative care. of note, we explored multiple public and private databases to identify medicare beneficiaries who underwent the mis procedure in the hospital inpatient setting ; however, these databases did not contain sufficient sample sizes (due to the lack of available reimbursement codes to uniquely identify mis patients in the datasets and/or due to the limited availability of more recent data). therefore, the cost of mis hospitalization was based on the estimated national average drg payments for drg 459 and drg 460, and the professional fee for the procedure was based on the 2012 medicare payment for cpt 27280. the follow - up medical resources and costs for mis were based on inputs from three surgeons (dwp, th, and jc, who have collectively treated over 360 patients using mis) and standard medicare fee schedules. where differences in clinical practice management were identified, the more conservative values (ie, higher costs) were used in the economic model in order to minimize any potential cost savings associated with use of mis. additional research should include prospective multicenter studies, medical chart reviews, or analysis of health insurance claims made by mis patients. two trials have been initiated to further characterize the safety and effectiveness of mis si joint fusion : a large single arm trial (sifi : nct01640353)38 and a randomized controlled trial (insite : nct01681004).39 since it will be several years before those trials are complete, the values used in the economic model were based on the best evidence that is currently available. as more robust evidence emerges first, the mis treatment success rate of 82% at 1 year is based on two studies a retrospective study of 50 consecutive patients treated with a specific mis system,14 and a second retrospective study of 40 consecutive patients.15 despite the small sample size, clinically and statistically significant improvements were reported by rudolf14 in seven out of nine quality of life domains, with 82% of patients reaching the mcid (> 2-point change in pain score) at 40 months postimplant. similarly, in a recent retrospective study with the same mis system, sachs and capobianco15 reported a clinically significant improvement (> 2-point change from baseline) in 39 out of 40 patients at 1-year follow - up. the rate of perioperative complications (20%) reported by rudolf14 was similar to other reports in the literature ; half of these events were minor (mild hematoma at the incision site and superficial cellulitis), requiring little to no intervention, whereas the remaining five patients experienced major events. specifically, three patients were brought back to the operating room for retraction of a misplaced implant (likely due to the learning curve early on during experience), one patient experienced a deep soft tissue wound infection that resolved after 6 weeks of intravenous antibiotics, and the final patient had a nondisplaced fracture that healed without intervention. beyond the perioperative complications reported by rudolf,14 only one additional complication occurred in months 1340 (implant loosening), which resulted in the placement of two additional implants. the revision rate in the 50-patient cohort was 8% after 3 years,14 which compares favorably to that reported in the literature for other types of mis approaches (8%15%).3237 more recently, a complication rate of 3.8% (204 of 5,319 patients) has been reported for a new mis system over a 4-year period.13 revision surgeries were performed in 1.8% of patients (n=94) at a median follow - up of 4 months ; early revisions primarily for symptomatic malposition were performed at a median of 19 days postoperatively, whereas late revisions primarily for symptom recurrence or continued pain of undetermined etiology were performed at a median of 279 days postoperatively.13 these more recent findings from miller and sachs and capobianco15 further suggest that the 1-year mis treatment success rate of 82% is a reasonable base case value for the economic model (where treatment failure has been defined as having one or more of the following : implant failure, loosening, and/or malposition ; failure to relieve pain requiring repeat intervention ; and infection requiring reoperations). nevertheless, because long - term durability data for mis are limited to up to 4 years, we can only estimate the lasting effects of mis treatment on symptoms of si joint disruption / degeneration.14,15 nonetheless, based on clinical experience to date, mis failures have occurred within the first 12 months, which has been reflected in the 18% mis treatment failure rate incorporated into the economic model. in light of the small sample sizes in the rudolf14 and sachs and capobianco15 studies (50 and 40 patients, respectively), and given the question surrounding longer - term durability of mis, a sensitivity analysis was performed by varying the 1-year mis treatment success rate from 72% to 92%, which resulted in cost neutrality at a ~79% success rate at 1 year (figure 2). of note, the mis treatment success rate where cost neutrality is achieved is dependent on the medicare reimbursement for the mis procedure (that is, drg payments for drgs 459 and 460) ; for instance, if the drg payment were to increase, then the mis treatment success rate where cost neutrality is achieved would also increase. second, because the medicare saf does not include pharmacy data, the economic model estimated retail pharmacy pain medication costs for nonoperative care using data from younger, privately insured patients, which is notably imperfect. to address this limitation while the exclusion of all retail pharmacy pain medication use is not realistic clinically, the results do provide insights into the economic burden of retail pharmacy pain medications as a component of nonoperative care in patients with degenerative sacroiliitis and si joint disruption. third, the percentage of mis index hospitalizations that fall under drg 459 (with major complications) was based on complaint reporting under a us food and drug administration - mandated postmarket product surveillance program,13 which may have underestimated the true incidence of events. as such, it is possible that the true rate of complications is higher than the 3.8% reported by miller.13 nevertheless, an analysis of drg 459 (with major complications) and drg 460 (without major complications) using the medicare provider analysis and review (medpar) 2011 data (a medicare database reflecting 100% of hospital inpatient stays for medicare beneficiaries) estimated the percent of patients with drg 459 at 5% and drg 460 at 95%. these drgs include lumbar spinal fusion in addition to mis si joint fusion. to address this limitation, a sensitivity analysis was conducted by increasing the complication rate from 3.8% to 15%. among medicare patients with degenerative sacroiliitis or si joint disruption, 3.7% underwent lumbar spinal fusion 1 year prior to or 5 years following diagnosis.18 other investigators have reported that between 18% and 48% of their patients treated with mis si joint fusion underwent lumbar spinal fusion,14,15 which suggests that the 6-year window may not have been long enough to fully capture those patients who previously or subsequently underwent lumbar spinal fusion. as the percentage of patients who underwent lumbar spinal fusion increases, the overall per - patient cost differential (cost of nonoperative care cost of mis) also increases. as noted earlier, the health - related quality of life (hrqol) effects of mis and nonoperative care were not included in the present analysis. nonetheless, among mis patients, rudolf14 reported early and sustained clinical improvement through 12 months of follow - up among multiple hrqol domains, including pain, activities (light, moderate, and vigorous), sleep, overall happiness, and pain effects on social interest. on the other hand, nonoperative care often requires continued therapy over time,18 which suggests that mis may lower lifetime costs and result in greater improvement in hrqol, as compared to nonoperative care. of note, we explored multiple public and private databases to identify medicare beneficiaries who underwent the mis procedure in the hospital inpatient setting ; however, these databases did not contain sufficient sample sizes (due to the lack of available reimbursement codes to uniquely identify mis patients in the datasets and/or due to the limited availability of more recent data). therefore, the cost of mis hospitalization was based on the estimated national average drg payments for drg 459 and drg 460, and the professional fee for the procedure was based on the 2012 medicare payment for cpt 27280. the follow - up medical resources and costs for mis were based on inputs from three surgeons (dwp, th, and jc, who have collectively treated over 360 patients using mis) and standard medicare fee schedules. where differences in clinical practice management were identified, the more conservative values (ie, higher costs) were used in the economic model in order to minimize any potential cost savings associated with use of mis. additional research should include prospective multicenter studies, medical chart reviews, or analysis of health insurance claims made by mis patients. two trials have been initiated to further characterize the safety and effectiveness of mis si joint fusion : a large single arm trial (sifi : nct01640353)38 and a randomized controlled trial (insite : nct01681004).39 since it will be several years before those trials are complete, the values used in the economic model were based on the best evidence that is currently available. as more robust evidence emerges, the economic model will be updated accordingly. recent us health care reform legislation focuses on improving quality of care and reducing costs. the economic burden of si joint disruption and degenerative sacroiliitis among medicare beneficiaries in the us is substantial and highlights the need for new mis therapies to treat this condition and to reduce health care expenditures. in patients who suffer from lbp due to si joint disruption or degenerative sacroiliitis, this economic analysis suggests that mis si joint fusion performed in the hospital inpatient setting could result in a cost savings to the medicare program of $ 660 million over medicare patients lifetimes by treating this population with mis fusion. | introductionthe economic burden associated with the treatment of low back pain (lbp) in the united states is significant. lbp caused by sacroiliac (si) joint disruption / degenerative sacroiliitis is most commonly treated with nonoperative care and/or open si joint surgery. new and effective minimally invasive surgery (mis) options may offer potential cost savings to medicare.methodsan economic model was developed to compare the costs of mis treatment to nonoperative care for the treatment of si joint disruption in the hospital inpatient setting in the us medicare population. lifetime cost savings (2012 us dollars) were estimated from the published literature and claims data. costs included treatment, follow - up, diagnostic testing, and retail pharmacy pain medication. costs of si joint disruption patients managed with nonoperative care were estimated from the 20052010 medicare 5% standard analytic files using primary international classification of diseases, ninth revision, clinical modification (icd-9-cm) diagnosis codes 720.2, 724.6, 739.4, 846.9, or 847.3. mis fusion hospitalization cost was based on diagnosis related group (drg) payments of $ 46,700 (with major complications - drg 459) and $ 27,800 (without major complications - drg 460), weighted assuming 3.8% of patients have complications. mis fusion professional fee was determined from the 2012 medicare payment for current procedural terminology code 27280, with an 82% fusion success rate and 1.8% revision rate. outcomes were discounted by 3.0% per annum.resultsthe extrapolated lifetime cost of treating medicare patients with mis fusion was $ 48,185/patient compared to $ 51,543/patient for nonoperative care, resulting in a $ 660 million savings to medicare (196,452 beneficiaries at $ 3,358 in savings / patient). including those with icd-9-cm code 721.3 (lumbosacral spondylosis) increased lifetime cost estimates (up to 478,764 beneficiaries at $ 8,692 in savings / patient).conclusiontreating medicare beneficiaries with mis fusion in the hospital inpatient setting could save medicare $ 660 million over patients lifetimes. |
the health benefits of the use of radiation in medical practice are nowadays widely acknowledged. nevertheless, for instance, the risks for pregnant women, more precisely for their embryo / fetus to develop structural and functional brain defects, have been described based on epidemiological studies from atomic bomb survivors. these observations are particularly relevant in the case of radiotherapy treatment or medical imaging during pregnancy, which often results in abortion, delay of maternal therapy, or preterm delivery when women were diagnosed with cancer during pregnancy. furthermore, the use of medical radiation modalities employing low doses, such as ct scans, has increased tremendously over the past decades. the advantages of this increasing use of low doses in the hospital are obvious, but a major concern exists regarding the cumulative exposure doses of repetitive tests and the inappropriate utilization of these imaging techniques [3, 4 ]. in all, the benefits of current medical practice can still be improved for pregnant women and their unborn child, for which a better understanding of radiation effects, especially in the low - dose range, is imperative. the human developing brain is extremely sensitive to radiation exposure, which is especially the case when exposure occurs within a specific developmental time - window, that is, between weeks 8 and 25 of gestation. this period is characterized by specific coordinated developmental processes and corresponds to embryonic days 11 (e11) to 17 in mice (see figure 1). in most cases, the mouse has been the experimental model of choice to investigate radiation - induced defects to the brain, but the high vulnerability of the fetal brain to environmental insults has been recognized in many other mammalian species, including primates and other rodents [68 ]. research on prenatal radiation - induced brain defects has mainly focused on the developing neocortex, of which the developmental hallmarks are depicted in figure 1. however, persistent brain defects are presumed to result from a concerted action of different brain regions, going beyond the neocortex and including other major brain structures such as the hippocampus, cerebellum, and striatum. most of these regions undergo neurogenesis during the gestational period, with the exception of the cerebellum and hippocampal dentate granule cells that display crucial developmental and neurogenic events during the early postnatal period and further. thus, to understand the full extent of developmental aberrations to the brain following irradiation, we need to be aware of the necessity to investigate all brain regions that might be affected by irradiation, which is currently still lacking. it is only then that we can identify the causative factors responsible for the observed brain damage in prenatally exposed survivors of the atomic bombings in hiroshima and nagasaki, which manifested as an increase in the frequency of mental retardation, lower iq and school performance, and unprovoked seizures. concomitantly, this knowledge would be highly beneficial for the medical sector, where the use of ionizing radiation is common standard and increasing constantly and where we are facing many uncertainties on the exact impact of (repeated) low - dose exposures. here, we review the current knowledge, but also the concerns and limitations, regarding early and persistent defects to the brain after in utero exposure to low- and moderate - to - high doses of irradiation. in 1929, goldstein and murphy reported on mental retardation and microcephaly resulting from prenatal radiation exposure, as revealed from 38 case reports of children born to mothers that received pelvic radiotherapy. decades later, this awareness was further strengthened and quantitative data were provided through the follow - up of the health of atomic bomb survivors, primarily performed and published by otake and schull. their study involved 1500 individuals exposed in utero to the radioactive fallout of the atomic bombs in hiroshima and nagasaki (mainly -radiation). apart from an excess cancer risk, a higher incidence in generalized growth retardation and microcephaly, mental disability, and seizures, as well as a decreased school performance and scoring on intelligence tests, was observed. these defects were all relatively linearly dose - dependent, with an increased risk for mental retardation of 43% and a decline of 2529 points in iq values per gy. no dose threshold was proposed for these observations, except for mental retardation, for which symptoms were detected at doses as low as 0.06 to 0.31 gy. important to note from these studies is that the developing brain is particularly sensitive to irradiation when exposure occurred between weeks 8 and 15 of pregnancy and to a lesser extent between weeks 16 and 25 [12, 14 ]. hence, the brain appears especially vulnerable to radiation during the period characterized by a massive neuron production and differentiation / migration (figure 1). the fallout of the chernobyl accident in 1986 has exposed many people to radioiodine (i) and radiocaesium (cs). also here, prenatally irradiated subjects were followed over time, but findings are much less consistent and are subject to debate [15, 16 ]. this might be due to the fact that people in the surrounding areas of the catastrophe were exposed to relatively low doses (between 0.01 and 0.25 sv), with, for instance, an important norwegian cohort receiving doses less than 0.10 sv. other limitations of these epidemiological studies were the potential confounding variables that could not be taken into account, the lack of accurate dose measures per individual, and the fact that cohorts were considerably smaller than those of the atomic bomb survivors [17, 18 ]. nevertheless, an increased occurrence of mental retardation and decrease in (verbal) iq scores could be noted in children and adolescents in utero exposed [1821 ]. neuropsychiatric problems were also reported but might as well be associated with the mother 's health and stress. noteworthy, consistent with previous findings, the radiosensitive period to develop such anomalies involved weeks 8 to 25. this was further corroborated by a recent study involving a cohort of prenatally exposed children of whom the mothers were exposed to diagnostic x - ray pelvimetry during late pregnancy, and thus beyond the peak of neurogenesis. during this late stage, no association of irradiation with school performance was found, although this might also be due to the low doses of radiation received by the fetus (0.59 mgy) in this study. in all, it is evident that, above a certain threshold, irradiation during the gestational period hampers normal brain development and functioning during later life when exposure occurred during the peak of neuronal expansion and differentiation. while experimental findings can vary depending on the study, most credible results were obtained by epidemiological investigations in atomic bomb survivors. yet the lack of knowledge about the underlying causes warranted a more in - depth research involving in vitro and in vivo animal models, which will be elaborately discussed in the following paragraphs. in search for defects that occur shortly after prenatal radiation exposure and that might account for long - lasting cognitive defects as observed in human cohorts, animal studies have been proven a highly valuable tool. the rodent brain is a widely used model system, given the obvious advantages of working with small rodents, the ease to genetically modify the mouse genome, and most importantly the high similarity in brain development, architecture, and interconnectivity when compared to humans. as a defense mechanism to radiation exposure, a series of biochemical pathways are activated to promote cell survival while maintaining genetic integrity. yet proliferating cells are considered much more vulnerable to radiation - induced damage because they require correct and intact dna for their progeny. in postmitotic cells on the other hand, the integrity of their transcribed genes is considered crucial rather than that of their whole genome. this is exemplified by the fact that the immature rodent brain is much more sensitive to radiation stress as opposed to the juvenile or adult brain that contains very few proliferating cells. during the radiosensitive embryonic period (figure 1), neuronal precursor cells appear even more susceptible to radiation damage than proliferating cells in other embryonic tissues. it is therefore imperative to closely examine the central nervous system in order to gain knowledge about prenatal radiation - induced health risks. a schematic overview of brain developmental processes that are altered after prenatal radiation exposure and that are described in detail in the following paragraphs is depicted in figure 2. differences in radiosensitivity between neural stem / progenitor cells and postmitotic neurons may be related to differences in the radiation - induced dna damage response (ddr) between these cell types. dna damage and the production of reactive oxygen species (ros) and reactive nitrogen species (rns) is a general hallmark after irradiation in mammalian cells. this is for example shown in cultured rat embryonic cells irradiated with a high dose of 2.0 gy, generating an excess of ros and rns within few hours after radiation. the high sensitivity of the developing brain to oxidative stress is thought to be due to its low concentrations of antioxidants, its particular lipid configuration of the cell membranes, its high rate of oxygen consumption, and the large proportion of sensitive immature cells. of note, however, the limited antioxidative capacity is also characteristic of postmitotic neurons in the developing brain, thus seeming to be in contrast with the proposed sensitivity of neural precursors to radiation - induced dna damage [31, 32 ]. the induction of dna double - strand breaks (dsbs) after irradiation of the developing mouse cortex has been explored by many groups over the years, by assessing different embryonic stages and postirradiation (pi) time points (summarized in table 1). irradiation - induced dsbs are immediate events that decrease between 1 h and 4 h after irradiation. at e14.5, dsb foci are formed homogenously throughout the irradiated neocortex, with an equal distribution in the ventricular zone (vz) and subventricular zone (svz), comprising stem and progenitor cells, and in the intermediate zone (iz) and cortical plate (cp), comprising maturing neurons (figure 2). foci are observed in a linear dose - dependent manner and can be already detected after a low - dose of 0.01 gy in the e13.5 irradiated cortex. interestingly, and in contrast to the appearance of dsb foci, dna damage is not repaired at a similar speed in cells of the vz / svz compared to cp cells. more specifically, in the mouse neocortex exposed to 2.0 gy of -radiation, the decrease in dsb foci was much more pronounced in differentiating neurons than in vz / svz cells between 1 h and 4 h pi, which was suggested to correlate with the high radiosensitivity of neuronal precursors. alternatively, this temporal difference in dna repair might be better anticipated from the fact that both cell types utilize their own ddr machinery (see next section), in which repair occurs much faster in postmitotic neurons as compared to proliferating cells. differences in dna repair kinetics depend not only on the cell type, but also on the administered radiation dose. this was shown in vitro using human cells but was recently also demonstrated in vivo by saha., who showed that, between 1 h and 6 h after irradiation, dna repair did not take place in mouse neocortical cells exposed to doses below 0.05 gy, while foci in 0.1-gy exposed neocortices returned to background levels within this time span. the implications of this finding dissociating the biological defense response between high and very low doses of radiation are not yet fully understood. it is however of particular interest in light of the increasing use of low - dose imaging modalities in current medical practice. dna damage repair requires dividing cells to arrest their cell cycle in order to activate the proper repair mechanisms, and this cell cycle block is again highly dependent on the administered radiation dose (see table 1). indeed, cell cycle arrest was induced in neocortical precursor cells exposed to 0.5 gy, while 0.1 gy was not sufficient to fully stop cell division and initiate dna repair, probably because of an insensitivity to a low number of dsbs in such low - dose irradiated cells (figure 2) [36, 47 ]. as a consequence of this escaped dna repair, such cells survive and further differentiate, rendering them highly susceptible to persistent damage and ultimately cell death. whereas the intra - s and g2/m checkpoints were shown to be activated in neural progenitors after radiation, the g1/s checkpoint was not. this was proposed to result from the fact that p21, a key activator of g1/s cell cycle arrest, is not activated in neocortical stem cells. contrarily, p21 was suggested to be activated in irradiated migrating cells of the iz, where it is believed to have a proapoptotic role. this finding is rather surprising, since a p53-dependent increase in p21 expression and a concomitant g1/s block were found in irradiated cultured neural progenitors by others [48, 49 ]. this discrepancy thus shows that we should always consider the influence of the extracellular environment and the specific structural organization of the neocortex that can only be recapitulated in vivo. in all, we have gathered a basic understanding of radiation - induced dna damage and repair over the past years, as summarized in table 1, indicating that the ddr in the irradiated developing brain is not homogenously distributed but instead depends on the cell type and the administered radiation dose. the impact of a defective ddr in the embryonic brain is exemplified by the symptoms observed in patients with mutations in crucial ddr genes. these patients have a high risk of developing immunodeficiency, genomic instability, and cancer, but, surprisingly, they also frequently display neurological disorders, neurodegenerative diseases, or brain tumors [5053 ]. amongst those neurological disorders is microcephaly, a disease that is commonly evident at birth and very often observed in patients suffering from ddr - defective disorders. one prominent example is microcephalin- (mcph1-) dependent primary microcephaly, in which a defective atr - dependent g2/m checkpoint arrest was evidenced [54, 55 ]. these observations prompted in vitro and in vivo research that disclosed substantial insight into the temporal use of ddr pathways, which is of vital importance during brain development to prevent an expansion of mutant progenitors and later occurring brain diseases [32, 53 ]. during the first days of neurogenesis, at which proliferation peaks, homologous recombination (hr) is the predominant dna repair mechanism. this was demonstrated by studies using xrcc2-deficient mice, lacking the gene crucial for an efficient hr, in which a massive apoptosis of neural stem and progenitor cells between e10 and e14.5 results from unrepaired dna damage. on the other hand, studies using ligiv- and xrcc4-deficient mice, which are characterized by impaired nonhomologous end - joining (nhej), unveiled the importance of nhej as a predominant repair modality during later phases of embryonic development [5860 ]. in fact, in e14.5 ligiv null embryos, an induction of dsbs was observed in all cortical layers, to a similar extent as in 0.1-gy irradiated control embryos (table 1). in line with the clear time - dependency in repair mechanisms, rad54 mice, lacking the crucial hr actor rad54, were hypersensitive to ionizing radiation at the embryonic but not at the adult stage. this demonstrates the importance of hr for the long - term survival of cortical progenitors irradiated during the s and g2 phases of the cell cycle, while it does not seem to be involved in a correct dna damage repair of postmitotic neurons or progenitors in the g1 or g0 phase, when no sister chromatid is available to undergo hr. as stated above, the adult brain is much less sensitive to radiation - induced damage in comparison to the developing brain, in which dividing cells show a higher susceptibility to dna damage. to note, astrocytes exhibit an even greater radioresistance than postmitotic neurons, as revealed by a strongly attenuated phosphorylation and reduced protein levels of atm, chk2, and p53, and a concomitant lack of apoptosis, even after an extremely high dose of 20 gy nevertheless, even though dna repair is not that crucial as in dividing cells, neurons still need to survive from genotoxic stress since they are irreplaceable, which is also true for postmitotic neurons that reside in the cp of the neocortex. differentiated cells remain capable of rejoining broken dna ends through the collaboration of atm and dna - pk, or via nucleotide excision repair mechanisms in case of single strand breaks. even hr might still be used by cells that reenter the cell cycle upon genotoxic stress, which however predisposes the cells to die by apoptosis. whether these mechanisms also apply for mature cells in the developing cortex has however not been established yet. radiation - induced neuronal apoptosis is well described in literature and is clearly evidenced in the developing neocortex (summarized in table 1). a dose - dependent increase in apoptosis was found in the e14.5 cortex from a dose of 0.05 gy onwards, with two to five dsbs being sufficient for a cell to initiate cell death. apoptosis is proposed to be the main outcome of unrepaired dna dsbs in the developing nervous system, since dividing cells can be quickly replaced by the extensive neuronal stem cell pool. with respect to the difference in radiosensitivity between proliferating and maturing neurons in the developing neocortex, radiation - induced apoptosis indeed seems to differ greatly between the vz / svz and the iz / cp. possibly related to the slower repair kinetics of vz / svz cells, these progenitors display the highest degree of apoptosis following radiation exposure. early after irradiation (36 h pi), apoptotic cells largely reside in the vz / svz and iz, with around 31% of all cells being apoptotic [31, 36 ]. at later time points (14 h and 24 h pi), a significant increase in apoptosis is noticeable in the iz [36, 44 ] and cp [31, 39 ]. whether apoptotic cells at this late stage are predominantly observed in these upper layers, or whether they are also still represented in the other proliferative layers, is however unclear. either way, this second wave of apoptosis seems to contradict the general assumption of extensive cell death in proliferating cells. yet it can be assumed that dying cells in the iz / cp originate from proliferating cells with irreparable damage which progressed through mitosis and migrated to the upper neocortical layers where they undergo apoptosis, as was indeed suggested by gatz.. this is for instance the case in the dorsal telencephalon, the site of origin for excitatory cortical neurons, and in the lateral ganglionic eminence (lge), a source of striatal projection neurons and interneurons of the olfactory bulb and amygdala [66, 67 ]. in particular, in the lge, intermediate progenitors of the svz were highly sensitive to radiation, whereas the radial stem cells were more resistant and entered self - renewal shortly after irradiation. this is in sharp contrast to the dorsal telencephalon in which the radial glia in the vz are the main targets of early radiation - induced apoptosis. this region - specific difference is likely correlated to a difference in the size of the svz, which is much more prominent in the lge than in the dorsal telencephalon at the stage of irradiation. mechanisms involved in this differential apoptotic response and functional consequences hereto are however not yet clarified. of note, it is also important to consider the large variation in parameters used by different authors to study radiation - induced apoptotic responses [31, 45 ]. for instance, differences in radiation dose, time points after exposure, and the delineation / definition of cellular layers all lead to a lack of unification of obtained results (see table 1). in addition, the developmental stage at which radiation exposure takes place, as well as the irradiation parameters such as the dose - rate or energy of the beam, might significantly influence the apoptotic response. nevertheless, radiation - induced apoptosis in the developing brain is highly dependent of p53 activation [42, 46 ], by the protein kinase atm. phosphorylation of p53 and subsequent transactivation of apoptotic genes occur already at 2 h after irradiation. however, p53-independent mechanisms also exist in the irradiated prenatal brain, which are able to induce cell cycle arrest but not apoptosis, as suggested from gene expression analysis of trp53-null mice exposed to 0.5 gy at e13. unfortunately, a general consensus on which molecular players are involved in radiation - induced cell death in the developing brain is lacking. the participation of atm and bax in this radiation response was described both in vivo and in vitro [71, 72 ] but was disputed by another study. furthermore, caspase-9 and caspase-3 were defined as crucial death effectors in the central nervous system in response to irradiation, while this was contradicted by others [71, 73 ]. these opposing findings might be accredited to the use of a wide range of doses, including extremely high doses (10.0 gy), in vitro set - ups, and irradiation at prenatal versus postnatal stages. a number of studies showed that radiation induces excitotoxic apoptosis, suggesting that n - methyl - d - aspartate (nmda) receptor antagonists might be used as neuroprotectors after radiation exposure. this was for the first time investigated in the early 1990s for postnatal radiation injury, revealing an ameliorating effect of nmda receptor blockage on radiation - induced hippocampal - dependent learning deficits and neuronal damage. more recently, it was shown that the nmda receptor antagonist mk-801 could attenuate radiation - induced apoptosis in immature primary cortical neurons and embryonic brains. however, other studies have reported an increased cell death in the mouse brain following postnatal delivery of mk-801 (reviewed in), thus urging for further investigation into the mediators of these processes and the likelihood of excitotoxicity in the irradiated brain. moreover, the administered dose of a particular nmda receptor blocker needs to be carefully considered, especially with regard to incidental health risks posed to the pregnant mother. this concern is justified, since staggering and hyperlocomotion were detected in adult mice injected with doses of 0.3 mg / kg mk-801 and above [78, 79 ]. despite the fact that the precise mechanisms remain unknown, it is generally accepted that radiation to the developing brain causes extensive apoptosis in a specific spatiotemporal manner. this radiation - induced apoptosis might contribute to the late occurring functional brain defects as observed in atomic bomb survivors, although the induction of early apoptotic events was never investigated in these cohorts. therefore, to fully elucidate such a link, a more in - depth analysis of the causal relationship between short- and long - term consequences is warranted. this can for example be achieved by using transgenic animals lacking a radiation - induced apoptotic mediator in the developing brain and investigating possible changes in long - lasting brain defects. besides dna damage, cell cycle arrest, and apoptosis, one other outcome of prenatal irradiation is the disturbance of neuronal migration. more specifically, a dose of 2.25 gy to the rat brain at e17 results in ectopic cell populations in the cerebral cortex and hippocampus, predisposing the animals to epileptic seizures, characteristic for the disease phenotype in human patients. also lower radiation doses were suggested to cause decelerated neuronal migration. for example, magnetic resonance imaging (mri) of brains of mentally retarded atomic bomb survivors revealed large regions of abnormally situated gray matter, which the authors proposed resulted from defective migration around the timing of irradiation [5, 12 ]. who performed a brdu pulse experiment in prenatally irradiated mice and discovered ectopic cells scattered over the svz, iz, and cp instead of being restricted to the cp at 3 days following irradiation, which was clearly dose - dependent. at early time points after birth, ectopic cells outside layer iv were still detected, whereas such cells were no longer observed at 8 weeks after birth, indicating that appropriate migration could be restored. similar migration defects in the embryonic brain were observed by others, with the severity depending on the dose [8284 ]. yet, of note, the observation of ectopic cells at later time points is only indirect evidence of disturbed neuronal migration. a first indication for an incorrect migration process was given by sun. who showed that radial glia fibers, which serve as an essential substrate for outward neuronal migration in the dorsal neocortex, were disorganized, as defined by a distorted orientation and marked reduction in the number of these fibers (figure 2). still, a more detailed investigation at multiple early time points is desirable to determine if and how radiation hampers the migration route towards the cp. in addition, a time - lapse follow - up of these migrating cells would be extremely informative to unravel possible radiation - induced changes in migration kinetics. the observation of microcephaly already within days after in utero radiation exposure of mice is believed to be largely attributable to the massive radiation - induced apoptosis (figure 2), but direct evidence linking the acute apoptosis with long - term brain anomalies is missing. as stated before, ddr pathways are fundamental to ensure a proliferative developmental nervous system, which is of importance after radiation exposure as well as in microcephalic disorders. unrepaired dna damage and a resulting apoptosis or loss of proliferative capacity, for instance, induced by irradiation, can as such be the main underlying factor to induce microcephaly. reduction of cortical thickness was already revealed 24 h after 1.0 gy exposure at e11 and a decreased thickness of the postnatal cortex irradiated at e15 was discovered from a dose of 0.5 gy onwards (figure 2). one possible key factor underlying radiation - induced microcephaly is the abnormal spindle - like microcephaly associated gene (aspm), which was downregulated in the ventricular zones 2 h after 2.0 gy exposure to the e12 mouse brain. the aspm gene was convincingly linked to microcephaly, as a homozygous mutation of this gene is responsible for autosomal recessive primary microcephaly (mcph) in humans. together with a downregulation of aspm, the induction of supernumerary centrosomes due to centrosomal overduplication was also described to result from radiation exposure, albeit only shown in human tumor cells after high irradiation doses. such centrosomal deficiencies are often observed in cells of patients with primary microcephaly, where they contribute to cell death or an aberrant proliferation. microcephaly is also often related to mitotic spindle defects, as described for mcph where spindle defects are the result of mutations in, for example, mcph1, wd repeat domain 62 (wdr62), cdk5 regulatory subunit - associated protein 2, and aspm (for an overview, see). here, mitotic spindle defects lead to a perturbation of the intricate balance between symmetric and asymmetric divisions, in favor of asymmetric cell divisions and thus of a premature differentiation, leading to a notable reduction in the number of neural precursors and ultimately in overall brain size. in light of the microcephalic phenotype in radiation - exposed brains, and the reduction in aspm expression in irradiated brains as described earlier, it would thus be meaningful to investigate the possibility of a premature differentiation in prenatally irradiated brains, which has not been studied in much detail before. recently, however, a thorough gene expression analysis suggested that in utero irradiation triggers a p53-dependent induction of genes associated with neuronal differentiation and mitotic spindle assembly, hinting for a possible premature differentiation following radiation exposure. this hypothesis was further strengthened by the very strong overlap between gene expression profiles of irradiated embryonic brains and those of magoh mice, which display microcephaly associated with premature neuronal differentiation [69, 93 ]. a follow - up of these mice showed that apoptosis, but not premature neuronal differentiation, was p53-dependent. a more detailed investigation should thus be performed to assess whether this also applies to irradiated mouse embryos. in all, microcephaly as a result of prenatal irradiation is starting to be further explored, with a growing awareness of similarities between radiation - induced and microcephaly disease genes that might converge to related mechanisms. yet in vivo evidence for radiation - induced mitotic spindle defects and premature differentiation remains to be provided, for example, via investigation of the division angle in neocortical stem cells. the prenatal radiation - induced microcephaly, as established both in humans and in animals, is mostly accompanied by overall growth retardation. this effect appears to be induced from a dose of 0.3 gy on. whether the reduction in brain size is associated with an overall decrease in the number or density of neurons the majority of animal studies are in agreement with a reduced cell number, for instance, evidenced for the e15 irradiated rat brain by means of mri analyses and histology [96, 97 ], and further substantiated for the irradiated rodent hippocampus, corpus callosum, cerebellar purkinje cells, and primary visual cortex [98103 ]. contrarily, other studies found no differences in cell number in the somatosensory cortex, in cortical layer v pyramidal neurons [104, 105 ], and in the visual cortex after irradiation, the latter being in sharp contrast with the study of vitral. who showed a strong neural reduction in the irradiated visual cortex. interestingly, a change in cell density can strongly depend on the cell type within a single brain region, as shown for the irradiated cerebellum. here, stereological investigations revealed a reduced purkinje cell number but an increased granule cell number after low - dose irradiation of the e13 embryo. the authors reasoned that this seeming paradox might be due to an increased granule cell production after radiation - induced ros production but also highlighted the crucial importance of timing of radiation exposure, given that both cell types were decreased in number after low - dose exposure at later stages of development (e17e19). apart from rodents, several studies on prenatally irradiated nonhuman primates have been performed in which consecutive developmental events are more spread over time, as compared to the overlapping sequence of events in the short gestational period of rodents. therefore, such data provided a better insight into the importance of developmental timing to develop irradiation - induced defects. specific depletion of cells born at the stage of irradiation was found and consequent volume reductions occurred only in the regions and/or layers constituted by those cells, after irradiation of macaques during early- or midgestation [108111 ]. for example, irradiation of the e11 mouse embryo, a stage at which cortical layer vi cells are being produced, caused a decrease in the thickness of layers i, ii, and iii. further, irradiation at e13, corresponding to formation of cells that will constitute layer v, resulted in a decrease in layers iv and vi cells. finally, irradiation at e15 or e17 surprisingly affected the thickness of all cortical layers. this finding is puzzling and implies that irradiation of progenitors during a specific developmental time - window might not only impact cells typical for its birth date, but instead might disrupt a more elaborate neuronal network and cytoarchitecture. over the years claims that the fate of a common progenitor changes over time to generate the different subtypes of neurons. this thus implies that early progenitors, normally producing lower layer neurons, are also capable of producing upper layer neurons (reviewed in), which explains to some extent our findings after prenatal radiation exposure. on top of that, this suggests that irradiation during an early developmental period has the greatest impact on cortical layer formation. this hypothesis would indeed favor the finding of a hampered juvenile hippocampal neurogenesis resulting from acute irradiation at the onset of neurogenesis, since hippocampal neurons start to be produced at an early stage of e10.5, followed by a peak of pyramidal neuron production only several days later [114116 ]. as mentioned before, the observed disruption of neuronal migration following irradiation causes the introduction of ectopic cells spread throughout the brain [117120 ]. such a disorganization of neurons can be accompanied by a defective neuronal orientation, morphology and arborization, resulting from an improper and disturbed maturation, as was suggested for irradiated corticospinal tract neurons, ca1 neurons, pyramidal cells of cortical layer v, and hippocampal mossy fibers. evidently, such a disturbed dendritic organization might also entail an improper neural circuit formation and synaptic communication. indeed, a decreased inhibition of excitatory cells was shown in the cortical dysplasia model, probably contributing to the epileptogenic development in these animals. likewise, an increase in excitatory synapse markers in specific layers of the cortex and an elevated glutamate release were demonstrated after radiation - induced cortical dysplasia. in neurons cultured from irradiated neocortices, a disturbed synaptic communication was further unveiled by an increased inhibitory versus excitatory input. yet a firm conclusion on the effects of radiation on synaptogenesis is hindered by the lack of evidence for the low - dose range and by many contradictory findings between studies. for example, no difference in the number of synapses was found in layer v cortical cells of the e15 embryo irradiated with different doses, and synapse density was normal in the somatosensory and visual cortex after 1.0 gy exposure. on the other hand, a proteomic study on hippocampal samples from 6-month - old prenatally irradiated mice revealed an enhanced expression of postsynaptic density protein 95 (psd95) after 1.0 gy exposure, suggesting a pronounced effect of moderate doses of irradiation on synaptic plasticity in hippocampal dendrites. thus, these findings demonstrate the necessity to further explore neuronal communication after prenatal irradiation and to investigate synaptogenesis and inhibitory neuron development at multiple time points following irradiation, using a broad range of irradiation doses. at high doses of exposure (1.0 gy), the dispersed cell masses supposedly associated with a defective migration are accompanied by large cytoarchitectural aberrations. as an illustration, ectopic gray matter was found at the ventricular boundaries in adult animals prenatally exposed to radiation, indicative of cells that have not migrated at all since development. moreover, the typical 6-layered cortical architecture and the hippocampal laminar formation of ca1, ca3, and/or dg often appear severely impaired after high - dose prenatal irradiation [131, 132 ]. notably, these high doses have been shown to produce such a large spectrum of defects in the postnatal, juvenile, and/or (young) adult brain, with structural changes that completely disrupt the brain 's integrity. examples for this are the underdevelopment or even complete loss of white matter structures such as the corpus callosum [100, 133 ], severe cerebellar deficits [99, 103 ], and extreme hydrocephalus [134, 135 ]. as such, it is not surprising that animals irradiated with doses 1.0 gy display a severely affected behavior. in the following paragraph and in line with our focus on low to moderate doses of irradiation, we will further report on mild changes that are induced by this lower dose range, unless findings in which higher doses were used can be informative to explain underlying mechanisms. the use of behavioral tests to assess brain function after in utero irradiation is frequent and is considered as the best available strategy to translate animal behavior to neurological changes seen in the atomic bomb survivors. ample evidence from rodent studies exists regarding alterations in activity, locomotor, exploratory, and anxiety - related behavior, as well as in cognition for doses 1.0 gy [95, 136140 ]. recently, a proteomics study uncovered changes in several signaling pathways, 6 months after 0.5 and 1.0 gy exposure at e11, which correlate well with these cognitive defects. however, likely resulting from interlaboratory variation in behavioral testing, but probably also due to differences in radiation doses and dose - rates used, many discrepancies exist between studies. in particular, no consistent conclusions can be drawn regarding the gestational age at which rodent behavior is mostly affected by irradiation. for example, 0.35-gy and 1.0-gy exposure to the e11-e11.5 mouse embryo induced changes in locomotor and anxiolytic behavior as well as in learning and memory, while no changes were observed when exposure occurred at earlier or later stages [39, 141 ]. on the other hand, exposure to an intermediate dose of 0.5 gy furthermore, sienkiewicz. postulated that the e15 and e18 embryo was most sensitive to learning and memory deficits after 1.0 gy exposure, when compared to embryos irradiated at e13, although only a limited number of tests were performed in this study. thus, it is clear that the exact functional deficits resulting from prenatal irradiation are far from completely understood and that additional research is required, preferably using the same behavioral test battery and similar radiation types and dose range for each gestational stage. nevertheless, many research groups appear to observe a threshold dose below which no behavioral changes can be detected. this threshold is believed to be around 0.3 gy in mice [95, 112, 138, 139 ]. noteworthy, in humans, a total dose of 0.2 gy is regarded as a threshold value above which a therapeutic abortion should be considered, while a fetal dose not exceeding 0.1 gy and preferably 0.05 gy is strongly advised [144, 145 ]. this might relate to a possible higher radiosensitivity of humans as compared to rodents but might also indicate a lack of sensitive protocols to assess functional deficits in prenatally irradiated animal models. other alterations that have been observed and that might contribute to persistent structural and functional deficits after in utero radiation exposure are, for instance, inflammation and vascular modifications. irradiation of rats at e11 with 1.3 gy or at e15 with 1.5 gy was shown to induce astrogliosis and astrocyte proliferation in the hindbrain and in the whole brain, respectively. in the context of vascular modifications furthermore, a dose of 1.5 gy resulted in an underdevelopment of the microvasculature, responsible for a decreased cerebral blood flow and angioarchitectonic abnormalities. however, the bbb and blood - cerebrospinal fluid barrier were not altered. of note, most research on radiation - induced bbb permeability has been focused on high doses, in the context of radiotherapy research where an increased permeability is desirable for the delivery of chemotherapeutics to the brain. as such, due to the poor amount of data, effects on bbb permeability after lower doses of irradiation might be overlooked and should be further explored. besides, since the blood - brain barrier is still immature in the developing embryo and more prone to drugs, toxins, and pathological conditions, special attention should be directed to effects of prenatal irradiation on bbb formation and associated neurological disorders in later life. in conclusion, brain functional and structural modifications following prenatal irradiation are far - reaching. yet, despite small brain size and ectopic neurons, there is no consensus about the exact brain defects or their link to the observed behavioral changes. then again, such a link might be difficult to establish, given that higher doses of irradiation generate such widespread changes. therefore, we suggest that the use of low doses, which is becoming extremely relevant in the medical sector, needs to be exploited further to detect more subtle effects on brain structure and function and to provide as such a better model to study underlying mechanisms and possible causative relationships. to our knowledge, the influence of prenatal irradiation on ageing and in particular brain ageing has never been studied. nonetheless, evidence exists to suggest that prenatal radiation might aggravate or at least influence to a certain extent ageing processes in the brain. this can be suggested by the fact that early - life dna damage and replicative stress are believed to control the onset of ageing [150152 ] and by the fact that the developing embryonic brain is by far the most sensitive organ to dna damage events [27, 153 ]. indeed, as described in previous paragraphs, irradiation induces a substantial amount of dna damage in the embryonic neocortex, stressing the predisposed risk of accelerated ageing. the link between genomic instability and ageing is further corroborated by the accelerated ageing or neurodegeneration observed in patients and mice with defective dna repair mechanisms [5153, 154 ]. although most of these models display chronic dna damage, which is in contrast to acutely irradiated mice, a mouse model for atr - seckel syndrome displayed accelerated ageing despite dna damage occurring only during embryonic development. other underlying pathways for a possible radiation - induced premature ageing might involve changes in tau expression, as recently demonstrated in primary cultured hippocampal cells exposed to 0.5 and 2 gy. still, a clear - cut answer as to whether senescence can be a mechanism that contributes to radiation effects in the developing brain is unknown. in favor of such a link, an induction of senescence / ageing markers could be revealed in various tissues after adult mouse irradiation, including dna damage foci, p21, and senescence - associated -galactosidase expression and the mitochondrial common deletion [156, 157 ]. also in adult brain tissue, albeit only after high radiation doses, ageing - like processes such as oxidative stress and inflammation were found to occur [158161 ]. finally, some prenatal radiation - induced alterations in behavior were investigated at multiple time points and worsened over time, indicative of a progressive decrease in brain function. this was also observed in nonhuman primates, in which cognitive impairment after in utero irradiation was not yet evident in young adulthood but did manifest at an adult age. all the above suggests that taking a closer look at in utero radiation - induced ageing might be very useful, particularly with respect to the increased use of low - dose exposures and the steady growth of the elderly population. in first instance, investigation of ageing parameters as has been done for adult high - dose irradiation should be performed, substantiated by an examination of cognition and brain functionality at the long term. atomic bomb survivors have now reached about 70 years of age and the follow - up of their health might already provide crucial information in this regard. interestingly, an accelerated occurrence of age - related changes, modelled in terms of cardiopulmonary and psychological age, was indeed shown in clean - up workers of the chernobyl area. unfortunately, no scientific consensus can be found in the atomic bomb survivor life - span study, due to an insufficient statistical power and a biased dose - mortality relationship caused by an incomplete knowledge on received doses. although the precise consequences of in utero radiation exposure are not yet fully understood, the risk for the unborn child to develop brain abnormalities following exposure is generally recognized. guidelines for pregnant women needing medical intervention using radiation currently apply the alara (as low as reasonably achievable) principle, and the exposure dose should not exceed 0.1 gy [144, 145 ], as implemented by the icrp. this threshold implies that no noncancer effects are expected to occur below 0.1 gy and that the increased risk to develop cancers is neglectable. however, the proposed sharp threshold is due to many uncertainties that still exist on the exact consequences of prenatal radiation exposure, warranting a better understanding of the broad range of radiation effects to improve the outcome for pregnant women and to better inform medical professionals. this is especially important given that the current use of high but especially of low - dose radiation rises continuously in the clinic. for example, in the united states, the collective radiation dose from medical imaging purposes has increased no less than 6 times over the last two decades. notably, whereas a single ct scan is not believed to administer a dose that can provoke a negative outcome to the patient, it is the cumulative dose from multiple scanning sessions and the unjustified use of scans that might pose a considerable risk to human health [3, 4, 168 ]. in case of exposure to the embryo / fetus, evidence indicates an increased cancer risk for doses as low as 6 mgy, which is in the range of those received by some ct examinations (see table 2 for an overview of doses received after various radiation types). ever since its discovery, ionizing radiation has proven to be a double - edged sword. its use in the medical field is of crucial importance, but it must be utilized with great caution since exposure to radiation might evoke serious health consequences, as pointed out for in utero exposure in this work. unfortunately, a detailed view on short- and long - term brain defects following prenatal exposure is incomplete, which is why it is necessary to integrate more animal studies, more radiation doses, and a broader range of developmental time points to be considered. indeed, epidemiological studies do not allow identifying subclinical health effects that can develop into more serious health risks later in life. in light of such long - lasting functional brain defects, a recent study argued that little information exists on the long - term outcome of radiotherapy - exposed fetuses, for which the use of a good mouse model might hold the key to better understand such a correlation and to achieve a correct health risk assessment. another uncertainty is the effect of low - dose irradiation and whether it would fit in a linear - nonthreshold (lnt) model or would rather induce a hormetic, or an aggravated, health status. in fact, estimates of low - dose risks are too often an extrapolation from high doses, while it is important to realize that low - dose risks should be based on a sufficient knowledge on the underlying biological mechanisms. in this respect, evidence is culminating over the years which disputes a lnt relationship for low radiation doses, in terms of both carcinogenic and noncarcinogenic effects [179182 ]. taken together, the growing awareness of radiation defects in the developing brain is an important step forward in a complete understanding of early and persistent brain defects occurring after in utero radiation exposure, which will help to improve the health care of expecting mothers and their unborn children after exposure to various types of radiation sources. | ionizing radiation is omnipresent. we are continuously exposed to natural (e.g., radon and cosmic) and man - made radiation sources, including those from industry but especially from the medical sector. the increasing use of medical radiation modalities, in particular those employing low - dose radiation such as ct scans, raises concerns regarding the effects of cumulative exposure doses and the inappropriate utilization of these imaging techniques. one of the major goals in the radioprotection field is to better understand the potential health risk posed to the unborn child after radiation exposure to the pregnant mother, of which the first convincing evidence came from epidemiological studies on in utero exposed atomic bomb survivors. in the following years, animal models have proven to be an essential tool to further characterize brain developmental defects and consequent functional deficits. however, the identification of a possible dose threshold is far from complete and a sound link between early defects and persistent anomalies has not yet been established. this review provides an overview of the current knowledge on brain developmental and persistent defects resulting from in utero radiation exposure and addresses the many questions that still remain to be answered. |
ec coupling relies on a supramolecular complex between dhpr, which is the tt voltage sensor, and the ryr1, which is the sr ca release channel. the distinctive spatial relationship between these two proteins was revealed using freeze fracture shadowing electron microscopy. on the sr terminal cisternae side of the triad junction, large proteins named these form ordered 2d arrays, whereby ryr1s contact each other through their corners forming two or three rows along the edge of the terminal cisternae. on the tt side, groups of four particles (tetrads), identified as four dhprs, correlated with the disposition of individual ryr1 particles on the sr. the tetrads of dhpr are formed by the specific interaction between each dhpr and each of the four corners of the much larger, prism - shaped homotetrameric ryr1, and do not form in its absence. further evidence of the tight interaction between dhpr and ryr1 is the change in intra - tetrad distance when applying an effector known to change the conformation of ryr1. the skeletal muscle dhpr is a heteropentamer formed by the 1s or cav1.1, 2-1, 1a, and subunits. cav1.1, an l - type voltage - activated ca channel, acts as the voltage sensor for ec coupling. since its ca channel activity is irrelevant in the ec coupling context, much effort has been dedicated to understand how the dhpr transmits the voltage - sensing signal induced by membrane depolarization to the ryr1. the main candidates that have emerged are the ii - iii loop of a1s and the 1a subunit : their presence is essential for ec coupling and tetrad formation. thus, it became important to see how these two entities are organized within the dhpr complex and how is their spatial relationship with respect to the ryr1. the dhpr has been visualized within the context of the muscle tissue by freeze fracture followed by rotary shadowing as described above. in this technique, the frozen tissue is fractured through the less cohesive membrane planes, exposing the protruding regions of membrane proteins. these protrusions can then be highlighted by heavy metal directional or rotary shadowing and then imaged on the transmission electron microscope (tem). the particles however are featureless and the resolution of this technique is limited by the size of the grain of evaporated heavy metal, to around 50. additionally, it is unclear which part of the molecule is represented by the particle since the particle size is the same in presence and absence of the 2-1 subunits even though these constitute about half the molecular weight of the whole complex. an approach to get higher resolution, at the expense of losing the triad context, has been to extract the dhpr complex from the membrane using detergent, and then to image the solubilized protein using tem and single - particle 3d reconstruction. purification and 3d structural determination of membrane proteins has many challenges when compared to soluble proteins, therefore structures of membrane proteins account for less than 2% of all protein structures available in the 3d structure databases. extraction of the protein from the membrane needs the right amount of detergent : too little detergent lowers the yield of protein, and too much detergent may denature the protein. the choice of detergent is also important ; digitonin has been the most widely used for dhpr. after extraction, the dhpr is purified from the other membrane proteins using chromatography that may be further combined with size separation techniques (gel filtration, sucrose gradient). since dhpr is a heteropentamer, the purification method also has to ensure that all subunits are present at the end of the purification process. simultaneous heterologous expression of all dhpr subunits yielded functional dhprs in cho cells, however producing sufficient quantity for protein purification could prove difficult. to overcome many of these challenges, one successful approach was to genetically engineer one of the dhpr subunits with a tag for purification, and a second tag for 3d identification. for 3d structural determination the purified dhpr in solution is then prepared for negative staining or cryoem, and imaged on the tem. negative staining uses heavy metals to provide high contrast of the dehydrated protein at the expense of resolution (up to ~20 resolution) ; cryoem has less contrast but records images of the protein in its frozen hydrated state and has the potential for higher resolution (up to near atomic resolution). however an additional complication for membrane proteins in the case of cryoem is the presence of detergent, which reduces image contrast. single - particle image processing uses the different views of the protein to reconstruct its original shape. this is done by finding the exact orientation in space of each dhpr particle, back projecting it to obtain its 3d reconstruction using algorithms related to these used in tomography, and by averaging multiple (thousands) of such particles. finding the spatial orientation is not trivial when the 3d structure of the protein is not known ; specialized algorithms and software tackle this problem., important factors favoring 3d structural determination are a large size, presence of distinctive features in the protein, and conformational rigidity. while the dhpr has distinctive features and its mw of ~450 kda is larger than most proteins, it is slightly small for single particle image processing, and has a flexible region. in this context ryr1, five times larger than dhpr and more rigid, is a much better candidate for this technique, which has enabled its 3d reconstruction to 10 resolution revealing secondary structure. for dhpr, both negative staining and cryoem have been applied ; the best resolution achieved so far is 19 by negative staining. the 3d structures of skeletal dhpr that have remained consistent with the increase in resolution,, show a main globular body with an appendage emerging from it. the structural description will be centered in our negative staining 3d reconstruction, which has the highest resolution to date (19) and enabled placing of the 1s and 1a subunits for the first time. the dhpr used to obtain these images had a heterologous 1a subunit that contained an yfp to aid in 3d sub - localization and a biotin - acceptor domain tag that was used for purification ; the engineered 1a subunit was expressed in transgenic mouse as indicated above. after purification from the muscle tissue, the dhpr sample was negatively stained, imaged by tem (fig. 1), and its 3d reconstruction was determined by single particle analysis, using around 12,000 particles. its dimensions are 17 x 11 x 8 nm for the main body and an appendage that extends 7 nm from the point of attachment to the main body (fig. the main body shows a flat oblong shape with five pronounced corners around its contour, and a small protrusion on its base, opposite to the side where the appendage is attached. the engineered yfp, which is directly recognizable in the structure, together with antibody labeling in this and previous works, identifies several subunits and functional regions within the complex. cav1.1 or 1s is the largest subunit (176 kda) and contributes the voltage - sensing feature. it has a similar molecular organization to that found in voltage sensitive k and na channels and it is assumed that the tertiary structure is also the same ; their canonical organization consists of four repeats with six transmembrane each, s1-s6, where s1-s4 form the peripheral voltage sensors, and s5-s6 from each subunit combine in the center to form the cation channel, forming an overall square prism structure with protruding corners, and a footprint of 9 x 9 nm. for each subunit, an additional pore helix between s5-s6 and an s4-s5 linker do not cross the membrane. we placed the atomic structure of a voltage gated cation channel in the main body of the tem derived structure, matching two contiguous square corners separated by 11 nm (fig. we attribute the slightly wider dimensions of the tem structure to the presence of detergent surrounding the transmembrane domain, and to the lower resolution of the tem structure. two voltage sensors would be totally exposed to the periphery of the structure while the other two are partially exposed to the periphery and partially interacting with the rest of the main body. a characteristic of mammalian cav channels is that the four repeated units (i - iv) are encoded in a single polypeptide chain of almost 1,900 residues. this allows for differences in sequence among the four repeats, and importantly, cytoplasmic loops connecting these repeats have distinct structural and functional roles. the i - ii loop (residues 334 - 432) has the alpha - interacting domain (aid) region (residues 357 - 374) that anchors the 1a subunit., the ii - iii loop (residues 671 - 790), longest and partially unfolded, is an important determinant of ec coupling ; the sequence formed by residues 720 - 760 is the critical sequence for ec coupling., the iii - iv loop (residues 1066 - 1117) influences cav1.1 current but does not appear to be involved in ec coupling interactions with the ryr1, however its importance is underscored by the fact that mutation at residue 1086 results in malignant hyperthermia. cav1.1 has an extended c domain of ~500 residues ; its distal part is proteolytically cleaved in vivo and in general remains associated through non - covalent interactions. tem images of the dhpr incubated with antibodies against the ii - iii loop showed that these bound to the protuberance emerging from the main body in the center of the region attributed to the transmembrane region of cav1.1, and on the opposite side of the appendage, indicating that this protuberance contains the ii - iii loop (fig. this region marks the intracellular side of cav1.1 and hence it is plausible that it also contains at least part of the neighboring ii - ii and iii - iv loops, and the c domain. 2 and 1 subunits the extracellular 2 subunit (147 kda) was previously assigned to the appendage emerging from the main body, using antibody labeling., together with the identification of the ii - iii loop within the cav1.1 subunit, this establishes the orientation of the dhpr 3d structure within the triad junction. compared with the main body of the dhpr, the size and shape of the appendage attributed to 2 is very threshold - dependent. this is indicative of variability within this region, which is compatible with its heavy glycosylation, and may also be an indicator of flexibility. the 2 and 1 subunits are encoded by a single gene ; the product is later cleaved but remains disulfide - bonded. thus the single - pass transmembranal 1 subunit (24 kda) must be at the base of the 2 subunit, anchoring it to the membrane. 1a subunit the position of the intracellular 1a subunit (56 kda) was established by identification of the typical barrel shape corresponding to yfp (fig., the atomic structure of the core region of the 2a isoform, encompassing the src homology 3 (sh3) domain and a guanylate kinase (gk) domain, with 84% identity with 1a, was docked into the 3d reconstruction of the dhpr complex obtained by tem taking several factors into account. the alpha - binding pocket of the subunit, which interacts with the aid binding region within the i - ii loop of 1s, was situated adjacent to 1s in a position appropriate for this inter - subunit interaction to occur. the sh3 in the n region of the subunit was placed adjacent to the identifiable yfp engineered in the n region. finally, the two prominent lobes of the gk domain were fit with the two outward spikes in the 3d volume of dhpr (fig. 2e - f). in addition that the choice of location and orientation satisfy all the above conditions, further confidence in this model comes from the fact that the docking is close fitting when seen from all directions (see supplemental movie) subunit the subunit (34 kda), with four transmembrane domains, must be in one of the empty spaces near 1s, as it is known to interact with this subunit. in the context of the quaternary interactions with the ryr1, the dhpr would be facing the ryr1 across the junctional triadic cleft. on the sr side, the ryr1 s cytoplasmic domain (i.e. the foot structure) occupies 120 of the cleft, practically bridging the space between the two membranes. on the tt side, the ii - iii loop of cav1.1 is sandwiched between the rest of the dhpr and the ryr1, in full agreement with its vital role in ec coupling. the 1a subunit location, adjacent to cav1.1 and sandwiched between the tt membrane and the ryr1, with potentially large areas of interface, is compatible with its functions of trafficking cav1.1 to the membrane and control of ec coupling, possibly indirectly by determining the dhpr - ryr1 association. figure 3 summarizes how the ryr1, the dhpr and its more important ec coupling domains would be located relative to each other in the context of the triad junction. it is expected that higher resolution structures, combined with functional studies and high electron tomography, will, in the future, provide a picture of the quaternary assembly of dhpr and ryr1 at the atomic level, and enable a more thorough understanding of the ec coupling mechanism at the molecular level. recent advancements in membrane protein stabilization (i.e. using lipid mimics or nanodisks) and in tem (most notably the installation of direct electron detectors) should help in the single - particle 3d structural determination of the dhpr and ryr1 at much higher resolution. while this paper was in press, the near - atomic structure of ryr1 was solved at 3.8 by cryoem. while this paper was in press, the near - atomic structure of ryr1 was solved at 3.8 by cryoem. | excitation contraction coupling, the rapid and massive ca2 + release under control of an action potential that triggers muscle contraction, takes places at specialized regions of the cell called triad junctions. there, a highly ordered supramolecular complex between the dihydropyridine receptor (dhpr) and the ryanodine receptor (ryr1) mediates the quasi - instantaneous conversion from t - tubule depolarization into ca2 + release from the sarcoplasmic reticulum (sr). the dhpr has several key modules required for ec coupling : the voltage sensors and ii - iii loop in the alpha1s subunit, and the beta subunit. to gain insight into their molecular organization, this review examines the most updated 3d structure of the dhpr as obtained by transmission electron microscopy and image reconstruction. although structure determination of a heteromeric membrane protein such as the dhpr is challenging, novel technical advances in protein expression and 3d labeling facilitated this task. the 3d structure of the dhpr complex consists of a main body with five irregular corners around its perimeter encompassing the transmembrane alpha 1s subunit besides the intracellular beta subunit, an extended extracellular alpha 2 subunit, and a bulky intracellular ii - iii loop. the structural definition attained at 19 resolution enabled docking of the atomic coordinates of structural homologs of the alpha1s and beta subunits. these structural features, together with their relative location with respect to the ryr1, are discussed in the context of the functional data. |
parathyroid glands are located behind the thyroid gland that produces parathyroid hormones (pth). pth has a main role in bone formation, vitamin d activation occurred in the kidney by pth that leads to intestinal absorption of calcium and phosphate re - absorption. the primary function of pth is to maintain the extracellular fluid calcium concentration, which is done by pth effect on bone and kidney directly and on the intestine through its effects on synthesis of 1, 25 (oh)d, indirectly. primary hyperparathyroidism (phpt) is a generalized disorder of calcium, phosphate, and bone metabolism due to excessive secretion of pth. secondary hyperparathyroidism (shpt) is caused by impaired phosphate excretion, and failure to activate vitamin d. hypocalcemia is caused by elevated levels of the fibroblast growth factor 23 (fgf-23), and reduced synthesis of calcitriol, which is the active form of vitamin d. elevated phosphate level, decreased ionized calcium level, and reduced serum calcitriol lead to continuous stimulation of the parathyroid glands that causes increased pth release. tertiary hpt is defined as long - time shpt leads to autonomous parathyroid glands that lead to hypercalcemia, pruritis and extra - skeletal calcification, bone involvement despite aggressive treatment to suppress pth. hpt leads to bone involvement that include generalized osteoporosis, multiple focal areas of demineralization of the skull, and brown tumor. the brown tumor or osteitis fibrosa cystica is a benign bone lesion that caused by hpt. pelvis, ribs, clavicle, mandible and the extremities are most commonly affected bones in brown tumor, whereas maxillary involvement is rare (0.1%). this case report was unique due to the presentation of brown tumors in the maxillofacial region. in july 2012, a 29-year - old man was referred to internal medicine clinic, al zahra hospital, isfahan, iran with chief complaint of generalized bone pain that started seven months ago and, swelling in the cheek regions, which initiated three months ago and gradually increased in size. the patient 's medical history revealed that he had end - stage renal disease (esrd) for about eight years, due to systemic lupus erythematous (sle) that suppressed after renal failure. hyperparathyroidism was diagnosed 15 months ago with elevated pth levels that vitamin d and calcium had been recommended for treatment. other medications were as follows : carbonate calcium 2 g / day, calciteriol 1g / day, vitamin d 50000 units weekly, losartan 50 mg 2 daily. on oral examination, displaced teeth without normal alignment was seen, teeth were loose [figure 1 ]. bilateral swelling in the cheek regions. increased distance between teeth and irregular alignment with displaced teeth the patient compliance was poor and his follow - up laboratory test programs were not periodically in accordance with his medical condition. result of routine laboratory tests were as follows : pth : 3552 pg / ml (8 - 60), calcium : 8.7 mg / dl (8.5 - 10), phosphorus : 6.3 mg / dl (2.5 - 5), alkaline phosphatase : 2800 u / l (55 - 200), hemoglobin : 8.5 mg / dl (14 - 18), creatinin : 6.5 mg / dl (0.7 - 1.2), potassium : 5.5 mg / dl (3.5 - 5), alb : 3.5 mg / dl (3.5 - 4). the level of pth began to increase from 15 months that was 350 pg / ml, and so the dose of carbonate calcium and and pearl vitamin d was added to the patient 's medications that did not respond to treatment. carbonate calcium was started with two tabs daily and increased every two week that reached to eight tabs in divided dose. tc-99 m sestamibi of the parathyroid glands scan was advised two weeks before admission, which showed a normal uptake inminute 30 and 240. panoramic radiograph, shows multiple small unilocular well - defined radioluscencies, suggestive of brown tumors in the body of the mandible bone [figure 2 ]. computed tomography (ct) scan of the head and neck region, showed multiple small unilocular well - defined radioluscencies, suggestive of brown tumors in zygoma, maxilla, palate and mandibular bones [figure 3 ]. also, a generalized diffuse severe osteoporosis in maxillofacial bone with salt and pepper pattern was seen [figure 3 ]. panoramic radiograph, shows multiple small unilocular radioluscencies, suggestive of brown tumors ct scan ; (a) axial view shows multiple small unilocular well - defined radioluscencies, suggestive of brown tumors in hard palate (arrow) ; (b) coronal view shows a well - defined radioluscency in zygoma (arrow) ; (c) axial view shows multiple small unilocular well - defined radioluscencies in the mandibular body (arrow) a radiograph of the forearm and elbow regions, showed a unilocular well - defined radioluscency, suggestive of brown tumor in ulnar bone, in addition to calcification of dialysis shunt and metastatic vascular calcification [figure 4 ]. radiograph of the forearm and elbow regions, showed a unilocular well - defined radioluscency, suggestive of brown tumor in ulnar bone. note to the calcification of dialysis shunt and metastatic vascular calcification finally, he underwent parathyroidectomy due to tertiary hpt diagnosis. the histopathological examination showed a chief cell proliferation of parathyroid with clear cytoplasm and small central nucleus with oxiphilic cells seen between them. the tissue was surrounded by a thin capsule penetrated by thin septa that divided that into labels, all of these suggested hpt. result of routine laboratory tests were as follows : pth : 3552 pg / ml (8 - 60), calcium : 8.7 mg / dl (8.5 - 10), phosphorus : 6.3 mg / dl (2.5 - 5), alkaline phosphatase : 2800 u / l (55 - 200), hemoglobin : 8.5 mg / dl (14 - 18), creatinin : 6.5 mg / dl (0.7 - 1.2), potassium : 5.5 mg / dl (3.5 - 5), alb : 3.5 mg / dl (3.5 - 4). the level of pth began to increase from 15 months that was 350 pg / ml, and so the dose of carbonate calcium and and pearl vitamin d was added to the patient 's medications that did not respond to treatment. carbonate calcium was started with two tabs daily and increased every two week that reached to eight tabs in divided dose. tc-99 m sestamibi of the parathyroid glands scan was advised two weeks before admission, which showed a normal uptake inminute 30 and 240. panoramic radiograph, shows multiple small unilocular well - defined radioluscencies, suggestive of brown tumors in the body of the mandible bone [figure 2 ]. computed tomography (ct) scan of the head and neck region, showed multiple small unilocular well - defined radioluscencies, suggestive of brown tumors in zygoma, maxilla, palate and mandibular bones [figure 3 ]. also, a generalized diffuse severe osteoporosis in maxillofacial bone with salt and pepper pattern was seen [figure 3 ]. panoramic radiograph, shows multiple small unilocular radioluscencies, suggestive of brown tumors ct scan ; (a) axial view shows multiple small unilocular well - defined radioluscencies, suggestive of brown tumors in hard palate (arrow) ; (b) coronal view shows a well - defined radioluscency in zygoma (arrow) ; (c) axial view shows multiple small unilocular well - defined radioluscencies in the mandibular body (arrow) a radiograph of the forearm and elbow regions, showed a unilocular well - defined radioluscency, suggestive of brown tumor in ulnar bone, in addition to calcification of dialysis shunt and metastatic vascular calcification [figure 4 ]. radiograph of the forearm and elbow regions, showed a unilocular well - defined radioluscency, suggestive of brown tumor in ulnar bone. note to the calcification of dialysis shunt and metastatic vascular calcification finally, he underwent parathyroidectomy due to tertiary hpt diagnosis. the histopathological examination showed a chief cell proliferation of parathyroid with clear cytoplasm and small central nucleus with oxiphilic cells seen between them. the tissue was surrounded by a thin capsule penetrated by thin septa that divided that into labels, all of these suggested hpt. the brown tumor is a bone lesion that is caused by osteoclast activity in hpt condition. the incidence of skeletal brown tumors in esrd ranges from 1.5 to 13%, but this complication has been decreased with novel diagnostic pth radioimmunoassay techniques and successful treatment of the disease. this unusual complication of secondary hpt is more commonly seen in young female patients, and so the patient in this report was unusual for his gender. pelvis, ribs, clavicle, mandible and the extremities are most commonly affected bones in brown tumor, whereas involvement of the maxilla is considered rare. elevated pth is the cornerstone of all hpt, but they differ in calcium and phosphate level. primary hpt presented with an elevated calcium level, normal or low phosphate level, secondary hpt hypocalcemia and hyperphosphatemia are dominant, while hypercalcemia and hyperphosphatemia occurred in tertiary. hypocalcemia, hyperphosphatemia and calcitriol deficiency may be found in chronic kidney disease (ckd), that can lead to pth, directly affecting the skeletal system. periapical radiographs reveal loss of lamina dura in 10% of patients with hpt. depending on duration and severity of disease, loss of lamina dura may occur around one tooth or all remaining teeth. the result of lamina dura loss may give the root a tapered appearance because of loss of image contrasts. the major clinical manifestations of hpt in the oral cavity are brown tumor, malocclusions, loss of bone density, soft - tissue calcifications, weak teeth, and dental abnormalities of such developmental defects, alterations in dental eruption and widened pulp chambers. according to the examination and using panoramic radiography, the differential diagnosis is multiple myeloma, bone metastases of a carcinoma, osteosarcoma, osteomyelitis or osteonecrosis secondary to bisphosphonate therapy and paget 's disease that elevated pth level differentiate hpt from others. brown tumor is a late manifestation and happens in advanced shpt, while they can rarely occur as an early sign of hpt. in the jaw bones, it may present sometimes painful, hard and clearly palpable swellings, if a large deformity in affected bone occurred. also an altered masticatory function and facial deformities can be seen, as was seen in the presented case, but it may be completely asymptomatic., showed that the diagnosis of brown tumor in esrd condition is suggested clinically and confirmed by the endocrinological status of the patient, and so based on history and labarotary test, brown tumor was his diagnosis. but tc-99 m sestamibi scan can be useful for diagnosis of hpt, but normal scan in 30 and 240 minutes can be falsely negative. the scan must be done every 15 minutes, and false negative can be due to the fact that, it has been done only in 30 and 240. chang cw., showed that imaging of parathyroid glands should be done only before to reoperation and is not usually required before initial parathyroidectomy. according to the fact that hypocalcemia has the main role in shpt, correction of hypocalcemia and hyperphosphatemia with calcium and phosphate binders, and 1,25 dihydroxy vitamin d, is the cornerstone of treatment and prevention of secondary hpt and its complication. patients with very severe shpt may be unresponsive to high - dose vitamin d and require parathyroidectomy as seen in the presented patient. ishikawa y., presented parathyroidectomy was a good choice for patients with high pth levels and diffuse brown tumor. secondary hyperparathyroidism is a common complication of end - stage renal disease, on long time hemodialysis. the scan is not required to confirm the hpt, and diagnosis of brown tumor is clinically in esrd. it is important for general practitioner dentists, oral and maxillofacial surgeon, oral and maxillofacial radiologist, an endocrinologist and internist to be aware of shpt to diagnosis and treatment. dentists should also be aware of possible presence of brown tumors in the jaws of patients having hpt or presented with radiologic finding of hpt to prevent fatal complications. panoramic radiographs and ct scans were all useful radiographic methods for the correct diagnosis of brown tumour. njp contributed in the conception of the work, conducting the study, revising the draft, approval of the final version of the manuscript, and agreed for all aspects of the work. makh contributed in the conception of the work, drafting and revising the draft, approval of the final version of the manuscript, and agreed for all aspects of the work. shjp contributed in the conception of the work, conducting the study, revising the draft, approval of the final version of the manuscript, and agreed for all aspects of the work. makh contributed in the conception of the work, revising the draft, approval of the final version of the manuscript, and agreed for all aspects of the work. | parathyroid glands produce parathyroid hormone (pth). pth has a main role in bone formation. hyperparathyroidism (hpt) is explained as primary, secondary and tertiary types defined as overproduction of pth. the brown tumor or osteitis fibrosa cystica is a benign bone lesion that is caused by hpt. this complication has been decreased by diagnosis and successful treatment of secondary hyperparathyroidism. pelvis, ribs, clavicle, mandible and the extremities are most commonly affected bone in brown tumor, whereas maxillary involvement is rare. the present article report a 29-year - old man with chief complaints of bone pain, swelling cheeks and teeth displacement with secondary hpt. parathyroidectomy was done due to bone disorder. it is important for dentists and endocrinologists to understand maxillofacial manifestation of secondary hpt to prevent its complication. |
shigellosis is among the most common causes of bacterial diarrheal disease in both developing and developed countries. shigella consists of four serogroups including serogroup a (s. dysenteriae), serogroup b (s. flexneri), serogroup c (s. boydii) and serogroup d (s. sonnei). it is one of the major causes of morbidity in children with diarrhea in iran (1, 2). it has been recently reportedit as the prevalent shigella serotype in iran (4, 5). treatment using adequate antibiotics is effective for shigellosis particularly for the children and immunosuppressed patients because it may shortens the clinical course of the disease, reduce the risk of transmission and prevent potentially lethal complications., the conventional bacterial typing methods such as antimicrobial resistance pattern, bacteriophage typing, or serotyping have been replaced by molecular techniques such as ribotyping, pulsed - field gel electrophoresis (pfge), and pcr - based methods (1, 11, 12). an entero - bacterial repetitive intergenic consensus (eric) sequence is an imperfect palindrome of 127 bp. eric sequences have been found only in intergenic regions, apparently only within transcribed regions. pcr- mediated genomic fingerprinting based on eric sequences has been found to be useful for subtyping gram - negative enteric bacteria and differentiation of their strains (13). the present study was conducted to determine the antimicrobial susceptibility and genetic relationship among s. sonnei strains isolated from pediatric patients in tehran, iran. the study included all s. sonnei strains isolated from pediatric patients with diarrhea who were admitted to several hospitals in tehran, iran, during 2008 - 2010. a single specimen was obtained from each patient, and rectal swabs were collected from patients on the day of admission at the hospital. when the isolates were identified as shigella by the conventional methods (14), these were serotyped using slide agglutination with specific antisera (mast group ltd, merseyside, uk). all ethical issues were considered. the name and characters, personal information and even patients illnesses and their medical information remained secret and the life, health, dignity, integrity, rights to self - determination, privacy, and confi - dentiality of personal information of research subjects were protected in this study. antimicrobial susceptibility testing was performed according to clinical and laboratory standards institute (formerly national committee for clinical laboratory standards) (15). the following antimicrobial agents were tested : ampicillin (am, 10 g), amoxycillin - clavulanic acid (amc20 + 10 g), amikacin (an 30 g), ceftazidime (caz 30 g), cephalothin (cf 30 g), ceftriaxone (cro 30 g), cefotaxime (ctx 30 g), kanamycin (k 30 g), nalidixic acid (na 30 g), streptomycin (s 10 g), trimethoprim - sulfamethoxazole (sxt 23.75 + 1.25 g), tetracycline (te 30 g), ticarcillin (tic 75 g), tobramycin (tob 10 g), gentamicin (gm 10 g), ceftizoxime (ct 30 g), ciprofloxacin (cp 5 g), chloramphenicol (c 30 g). escherichia coli atcc 25922 were used as control strain. one colony from overnight culture was inoculated into lb - broth (tryptone 1%, nacl 1% and yeast extract 0.5%) and was grown with shaking overnight at 37c. eric - pcr was performed using the primers eric 1r (5-atgtaagctcctggggattcac-3) and eric2 (5-aagtaagtgactggggtgagcg-3) with minor modifications (16, 17). the pcr reaction mixture (20l) consisted of 10x reaction buffer [750 mm tris - hcl (ph 8.8), 200 mm (nh4)2so4, 0.1% tween20 ], a 250 m concentration of each dntp, 20 pmol of primer eric 1r, 20 pmol of primer eric 2, 3.75 mm mgcl2, 100 ng of template dna and 2 u of taq polymerase. the reaction mixture was denatured for 7 min at 95 c and then subjected to 30 cycles of denaturation for 30s at 90 c, annealing for 1 min at 52 c, extension for 3 min at 65 c, and a final extension for 16 min at 65 c. negative controls without template dna were included in each run. amplified products were resolved by electrophoresis of 5 l samples on 1% agarose gels in 1x tbe buffer at 70 v for 3 h and were visualized by ethidium bromide staining. the pcr patterns were visually compared and were considered to be identical on the basis of similar numbers and matching positions of all major bands. the type strain of s. sonnei, atcc 9290, was also included in this study for the comparison. the study included all s. sonnei strains isolated from pediatric patients with diarrhea who were admitted to several hospitals in tehran, iran, during 2008 - 2010. a single specimen was obtained from each patient, and rectal swabs were collected from patients on the day of admission at the hospital. when the isolates were identified as shigella by the conventional methods (14), these were serotyped using slide agglutination with specific antisera (mast group ltd, merseyside, uk). all ethical issues were considered. the name and characters, personal information and even patients illnesses and their medical information remained secret and the life, health, dignity, integrity, rights to self - determination, privacy, and confi - dentiality of personal information of research subjects were protected in this study. antimicrobial susceptibility testing was performed according to clinical and laboratory standards institute (formerly national committee for clinical laboratory standards) (15). the following antimicrobial agents were tested : ampicillin (am, 10 g), amoxycillin - clavulanic acid (amc20 + 10 g), amikacin (an 30 g), ceftazidime (caz 30 g), cephalothin (cf 30 g), ceftriaxone (cro 30 g), cefotaxime (ctx 30 g), kanamycin (k 30 g), nalidixic acid (na 30 g), streptomycin (s 10 g), trimethoprim - sulfamethoxazole (sxt 23.75 + 1.25 g), tetracycline (te 30 g), ticarcillin (tic 75 g), tobramycin (tob 10 g), gentamicin (gm 10 g), ceftizoxime (ct 30 g), ciprofloxacin (cp 5 g), chloramphenicol (c 30 g). escherichia coli atcc 25922 were used as control strain. one colony from overnight culture was inoculated into lb - broth (tryptone 1%, nacl 1% and yeast extract 0.5%) and was grown with shaking overnight at 37c. eric - pcr was performed using the primers eric 1r (5-atgtaagctcctggggattcac-3) and eric2 (5-aagtaagtgactggggtgagcg-3) with minor modifications (16, 17). the pcr reaction mixture (20l) consisted of 10x reaction buffer [750 mm tris - hcl (ph 8.8), 200 mm (nh4)2so4, 0.1% tween20 ], a 250 m concentration of each dntp, 20 pmol of primer eric 1r, 20 pmol of primer eric 2, 3.75 mm mgcl2, 100 ng of template dna and 2 u of taq polymerase. the reaction mixture was denatured for 7 min at 95 c and then subjected to 30 cycles of denaturation for 30s at 90 c, annealing for 1 min at 52 c, extension for 3 min at 65 c, and a final extension for 16 min at 65 c. amplified products were resolved by electrophoresis of 5 l samples on 1% agarose gels in 1x tbe buffer at 70 v for 3 h and were visualized by ethidium bromide staining. the pcr patterns were visually compared and were considered to be identical on the basis of similar numbers and matching positions of all major bands. the type strain of s. sonnei, atcc 9290, was also included in this study for the comparison.. strains were isolated which were distributed as following ; s. sonnei, 54 (60.7%), s. flexneri 28 (31.5%), s. boydii 5 (5.6%) and s. dysenteriae 2 (2.2%). the season distribution of the isolated strains was winter, 10 ; spring, 17 ; summer, 38 and fall, 24. of all patients, shigella was isolated frequently from children under 5 years of age, who accounted for 62.9.7% of all isolates. about 35.1 % of all isolates came from persons aged 5 - 12 years, and 1.8% from persons aged over 12 years of age. results of further examination of s. sonnei strains showed that the majority of the isolates (90%) were resistant to trimethoprim - sulfamethoxazole, tetracycline and streptomycin, 37% to ampicillin, 27.8% to nalidixic acid, 9.3% to amoxycillin - clavulanic acid and 11.3% tobramycin. the most of the isolates were susceptible to cephalothin, ticarcillin, cefotaxime, ceftriaxone, amikacin, ceftazidime, kanamycin and gentamicin. all strains were fully susceptible to ciprofloxacin, ceftizoxime and chloramphenicol. as shown in table 1, the majority of the isolates demonstrated multiple drug resistance profile, among which, 1.8% were resistant to 2, 57.4% to 3, 3.7% to 4, 16.7% to 5, 7.4% to 6, 1.8% to 9, 5.5% to 11 and 1.8% were resistant to 12 tested antibiotics. eleven antibiotic resistance patterns (r1-r11) were observed among the strains, r1 (tetracycline / streptomycin / trimethoprim - sulfamethoxazole) was however found as most prevalent (51.8%) resistance pattern. the number of eric - pcr bands produced for a given primer ranged from 10 to 15, with molecular sizes ranging from 100 to 3500 bp. eric - pcr analysis of the isolates resulted in five different patterns (e1-e5) with 9 - 13 dna bands (fig. 1.) ; however the half of the isolates was clustered in e4 pattern. no similarity was observed in the eric - pcr patterns between clinical isolates and atcc type strain 9290 (fig. lanes 1 - 5 are representative patterns e1, e2, e3, e4 and e5 respectively. the infections caused by shigella spp. has been increasing continuously and has turned into a prominent public health concern worldwide. is one of the major causes of diarrheal disease among children in iran (18). shigella strains were isolated frequently from children under 5 years of age, who accounted for 62.9.7% of all isolates. previous studies have also demonstrated the fact that age can be a risk factor in shigellosis where children are in the high risk zone for shigella infections (19). the typical seasonal increase in shigellosis occurred during the summer, with peak incidence in august that confirmed the warm months of the year can intensify the frequency of shigella associated infections. characteristics of s. sonnei isolates included in the study assessment of antibiotic resistance patterns among shigella isolates revealed that the antibiotic resistance rates have increased in comparison with previous reports published from iran. for examples, when compared to a previous study in tehran, the percentage of resistance against ampicillin, and nalidixic acid has increased from 10% and 8.3% to 37% and 27.8% respectively in our study (20). moreover, to our knowledge, resistance to ceftriaxone and cefotaxime has not been reported in clinical strains of s. sonnei in iran to date. only one strain was resistant to kanamycin where also more than half of isolates showed intermediate resistance to this antibiotic which can be result in the probable increase in shigella resistance to this antibiotic in the future. moreover, in a ten - year study conducted by ashtiani, in tehran, iran, shigella spp. were found to show noticeably increasing resistance to kanamycin between 2001 and 2005 (21). these finding is consistent with those reported by vrints. who showed that the all shigella isolates were sensitive to ciprofloxacin as well as gentamicin in a 18-years study from 1990 to 2007 in belgium (22). subtyping using phenotypic methods is hindered by the homogeneity of circulating strains causing infectious diseases. molecular typing of microbial strains helps us to accurately interpret epidemiological evolution of infectious diseases in the communities. several different molecular methods have been applied to study the molecular epidemiology of s. sonnei isolates. pcr - based typing methods have been applicable to many organisms including s. sonnei and can be completed easily within a single day (18, 23). eric - pcr is less laborious and time - consuming than other dnabased typing techniques. this method has been widely used for the molecular typing of different bacteria in epidemiological studies, and its advantages and disadvantages are well known (24). however a few limited studies have previously evaluated the eric - pcr for molecular typing of shigella strains., this technique has been established to be a reliable and rapid genotyping approach with high discriminatory power for the differentiation of shigella strains (25). here we used eric - pcr to study the genetic relatedness among endemic s. sonnei strains isolated from pediatric patients in tehran, iran. using one pair of eric - pcr primers in a study carried out by penatti, three genetic patterns were reported from s. flexneri and s. sonnei strains isolated from bacillary dysentery cases in southeast brazil (26). this method has been also used for subtyping of epidemic s. flexneri strains in iran previously. in a recent outbreak of shigellosis occurred among prisoners in isfahan, eric - pcr showed to be a powerful method for molecular typing of shigella strains. this technique differentiated the epidemic causative agent of outbreak from endemic and type standard strains and showed that a single clone of s. flexneri serotype 3a was responsible for the outbreak since all tested isolates had a single pattern (27). also reported that this method was highly reproducible and could provide highly similar and supplementary data compared with serotyping regarding the transmission dynamics of shigellosis in the community studied (28). otherwise, in another study for investigation of shigellosis outbreaks occurred in school children in lungtan and bade in taoyuan county in northern taiwan, this technique showed poor discriminatory power where eric - pcr analysis could not discriminate an epidemiologically unrelated strain from some outbreak strains. epidemic s. sonnei strains could not be differentiated from type strain atcc 9290 (29). here we also used type strain atcc 9290 for the comparison ; however eric - pcr was capable to differentiate endemic s. sonnei strains from this standard strain and from each other clinical strains distributed in different clusters. used another pcr - based technique, in which the amplication of the regions between repetitive extragenic palindromic (rep) sequences gave a ngerprinting pattern valid for epidemiological typing of different species of shigella (30). also in a study carried out on 60 s. sonnei strains isolated from children hospitalized at five hospitals in tehran during 2003, a similar technique, known as arbitrarily primed pcr (ap - pcr), was evaluated for subtyping of endemic s. sonnei isoaltes. only a single ap - pcr pattern was observed among all s. sonnei strains (23). when reviewing the previous reports on the distribution of molecular types of endemic s. sonnei in iran, our findings further confirm the involvement of our geographic area within an epidemiological global picture of dissemination of a limited number of well - defined clones of s. sonnei. the results obtained from our previous global project on 1,672 s. sonnei isolates obtained since 1943 from 50 countries including iran revealed that three major s. sonnei groups were responsible for shigellosis caused by this serogroup in which two groups were globally spread (3). considering that more than half of isolates were clustered into e4, it is concluded that one predominant clone or limited clones of s. sonnei are responsible for shigellosis caused by this shigella species in pediatric patients during our research period. we hope our finding could be helpful for further epidemiological surveillance of s. sonnei in our country in the future. continuous studies using more discriminating molecular methods is needed to be conducted in tehran and other parts of iran in order to determine molecular subtypes of s. sonnei and other shigella species in the future. all ethical issues including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc. have been completely observed by the author. | abstractbackgroundshigella sonnei is considered as a major cause of diarrheal disease in both developing and developed countries. iran is one of the endemic areas of shigellosis. the present study was undertaken to investigate the antibiotic susceptibility and genetic relatedness of s. sonnei strains isolated from pediatric patients in tehran, iran.methodsthe study included all s. sonnei strains isolated from pediatric patients with diarrhea admitted to several hospitals in tehran, iran, during 2008 - 2010. shigella spp. strains were recovered from patients using standard microbiological methods. s. sonnei strains were further studied by antimicrobial susceptibility testing and enterobacterial repetitive intergenic consensus (eric) - pcr analysis.resultseighty nine shigella isolates were isolated. s. sonnei was themost prevalent shigella species (60.7%) followed by, s. flexneri (31.5%). eleven antimicrobial resistance patterns (r1-r11) were identified among s. sonnei isolates. the majority of the strains were resistant to trimethoprim - sulfamethoxazole, tetracycline and streptomycin. all isolates were susceptible to ciprofloxacin, ceftizoxime and chloramphenicol. all strains were typable by eric - pcr. five eric - pcr patterns (e1-e5) were found among s. sonnei isolates ; however the half of the isolates was clustered in e4 pattern.conclusionthe antibiotic resistance rates are increasing among s. sonnei strains. moreover, a predominant clone or limited clones of s. sonnei were responsible for shigellosis caused by this shigella species in pediatric patients in tehran, iran. |
the efficacy of antitumor necrosis factor (tnf-), infliximab, has been shown to be effective in various disease treatment including crohn s disease (cd). however, there is limited information in the use of infliximab in patients on hemodialysis. although the highest incidence in cd is among younger generations, 1030 years old, older people are also affected. here we report a case of an old man on hemodialysis who developed cd and was successfully treated with infliximab. a 76-year - old man, 166 cm tall and weighing 47.1 kg, receiving hemodialysis for 6 years was referred to our division of gastroenterology, akita city hospital, akita city, japan, in the middle of november 2013, because of diarrhea, abdominal pain, and fever for 1 week. he was diagnosed with wolff - parkinson - white syndrome in 1973, paroxysmal atrial fibrillation in 1982, myocardial infarction in 1993, and immunoglobulin a (iga) nephropathy in 2005. tenderness in the left lower abdomen and a marked edema in both sides of the pretibia and ankle were observed. laboratory data were as follows : an increase of c - reactive protein (crp), 10.73 mg / dl (normal range 0.19) ; an increase of erythrocyte sedimentation rate, 46 mm / h ; hypoalbuminemia, 2.0 g / dl ; hypocholesterolemia, 107 mg / dl ; and moderate anemia, hemoglobin 9.0 g / dl (figure 1). ultrasonography and computed tomography (ct) found wall thickness in continuous form in the left colon. sigmoidoscopy revealed multiple irregular - shaped ulcers in the sigmoid colon (figure 2). barium enema study exhibited collar button ulcers, longitudinal ulcers, and coarse mucosa in the left colon (figure 3). negative results were obtained in the following tests : cytomegalovirus antigenemia, stool culture, and clostridium difficile toxin. rapid efficacy of infliximab in cd and reports of safety of infliximab in patients on hemodialysis encouraged us and our patient to use infliximab rather than steroid hormone that has an adverse osteoporotic effect. latent tuberculosis was excluded by normal roentgenogram of the chest and negative elispot assay for mycobacterium tuberculosis. hepatitis b infection was also excluded by negative test in surface antigen, surface antibody, and core antibody. written informed consent on the use of infliximab and publishing the study was obtained. the standard induction therapy (3 infusions at week 0, 2, and 6) of infliximab (300 mg) was initiated. infliximab was infused on alternative days to hemodialysis crohn s disease activity index (cdai) decreased from 747 to a remission level (less than 150) of 134 after 2 infusions of infliximab. crp became normal and increase of albumin was ascertained before discharge (figure 1). although albumin was further increased to 2.9 g / dl, fecal occult blood was more than 1000 ng / ml (figure 1) indicating that there is still active inflammation in the intestine. an easy tendency to arrhythmia and tachycardia and renal failure hampered invasive morphological studies of the bowel after the standard induction therapy. noninvasive studies with ultrasonography and ct found a disappearance of the wall thickness of the colon. crp (normal range 0.19 mg / dl) ; esr (10 mm / h) ; fecal occult blood (< 100 ng / ml) ; t chol (120 to 220 mg / dl). cdai = crohn s disease activity index ; crp = c - reactive protein ; esr = erythrocyte sedimentation rate ; ifx = infliximab ; t chol = total cholesterol. endoscopic pictures of sigmoidoscopy. inflamed mucosa with (a) multiple irregular - shaped ulcers (arrow) and (b) punched - out ulcer (arrow) clear collar button signs were observed in the (a) sigmoid colon (arrows) and the (b) descending colon (short arrows). collar button signs and longitudinal ulcers are observed in both cd and ulcerative colitis. in the present case, an absent hematochezia and a positive noncaseating epithelioid granuloma led to a diagnosis of cd. tnf- is a key molecule of the inflammatory response and is involved in the pathogenesis of various autoimmune and noninfectious inflammatory conditions. tnf- antagonists have been shown to be effective in cd, ulcerative colitis, rheumatoid arthritis, behcet s disease, ankylosing spondylitis, uveitis, psoriasis, pyoderma gangrenosum, and hidradenitis suppurativa. in cd, tnf- antagonists are effective in inducing and maintaining remission, systemic complications such as pyoderma gangrenosum and arthritis, and secondary amyloidosis. infliximab was the first biologic agent approved in japan for cd in 2002 and for rheumatoid arthritis in 2003. there are very few reports on the use of infliximab in patients on dialysis : rheumatoid arthritis, ankylosing spondylitis, sarcoidosis, and psoriatic arthritis. in cd, 3 case reports are available and all are from japan. in these reports, cd was first diagnosed followed by renal failure resulting in hemodialysis. infliximab was used for enterocutaneous fistula, relapse of cd, and uncontrolled activity with conventional treatment. conventional treatment such as 5-aminosalicylate and steroid hormone was also ineffective in the former 2 cases. therefore, in all the 3 cases of cd, infliximab was used after conventional therapy. adverse effects were described in 3 cases : postinfusion transient itching, deep vein thrombosis, and pneumocystis jivoreci pneumonia. except for these 2 cases, scheduled infliximab maintenance therapy was administered in these diseases. needless to say, pharmacokinetics of tnf- antagonists in patients on dialysis is not known. however, kume, in cd, measured the serum concentration of infliximab before and after hemodialysis and showed that the serum level of infliximab was essentially unchanged by hemodialysis. in our case, cd appeared in a patient on hemodialysis. the number of patients on regular dialysis treatment in japan is steadily increasing : 103,296 in 1990, 206,134 in 2000, and 309,946 in 2012. the primary disease for dialysis is diabetic nephropathy holding the first place since 1998 accounting for 44.1% followed by chronic glomerulonephritis in the second place (19.4%). the number of patients with cd is also steadily increasing in japan : 6609 in 1990, 19,651 in 2000, and 36,418 in 2012. both diabetes mellitus and cd are popular in wealthy nations and they are not restricted to western countries anymore but distributed worldwide and known as global diseases. it is anticipated that the number of cd patient on dialysis will increase in the future not only in japan but also worldwide. our case has contributed additional literature in accordance with previous reports supporting infliximab as effective and safe in patients on hemodialysis. | abstractthere is limited information in the use of antitumor necrosis factor, infliximab, in patients on hemodialysis. in crohn s disease (cd), only 3 cases are reported.a 76-year - old man on hemodialysis for renal failure caused by immunoglobulin a nephropathy developed diarrhea and abdominal pains. a marked edema was observed in the pretibia and ankle. an increase of c - reactive protein (crp) and erythrocyte sedimentation rate, hypoalbuminemia, hypocholesterolemia, and moderate anemia was found. ultrasonography and computed tomography (ct) found wall thickness in the left colon. sigmoidoscopy revealed multiple ulcers in the sigmoid colon and noncaseating epithelioid granuloma was found in the biopsy specimen. barium enema study exhibited collar button signs and longitudinal ulcers in the left colon.a severe form of cd was diagnosed. metronidazole seemed to decrease crp but was ineffective in ameliorating diarrhea. infliximab rather than steroid hormone was chosen for the treatment. standard induction therapy with infliximab was initiated. symptoms rapidly improved then disappeared. cd activity index decreased from 747 to a remission level of 134 after 2 infusions of infliximab. scheduled maintenance infliximab therapy was administered after the induction therapy. ultrasonography and ct showed a disappearance of the wall thickness of the colon. adverse reactions were not observed.infliximab was effective and safe in a patient with cd on hemodialysis. our case has added additional literature in accordance with previous reports supporting infliximab as effective and safe in patients on hemodialysis. |
cyanine dyes have been shown to undergo reversible photoswitching, where the fluorophore can be switched between a fluorescent state and a dark state upon illumination at different wavelengths. the photochemical mechanism by which switching occurs has yet to be elucidated. in this study, we have determined the mechanism of photoswitching by characterizing the kinetics of dark state formation and the spectral and structural properties of the dark state. the rate of switching to the dark state depends on the concentration of the primary thiol in the solution and the solution ph in a manner quantitatively consistent with the formation of an encounter complex between the cyanine dye and ionized thiol prior to their conjugation. mass spectrometry suggests that the photoconversion product is a thiolcyanine adduct in which covalent attachment of the thiol to the polymethine bridge disrupts the original conjugated -electron system of the dye. |
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gastrointestinal toxicity induced by non - steroidal anti - inflammatory drugs (nsaids) is one of the major causes of peptic ulcers. due to its widespread use, low - dose aspirin (lda), which is used for prophylaxis of atherothrombotic diseases, plays an important role in the occurrence of peptic ulcers. in japan, previous studies have shown that upper gastrointestinal bleeding caused by lda can be decreased with acid suppressants, such as proton pump inhibitors (ppi). unfortunately, these protective effects are incomplete, and new therapeutic options have not been developed [14 ]. many gastroduodenal mucoprotective drugs have not been evaluated in terms of efficacy for lda - related toxicity, although they are often used in clinical settings in japan. in addition, there is little clinical information and the current condition of lda - related gastrointestinal mucosal injury remains uncertain in japanese subjects [5, 6 ]. we conducted this study to clarify the current condition of mucosal injury in japanese patients taking lda and to identify potential effective treatments against lda - induced mucosal injury. this study involved a retrospective review of medical records. from january 2005 through december 2006, 5555 patients underwent upper gastrointestinal endoscopy at the department of internal medicine of teikyo university hospital (tokyo, japan). among these patients, 548 had taken lda for 1 month or more and were selected for inclusion in this study. patients with malignant diseases, hemorrhage from variceal lesions, and mallory - weiss syndrome were excluded because of difficulty in performing accurate endoscopic evaluations. two well - trained examiners with more than 10 years experience retrospectively evaluated endoscopic records to determine whether gastroduodenal mucosal injury and bleeding from the lesion were present ; examiners were blinded to patient data. if the two examiners differed in opinion, another examiner evaluated the record again to make a final diagnosis. bleeding was diagnosed on the basis of endoscopic evidence of current or recent hemorrhage with drop of hemoglobin of 2 g / dl or more, which was judged to require hospitalization. minor hemorrhages judged to be treatable on an outpatient basis, such as small clots attached to the gastric wall, were not classified as bleeding in the present study. mucosal injury was defined as obvious findings of gastroduodenal mucosal defects including both erosion (less than 5 mm in diameter) and ulcer (more than 5 mm). background characteristics of the subjects (age, gender, underlying disease, reason for undergoing examination, past history of gastroduodenal ulcer, and concomitant administration of warfarin, nsaids, ppi, histamine 2-receptor antagonists (h2ra), or other gastric mucoprotective (mp) agents) were ascertained from medical records. the presence of serum anti - helicobacter pylori (h. pylori) antibody was tested using a commercially available kit (e - plate, eiken chemical co., tokyo, japan) in participants who gave written informed consent. chart review was performed to confirm the present status of the infection in subjects that underwent past eradication treatment. unadjusted odds ratios (or) and 95% confidence intervals (ci) were calculated to clarify the significance of differences between two groups, if appropriate. prior to the study, the protocol was approved by the institutional review board of teikyo university. underlying diseases of subjects included ischemic heart disease (66%), cerebrovascular disease (30%), and systemic arteriosclerosis (8%). the influence of background characteristics on gastroduodenal bleeding and mucosal injury are shown in tables 1 and 2. the only factor significantly associated with reduced bleeding was co - administration of a ppi. in terms of mucosal injury, use of a ppi decreased the prevalence of mucosal injury, whereas the presence of h. pylori increased the prevalence (table 2). the impact of gastric protective medications on gastroduodenal toxicity is presented in table 3. compared to patients not receiving any gastroprotective medication, any treatment was associated with significantly lower rates of bleeding and mucosal injury (p<0.01 for both comparisons). similarly, use of a ppi alone was significantly more protective against bleeding and mucosal injury than non - use of any medicine (p<0.01 for both comparisons). in terms of combination therapy, patients taking a ppi or h2ra plus an mp agent showed significantly better outcomes in terms of mucosal injury compared to patients taking no medication. use of a ppi / h2ra plus rebamipide was associated with a better overall outcome (bleeding, mucosal injury) than acid suppressants plus other mps, although the rate of h. pylori infection, a significant confounding factor, did not differ between two groups (table 4). aspirin, even in low doses, induces gastrointestinal mucosal injury and hemorrhage, which limits its clinical use. lda - induced gastrointestinal toxicity has become a big problem in japan as well as other countries. a case - control study of hemorrhagic peptic ulcer patients indicated that the risk of lda for bleeding from ulcers is similar to other nsaids. other reports also support the high prevalence of gastroduodenal mucosal injury in lda users [5, 6 ]. although information is limited in japanese patients, data indicate that effective treatments are needed for lda - related gastrointestinal toxicity in japanese patients as well. however, in cases in which lda use is required, such as after placement of a drug - eluted coronary stent, protection of gastrointestinal mucosa against lda - injury is important. proven treatment for lda - related mucosal injury includes co - administration of a ppi and eradication of h. pylori. because these treatments are comparable in efficacy, but do not provide complete protection, other therapeutic options are needed. co - administration of acid suppressants and other gastroprotective medicines is one possible treatment, although we can find only one report regarding this treatment. the present data clearly showed the effectiveness of acid suppressants, especially ppi, for suppressing lda - induced mucosal damage, which is consistent with previous data. patients receiving any gastroprotective medicine suffered from mucosal injury significantly less frequently than those who did not receive any medication. rebamipide provides it gastric mucoprotective effect via different mechanisms from acid suppressants, which affect not only the upper gastrointestinal tract but also other intestinal organs [1015 ]. rebamipide stimulates the production of prostaglandins and epidermal growth factor, preventing h. pylori - elicited neutrophil - induced mucosal injury and decreasing free radical levels [16, 17 ]. clinically, the efficacy against nsaid - induced gastric injury is reported as comparable to that of low - dose famotidine, one of h2ra. the present results suggest the new potential for use of rebamipide with a ppi and h2ra to protect against lda - related intestinal damage. the limitations of the present study include the retrospective study design, the small sample number, and the fact that all patients were drawn from a single institute. because selection bias may have affected the present results, data should be interpreted carefully. a prospective trial is needed to clarify the effect of rebamipide added to ppis and h2ra therapy on the prevention of aspirin - induced gi injury. | the present study was conducted to investigate the prevalence of mucosal injury in patients taking low - dose aspirin in japan and examine the effect of gastric mucoprotective drugs on aspirin - related gastroduodenal toxicity. we selected 530 patients who had taken low - dose aspirin for 1 month or more after undergoing esophagogastroduodenoscopy from 2005 through 2006 at teikyo university hospital, tokyo, japan. endoscopic records were retrospectively reviewed to determine the presence of massive bleeding and mucosal injury (ulcer or erosion). the influence of clinical factors, including co - administration of gastroprotective drugs, was also examined. hemorrhage was observed in 25 patients (3.7%) and mucosal injury (36.2%) in 192 patients. the presence of helicobacter pylori antibody was a significant risk factor associated with mucosal injury. patients taking any gastroprotective drug showed a significantly lower rate of mucosal injury than those not taking these drugs. patients taking rebamipide concomitantly with proton pump inhibitors or histamine 2 receptor antagonists had mucosal injury less frequently than those taking acid suppressants plus other mucoprotective drugs. in conclusion, these results show the possible gastroprotective effects of rebamipide, suggesting that it may be a good choice in aspirin users with gastroduodenal toxicity that is not suppressed by acid suppressants alone. |
the axillary crutch is an assistive device that supports the gait of patients with lower limb impairments. however, crutch walking has several unresolved issues, including the appropriate length of the crutch1, crutch palsy2, palmar pain during crutch weight bearing3, and deviation of the axillary pad from the axilla during the crutch stance phase4. in particular, the axillary pad often deviates from the axilla during the crutch stance phase, and this is one of the main issues for safe crutch walking. if the axillary pad is not held firmly against the chest wall, the crutch tends to be easily displaced from the axilla, possibly leading to falls. therefore, the cause of the axillary pad deviation from the axilla needs be identified to ensure user safety. we previously showed that external rotation of the crutch throughout the crutch stance phase ensured that the axillary pad was held tightly against the chest wall during single - crutch walking5. however, this observation is thought to apply only to single - crutch walking. thus, the aim of this study was to analyze the crutch position in the horizontal plane to confirm the stability of the axillary pad during double - crutch walking. twelve healthy young subjects were enrolled in this study (6 males and 6 females ; mean age, 22.1 0.5 years ; mean height, 168.7 8.0 cm ; mean weight, 63.3 11.3 kg). this study was performed in accordance with the ethical principles of the declaration of helsinki and was approved by the ethics committee of kyushu university of nursing and social welfare. all subjects received a description of this study and provided their written, informed consent before participating in this study. before carrying out the measurements, the crutches were adjusted to the appropriate length for each subject, and adequate practice of crutch walking was done. the appropriate crutch length was determined using the standard method described by rusk and lowman6. double - crutch walking was performed using a three - point gait (swing - through gait) with a pseudo - affected left leg. for this study, a new crutch was created that had stepless adjustments for the height of the hand grip and the crutch length to enable its adaptation to all subjects. the new crutch had an extension that was attached to the axillary pad so that a straight line connecting the two infrared reflective markers indicated the long axis of the axillary pad (fig. schematic diagram of the crutch position the measuring system consisted of a three - dimensional motion capture system (vicon nexus, uk), comprising nine infrared cameras and six force plates (amti, usa) and a digital video camera (jvc, japan). the sampling rates were 100 hz. the crutch position in the horizontal plane was defined as the angle between the sagittal (yz) plane and the long axis of the axillary pad. the direction of the crutch position was determined as internal rotation when the crutch turned toward the body, and external rotation when the crutch turned away from the body (fig. the crutch position data were obtained using four infrared reflective markers attached to both crutches (two markers each on the right and left crutches), and the results were calculated using the data collected by the motion capture system. the positions of both crutches, that were measured during the crutch walking, were calculated using the data of the ground reaction forces. all subjects were asked to perform a 5-m straight walk using double - crutch walking. repeated - measures analysis of variance and tukey s honest significance test were used to compare crutch angles across three time points during the crutch stance phase (crutch contact, mid stance, and crutch off) of each subject. cross - correlation coefficients were used to examine the symmetry of the left and right crutch angles during the crutch walking cycle of each subject. table 1table 1.crutch angles of each phase of the crutch walking of each subjectsubjectcrutch angle (degrees)direction of crutch rotationbetween crutch contact and crutch offcrutch contactmid stancecrutch off1rt.9.3 1.93.9 2.1 0.3 2.3external rotationlt.7.8 2.06.7 0.92.7 2.6 2rt.9.2 1.70.0 1.1 5.4 1.4lt.11.7 2.25.0 1.50.3 4.9 3rt.14.0 1.55.2 1.21.4 2.0lt.12.8 1.53.2 1.2 0.7 2.0 4rt.5.2 1.42.0 1.9 2.6 1.2lt.5.6 1.31.7 2.1 2.6 4.1 5rt.1.5 2.73.8 1.4 6.1 2.7lt.1.0 2.13.9 1.7 9.9 2.1 6rt.4.8 1.90.1 5.9 5.9 3.4lt.7.5 2.41.1 1.1 4.7 2.0 7rt.6.3 1.90.7 2.7 3.9 3.1lt.11.5 3.62.9 4.6 2.4 3.9 8rt.9.1 3.13.2 2.5 9.2 2.9lt.3.1 2.01.6 2.09.5 2.4 9rt.3.8 2.13.9 1.7 12.2 5.2lt.5.0 2.92.1 1.2 6.2 1.6 10rt.7.0 1.02.0 3.5 2.8 2.0lt.4.7 0.52.0 2.4 12.8 1.7 11rt.12.9 1.710.4 1.25.4 2.2lt.14.5 0.511.4 1.110.2 3.4 12rt.7.3 2.51.8 3.5 3.9 5.1external rotationlt.0.7 2.05.9 5.05.6 3.6no rotationdata are means sd. shows the crutch angles of both sides in each phase of the crutch stance of each subject. crutch angles of each of the two crutches were significantly different across the crutch stance phase of 11 of the 12 subjects. post hoc analysis showed significant differences for 6 of the 11 subjects (subjects 2, 3, 6, 7, 9, and 10) in three comparisons (crutch contact vs. mid stance, crutch contact vs. crutch off, and mid stance vs. crutch off) on both sides. four other subjects (subjects 1, 4, 5, and 8) showed significant differences in three comparisons on one side, whereas the other side showed significant differences in two of three comparisons (significance was always observed in the comparison of crutch contact vs. crutch off). the remaining subject (subject 11) showed significant differences in one or two of three comparisons on both sides (significance was always observed in the comparison of crutch contact vs. crutch off). finally, subject 12 showed significant differences in three comparisons on one side, but the other side did not show significant differences in these three comparisons. correlation coefficients and phasic differences between the left and right crutch angles of each subjectsubjectccphasic difference10.851%20.950%30.950%40.906%50.911%60.930%70.930%80.980%90.950%100.890%110.901%120.870%cc : correlation coefficient. phasic difference as a percentage of the whole crutch - walking cycle. shows the correlation coefficients and phasic differences between the left and right crutch angles during the crutch - walking cycle of each subject. a strong correlation was observed between the left and right crutch angles in all subjects. however, phasic differences were shown by 4 of the 12 subjects (subjects 1, 4, 5 and 11), and subject 4 had an especially large phasic difference of 6%. sasaki. reported that, when the shoulder joint was positioned in an internally rotated position, the axillary pad was not fixed against the chest wall and could easily slide backward from the axilla4. in other words, it is very important for safe crutch walking that the axillary pad is kept in the axilla during crutch walking. in an earlier study, we suggested that analysis of the crutch position in the horizontal plane during single - crutch walking would be necessary to ensure safe crutch walking5. we also showed that external rotation of the crutch throughout the crutch stance phase ensured that the axillary pad was held tightly against the chest wall during single - crutch walking. the reason for this is that external rotation of the crutch throughout the crutch stance phase is related to the shape of the chest wall (fig. 2.relationship between the chest wall and the position of the axillary pad during the crutch stance phase). that is, because the chest wall is rounded, the axillary pad moves along the chest wall. therefore, the aim of this study was to analyze the crutch position in the horizontal plane to confirm the stability of the axillary pad during double - crutch walking. relationship between the chest wall and the position of the axillary pad during the crutch stance phase this study found that the crutch rotated externally in the horizontal plane during the crutch stance phase of 11 subjects, although there was inter - subject variation in the angle. we consider that these variations are dependent on : crutch tip contact position, transfer and/or position of center of gravity, and difference of crutch walking pattern (step length, walking speed). regarding the crutch tip contact position, the crutch angle is posited to become externally rotated when the width between the left and right crutch tips becomes wider. conversely, when the width becomes narrower, the crutch angle tends to be internally rotated. in both cases, external rotation of the crutch throughout the stance phase probably ensured that the axillary pad was held tightly against the chest wall during double - crutch walking, as in single - crutch walking. in the one atypical subject (subject 12), the crutch rotated externally on one side, but did not rotate on the other side during the crutch stance phase. in this case, it is likely the crutch deviated from the chest wall on the side that did not rotate externally, and the subject s crutch gait was unstable. in a review of the video recordings, subject 12 was one of the subjects who lacked skill in crutch walking, even though sufficient training was performed in advance ; i.e., subject 12 exhibited many failures of the measurement and large body sway during crutch walking. therefore, external rotation of the crutch throughout the stance phase appears to be very important for keeping the axillary pad in the axilla, not only during single - crutch walking, but also during double - crutch walking. in double - crutch walking, the symmetry of the right and left crutch positions appears to be an important factor of safe crutch walking, in addition to external rotation of the crutches. 3.the ideal crutch transition during crutch walking (conceptual illustration) shows the ideal crutch transition during double - crutch walking. as shown in fig. 3, the symmetry of the right and left crutch positions is required for straight walking throughout double - crutch walking. if the positions of both crutches are not synchronized, straight walking becomes unstable because the direction of the force holding the axillary pad against the chest wall becomes asymmetrical. the present study confirmed that there was a similarity between the left and right crutch angles during crutch walking of all subjects. incidentally, assuming the crutch - walking cycle is the same as the normal walking cycle, the phase from crutch contact to mid stance is about 30% of whole crutch - walking cycle. further, the length of this phase is less than one second. as shown in table 2, a phasic difference of 6% it means that there was a considerable discrepancy in the symmetry of the left and right crutch angles in this phase for subject 4. therefore, it is likely that the crutch stance phase of subject 4 was negatively impacted because this subject lacked skill in crutch walking. it is our opinion that symmetry of crutch positioning is one of the important factors for safe double - crutch walking. the ideal crutch transition during crutch walking (conceptual illustration) in conclusion, the crutch positions of both sides were measured in the horizontal plane in order to confirm the stability of the axillary pad during double - crutch walking. external rotation of the crutch throughout the stance phase is very important for keeping the axillary pad in the axilla, not only during single - crutch walking, but also during double - crutch walking. in addition, symmetry of crutch positioning appears to be one of the important factors for safe double - crutch walking. however, we think our findings are not sufficient to recommend their application in clinical settings, because the subjects were healthy. therefore, the selection of subjects requiring crutches is needed to confirm our findings in further studies. | [purpose ] the aim of this study was to analyze the crutch position in the horizontal plane to confirm the stability of the axillary pad during double - crutch walking. [subjects ] twelve healthy young subjects (6 males and 6 females). [methods ] the subjects were asked to walk in a straight line, using double crutches, for a distance of 5 m, 5 times. crutch position data were obtained using four infrared reflective markers attached to both crutches (two markers each on both crutches). the crutch angles of each subject were compared across three time points during the crutch stance phase (crutch contact, mid stance, and crutch off). [results ] crutch angles of each of the two crutches were significantly different across the crutch stance phase of 11 of the 12 subjects. phasic differences were shown by 4 of the 12 subjects, and a strong correlation was observed between the left and right crutch angles of all subjects. [conclusion ] external rotation of the crutch throughout the stance phase was very important for keeping the axillary pad in the axilla, not only for single - crutch walking, but also for double - crutch walking. in addition, symmetry of the crutch positioning is one of the most important factors for safe double - crutch walking. |
breast cancer is one of the leading causes of death in women worldwide (1, 2) and is the second most common malignancy newly diagnosed in korean women. more than 15000 women were newly diagnosed with breast cancer in 2011, and breast cancer was in second (21.5%) place among all newly diagnosed cancers in korean women (www.ncc.re.kr). moreover, the 5-year survival rate of breast cancer has increased from 83.2% to 91.3% over the last 10 years due to advances in postoperative treatment modalities and medications. the overall incidence of breast cancer in korean women has increased from 1999 to 2011, and the prevalence of breast cancer during this period is 19.3% of all cancers in women (3), which agrees with reports on the western population (1, 4). as the number of breast cancer survivors increases, patient management and surveillance after primary treatment has come under the spotlight. women who have been treated for breast cancer are at risk for second breast cancers, such as tumor recurrence in the ipsilateral breast or a newly developed cancer in the contralateral breast (2, 4, 5). reported risks for locoregional tumor recurrence range from 5 - 27%, whereas the risk for development of contralateral breast cancer is 5 - 10%, with a two six - fold increased risk (4, 6, 7, 8, 9, 10). in addition, recent studies have demonstrated that local recurrence is an independent predictor of survival, an high relative risks for developing distant metastases or breast cancer - related deaths in patients with local recurrences have been shown when compared to patients without a recurrence (10). considering these risks, a well - designed, evidence - based post - treatment surveillance protocol is needed to manage patients with breast cancer after their primary treatment. the surveillance program would be intended to detect second breast cancers at an early stage when curative intervention is possible. up to now, mammography has been the only evidence - based imaging modality with demonstrated efficiency for detecting asymptomatic tumor recurrence or a second breast cancer in women who have been treated for primary breast cancer (2, 4, 11, 12, 13, 14, 15, 16, 17). ultrasonography (us), magnetic resonance imaging (mri) and f - fluorodeoxyglucose (fdg) positron emission tomography (pet) have been utilized in many institutions to increase detection of second cancers at an early stage. screening mammography for women with an average risk of breast cancer results in early detection of breast cancer, leading to reduced mortality and improved patient outcome. many case - controlled or non - randomized controlled trial (rct) studies show a 20 - 30% reduction in breast cancer mortality after screening (18, 19). hence, we assume that women with an elevated risk for breast cancer, including those who have already been treated for primary breast cancer, may benefit even more from screening mammography. at present, mammography is the only imaging modality commonly recommended for breast cancer surveillance (table 1) (1, 11, 20). as mammography enables detection of an early asymptomatic recurrence, early intervention or treatment is also possible (fig. several recent studies have demonstrated that early detection of a recurrence in asymptomatic patients during post - treatment follow - up improves survival (4, 22, 23), supporting the role of routine mammography for post - treatment surveillance of breast cancer. based on a literature review, houssami and ciatto (4) reported that the proportion of ipsilateral breast recurrences detected on mammography is 50 - 80%, and mammography detects 45 - 90% of contralateral metachronous breast cancers. (22) reported that surveillance mammography is associated with a significant reduction in the hazard for death related to breast cancer. similarly, surveillance mammography helps detect asymptomatic tumor recurrence, resulting in improved patient survival, but most recommendations are based on consensus rather than evidence supported by rcts. in another study, the proportion of ipsilateral breast recurrences detected with mammography was 8 - 51% of lesions detected on mammography only, but approximately three - fifths of the participating hospitals perform mammography surveillance at 6-month intervals for 2 to 5 years in patients with breast conservation surgery (24). such semiannual mammographic surveillance allows the detection of a significantly higher proportion of cancer recurrences at an earlier stage than that of annual surveillance (25). however, these results do not support establishing intensive surveillance because no significant differences was found in tumor size or nodal status between the semiannual surveillance and the annual surveillance groups, and the follow - up intervals were 3 - 18 months, which was too long to strictly separate the patients into two groups (26). as seen in the various reports mentioned above, although most studies include mammography for post - treatment surveillance of women who have been treated for breast cancer, two important issues remain unsolved. the mammography follow - up interval and the follow - up duration need to be defined. the more popular post - treatment surveillance recommendations for patients with breast cancer are summarized in table 1. the american society of clinical oncology clinical practice guidelines recommend a post - treatment mammogram 1 year after initial diagnosis or at least 6 months after completion of radiation therapy, and yearly mammography follow - up thereafter (11). similarly, the national comprehensive cancer network (www.nccn.org, version 2013.03) and the national institute for clinical excellence guidelines (www.nice.org.uk, 2011) recommend mammography every 12 months in addition to routine history and physical examinations obtained at regular visits. as in the prior recommendations, we agree that annual mammography should be performed for 5 years after treatment, and a mammography every 1 - 2 years thereafter may be a reasonable compromise. it should be emphasized that there is insufficient evidence regarding mammography follow - up intervals in post - treatment surveillance. additionally, quantifying the actual impact of screening mammography in these patients excluding bias, specifically lead - time and length - time bias, is difficult using mostly non - randomized retrospective studies. rcts are the most appropriate method to estimate the effect of early detection of ipsilateral or contralateral breast cancer recurrence, but applying this study design to clinical practice is not feasible or ethical, as patients who have been treated for breast cancer are already at high risk for developing second breast cancers. further prospective investigation that includes a large data set showing how women with a personal breast cancer history benefit from surveillance mammography is anticipated in the future, along with evidence - based meticulous screening programs that will build on the results. ultrasonography is a widely available, relatively inexpensive imaging method that is easy to perform, has no radiation hazards, does not require a contrast agent, and enables biopsy under image guidance. breast us has been popularly used to characterize lesions and differentially diagnose breast masses as an adjunctive tool to mammography, particularly in women with dense breasts (5, 27). preoperative bilateral whole - breast us also provides complementary information to mammography (28, 29), and detects up to 88% of contralateral synchronous cancers, among which 43% are occult on a mammogram (28). based on the results of preoperative us, one study showed that approximately 16% of women who had undergone preoperative us had changes in treatment plans set by mammography alone (29). moreover, us has drawn attention as a useful surveillance imaging method in addition to mammography in women who have been treated for breast cancer. us detects ipsilateral recurrent or contralateral metachronous breast cancers with higher sensitivity (91 - 97%) (table 2) than that of palpation or mammography, which have sensitivity values of 45.5 - 79% and 45 - 87%, respectively (5, 8, 28, 30, 31, 32). adding us to mammography in the american college of radiology imaging network (acrin) 6666 trials yielded an additional 1.1 - 7.2 cancers per 1000 high - risk women, of which 53% of the 2637 enrolled women had a personal history of breast cancer (27). other than the breast, us is an excellent modality to evaluate chest wall and axillary areas, which can not be easily approached by mammography. one of the most common sites for post - treatment breast cancer recurrence is the chest wall (fig. 2), either from direct extension of the tumor, indirect extension via interpectoral nodes, or from undissected lymphatics (32). approximately 10 - 35% of patients who have been treated for breast cancer have a metastasis in the axillary, internal mammary, and supraclavicular nodes (33, 34). among the occult regional recurrences after surgical treatment for breast cancer, only 21.4% occur at the axillary area lateral to the pectoralis muscle, which may be included within the fields of mammography and surgical sampling, while the remaining 78.6% of regional recurrence was detected by us alone at areas other than the axilla (34, 35, 36). in other studies, the sensitivity of mammography is only 10% (0.0 - 14.3%), whereas that of us was around 90% (81.1 - 100.0%) to detect regional or locoregional recurrences (24, 34, 37, 38, 39, 40). several studies have shown surveillance results of us applied to women who were treated for breast cancer (34, 37, 39, 40, 41). the reported cancer detection rates were 1.7 - 5.1% per patient, and the positive predictive value (ppv) was 21.5 - 52.6%, with percentages varying according to the area involved. because isolated recurrences are associated with distant metastasis and/or poor outcome, early detection and targeted treatment for recurrences are critical to improve patient outcome (41, 42, 43). early detection of a locoregional recurrence of breast cancer after primary treatment by us can help guide patient management by sorting out those who may benefit by early therapeutic intervention or curative treatment of local disease. patients who received a mastectomy and who had a locoregional recurrence detected at an early stage or an isolated regional recurrence had better survival with short follow - up (23, 29, 41), but controversy remains about whether post - treatment surveillance us actually affects long term survival in patients with breast cancer (40) and whether all asymptomatic regional recurrences will be detected by us or by pet - ct or another imaging modality in clinical practice (42). many studies agree that mammography is the basic imaging modality for breast cancer surveillance (5), and the usefulness of us surveillance can be amplified under conditions in which the benefits of mammographic surveillance are reduced such as in dense breasts. the sensitivity of us for detecting metachronous breast cancer is 94%, regardless of breast density, whereas sensitivity of mammography in women with dense breasts is lower than that of women with scattered fibroglandular tissue (73% vs. 80%) (5). in contrast, results of another study show that the differences in sensitivity between mammography and us for non - palpable second breast cancers is not noticeably different for ipsilateral breast tumor recurrence (14%) compared to that of contralateral breast recurrence (28%), and that the sensitivity of us for non - palpable breast tumor recurrences drops by about 10% in the ipsilateral breast, which may due to sonographically architectural distortion obscuring detection of an ipsilateral breast tumor recurrence (38). additionally, the main shortcoming of us in the acrin 6666 trial was the substantial false - positive rate ; the ppv of the biopsy recommendation after us examinations was 20 - 25% by the american cancer society (20), the american college of radiology (47), and the society of breast imaging (48), these recommendations only state that mri " may be considered " for women who have a personal history of breast cancer ; that is, women with intermediate risk (lifetime breast cancer risk > 15 - 20%) for breast cancer (47). therefore, little is known about the role of breast mri in post - treatment surveillance programs. breast mri shows high sensitivity and specificity for differentiating between post - treatment changes in the breast from recurrent malignancies (table 2) (31, 49), particularly when performed later than 12 months after treatment (50). in a study by brennan. (49), breast screening with mri detected cancer in 12% of the study population, including women with a personal history of breast cancer only, and the ppv of the biopsy recommendations from mri was acceptable at 39%. based on their results, the authors claimed that mri screening of women with a personal history of breast cancer is clinically valuable because cancers discovered from screening mri benefit from early detection, as more than half were minimal breast cancers (49). (51) emphasize the importance of a personal history of breast cancer treatment as an indication for mri screening. the ppv of biopsy recommendations based on mri features is 32% in women with a family history of breast cancer and it increases to 50% in women who have also been diagnosed and treated for breast cancer (51). breast mri screening has the highest yield in women with both a family and personal history of breast cancer, particularly in those who have had breast conservation surgery (51). additionally, high negative predictive values of breast mri have been reported in women who had breast conservation therapy ; belli. (31) concluded that the absence of enhancing foci in post - treatment breasts has 100% reliability for predicting the absence of tumor recurrence. there may be no need for additional invasive procedures such as biopsy if there are no enhancing foci by correlating lesions that are suspected to be recurrences on conventional imaging modalities to breast mri. patients may benefit from screening mri, with its high npv, by reducing many benign biopsies, which is supported by the results of another study (52) concluding that a negative mri is more useful and conclusive than a positive mri, as positive features warrant further investigation. this is also supported by a recent study that included a large proportion of women who had been treated for breast cancer with additionally detected early stage breast cancers on supplemental screening breast mri performed in addition to mammography and us ; 14.7 more cancers per 1000 women were additionally detected by mri (25). mri for surveillance or screening purposes is limited, as this imaging method is expensive, lacks availability, requires contrast media injection for adequate imaging, and neither the technique nor interpretive criteria for breast mri are standardized (51). despite its shortcomings, breast mri is a highly sensitive imaging modality. however, when and in which circumstances screening breast mri should be applied is still a question for women who have been treated for breast cancer. as most postoperative women undergoing surveillance are under a hormonal therapy that suppresses the ovary, use of mri is supported in postoperative surveillance programs. as in the many studies on mammography or us, studies evaluating the efficacy of mri for post - treatment surveillance in women who have been treated for breast cancer are of retrospective design and include a limited number of patients. therefore, further randomized prospective studies are needed to properly assess the role of breast mri in post - treatment surveillance programs. positron emission tomography / ct has been used for preoperative staging and the treatment response of patients with breast cancer. this hybrid imaging method has a strong point as it enables anatomic localization of the pet signal via ct (53). pet / ct is particularly useful in patients who are suspected to have or who are exhibiting a recurrence on physical examination or conventional imaging methods (53, 54). because pet / ct is highly sensitive for detecting lesions and determining whether a recurrence is solitary or disseminated, information gained from pet / ct has a significant impact on the decision for upcoming treatment and post - treatment patient outcome. sensitivity of pet / ct for detecting locoregional recurrence or metastasis among patients with breast cancer is approximately 97%, with a diagnostic accuracy of 95% in one study (55), supporting the efficacy of pet / ct for patients diagnosed or suspected of having recurrent breast cancer (fig. however, there is a lack of evidence demonstrating the efficacy and cost - effectiveness of this modality, along with the hazard of radiation exposure and the absence of specific clinical indications (56). screening mammography for women with an average risk of breast cancer results in early detection of breast cancer, leading to reduced mortality and improved patient outcome. many case - controlled or non - randomized controlled trial (rct) studies show a 20 - 30% reduction in breast cancer mortality after screening (18, 19). hence, we assume that women with an elevated risk for breast cancer, including those who have already been treated for primary breast cancer, may benefit even more from screening mammography. at present, mammography is the only imaging modality commonly recommended for breast cancer surveillance (table 1) (1, 11, 20). as mammography enables detection of an early asymptomatic recurrence, early intervention or treatment is also possible (fig. several recent studies have demonstrated that early detection of a recurrence in asymptomatic patients during post - treatment follow - up improves survival (4, 22, 23), supporting the role of routine mammography for post - treatment surveillance of breast cancer. based on a literature review, houssami and ciatto (4) reported that the proportion of ipsilateral breast recurrences detected on mammography is 50 - 80%, and mammography detects 45 - 90% of contralateral metachronous breast cancers. (22) reported that surveillance mammography is associated with a significant reduction in the hazard for death related to breast cancer. similarly, surveillance mammography helps detect asymptomatic tumor recurrence, resulting in improved patient survival, but most recommendations are based on consensus rather than evidence supported by rcts. in another study, the proportion of ipsilateral breast recurrences detected with mammography was 8 - 51% of lesions detected on mammography only, but approximately three - fifths of the participating hospitals perform mammography surveillance at 6-month intervals for 2 to 5 years in patients with breast conservation surgery (24). such semiannual mammographic surveillance allows the detection of a significantly higher proportion of cancer recurrences at an earlier stage than that of annual surveillance (25). however, these results do not support establishing intensive surveillance because no significant differences was found in tumor size or nodal status between the semiannual surveillance and the annual surveillance groups, and the follow - up intervals were 3 - 18 months, which was too long to strictly separate the patients into two groups (26). as seen in the various reports mentioned above, although most studies include mammography for post - treatment surveillance of women who have been treated for breast cancer, two important issues remain unsolved. the mammography follow - up interval and the follow - up duration need to be defined. the more popular post - treatment surveillance recommendations for patients with breast cancer are summarized in table 1. the american society of clinical oncology clinical practice guidelines recommend a post - treatment mammogram 1 year after initial diagnosis or at least 6 months after completion of radiation therapy, and yearly mammography follow - up thereafter (11). similarly, the national comprehensive cancer network (www.nccn.org, version 2013.03) and the national institute for clinical excellence guidelines (www.nice.org.uk, 2011) recommend mammography every 12 months in addition to routine history and physical examinations obtained at regular visits. as in the prior recommendations, we agree that annual mammography should be performed for 5 years after treatment, and a mammography every 1 - 2 years thereafter may be a reasonable compromise. it should be emphasized that there is insufficient evidence regarding mammography follow - up intervals in post - treatment surveillance. additionally, quantifying the actual impact of screening mammography in these patients excluding bias, specifically lead - time and length - time bias, is difficult using mostly non - randomized retrospective studies. rcts are the most appropriate method to estimate the effect of early detection of ipsilateral or contralateral breast cancer recurrence, but applying this study design to clinical practice is not feasible or ethical, as patients who have been treated for breast cancer are already at high risk for developing second breast cancers. further prospective investigation that includes a large data set showing how women with a personal breast cancer history benefit from surveillance mammography is anticipated in the future, along with evidence - based meticulous screening programs that will build on the results. ultrasonography is a widely available, relatively inexpensive imaging method that is easy to perform, has no radiation hazards, does not require a contrast agent, and enables biopsy under image guidance. breast us has been popularly used to characterize lesions and differentially diagnose breast masses as an adjunctive tool to mammography, particularly in women with dense breasts (5, 27). preoperative bilateral whole - breast us also provides complementary information to mammography (28, 29), and detects up to 88% of contralateral synchronous cancers, among which 43% are occult on a mammogram (28). based on the results of preoperative us, one study showed that approximately 16% of women who had undergone preoperative us had changes in treatment plans set by mammography alone (29). moreover, us has drawn attention as a useful surveillance imaging method in addition to mammography in women who have been treated for breast cancer. us detects ipsilateral recurrent or contralateral metachronous breast cancers with higher sensitivity (91 - 97%) (table 2) than that of palpation or mammography, which have sensitivity values of 45.5 - 79% and 45 - 87%, respectively (5, 8, 28, 30, 31, 32). adding us to mammography in the american college of radiology imaging network (acrin) 6666 trials yielded an additional 1.1 - 7.2 cancers per 1000 high - risk women, of which 53% of the 2637 enrolled women had a personal history of breast cancer (27). other than the breast, us is an excellent modality to evaluate chest wall and axillary areas, which can not be easily approached by mammography. one of the most common sites for post - treatment breast cancer recurrence is the chest wall (fig. 2), either from direct extension of the tumor, indirect extension via interpectoral nodes, or from undissected lymphatics (32). approximately 10 - 35% of patients who have been treated for breast cancer have a metastasis in the axillary, internal mammary, and supraclavicular nodes (33, 34). among the occult regional recurrences after surgical treatment for breast cancer, only 21.4% occur at the axillary area lateral to the pectoralis muscle, which may be included within the fields of mammography and surgical sampling, while the remaining 78.6% of regional recurrence was detected by us alone at areas other than the axilla (34, 35, 36). in other studies, the sensitivity of mammography is only 10% (0.0 - 14.3%), whereas that of us was around 90% (81.1 - 100.0%) to detect regional or locoregional recurrences (24, 34, 37, 38, 39, 40). several studies have shown surveillance results of us applied to women who were treated for breast cancer (34, 37, 39, 40, 41). the reported cancer detection rates were 1.7 - 5.1% per patient, and the positive predictive value (ppv) was 21.5 - 52.6%, with percentages varying according to the area involved. because isolated recurrences are associated with distant metastasis and/or poor outcome, early detection and targeted treatment for recurrences are critical to improve patient outcome (41, 42, 43). early detection of a locoregional recurrence of breast cancer after primary treatment by us can help guide patient management by sorting out those who may benefit by early therapeutic intervention or curative treatment of local disease. patients who received a mastectomy and who had a locoregional recurrence detected at an early stage or an isolated regional recurrence had better survival with short follow - up (23, 29, 41), but controversy remains about whether post - treatment surveillance us actually affects long term survival in patients with breast cancer (40) and whether all asymptomatic regional recurrences will be detected by us or by pet - ct or another imaging modality in clinical practice (42). many studies agree that mammography is the basic imaging modality for breast cancer surveillance (5), and the usefulness of us surveillance can be amplified under conditions in which the benefits of mammographic surveillance are reduced such as in dense breasts. the sensitivity of us for detecting metachronous breast cancer is 94%, regardless of breast density, whereas sensitivity of mammography in women with dense breasts is lower than that of women with scattered fibroglandular tissue (73% vs. 80%) (5). in contrast, results of another study show that the differences in sensitivity between mammography and us for non - palpable second breast cancers is not noticeably different for ipsilateral breast tumor recurrence (14%) compared to that of contralateral breast recurrence (28%), and that the sensitivity of us for non - palpable breast tumor recurrences drops by about 10% in the ipsilateral breast, which may due to sonographically architectural distortion obscuring detection of an ipsilateral breast tumor recurrence (38). additionally, the main shortcoming of us in the acrin 6666 trial was the substantial false - positive rate ; the ppv of the biopsy recommendation after us examinations was 20 - 25% by the american cancer society (20), the american college of radiology (47), and the society of breast imaging (48), these recommendations only state that mri " may be considered " for women who have a personal history of breast cancer ; that is, women with intermediate risk (lifetime breast cancer risk > 15 - 20%) for breast cancer (47). therefore, little is known about the role of breast mri in post - treatment surveillance programs. breast mri shows high sensitivity and specificity for differentiating between post - treatment changes in the breast from recurrent malignancies (table 2) (31, 49), particularly when performed later than 12 months after treatment (50). in a study by brennan. (49), breast screening with mri detected cancer in 12% of the study population, including women with a personal history of breast cancer only, and the ppv of the biopsy recommendations from mri was acceptable at 39%. based on their results, the authors claimed that mri screening of women with a personal history of breast cancer is clinically valuable because cancers discovered from screening mri benefit from early detection, as more than half were minimal breast cancers (49). (51) emphasize the importance of a personal history of breast cancer treatment as an indication for mri screening. the ppv of biopsy recommendations based on mri features is 32% in women with a family history of breast cancer and it increases to 50% in women who have also been diagnosed and treated for breast cancer (51). breast mri screening has the highest yield in women with both a family and personal history of breast cancer, particularly in those who have had breast conservation surgery (51). additionally, high negative predictive values of breast mri have been reported in women who had breast conservation therapy ; belli. (31) concluded that the absence of enhancing foci in post - treatment breasts has 100% reliability for predicting the absence of tumor recurrence. there may be no need for additional invasive procedures such as biopsy if there are no enhancing foci by correlating lesions that are suspected to be recurrences on conventional imaging modalities to breast mri. patients may benefit from screening mri, with its high npv, by reducing many benign biopsies, which is supported by the results of another study (52) concluding that a negative mri is more useful and conclusive than a positive mri, as positive features warrant further investigation. this is also supported by a recent study that included a large proportion of women who had been treated for breast cancer with additionally detected early stage breast cancers on supplemental screening breast mri performed in addition to mammography and us ; 14.7 more cancers per 1000 women were additionally detected by mri (25). mri for surveillance or screening purposes is limited, as this imaging method is expensive, lacks availability, requires contrast media injection for adequate imaging, and neither the technique nor interpretive criteria for breast mri are standardized (51). despite its shortcomings, breast mri is a highly sensitive imaging modality. however, when and in which circumstances screening breast mri should be applied is still a question for women who have been treated for breast cancer. as most postoperative women undergoing surveillance are under a hormonal therapy that suppresses the ovary, use of mri is supported in postoperative surveillance programs. as in the many studies on mammography or us, studies evaluating the efficacy of mri for post - treatment surveillance in women who have been treated for breast cancer are of retrospective design and include a limited number of patients. therefore, further randomized prospective studies are needed to properly assess the role of breast mri in post - treatment surveillance programs. positron emission tomography / ct has been used for preoperative staging and the treatment response of patients with breast cancer. this hybrid imaging method has a strong point as it enables anatomic localization of the pet signal via ct (53). pet / ct is particularly useful in patients who are suspected to have or who are exhibiting a recurrence on physical examination or conventional imaging methods (53, 54). because pet / ct is highly sensitive for detecting lesions and determining whether a recurrence is solitary or disseminated, information gained from pet / ct has a significant impact on the decision for upcoming treatment and post - treatment patient outcome. sensitivity of pet / ct for detecting locoregional recurrence or metastasis among patients with breast cancer is approximately 97%, with a diagnostic accuracy of 95% in one study (55), supporting the efficacy of pet / ct for patients diagnosed or suspected of having recurrent breast cancer (fig. however, there is a lack of evidence demonstrating the efficacy and cost - effectiveness of this modality, along with the hazard of radiation exposure and the absence of specific clinical indications (56). current post - treatment surveillance guidelines for patients with treated breast cancer do not recommend intensive surveillance, such as routine chest radiography, bone scans, or laboratory tests, to evaluate distant recurrence or metastatic diseases. studies have reported prolonged survival in patients who had asymptomatic metastatic lesions detected at an early stage but early detection was not advantageous to patient survival when the time of initial breast cancer diagnosis was applied. in other words, lead - time bias or length - time bias may have misled thinking that these intensive surveillance programs may prolong patient survival (57). nevertheless, post - treatment surveillance programs applied to patients with breast cancer vary among organizations and countries, mostly due to the lack of a standardized protocol, and studies evaluating distant metastasis are quite often performed along with mammography and physical examinations. a recent study based on the texas cancer registry (56) represents the current status of imaging modalities in post - treatment surveillance programs ; only 55.3% of patients treated for breast cancer showed strict adherence to the current surveillance program, including a routine physical examination and mammography. during the 2001 - 2007 study period, use of mammography and bone scans decreased (81% to 75% and 21% to 13%, respectively), whereas use of mri and pet - ct increased significantly (0.5% to 7.0% and 2% to 9%, respectively) (56). based on their report, it is evident that clinicians and patients do not feel that the current surveillance program with mammography as the only imaging modality is sufficient. although evidence does not yet demonstrate that early detection of a distant metastasis improves patient survival, early detection provides a chance for curative intervention, which may affect quality of life or long - term survival of patients, and this may be the cause for the current trend in which supplemental surveillance imaging modalities other than just mammography are used. however, not all patients benefit from these extensive and rather costly studies repeated annually, and the accuracy of the additional imaging modalities has not yet been confirmed. additionally, these additional modalities may reveal many false - positive lesions, particularly mri and pet scans (20, 54, 55), and may provoke unnecessary interventional procedures and patient anxiety. considering the development of new imaging technologies and the desire of clinicians and patients for better supplementary surveillance methods, it is difficult to insist on patient compliance with the current surveillance program. therefore, discrete evidence on the cost - effectiveness and accuracy of applying supplemental modalities to mammography in terms of patient survival is required when used for post - treatment surveillance of breast cancer. currently, mammography is the single imaging modality recommended for routine follow - up surveillance in women who have been treated for breast cancer. the role of additional imaging modalities, such as us, mri, and pet - ct, as post - treatment surveillance in women treated for breast cancer has not yet been established, but they are potentially useful and show high sensitivity and accuracy for detecting recurrences or distant metastases. although many studies have demonstrated the efficacy of these additional imaging modalities when applied to post - treatment surveillance, they are currently used in clinical practice without specific clinical indications or organized programs due to a lack of concrete evidence. an evaluation of the cost - effectiveness of these imaging modalities should be considered because of their additional costs. a number of different guidelines regarding post - treatment surveillance of patients with breast cancer have been produced worldwide. this is mostly from efforts to ensure that patients should undergo the most appropriate follow - up to decrease patient morbidity and mortality and enable long - term survival after treatment. the heterogeneity regarding post - treatment patient management may arise from the lack of solid evidence on the potential benefits of each follow - up imaging modality. the multiplicity of guidelines or recommendations may reflect that socioeconomic conditions, mostly financial causes such as insurance policies, vary among countries and institutions. this is an important matter that should be considered when investigating the most appropriate and effective method for post - treatment surveillance of patients with breast cancer. further prospective studies including a large number of patients are expected in the future to demonstrate the role of various imaging modalities in post - treatment surveillance programs and how they affect survival in patients treated for breast cancer. | women who have been treated for breast cancer are at risk for second breast cancers, such as ipsilateral recurrence or contralateral metachronous breast cancer. as the number of breast cancer survivors increases, interest in patient management and surveillance after treatment has also increased. however, post - treatment surveillance programs for patients with breast cancer have not been firmly established. in this review, we focus on the imaging modalities that have been used in post - treatment surveillance for patients with breast cancer, such as mammography, ultrasonography, magnetic resonance imaging, and positron emission tomography, the effectiveness of each modality for detecting recurrence, and how they can be applied to manage patients. |
from 1994 through 2005, epidemiologic data on animal - related injuries and associated postexposure prophylaxis (pep) treatment were prospectively collected for marseille rabies treatment centre patients. only patients who had been injured in france were selected ; rabies pep for travelers who were injured abroad is detailed elsewhere (6). of the 4,965 eligible patients, 4,367 were outpatients or inpatients (192488/year), and from 2001 through 2005, a total of 598 were managed by teleconsultation only because their exposure risk was considered to be zero. the number of inpatients and outpatients decreased markedly from 1999 to 2001 (figure 1), which is consistent with the general decrease in the number of pep treatments in france after the elimination of terrestrial mammal rabies (7). furthermore, prescreening of persons by telephone also contributed to this decrease. the increase observed during 20042005 is likely an effect of the international alert in relation to the cases of rabid dogs imported from morocco ; these cases were intensively reported by the french media. the proportion of animal - related injuries tended to increase in late spring / early summer (figure 2), probably as a result of increased outdoor activities in southern france, which makes contact with animals more likely. number of injured patients per year seeking care for rabies postexposure prophylaxis, marseille centre, marseille, france, 19942005. average proportions of injured patients seeking care for rabies postexposure prophylaxis, by month, marseille centre, marseille, france, 19942005. the overall annual incidence of injured patients seeking care for rabies pep was 16/100,000, which is consistent with incidence recently reported in united states (8,9) (where rabies is enzootic in bats and raccoons) but far less than that reported in recently available studies from the canine rabies endemic countries of turkey (467/100,000) (10) and india (1,700/100,000) (11). the overall mean annual incidence in our study was 20/100,000 before 2001 and 11/100,000 after 2001. by contrast, a recent study on pet demographics in france indicated that dog and cat populations are nearly similar at 8.51 million and 9.94 million, respectively (12). this finding suggests that dogs, more often than cats, are responsible for severe injuries that lead persons to seek care for rabies pep. the mean annual incidence of animal - related injuries was lower in rural than in urban communities (technical appendix). because an estimation of the dog population in france indicated that 41% live in urban areas (12), our results suggest that a high human population density increases the probability of human dog interactions and risk for injuries. among patients seeking care for rabies pep, most were male (male : female ratio 1.49) and mean age was 31.5 (median 29, range 096) years. the likelihood for animal - related injuries among male patients was also dependant on the animal species involved ; dogs, bats, and monkeys accounted for most injuries (technical appendix). in contrast, female patients were more likely to be injured by cats, a finding consistent with previous reports (13). the mean time between injury and consultation was 2.6 days (range 0365 days) and did not statistically vary by sex or age group. time was longer in patients who were injured by bats (p 7 days (15). if the treatment can not be delayed, it should include both vaccination and rabies immunoglobulin in cases of category iii injury. from 2001 through 2005, not vaccinating the patient when the animal was under observation by its owner or a veterinarian would have represented an overall savings of 177,600 euros. to minimize overprescription of vaccination for rabies pep when treatment may be unjustified, we recommend delaying the initiation of rabies treatment in injuries involving an apparently healthy indigenous dog or cat that can be kept under veterinary or animal - owner observation for 2 weeks, which is the maximum rabies incubation time in these animals. however, when animals are not available for observation, complete rabies pep treatment should be initiated. given the risk for importation of rabid animals from nearby rabies - endemic countries, immediate rabies pep treatment according to who guidelines should be given when the following are involved : indigenous bats ; animals illegally imported from rabies - endemic countries ; or animals found in railway stations, trains, or other ports of entry. if the animal is suspected of being rabid at the time of exposure, confirmatory testing should be conducted (technical appendix). all travelers visiting countries where rabies is enzootic should be informed about the risks of bringing animals back to their home country and about the who recommendations regarding rabies vaccination of imported animals (14). | the administration of human rabies postexposure prophylaxis near marseille (southern france) has changed since the eradication of terrestrial mammal rabies in 2001. most injuries were associated with indigenous dogs ; rabies vaccine was overprescribed. we suggest that the world health organization guidelines be adapted for countries free of terrestrial mammal rabies. |
leflunomide (lef) is a disease - modifying antirheumatic drug for treating rheumatoid arthritis (ra). its efficacy was established by double - blind, randomized controlled trials. a 5-year follow - up study demonstrated comparative safety profile as in the phase iii trial, showing no excessive pulmonary adverse event. however, lef - related interstitial lung disease (ild) was increasingly reported after introduction of lef in japan in 2003, and diffuse alveolar damage (dad) was the principal histopathological change in autopsied cases. here an 80-year - old taiwanese woman was admitted to our hospital because of shortness of breath. her medical history was notable for remote pulmonary tuberculosis, which had been cured for 5 years and ra, which had been diagnosed for 11 years. follow - up chest radiograph taken 2 years earlier showed mild fibrocalcified lesion in right upper lung zone without obvious interstitial changes. she was a nonsmoker and her current medication included oral prednisolone (10 mg / day), cyclosporine (100 mg / day), and lef (20 mg / day). methotrexate (mtx) (7.5 mg / week) was prescribed initially after the diagnosis of ra. due to poor disease control, she had been in her usual state of health until 2 days before admission, when progressive dyspnea on exertion developed, along with productive cough, fever, and chills. at admission, her vital signs were : temperature 37.3 c, heart rate 110 beats / min, blood pressure 147/89 mm hg, respiratory rate 28 breaths / min, and oxygen saturation 95% while breathing supplementary oxygenation at a rate of 10 l / min through a face mask. laboratory examinations showed normal leukocyte count (5110/l), elevated blood urea nitrogen (57 mg / dl), creatinine (1.6 mg / dl), aspartate aminotransferase (93 u / l), c - reactive protein level (11.7 mg / dl), and mildly increased rheumatoid factor level (44 iu / ml) (normal range, 020.1 iu / ml). she was intubated with ventilator support on day 2 for acute hypoxic respiratory failure. a high - resolution computed tomography of the chest on day 3 (fig. 2a) showed diffuse ground - glass opacities with thickened interlobular septa and patchy consolidations at bilateral lower lobes. hrct of the chest on day 3 (a) showed diffuse ground - glass opacities with thickened interlobular septa, accompanied by patchy consolidations at bilateral lower lobes. a follow - up hrct of the chest on day 16 (b) showed persistent diffuse bilateral ground - glass opacities and interlobular thickening without improvement. hrct, high - resolution computed tomography. besides empirical intravenous ceftriaxone (2000 mg / d) and levofloxacin (750 mg / d) for presumed severe community acquired pneumonia, intravenous trimethoprim / sulfamethoxazole (trimethoprim component 15 mg / kg / d), and methylprednisolone (60 mg / d) were administered for suspicious pneumocystis jirovecii pneumonia and ra - associated ild. cultures of blood, tracheal aspirate, and bronchoalveolar lavage fluid revealed no evidence of bacteria, fungus, or mycobacterium infection. the results of rapid influenza diagnostic test and cytomegalovirus antigenemia assay were both negative. a follow - up high - resolution computed tomography of the chest on day 16 (fig. 2b) showed persistent diffuse bilateral ground - glass opacities and interlobular thickening without improvement. due to poor treatment response, she received thoracoscopic wedge resection of the right upper and middle lung on day 18. microscopically, the airspaces were lined by hyaline membranes, with thickened alveolar septa containing inflammatory exudates, but no obvious fibrotic change. there was no evidence of pneumocystis jiroveci, cytomegalovirus, or other microbial infection (fig. her condition deteriorated and she died of superimposed vancomycin - resistant enterococcal bacteremia and septic shock on day 24. microscopic examination of the lung (hematoxylin and eosin stain, 40) revealed the airspaces lined by hyaline membranes, with thickened alveolar septa containing inflammatory exudates, compatible with diffuse alveolar damage in the acute exudative stage. patients with ra often have pulmonary involvement, and ild is the primary manifestation. besides the underlying ra - associated ild, the other differential diagnoses include opportunistic infection such as pneumocystis jiroveci or cytomegalovirus infection due to immunosuppressive therapy or drug - induced lung disease by disease - modifying antirheumatic drugs or biologic agent. in our case, the ild may be related with lef, after excluding possible infectious causes and underlying ra - associated ild. the prevalence of ra - associated ild varies according the different diagnostic modalities and is 19% to 56% by the use of high - resolution computed tomography of the chest, which correlates with the histopathological patterns. lee found that usual interstitial pneumonia pattern is the most frequent histopathological pattern in patients with ra - associated ild (56%), followed by nonspecific interstitial pneumonia (33%) and organizing pneumonia (11%). when a total of 56 patients were reviewed from 4 studies, the histopathological patterns included usual interstitial pneumonia pattern in 34 patients, nonspecific interstitial pneumonia in 21 and lymphocytic interstitial pneumonia in another one, with no dad pattern was identified. parambil found 5 patients with ra had dad on surgical lung biopsy during a 7-year period from 1996 to 2002 at mayo clinic, but 1 patient was confounded by lef treatment. to date, dad is still a rare histopathological pattern in ra - associated ild, and the dad pattern seen in our patient is less likely due to the underlying ra - associated ild. lef is an isoxazole immunomodulatory agent that inhibits dihydroorotate dehydrogenase and thus de novo pyrimidine synthesis after being metabolized to its active metabolite a771726, which has a half - life of 2 weeks. lef was launched in the usa in 1998 and in europe the following year as a disease - modifying antirheumatic drug and approved by japanese government for treatment of ra in april 2003. although ild was rarely reported as a complication of lef usage worldwide (0.02%), post - marketing surveillance in japan revealed that, during the first few months after approval, 16 patients developed de novo or exacerbated ild, 9 of whom died. case reports in japan were published successively, and dad was the histopathological pattern in 2 autopsied cases. after that, the study committee for leflunomide - induced lung injury organized by the japan college of rheumatology analyzed the data of post - marketing surveillance obtained from sanofi - aventis japan. during the period from september 2003 to june 2006, 61 (1.2%) of 5054 ra patient receiving lef the duration of lef usage before symptom onset was 138.1 174.7 (61204) days. cholestyramine wash - out therapy, given orally at 8 g 3 times a day, can reduce plasma level by 40% in 24 hours, and to undetectable plasma levels in 11 days. multivariable logistic regression analysis identified pre - existing ild, cigarette smoking, a low body weight (50 kg) and the use of a loading dose as independent risk factors for lef - related ild, among which pre - existing ild was the most important one. a canadian nested case - control study showed that lef increased the risk of ild, but the effect was restricted in subjects with previous mtx use or pre - existing ild. patients with a fatal outcome were more likely to have pre - existing ild, extremely high serum c - reactive protein and low albumin levels, severe hypoxemia and mechanical ventilation and persistent blood lymphopenia throughout the course. based on the findings, the study committee for leflunomide - induced lung injury proposes that lef should not be used in patients with pre - existing ild and a loading dose is not recommended. in vitro studies showed lef could induce epithelial - mesenchymal transition of mouse pulmonary epithelial cell, which subsequently promoted pulmonary fibrosis. the phenomenon occurs in the presence of other fibrosis - inducing stimuli such as bleomycin but does not do this in the absence of these stimuli. this may explain why previous mtx use or pre - existing ild is a risk factor for lef - related ild. outside japan, cases of lep - related ild were reported in korea, canada, new zealand, and australia with similar higher prevalence rate in korea (10/1010 = 1%) than in other countries. thus, racial or genetic difference may further predispose the development of lef - related ild, and taiwanese ra patients may also have higher risk due to similar racial and genetic background. in taiwan, lef was licensed in september 2003 for the treatment of mtx - unresponsive or mtx - intolerable ra patients. theoretically, lef is more likely to be prescribed in ra patients with previous mtx use or pre - existing ild (to avoid mtx exposure), who then have increasing risk for lef - induced ild. in our patient, lef may be a potential cause of dad, supported by the history of previous mtx exposure, lack of infection evidence after extensive survey, and dad pattern on pathological examination. however, due to prolonged lef treatment (3291 days), the causative role of lef in dad in our patient can not be confirmed. nevertheless, our case still hints that lef - related dad may occur even if lef has been prescribed for a prolonged period. when acute ild develops in a ra patient, surgical lung biopsy may be needed early in the course to help diagnosis and prognosis. | abstractpatients with rheumatoid arthritis (ra) often have pulmonary involvement, and interstitial lung disease (ild) is the primary manifestation, in which diffuse alveolar damage (dad) is a rare histopathologic pattern. leflunomide (lef) is a frequently prescribed disease - modifying antirheumatic drug for treating ra. lef - related ild in the form of dad has been reported in patients with ra, with the duration of lef treatment before symptom onset ranging from 6 to 1204 days.we present a case of elderly woman with ra under prolonged lef treatment for > 9 years (3291 days), who had acute respiratory failure with the initial presentation of exertional dyspnea, fever, chills, and productive cough for 2 days. the histopathologic result of surgical lung biopsy was compatible with dad. she was diagnosed as having lef - related ild, based on correlated clinical history, compatible histopathologic examination and excluding possible infection after extensive survey.although the causative role of lef can not be confirmed, this case still hints that lef - related dad may occur even if lef has been prescribed for a prolonged period. |
to report a case of choroidal excavation accompanied by vogt - koyanagi - harada disease (vkh). a 54-year - old japanese woman who was complaining of bilateral blurring of vision associated with headache underwent optical coherence tomography (oct), fluorescein angiography, and indocyanine green angiography as well as a routine ophthalmological examination. oct of the left eye revealed the presence of a unilateral choroidal excavation under the fovea and subretinal fibrin over the site of the excavation. treatment successfully resolved vkh symptoms with gradual resolution of subretinal fibrin and fluid ; however, the choroidal excavation remained. our results suggest that choroidal excavation can be induced by choroidal inflammation caused by vkh. due to the increased availability of optical coherence tomography (oct) imaging, the entity of focal choroidal excavation has gained interest in recent years. it has been well - described in healthy individuals with otherwise normal eyes [1, 2, 3, 4 ]. focal choroidal excavation has been defined as an area of choroidal excavation in the macular area detected with oct without the existence of a posterior staphyloma or scleral ectasia. we report a case of unilateral choroidal excavation in a patient with vogt - koyanagi - harada disease (vkh). a 54-year - old japanese woman presented with bilateral blurring of vision associated with headache that had started 2 weeks earlier. her general family medical history and her personal medical history, including her history of ocular disease, were unremarkable. the best - corrected visual acuity on presentation was 0.6 in the right eye and 0.1 in the left eye. slit - lamp biomicroscopy showed bilateral anterior chamber cells and posterior synechiae, and fundus examination showed bilateral disc swelling with exudative retinal detachment (fig. fluorescein angiography detected bilateral multifocal leakage with pooling of dye in the subretinal space in the late phase (fig. 1d). a spectral - domain oct (cirrus hd - oct 4000 ; carl zeiss meditec inc.) image of the eyes showed the bilateral presence of subretinal fluid and pigment epithelial detachment. moreover, only in the left eye was unilateral choroidal excavation detected under the fovea, and the contour of the retinal pigment epithelium appeared to be conforming to the shape of the fibrin (fig. thus, our patient was treated with intravenous pulse methylprednisolone followed by oral prednisolone (initial dose of 40 mg / day) with tapering over a course of 6 months. treatment successfully resolved the vkh symptoms, and serial oct showed a gradual reduction in size and a subsequent disappearance of the subretinal fibrin and fluid (fig. d). however, 1 month after having started treatment, the eye was left with an area of choroidal excavation at the fovea despite a normal contour in the right eye (fig. 2d). there was a good recovery of visual acuity, with 1.2 and 0.9 in the right and the left eye, respectively. since then, many reports have been published, discussing the association between choroidal excavation and central serous chorioretinopathy [2, 3, 4 ]. however, the current case of choroidal excavation associated with vkh is the first report in which a preexisting choroidal inflammation caused these conditions. concerning the pathogenesis of choroidal excavation in our case, it has been speculated that subretinal fibrin might exert a direct pressure effect on the choroidal layer by disrupting its integrity. our case suggests one of the possible mechanisms of developing choroidal excavation as a result of ocular inflammation. it is likely that the inflammation induced focal choroidal atrophy, and thinning could also contribute to the development of the choroidal excavation. a further study should be warranted to elucidate the clinical course and possible mechanism of choroidal excavation associated with vkh. in conclusion the pathogenesis of choroidal excavation still remains to be addressed ; however, the current case highlights the fact that choroidal excavation can be induced by choroidal inflammation caused by vkh. the authors have no proprietary or commercial interest in any of the material discussed in this article. | purposeto report a case of choroidal excavation accompanied by vogt - koyanagi - harada disease (vkh).methodsa 54-year - old japanese woman who was complaining of bilateral blurring of vision associated with headache underwent optical coherence tomography (oct), fluorescein angiography, and indocyanine green angiography as well as a routine ophthalmological examination.resultsfundoscopy showed papilloedema and serous retinal detachment in both eyes. fluorescein angiography detected bilateral multifocal leakage with pooling of dye in the subretinal space. indocyanine green angiography showed patches of hyperfluorescence and hypofluorescent spots bilaterally. a diagnosis of vkh was reached soon afterwards. oct of the left eye revealed the presence of a unilateral choroidal excavation under the fovea and subretinal fibrin over the site of the excavation. treatment successfully resolved vkh symptoms with gradual resolution of subretinal fibrin and fluid ; however, the choroidal excavation remained.conclusionsthis case is the first report of choroidal excavation associated with vkh. our results suggest that choroidal excavation can be induced by choroidal inflammation caused by vkh. |
the metabolic syndrome (ms) is a complex metabolic disturbance that includes several risk factors for cardiovascular diseases, such as systemic artery hypertension, atherogenic dyslipidemia, insulin resistance (with or without glucose intolerance) and prothrombotic and proinflammatory tendencies. the syndrome is due to a complex interaction between genetic factors, poor dietary habits and physical inactivity. population studies reported by the national health and nutrition examination survey (nhanes) have shown that the ms, as defined by the international diabetes federation (idf), has a prevalence of 39% among the us adult population. the estimated prevalence of ms in the brazilian population is 29.8% (ci95 = 28 - 32%) and prevalence increased from the youngest (26 - 34 y) to the oldest (55 - 64 y) group (15.8% and 48.3%, respectively). there are scientific evidences showing that some lifestyle behaviors are associated to the risk of having the ms : lack of physical activity, smoking and poor dietary habits, involving particularly excessive intake of carbohydrates and lipids. high carbohydrate ingestion has been correlated with low levels of hdl cholesterol and increased concentration of triglycerides, whereas increased intake of saturated fat associates with hyperinsulinemia. nevertheless, the precise role of dietary habits in the etiology of the ms is still poorly understood. investigations have been restricted to a few observational studies in adults, not particularly in the elderly population. the present study investigated the influence of nutritional components on the prevalence of ms in a population of elderly women, in the city of passo fundo, brazil. the aim was to contribute for the establishment of the role of dietary habits in the risk for the metabolic syndrome among the elderly. the population studied was composed of 284 elderly women, aging 60 years or over, socially active and participating of a third age group for recreational activities (diviso de apoio ao idoso) supported by the city hall of passo fundo, south brazil. at the time of this study, there were 1204 participants and the elders were invited to take part of the study. this study was approved by the research ethics committee of pontifical catholic university of rio grande do sul, brazil, and all participants signed an informed consent form. all questionnaires were recorded by trained dieticians. besides recording the food ingested during the day, information was collected on the specific characteristics of each meal, such as the way they were prepared and stuffed, use of sugar or sweetener. to reduce the possibility of errors in the research, particularly relating to the representativeness of the dietary habits recorded, soon after the interview the participants were asked if the reported food ingestion represented a usual pattern. in cases in that answers did not match, the participant was asked to provide another dietary recall, so that the usual dietary ingestion could be recorded. nutritional calculations were performed with the dietwim professional software version 2.0 (porto alegre, rs, brazil). nutritional variables selected for this study included total calories, carbohydrates, proteins, lipids, sodium, potassium, magnesium, calcium, zinc and vitamins (a, d, e, c, b6, b9 and b12). the educational level was classified in three categories (8 years of formal study). physical activity was categorized according to type, duration and periodicity, into four groups (4x / week). according to smoking habits, the participants were classified as smokers, ex - smokers (if quitting smoke for more than two years) and non - smokers. for measurement of weight (kg) and height (m), performed in an electronic anthropometric scale validated with a fixed stadiometer, individuals were barefoot and wearing light clothes. waist circumference was measured midway between the iliac crest and the lower rib, with relaxed breathing, at approximately 0.1 cm above the umbilicus. body mass index (bmi) was determined by weight divided by the square height. blood pressure was measured with an aneroid sphygmomanometer, validated by an institution affiliated to inmetro (the brazilian regulatory agency). the mean of two measurements was used to express the systolic and diastolic blood pressures. hypertension was defined as systolic pressure 140 mmhg and/or diastolic pressure 90 mmhg, or by the use of antihypertensive medications. analyses were done through enzymatic colorimetric method with commercial kits for total cholesterol (cod- ana labtest), hdl cholesterol (precipitating hdl - c, labtest), triglycerides (tg) (gpo - ana) and glucose (pap labtest). ldl cholesterol was calculated with the friedewald equation in individual with tg levels 8 years of formal study). physical activity was categorized according to type, duration and periodicity, into four groups (4x / week). according to smoking habits, the participants were classified as smokers, ex - smokers (if quitting smoke for more than two years) and non - smokers. for measurement of weight (kg) and height (m), performed in an electronic anthropometric scale waist circumference was measured midway between the iliac crest and the lower rib, with relaxed breathing, at approximately 0.1 cm above the umbilicus. body mass index (bmi) was determined by weight divided by the square height. blood pressure was measured with an aneroid sphygmomanometer, validated by an institution affiliated to inmetro (the brazilian regulatory agency). the mean of two measurements was used to express the systolic and diastolic blood pressures. hypertension was defined as systolic pressure 140 mmhg and/or diastolic pressure 90 mmhg, or by the use of antihypertensive medications. analyses were done through enzymatic colorimetric method with commercial kits for total cholesterol (cod- ana labtest), hdl cholesterol (precipitating hdl - c, labtest), triglycerides (tg) (gpo - ana) and glucose (pap labtest). ldl cholesterol was calculated with the friedewald equation in individual with tg levels 8 years of formal study). physical activity was categorized according to type, duration and periodicity, into four groups (4x / week). according to smoking habits, the participants were classified as smokers, ex - smokers (if quitting smoke for more than two years) and non - smokers. for measurement of weight (kg) and height (m), performed in an electronic anthropometric scale validated with a fixed stadiometer, individuals were barefoot and wearing light clothes. waist circumference was measured midway between the iliac crest and the lower rib, with relaxed breathing, at approximately 0.1 cm above the umbilicus. body mass index (bmi) was determined by weight divided by the square height. blood pressure was measured with an aneroid sphygmomanometer, validated by an institution affiliated to inmetro (the brazilian regulatory agency). the mean of two measurements was used to express the systolic and diastolic blood pressures. hypertension was defined as systolic pressure 140 mmhg and/or diastolic pressure 90 mmhg, or by the use of antihypertensive medications. analyses were done through enzymatic colorimetric method with commercial kits for total cholesterol (cod- ana labtest), hdl cholesterol (precipitating hdl - c, labtest), triglycerides (tg) (gpo - ana) and glucose (pap labtest). ldl cholesterol was calculated with the friedewald equation in individual with tg levels < 400 mg / dl, (ldl - c = (tg) - total cholesterol hdl - c - tg/5). the metabolic syndrome was defined according to criteria proposed by the international diabetes federation (idf), which include altered abdominal circumference and two or more of the following components : plasma lipid levels 100 mg / dl ; systolic blood pressure 130 mmhg or diastolic blood pressure 85 mmhg (or the use of antihypertensive medication) ; hdl cholesterol < 50 mg / dl for women ; triglycerides 150 mg / dl. since the population analyzed had european origin, the cutoff for waist circumference used in this study was 80 cm. quantitative data are presented as mean and standard deviation, and categorical data, as frequency and percentages. the chi - squared test and variance analysis (anova) the groups with or without metabolic syndrome were compared for macro- and micronutrients intake, with adjustment of all nutrient values by total energy intake using the residue method. this method also allows the establishment of a regression equation between nutrient intake (as dependent variable) and total caloric intake (as independent variable). the presence of ms considering the quartile of nutrients was evaluated by the multiple logistic regression model with ors adjusted for age, smoking, physical activity, educational level, total energy intake and fiber contents of the diet. data were analyzed with the spss software version 11.5 (spss chicago, il). the sample included 284 women. according to the idf definition 91 (32% of the women) presented ms and 193 (68%) did not. women with the ms presented anthropometric measurements such as abdominal circumference and bmi higher than those without the ms (p<0.005 and p<0.04, respectively). biochemical analyses of total cholesterol, ldl cholesterol, triglycerides, hdl cholesterol and glucose also had less favorable results among women with the ms. characteristics of the study participants total energy intake and intake of all macronutrients and several micronutrients, adjusted or not adjusted for total energy intake, were not significantly different among elderly women with or without the metabolic syndrome (tables 2 and 3). daily intake energy and selected nutrients of the study participants daily intake energy and selected nutrients of the study participants the risk for the metabolic syndrome was not statistically different in relation to quartiles of intake of all macronutrients and several diet micronutrients adjusted for total energy intake, age, smoking, educational level, physical activity and fibers in the diet (tables 4 and 5). ors of metabolic syndrome according to quartiles of total energy intake ors of metabolic syndrome according to quartiles of total energy intake in this study, we evaluated the relationship between dietary components and the metabolic syndrome in the elder women and found no associations between the syndrome and intake of all macronutrients and several micronutrients. the prevalence of the ms defined according to idf criteria in the sample of 284 elderly women with mean age of 68.9 years was 32%, similar to results in industrialized countries such as the usa (22.8% to 39.0%). the etiology of the ms is still poorly understood, but it seems to be due to a complex interaction among genetic and environmental factors, which include dietary habits. recent studies have shown that increased total carbohydrate intake may result in altered levels of plasma lipids, lipoprotein concentration and glucose metabolism, leading to the establishment of the ms in some individuals. although dietary factors are associated to individual components of the ms, such as diabetes mellitus type 2, blood hypertension, dyslipidemia and obesity, their role in the etiopathogeny of the combination of these components - the metabolic syndrome - is not well known. some studies have investigated this question in adult populations, observing some associations between dietary components and the ms. no association was observed between total carbohydrate intake and fiber contents of the diet and the ms. similar results were reported by mc keown, who however observed a relationship between the intake of integral grains and a low prevalence of the ms. since the present study investigated the association between the ms and nutrients, but not specific types of meals, we could not corroborate those findings. interestingly, individuals with or without the metabolic syndrome presented similar intake of carbohydrate, which was higher than the recommended dietary allowances (rdis), suggesting some type of gene - nutrition interaction. concerning lipids, some studies have shown an association between the intake of linoleic acid and reduced risk for the ms. a similar association was reported by freire and colleagues, who also suggested that total dietary fat may increase the risk for the ms. in humans, a detrimental role for saturated fat and a beneficial effect of polyunsaturated fats are still under debate. the present study did not find any association ; both groups presented similar levels of intake of total lipids and linoleic acid, which was in agreement with nutritional recommendations. in this study, the intake of antioxidant vitamins (a, e, c) by individuals with or without the ms was also investigated. epidemiological evidences show that antioxidants have a protective role in the physiopathology of several diseases. ford (2003) examined the plasma concentration of antioxidants and their dietary ingestion. the results showed an association between the presence of ms and a low plasma concentration of antioxidants, but not with the intake of vitamins c, e and a. the results of this study corroborate these findings, since no relationship was observed between the ms and ingestion of vitamins c, e and a. the plasma concentration of these vitamins was not analyzed. some studies have shown an inverse association between the serum concentration of vitamin d and the prevalence of the metabolic syndrome. low serum concentrations of vitamin d are associated to dysfunction ofpancreas cells and deficient secretion and activity of insulin. in a study with 126 individuals, those with hypovitaminosis d presented a 3-fold higher probability to have ms than individuals with normal concentrations of vitamin d. in another study, by liu (2005), no significant associations were seen between the intake of vitamin d and the prevalence of ms, but a relationship between the ingestion of calcium and milk products and low prevalence of the ms was observed. the present study did not observe associations between the ingestion of calcium and vitamin d and the prevalence of the metabolic syndrome. several studies have shown a positive effect of magnesium in several mechanisms involved with the lipidic metabolism, such as glucose intolerance and insulin homeostasis. song (2005) showed an inverse association between the intake of magnesium and prevalence of the ms. the results of this study did not show a significant relationship between magnesium ingestion and the ms. according to most literature reports, the prevalence of ms is not associated with macronutrients and micronutrients present in the diet, but with the type of food ingested. the results of the present study did not disagree with other studies evaluating the relationship between dietary components and the prevalence of the metabolic syndrome among younger adults. the absence of a relationship between dietary components and the ms in older individuals, shown by these results, may be due to particular characteristics of this age group, which had not been previously investigated. it is possible that the nutritional factors associated to ms have an influence in younger ages (even since intrauterine life, as suggested by some studies), but not in more advanced age as in older individuals. as suggested by barker (1989), environmental factors and particularly nutrition may exert their effects in earlier phases of the development, when they become associated to risks for early onset of cardiovascular disease and metabolic alterations, or premature death (young adults). according to this hypothesis, alterations in nutrition and in the endocrine situation during fetal life result in developmental adaptations which permanently modify structure, physiology and metabolism later in life. it is also possible that the lack of association between nutritional factors and the ms, observed in the present study, is due to a modification induced in the dietary habits of the participants by previous medical recommendation, motivated by the risk of diseases such as the ms, but which have not been able to prevent the establishment of the syndrome. the method used in this work to analyze dietary habits, the 24-hour dietary recall, may be a limiting factor for not representing the global diet of the participants. the results of this study suggest that dietary factors do not have an important influence on the risk for the metabolic syndrome in elderly women, and that the condition is due to more a complex interaction of other factors such as lifestyle, genetic susceptibility and metabolism. multidimensional, longitudinal prospective studies involving groups in different ages, and with the investigation of several aspects such as nutritional habits, lifestyle, genetic factors and metabolism, are necessary for a better understanding of the etiology of the metabolic syndrome. | background : the metabolic syndrome is a complex metabolic disturbance due to an interaction between genetic factors, poor dietary habits and physical inactivity.aims:to investigate the role of dietary intake on the prevalence of the metabolic syndrome in a population of elderly, socially active women in brazil.patients and methods : a total of 284 women with mean age 69.3 6.3 years were evaluated in a cross - sectional retrospective study. the metabolic syndrome was diagnosed according to criteria of the international diabetes federation. the dietary intake was evaluated through a questionnaire for 24-hour dietary recall. the groups with or without the metabolic syndrome were compared for dietary intake and risk factors for metabolic syndrome by the multiple regression model adjusted for age, smoking, physical activity, educational level, total energy intake and fiber contents of the diet. the odds ratio for the presence of the metabolic syndrome was calculated for each nutrient by quartile for total energy intake adjusted by the residue method.results:the prevalence of metabolic syndrome was 32% in the sample. there was not found any association between dietary intake, including all macronutrients and several micronutrients, and the presence of metabolic syndrome.conclusion:no associations were observed between nutritional factors and the prevalence of the metabolic syndrome in elderly women, a result possibly due to the fact that these factors have an influence in earlier phases of life, or to a recent modification of dietary habits, which however was not able to prevent the establishment of the syndrome. |
many headache sufferers complaint that headache leads to sleep disruption in them while on the other hand, inadequate sleep leads to increased frequency, severity, and chronicity of headache. this relationship is well documented in primary headache disorders, particularly in chronic headache sufferers as compared to episodic variety. chronic daily headache (cdh) comprises a group of headache disorders occurring at least 15 days per month for 3 or more consecutive months. around 5% of the normal population suffers from cdh. among the many sleep disorders described in the international classification of sleep disorders, insomnia, daytime sleepiness, and obstructive sleep apnea including snoring insomnia had been suggested as an independent risk factor for chronification of headache. increased snoring incidence had been observed in chronic headache sufferers compared to episodic ones. sleep disordered breathing was found to be associated with migraine but not with tension headache. these disorders are commonly left under diagnosed and when treated helps in better control of headache. polysomnography (psg) is used for objectively assessing sleep macro- and micro - architecture and aids in detecting sleep disorders. the majority of the earlier studies had studied migraine population, few had studied tension - type headache, and very few both. we performed the study to evaluate subjectively and objectively sleep disorders in patients of primary cdh. a secondary subgroup analysis was performed in between chronic migraine (cm) and chronic tension - type headache (ctth) patients and polysomnographic variables among them were compared. this was a prospective, hospital - based observational study carried out at our tertiary care hospital over a span of 2 years from october 2013 to december 2015. 83 patients of primary cdh were selected from patients attending outpatient department (opd). these patients were evaluated by history, clinical examination, sleep questionnaires, and overnight psg study. clinical details regarding total disease duration, headache type, location, character, duration of attack, and severity were assessed. the epworth sleepiness scale (ess) was used for assessment of excessive daytime sleepiness, and a score of 10 or more was considered abnormal. sum of individual 's response in eight different situations over a scale of 03 is total score. each patient was asked about difficulty falling asleep, intermittent night arousals, and early morning awakenings. the patient was asked to respond according to usual perceivement felt on most of the days (4) of week. any positive reply was given a score of 1, making total score range of 03. all the patients were explained prior about the procedure of study and risks involved. the patient was asked to report in the night after washing hairs that morning. the overnight recording was performed using somnologica software (embla systems, broomfield, co, usa). the recording was continued till the patient wakes up which is lights on time. the recording analysis was done according to the american academy of sleep medicine guidelines 2007. the characteristics noted were time in bed, total sleep time, sleep efficiency, sleep onset latency, duration of sleep stages, apnea hypopnea index (ahi), periodic limb movement index (plmi), isolated limb movement index (ilmi), number of awakenings, oxygen saturation, and heart rate. statistical analysis was done using spss software of windows version 16.0 (chicago, il, usa). all categorical variables were expressed as percentages, and continuous variables were expressed as mean standard deviation. clinical details regarding total disease duration, headache type, location, character, duration of attack, and severity were assessed. the epworth sleepiness scale (ess) was used for assessment of excessive daytime sleepiness, and a score of 10 or more was considered abnormal. sum of individual 's response in eight different situations over a scale of 03 is total score. each patient was asked about difficulty falling asleep, intermittent night arousals, and early morning awakenings. the patient was asked to respond according to usual perceivement felt on most of the days (4) of week. any positive reply was given a score of 1, making total score range of 03. the overnight recording was performed using somnologica software (embla systems, broomfield, co, usa). the recording was continued till the patient wakes up which is lights on time. the recording analysis was done according to the american academy of sleep medicine guidelines 2007. the characteristics noted were time in bed, total sleep time, sleep efficiency, sleep onset latency, duration of sleep stages, apnea hypopnea index (ahi), periodic limb movement index (plmi), isolated limb movement index (ilmi), number of awakenings, oxygen saturation, and heart rate. statistical analysis was done using spss software of windows version 16.0 (chicago, il, usa). all categorical variables were expressed as percentages, and continuous variables were expressed as mean standard deviation. female patients were more than male (female = 69.9%, male = 30.1%). the mean duration of headache of all patients was 24.99 (20.72) months, with a range of 4144 months. out of total 83 patients, 50 (60.2%) were diagnosed as ctth, 31 (37.3%) as cm, and only 2 (2.4%) were of chronic cluster headache (cch). the mean iss and ess score of all patients were 0.73 (0.9) and 5.35 (3.23), respectively. both cm and ctth patients had complaints of insomnia (14 vs. 20, p = 0.65) and daytime sleepiness (1 vs. 20, p 30 min) were comparable into two groups. demographic, headache, and sleep - related data including questionnaire for all patients : counts or mean (standard deviation) table 2 summarizes the comparison of polysomnographic parameters among cm and chronic tension - type headache. total sleep time, sleep efficiency, stage n3 sleep, and rapid eye movement sleep was prolonged in ctth group though not statistically significant. sleep onset latency, awakenings per hour, and wake time was more in cm group but not statistically significant. ilmi, plmi, ahi, desaturation index, and heart rate were comparable among the two groups. comparison of polysomnographic parameters among chronic migraine and chronic tension - type headache patients : mean (standard deviation) table 3 summarizes the psg parameters comparison among those complaining of sleep disturbance with those having no complaints. total sleep time, sleep efficiency, stage n3 sleep, and sleep onset latency were comparable statistically among the groups along with ilmi, plmi, ahi, heart rate, and o2 saturation. comparison of polysomnographic parameters among patients with / without symptom of sleep disturbances : mean (standard deviation) we studied 83 cases of cdh subjectively by history and questionnaire while psg provided objective sleep parameters. among those, more than half were of tension - type and females being more commonly affected which is similar to general population data. nearly, half complained of sleep disturbances, insomnia being more common than excessive daytime sleepiness. both iss and ess score were more in ctth group with ess score statistically significant. sleep diaries revealed comparable average sleep duration and psg showed comparable total sleep time and sleep onset latencies into two groups. it is well known that chronic insomniacs not always have poor eeg sleep. in psg, ctth had more stage n3 sleep with lesser awakenings that is better sleep quality in comparison to cm group. as de gennaro. in his study showed decreased arousals with increased slow wave sleep after sleep deprivation, our psg findings could indicate more of sleep deprivation among chronic tension - type headache compared to cm. various psg parameters such as ilmi, plmi, ahi, and desaturation index did not differ significantly. we did not have control group in our study, but that is also the strength of study, for the first time a study comparing two major divisions of cdh had been performed. second, we did not have follow - up of patients. a study design with repeating psgs on follow - up our study group was population attending hospital opd, thus more of chronic and severe sufferers. calhoun and ford in his study, over migraine patients found that patients who received behavioral sleep therapy are more likely to revert to episodic migraine. thus, earlier evaluation and timely management of these may help in better control of headache, improving its outcome, and possibly preventing chronification. | objective : studies related to sleep disorders and polysomnography (psg) among chronic daily headache patients are rare. we studied this and compared chronic migraine (cm) with chronic tension - type headache.methods:eighty-three patients were recruited. they were evaluated by semi - structured interview, headache, and sleep diaries along with epworth sleepiness scale score and insomnia symptom score. overnight psg was performed and data compared.results:chronic tension - type headache was more common than cm, both having female preponderance. insomnia followed by excessive daytime sleepiness was prevalent sleep disorder. sleep efficiency and stage 3 sleep were lower in cm compared to chronic tension - type. esss was significantly increased among chronic tension - type patients. no significant correlation was found among psg parameters in patients with or without sleep disorders.conclusion:insomnia being most common sleep disorder among chronic headache population. chronic tension - type headache had slightly better slow - wave sleep than cm and significantly increased daytime sleepiness. |
osteoporosis associated with hemochromatosis in men was first described by delbarre in 1960 and confirmed by pawlotsky. in 1979. hemochromatosis due to excess tissue iron storage is frequently due to a homozygous mutation, cysteine to tyrosine, c282y, in the hfe gene. the prevalence of osteoporosis in hemochromatosis and the pathogenic mechanisms involved are not completely understood, for example, whether there is a relationship between the serum ferritin and bone mineral density. in men with hemochromatosis and a serum ferritin between 350 and 8410 g / l, the presence of osteoporosis was associated with a lower serum free testosterone. however, in men with the homozygous hfe mutation, the serum ferritin and the serum testosterone did not differ between those with and without osteoporosis. most osteoporotic men were not hypogonadal (76.9%). interestingly, in men heterozygous for the hfe mutation, c282y, but without iron overload, liver disease was associated with a lower bone mineral density in men of all ages and in women older than 60 years. in a three - year longitudinal study of postmenopausal women, however, in postmenopausal women a higher serum ferritin was not correlated with a lower bone mass [8, 9 ]. in postmenopausal women with and without osteoporosis the serum ferritin was not significantly different between the two groups, but in another study of postmenopausal women, mean age 73 years, who had sustained a hip fracture, those who had a serum ferritin above 150 g / l had a lower bone density than those in whom the serum ferritin was less than 150 g / l. in a study that looked at transiliac biopsy samples the concentration of iron in the cortical bone was greater in osteoporotic participants than in nonosteoporotic participants (p 0.01). in a rat study, ovariectomy was associated with an increased concentration of iron in bone and the loss of bone mass and the deterioration of bone microarchitecture was ameliorated by oral treatment with an iron chelator. in postmenopausal women, the serum ferritin was higher, 71 g / l, than in premenopausal women, 37 g / l. a question arises as to what happens to the serum ferritin in postmenopausal women with the heterozygous c282y mutation in the hfe gene and whether the serum ferritin is related to bone mineral density in this group of women. the serum ferritin levels were not different between those heterozygous for the c282y hfe mutation and those without the mutation between the ages of 20 and 70. in women aged 5079 the geometric mean for the serum ferritin was 87 g / l. in postmenopausal women aged 5464 there was a slight increase in the serum ferritin in women heterozygous for the hfe mutation, 113.5 g / l (median), compared to 101.0 g / l (median) in those without the mutation. in another study in women aged 6190 with the heterozygous c282y hfe mutation the serum ferritin was significantly higher than in women without the mutation (geometric mean 79 g / l compared to 48 the 95% confidence interval for serum ferritin in women with the heterozygous c282y hfe mutation was 6198 g / relatively small increases in the serum ferritin in postmenopausal women may be associated with an increased rate of loss of bone mineral density. with this in mind we studied bone mineral density in postmenopausal women heterozygous for the c282y hfe mutation. all postmenopausal women in our institution who had had an hfe gene analysis between 1999 and 2012 were identified. women homozygous for the c282y mutation and women who were either homozygous or heterozygous for the h63d (his asp at residue 63) mutation were excluded. women with celiac disease, hyperparathyroidism, or hyperthyroidism were also excluded. at the same time, the serum iron and serum ferritin were measured and liver function tests were carried out. between 1999 and 23 march 2001, the serum ferritin was measured by the roche immunoturbidimetric assay method (reference range 15150 g / l), and between 23 march 2001 and 26 october 2012, roche e170 chemiluminescent assay was used (reference range 15400 g / l). between 1999 and 23 march 2001 the serum iron was measured by roche hitachi 747 method (reference range 830 m / l) and between 23 march 2001 and 26 october 2012 roche modular was employed (reference range 830 m / l). of the 137 postmenopausal women who were heterozygous for c282y, 26 also had bone density measurements of the lumbar spine (l2l4) and femoral neck. the measurements were made on a norland xr26 bone densitometer and converted to hologic equivalents using the formula of genant.. using the data from the national health and nutritional examination survey 20052008 for white non - hispanic women, z - scores for the lumbar spine and femoral neck bone densities were calculated to determine if the age matched bone density measurements (z - scores) were significantly different on average from the general population. bone mineral density measurements in australian women were previously found to be very similar to those of women in the usa using nhanes data. the relationship between the serum iron and serum ferritin and bone mineral density at the lumber spine and femoral neck was also studied. two - sided t - tests were used to compare z - scores with z - scores for the general population. the pearson correlation test was used to study the relationship between the serum iron and serum ferritin and bone mineral density unless otherwise specified, the data are presented as mean standard deviation (sd). the study was approved by the northern sydney local health district human research ethics committee (nslhd hrec). because this was a retrospective study of data already accumulated by treating clinicians as part of general medical care, informed consent from individual participants the age of the study participants ranged between 48 and 81 years (average 62.3 9.3 sd). women over the age of 60 or in whom one year had elapsed since the last period were assumed to be menopausal. those under the age of 60 not receiving oestrogen replacement had serum oestradiol levels of 38 pmol / l, 39 pmol / l, and 45 pmol / l, within the menopausal range. of the 26 women, 6 were currently receiving estrogen therapy, 3 had previously received estrogen therapy, and 8 were currently on bisphosphonate therapy (alendronate, risedronate, or etidronate). one participant had received regular venesection because of raised serum iron and raised iron binding saturation. three women with significant liver function abnormality and two who were currently receiving prednisone were excluded from the study. finally 21 women were included in the analysis. the z - scores for bone density at the lumbar spine and femoral neck are shown in table 1. the average z - score for the lumbar spine, l2l4, was 0.44 0.77 and was significantly different from 0 (p = 0.016). the average z - score for the femoral neck was 0.19 0.69, not significantly different from 0 (p = 0.221) (tables 2 and 3). the correlation coefficients relating the serum ferritin to the bone mineral density of the lumbar spine, l2l4, and femoral neck were not significant for the lumbar spine (r = 0.019, p = 0.936) or for the femoral neck (r = 0.093, p = 0.688) (table 4). the correlation coefficients relating the serum iron to the bone mineral density of the lumbar spine and femoral neck were not significant for the lumbar spine (r = 0.236, p = 0.303) but were significant for the femoral neck (r = 0.436, p = 0.048) (table 4). in this study, women receiving estrogen replacement or bisphosphonate therapy were included in the analysis. therapy with either of these agents would have a tendency to increase bone mineral density so that any reduction in bone density is likely to have been greater before therapy. the link between excess tissue iron stores and osteoporosis was first noted by delbarre in 1960. excess tissue iron accumulation as reflected in an elevated serum ferritin is frequently seen in persons homozygous for the c282y hfe mutation and many of these patients may develop osteoporosis. additional risk factors for the development of osteoporosis are hypogonadism in men and chronic liver disease in men and women. the question arises as to whether milder degrees of tissue iron storage are associated with reduced bone density. in a group of postmenopausal women, the serum ferritin in those with osteoporosis was not different from those who were not osteoporotic. also in postmenopausal women a higher serum ferritin was not correlated with a lower bone mass [8, 9 ]. however, in a longitudinal study over 3 years in postmenopausal women, the serum ferritin was correlated with an increased loss of bone mineral density in the femoral neck with serum ferritin levels within the normal range, and in another study of postmenopausal women, mean age 73 years, who had sustained a hip fracture, those with a serum ferritin above 150 g / l had a lower bone density than those in whom the serum ferritin was less than 150 g / l. in the present study of postmenopausal women heterozygous for the c282y hfe mutation, the spinal bone mineral density of l2l4 was significantly less than that of the average population mean corrected for age. the femoral neck z - scores for bone mineral density were not significantly lower than the average population mean. the serum ferritin was not correlated with the bone mineral density at the lumber spine or the femoral neck, a finding similar to that found in postmenopausal women studied by lee. the average serum ferritin in the present study was 220.1 g / l, considerably higher than that found in the study of kim. and higher than average serum ferritin levels of 64 g / l found in women with average age 52.8 years heterozygous for the c282y hfe mutation in the study of rossi.. in another study of women aged 6190 with the heterozygous c282y hfe mutation the serum ferritin was 79 hfe simple heterozygotes followed over 12 years the serum ferritin levels of postmenopausal women did not change significantly and were not significantly different compared to the serum ferritin levels of postmenopausal women without the c282y hfe mutation. in a population study of australian women greater than 50 years the median serum ferritin was 80.5. a further study of postmenopausal women with osteoporosis found no difference in the serum ferritin between those with and without osteoporosis but the serum iron was slightly lower in those with osteoporosis (p = 0.047). this was linked to a variation in the haptoglobin molecule which binds heme more avidly and reduces the amount of free iron available to tissues and which protected against fracture in postmenopausal women with osteoporosis. excess tissue iron has been implicated in impairment of bone metabolism. in the zebra fish exposure to ferric iron increased the expression of tracp-5b of osteoclast origin and treatment with deferoxamine increased bone mineralization. in mice homozygous for the c282y hfe mutation excess iron was seen on the surfaces of bone trabeculae and there was a decrease in the number and thickness of trabeculae. there was no change in osteoblast surface but osteoclast numbers were increased. in a rat study, ovariectomy was associated with an increased concentration of iron in bone and the loss of bone mass and the deterioration of bone microarchitecture was ameliorated by oral treatment with an iron chelator. the serum ferritin was not measured in this study. in a study which looked at transiliac bone biopsy samples, the concentration of iron in cortical bone was greater in osteoporotic participants than in nonosteoporotic participants. bmp-2 increased hepcidin mrna expression in liver cells and this was enhanced by hemojuvelin which acts as a coreceptor for bmp-2 and which was independent of the effect of hfe. bmp-2 has a role in the differentiation and function of osteoblasts in bone so that diminished function may reduce bone mass and diminish hepatic hepcidin secretion. serum hepcidin was not measured in the present study. in a mouse study using c2c12 preosteoblasts in culture, increased iron and ferritin in the cell culture media in combination with low estradiol levels inhibited the effect of bmp-2 on cell differentiation. also mice heterozygous for the hfe mutation had lower femoral bone mineral density compared to wild type mice. the present study is the first to examine bone mineral density in postmenopausal women heterozygous for the c282y hfe mutation which showed a significant reduction in the lumbar spine bone mineral density when compared to age matched community controls. however, there are several important limitations in this study which must be regarded only as exploratory. firstly, the c282y hfe heterozygotes were identified from an already established data base and it is not clear why in individual cases the gene analysis was done in the first place. because the mean serum ferritin was 220.1 g / l, higher than the average values expected in postmenopausal women, it is likely that the hfe mutation was sought following the finding of a higher than average serum ferritin. also it is likely that the hfe analysis was carried out in relatives of known c282y hfe homozygotes. if the hfe mutation was sought specifically in postmenopausal women with known low bone mineral density then this would introduce substantial bias. women receiving estrogen or bisphosphonates were included in the analysis even though these medications were likely to have increased the bone mineral density and make it less likely to detect a significant reduction in age matched bone mineral density. even so, despite the inclusion of these participants, in this small number of 21 postmenopausal women there was a highly significant (p = 0.016) reduction in the l2l4 bone mineral density compared to age matched controls, strengthening the concept that c282y hfe heterozygosity is associated with reduced bone mineral density in the lumbar spine. nevertheless, in order to confirm or refute the results of the present study, postmenopausal women selected randomly from the population at large should have measures of bone mineral density, serum iron, and serum ferritin and also studies for the heterozygous c282y hfe mutation so that unbiased comparisons could be made between those with and those without the mutation. it is not known if there is a threshold for serum ferritin above which there is an impairment of bone formation or an increase in bone resorption or if c282y hfe heterozygotes who are osteoporotic respond to therapy designed to increase bone mineral density in the same way as persons without the mutation. g / l in a separate study was associated with a significant increase in the bone mineral density of the lumbar spine and the femoral neck after one year, additional studies are required to determine at what levels of serum ferritin venesection may be helpful in increasing bone mineral density in postmenopausal women with osteoporosis and mild increases in the serum ferritin. in the present study, the serum ferritin in c282y hfe heterozygotes ranged between 12 and 543 g / l. three patients were excluded from analysis because abnormal liver function tests had serum ferritin levels of 540, 595, and 634 g / l. in conclusion, in the present study, c282y hfe heterozygous postmenopausal women were shown to have significantly lower bone mineral density of the lumber spine, l2l4, compared to age matched community controls. in this small study the serum ferritin was not correlated with measures of bone mineral density in the spine or femoral neck. | mutations in the hfe gene may be associated with increased tissue iron stores reflected in an elevated serum ferritin. with homozygous mutation c282y, the increase in serum ferritin may be associated with tissue damage in the liver, pancreas, and pituitary and with a reduced bone mineral density. with heterozygous mutation c282y, the degree of iron retention is less but information relating to how a heterozygous c282y mutation might impact bone mineral density is uncertain. the present study was undertaken to study the relationships between bone mineral density measured by dual energy x - ray absorptiometry and the serum ferritin and serum iron in postmenopausal women heterozygous for the c282y mutation. the spinal bone mineral density, l24, was significantly less than age matched community controls (p = 0.016). there was no significant change in the femoral neck bone mineral density compared to age matched community controls. the correlation between the spinal bone mineral density, l24, the femoral neck bone mineral density, and the serum ferritin was not significant. the serum iron correlated significantly inversely with the femoral neck bone mineral density (p = 0.048). the heterozygous c282y mutation may be associated with impairment of bone cell function in postmenopausal women when only small increases in the serum iron or serum ferritin have occurred. |
osteoarthritis of the knee is a common problem in the elderly patients with a lifetime risk of symptomatic knee osteoarthritis approaching 45%. intraarticular corticosteroid injection is a common conservative management modality in treatment of osteoarthritis of knee. bellamy. in 2008 showed a well - established short - term benefit with intraarticular corticosteroid injection in treatment of knee osteoarthritis with some side - effects. the incidence of septic arthritis of knee after intraarticular corticosteroid injection ranges from one in 3000 to one in 50,000 and may be higher in immunocompromised patients. the treatment of iatrogenic septic arthritis necessitates multiple joint washout and debridement, long - term antibiotic therapy and prolonged inpatient hospital stay. a higher rate of infectious complications following intraarticular injection can be expected in immunocompromised patients. we would like to report an extremely rare case of simultaneous bilateral septic arthritis of knee as a result of intraarticular corticosteroid injection to both knees in a rheumatoid arthritis patient with end stage renal disease on dialysis. to the best our knowledge, a 66-year - old male patient was admitted to hospital under the medical team for investigation of recent unexplained weight loss of approximately one stone in 6 months. patient had severe bilateral arthritis of both knees that was being treated conservatively by the rheumatologist. as a part of his conservative management, the patient received bilateral knee intraarticular corticosteroid injections. a combination of 80 mg of methylprednisolone and 8 ml of 1% lignocaine was injected into each knee under aseptic precautions. after 6 days, the patient complained of severe bilateral knee pain and was unable to weight bear. after 2 days, his both knees were aspirated revealing purulent fluid, which was sent to microbiology for culture and sensitivity. he was referred to the orthopedic service for review. on examination, he was febrile with a temperature of 38.2c and tachycardic examination of both knees revealed local increased temperature, tenderness on palpation of the patello - femoral joint, medial and lateral joint line. a large effusion was noted in both knees ; left knee more than the right with a painful decreased range of movements in both knees. the inflammatory markers were raised with a white cell count of 35.19 10/l, neutrophil count of 33.27 10/l, erythrocyte sedimentation rate 159 mm at the end of 1 h and c - reactive protein 465. antero - posterior view [figure 1 ] and lateral views [figures 2 and 3 ] of bilateral knee radiographs showed advance osteoarthritis in both knees with large effusions. history, clinical examination and laboratory findings were highly suggestive of simultaneous bilateral septic arthritis of the knees secondary to intraarticular steroid injections. patient was taken to the operating theatre for arthroscopic washout and debridement of both knees. 270 ml of frank pus was drained from left knee while 120 ml drained from right knee. a thorough debridement was performed and both knee joints were washed out with 6 l of normal saline. the fluid from both knees and tissue samples taken were sent for culture and sensitivity. patient was commenced empirically on intravenous flucloxacillin 2 g 4 times daily and fucidic acid 500 mg twice - a - day. culture and sensitivity revealed staphylococcus aureus, which was sensitive to flucloxacillin. 6 weeks post - operatively white cell count and inflammatory markers had normalized and articular symptoms had resolved. anteroposterior radiograph of bilateral knee showing advance osteoarthritis in both knees with large effusions lateral radiograph of left knee showing advance osteoarthritis in the knee with large effusion lateral radiograph of right knee showing advance osteoarthritis in the knee with large effusion bellamy. in 2008 showed a well - established short - term benefit with intraarticular corticosteroid injection in the treatment of knee osteoarthritis with some side - effects. septic arthritis in rheumatoid patients has worse outcomes when compared with normal people primarily because of the delay in diagnosis. this delay in diagnosis occurs because the painful and hot joints can be often confused with exacerbations of rheumatoid arthritis. blackburn. in their study reported that there was an average delay of 13.7 days in diagnosing septic arthritis in rheumatoid arthritis patients. ultrasound guided arthrocentesis is a key to success in aspirating difficult joints such as shoulder or hip joint. microscopic analysis and culture and sensitivity of the aspirated synovial fluid are the most accurate tool in the diagnosis of septic arthritis. magnetic resonance imaging (mri) is another useful modality in diagnosing septic arthritis as mri allows the delineation of soft - tissue infection and osteomyelitis. increased susceptibility of infection in rheumatoid arthritis was caused by defective phagocytosis in blood and synovial fluid. previously, turner. and wilton. postulated that increased susceptibility to infection in rheumatoid arthritis is because of abnormal immune complexes and polymorphonuclear cell function but breedveld. failed to show defect in phagocytosis of polymorphonuclear cells and found that intracellular killing of s. aureus was intact in polymorphonuclear cells present in synovial fluid and peripheral blood. an animal study carried out by mahowald concluded that infection rapidly extends in rheumatoid arthritis joints along the pannus to the subchondral bone. subchondral neovascularisation carries the bacterial load into the subchondral bone and blocks the vasculature leading to subchondral bone and synovial ischemia. inhibition of tumour necrosis factor alpha has been implicated as one of the risk factors for increasing the chances of infection in rheumatoid arthritic joint. the incidence of septic arthritis of knee after intraarticular steroid injection varies between one in 3000 and one in 50,000. in rheumatoid patients on long - term immunosuppressive skin is the most common source of infection in rheumatoid patients, which accounts for 75% of the infection. skin in rheumatoid patients is vulnerable because of rheumatoid nodules and ulcerated callus in rheumatoid foot. other group of organisms like streptococcus pneumoniae, group b, c, g streptococcus, gram - negative bacilli, hemophilus have been reported to cause septic arthritis in rheumatoid arthritis patients. once the diagnosis of septic arthritis is established prompt arthrotomy, aggressive debridement, surgical drainage and commencement of appropriate antibiotic therapy according to culture and sensitivity is an acceptable treatment. gram - positive cocci are treated with vancomycin or third generation cephalosporin, whereas gram - negative organisms are treated with third generation cephalosporin and an aminoglycoside. goldenberg and reed reported that outcome of septic arthritis in rheumatoid arthritis patient is worse than a non - rheumatoid patient. the chances of recurrence of sepsis are more common in rheumatoid patients compared to patients without rheumatoid arthritis. this rare case of bilateral knee septic arthritis after intraarticular steroid injection in a rheumatoid arthritis patient has not been previously described in the literature and emphasizes the increased risk of infection in these patient groups. this increased risk of septic arthritis in rheumatoid arthritis patient requires risk benefit assessment on individual basis before carrying out intraarticular steroid injection and patient should be aware of the increased risk posed with it. | osteoarthritis of knee is one of the common problems in elderly population. intraarticular corticosteroid injection is a conservative management modality in osteoarthritis of knee. septic arthritis is an infective complication of intraarticular corticosteroid injection. septic arthritis in rheumatoid arthritis patients have worse prognosis because of delay in diagnosis. a higher rate of infectious complications following intraarticular injection is expected in immunocompromised and rheumatoid patients. we would like to report an extremely rare case of simultaneous bilateral knee septic arthritis after bilateral knee intraarticular steroid injection in a rheumatoid arthritis patient. patient was treated successfully with multiple bilateral knee arthroscopic washouts and long - term intravenous antibiotics. this case report emphasizes the increased risk of infection in rheumatoid arthritis patients and a risk benefit assessment on individual basis before carrying out intraarticular steroid injection. patient should be aware of this increased risk of infection. |
derived from the foxglove plant, cardiac glycosides have been used for the treatment of heart failure for more than 200 years and are continued to be used today for patients with atrial fibrillation and heart failure. currently, cardioactive steroid toxicity has become a well - known entity that even now, continues to be an issue. according to the national poison data system, in 2013, there were 26 deaths in the u.s. directly attributable to digoxin overdose, with many more non - fatal cases reported. bufadienolides are a group of cardioactive steroids with a chemical structure similar to digoxin. they are used in multiple traditional chinese medications and aphrodisiacs. because they are structurally similar to digoxin, they also display a similar toxicity profile. there have been multiple case reports of overdose with clinical presentations similar to digoxin toxicity, with death occurring owing to arrhythmia. currently, the only available treatment is the same digoxin - specific antibody fragments used to treat digoxin overdose. in this paper, we will discuss the clinical and electrocardiographic similarities between digoxin and bufadienolide - containing substances. a 39-year - old man presented to the emergency department with vomiting and diaphoresis after eating a half block of vital signs at the time were within normal limits ; heart rate was 65 bpm and regular, blood pressure was 157/66 mmhg, and respiratory rate was 20 breaths / min. shortly after arrival, ecg revealed sinus rhythm at 100 bpm with complete heart block (fig. potassium was 4.6 meq / l and digoxin concentration was 1.14 ng / ml (normal=0.50.8 ng / ml). 2 shows a qrs complex that is mildly wider than baseline, with 3:2 grouped beating ; this would indicate supraventricular tachycardia (atrial or junctional with 3:2 block) or bidirectional ventricular tachycardia (vt) with 3:2 exit block. ultimately, he deteriorated to ventricular fibrillation and underwent 90 min of resuscitation. treatment included defibrillation, epinephrine, atropine, amiodarone, procainamide, and three repeated boluses of digoxin - specific antibody fragments. after each dose of antibody fragments (10 vials, 12 vials, and 11 vials respectively ; 38 mg / vial), his rhythm converted to normal sinus with heart rate 7080 bpm. these improvements were transient and, despite aggressive care, the patient repeatedly deteriorated to ventricular fibrillation, which eventually led to asystole. digoxin has multiple effects on the myocardium and has multiple cardiac - related uses. in the treatment of atrial fibrillation, digoxin increases vagal tone to prolong the refractory period at the av node, decreasing ventricular response to higher atrial rates. toxic doses of digoxin can lead to an automatic atrial tachycardia, commonly termed pat, which, in the context of excessive av nodal blockade, produces the classic arrhythmia called pat with block. in practice, digoxin can produce a number of dangerous arrhythmias other than pat. as shown in fig. 1, the patients initial ecg shows a sinus rhythm with a high degree of av block. without treatment, the patient developed supraventricular tachycardia (atrial or junctional with 3:2 block) or bidirectional vt with 3:2 exit block, as seen in fig. 2 ; either of these arrhythmias are pathognomonic of digoxin. like digoxin, bufadienolide - containing substances are known to inhibit the na / k atpase pump. bufadienolides are structurally similar to digitalis, and as such, are able to replicate the electrophysiologic toxicity of digitalis. the structural similarity also explains the patients mild elevation in digoxin level. as in this case although not a one - to - one comparison of drug levels or toxicity, previous reports indicate that bufadienolide ingestion results in a detectable serum digoxin concentration. this patient had a mildly positive immunoassay, supporting the hypothesis of bufadienolide ingestion. with a combination of a mildly positive assay and waveforms consistent with those produced by digoxin toxicity, the physicians attempted treatment with digoxin - specific antibody fragments. with every administration, the patient converted to sinus rhythm ; however, the effect could not be sustained. once the supply of antidote was depleted, the patient relapsed into a terminal rhythm and eventually succumbed. this patient provides an example of how bufadienolide toxicity and digoxin toxicity can progress in a similar manner. repeated reversion of the arrhythmia using digoxin - specific antibody fragments demonstrates that the treatment has some binding compatibility with bufadienolide - containing substances, which has been supported in the literature. unfortunately, for this patient, there was not enough of the antidote on hand to yield a sustained effect. whereas digoxin is not dialyzable due to its size, bufadienolides lack one of the side chains found on digoxin, potentially allowing them to be dialyzed. in summary, there is increasing recognition that naturally produced substances, which may be used for recreational purposes, can have toxic effects. we discussed a case of a bufadienolide - containing aphrodisiac ingestion that resulted in a toxic profile similar to that of digoxin as demonstrated by his progressive electrophysiologic deterioration seen on ecg. | classically derived from toad venom, bufadienolides are a group of cardioactive steroids with properties similar to digoxin. some traditional chinese medications, including several aphrodisiacs, contain bufadienolides. owing to their physiologic similarities to digoxin, bufadienolides have been shown to produce a toxic profile similar to that of digoxin and there have been multiple case reports of the use of these aphrodisiacs resulting in death. this report will describe a case that illustrates the electrophysiologic similarities between bufadienolide toxicity and digoxin toxicity as well as the treatment of bufadienolide toxicity. |
our patient was an 8-month - old male child of indian origin and the first issue of a nonconsanguineous marriage. he presented to our center with the complaints of cough, cold, progressively increasing breathlessness, and refusal to feed over the past 3 days. past history was elicited that the child was a full - term baby weighing 2.4 kg. his important clinical course is summarized in table 1. on presentation to us, he had tachycardia (160 beats per minute), tachypnea (64 breaths per minute), capillary refill time of 3 s, and blood pressure of 76/60 mmhg in the right arm supine position. the child had increased work of breathing, as evidenced by the subcostal and intercostal retractions. cardiac examination revealed a pansystolic murmur (grade iii), in the third and fourth intercostal spaces. his weight, length, and head circumference were below the third percentile for his age (weight 3.5 kg, length 61 cm, head circumference 37 cm). he had narrow and upslanting palpebral fissures, epicanthal folds, and prominent ears [figure 1 ]. past clinical course of the patient hgt = hemoglucotest ; pcv = packed cell volume ; cbc = complete blood count ; tlc = total leucocyte counts ; dlc = differential leucocyte count ; anc = absolute neutrophil count ; alc = absolute lymphocyte count ; crp = c - reactive protein ; tof = tetralogy of fallot ; ps = pulmonary stenosis ; vsd = ventricular septal defect ; dose of the iv calcium 2cc / kg / dose, tds for 3 days facial profile of the child. note the narrow and upslanting palpebral fissures, epicanthal folds, and prominent ears q1 : name some hereditary and nonhereditary conditions in which the facial dysmorphisms associated with the underlying heart disease can give a clue to the etiology of the condition. ans. : table 2 elaborates the conditions in which facial dysmorphisms associated with underlying heart disease can give a clue to the etiology of the condition. conditions in which facial dysmorphisms are associated with a heart disease ecd = endocardial cushion defect ; asd = atrial septal defect ; pda = patent ductus arteriosus ; ar = aortic regurgitation ; mr = mitral regurgitation ; cad = coronary artery disease ; as = aortic stenosis ; pa = pulmonary artery ; dgs = digeorge syndrome case details (continued) : in view of the association of conotruncal cardiac anomaly, hypocalcemia with hypoparathyroidism [table 1 ] in the infantile period, and typical dysmorphic facies, we were prompted to suspect digeorge syndrome (dgs). : dgs is a 22q11.2 microdeletion syndrome, associated with the classic triad of conotruncal cardiac anomalies, hypoplastic thymus (resulting in immunodeficiencies), and hypoplasia of the parathyroid glands (resulting in hypoparathyroidism and hypocalcemia). the other synonyms for dgs are 22q11.2 deletion syndrome (preferred terminology), velocardiofacial syndrome, conotruncal anomaly face syndrome, sedlackova syndrome, and cayler cardiofacial syndrome. in less than 1% of all patients with dgs, there is complete athymia, resulting in severe combined immunodeficiency (scid). this is called complete dgs ; the remaining 99% are partial dgs, having some thymic function preserved. in our patient, repeated blood investigations failed to show lymphopenia [table 1 ]. detailed immunological tests could not be carried out in our patient. with a worldwide prevalence of between 1/2000 and 1/4000 live births, the actual prevalence may be higher due to high clinical expressivity, accounting for underrecognition. : dgs is caused due to the deletion of the genes in the digeorge chromosomal region (dgcr) on chromosome 22q11.2, secondary to a nonallelic meiotic recombination during spermatogenesis or oogenesis. a great percentage (93%) of the probands have a de novo deletion of 22q11.2, while some (7%) inherit the 22q11.2 deletion from a parent in an autosomal dominant fashion. rarely, less than 1% of the individuals with dgs have chromosomal rearrangements, such as a translocation between chromosome 22 and another autosome, involving 22q11.2. ? ans. : the clinical features of dgs [table 3 ], are exceedingly variable, with over 190 features described in the literature, involving almost every organ system thus underlining the need for sensitization among physicians and surgeons from all specialties. of note, no phenotype occurs in 100% of the patients, as the penetrance of each clinical feature is diverse. thus, one should be wary of excluding differential of dgs solely on the basis of absence of a particular feature. the presence of congenital heart disease (chd ; especially conotruncal anomalies), neonatal hypocalcemia (with hypoparathyroidism), and palatal defects can usually be picked up in the neonatal period, and should serve as red flags for early diagnosis. authorities in this subject recommend that, given the high frequency of dgs among individuals with conotruncal anomalies, newborns with these heart anomalies should be routinely screened with fluorescence in situ hybridization (fish) for 22q11.2 deletion, once other causes (down syndrome, trisomy 13) have been ruled out. other pointers for this condition, such as dysmorphisms, intellectual disability, and psychiatric disorders may appear only years later, often delaying the diagnosis. an emerging area of great interest is the high predilection of psychotic disorders (including schizophrenia and bipolar disorders) in adolescents and adults with dgs (25 times higher than the general population). other than serving as a reminder to psychiatrists, this also underlines that pediatricians and geneticists dealing with dgs / suspected dgs should diligently excavate the psychiatric history of the pedigree in detail, along with the other relevant particulars. clinical features of digeorge syndrome scid = severe combined immunodeficiency ; chd = congenital heart disease ; jra = juvenile rheumatoid arthritis case details (continued) : in view of suspected dgs, fish was performed using tuple1 probes on 200 interphase nuclei and 7 metaphases of the cultured blood. all the cells analyzed showed a heterozygous deletion in the 22q11.2 region [figures 2a and b ]. presence of two green signals (control arsa probe, locus 22q13) one orange signal (tuple1, locus 22q11.2) denotes hemizygous deletion in the 22q11.2 region (b) fish image of the patient : metaphase cell, each cell has two chromatids. presence of two pairs of green signals (control arsa probe, locus 22q13) and only one pair of orange signals (tuple1 locus 22q11.2) denotes hemizygous deletion in the 22q11.2 region q5 : what is the diagnostic modality for digeorge syndrome ? : the most regularly and widely used diagnostic test for detecting the 22q11.2 deletion is fish using probes for the commonly deleted region. however, around 5% of the cases may require more sophisticated and costlier methods such as multiplex ligation - dependent probe amplification (mlpa) and array comparative genomic hybridization (acgh) to detect the underlying genetic aberration. the presence of a chd in association with one or more of the following should serve as an important red flag for considering an underlying chromosomal imbalance as the possible genetic etiology : growth failure, intellectual disability, other malformations affecting a second organ, multiple anomalies, and dysmorphic features. in such cases, when the conventional karyotype fails to pick up the underlying genetic condition, an acgh can lead to increased detection of the underlying causal chromosomal imbalance. the major advantage of this acgh technique is its increased resolution, which surpasses that of conventional karyotyping by at least fivefold. in an excellent study by breckpot., 150 children with an underlying chd and one or more of the red flags mentioned above were subjected to acgh, after a genetic diagnosis could not be reached despite a conventional karyotype and ruling out well - defined genetic disorders. of these 150 children, 24 (17.3%) were detected as having an underlying copy number variation (cnv), that is, a deletion or duplication, as responsible for their syndromic chd, as detected by acgh. some examples of the cnvs detected in this study include 1p36 deletion, 1q21.1 duplication, 8q deletion, 14q32 deletion, and 16p13.3 duplication. acgh can also diagnose, among many other cnv conditions, williams syndrome and some cases of charge syndrome. case details (continued) : on admission in our center, the child had leukocytosis (total leukocyte count = 32 10/l) and radiological evidence of bronchopneumonia. however, the child 's condition worsened on day 9, due to congestive cardiac failure precipitated by the infection. in spite of ionotropic support and mechanical ventilation, the child timely diagnosis of this condition can alert the treating physician about the possibility of the associated comorbities known to occur with this disease. acute management in neonates and during infancy is mainly focused on the evaluation and treatment of hypocalcemia and the underlying chd as in our case. proactive prevention of the secondary complications and a watchful system - wise surveillance plan can go a long way in ensuring a healthy life for most of these individuals [table 4 ]. however, the lack of centers performing thymic transplantation in india and the immunosuppression following the transplant make that a difficult option. prevention of secondary complications and the system - wise surveillance in patients with digeorge syndrome case details (continued) : the parents were offered genetic counseling, the main focus being on the future risk of recurrence, as our patient was the first issue. ans. : following the detection of 22q11.2 deletion in the proband, it is of vital importance to screen both the parents for the same (using fish or mlpa) in order to ascertain the origin of the deletion, sporadic or inherited. if either parent is detected with the deletion, then the risk of recurrence in the next pregnancy is 50% irrespective of the sex of the child (autosomal dominant inheritance). however, if the parents of an individual with 22q11.2ds have normal studies, the risk of recurrence in the subsequent pregnancy is low, though greater than that of the general population. this is due to the possibility of germline or low - level somatic mosaicism in the parents. report retrospectively detecting 30 relatives of probands having a deletion in the 22q 11.2 region, following a diagnosis in the proband. of note, only 32% of the adults and 55% of the children in this case series ever had any major medical problems warranting care. thus, the absence of obvious clinical features should not be a reason to exclude the screening for the 22q11.2 deletion in the parents, if their child has been proven to carry the same. unfortunately, in our case the parents were unwilling to investigate themselves further. in the scenario of one child having dgs, prenatal testing by fish or mlpa can be offered in the next pregnancy, using fetal cells obtained by chorionic villus sampling (10 - 13 weeks of gestation) or amniotic fluid analysis (15 - 18 weeks of gestation.) in pregnancies without any family history of the disorder, the sonographic markers that should prompt one to request for prenatal diagnosis are : chd (especially conotruncal anomalies) and/or cleft lip and/or cleft palate, congenital diaphragmatic hernia, umbilical or inguinal hernia, tracheoesophageal fistula / esophageal atresia / laryngeal atresia, polydactyly, craniosynostosis, polymicrogyria, and renal anomalies. the prognosis for patients with dgs depends mainly on whether the dgs is partial or complete and on the severity of the underlying cardiac defect. most children with complete dgs who do not undergo a thymic transplant usually die in infancy. in contrast, the overall mortality rate in children with partial dgs is reported to be < 10%, most of them due to the underlying heart disease. however, if the child with an underlying heart disease has a successful repair, then the prognosis may be much better, the majority of the immunological problems settling with time. the endocrinological problems, too, do not tend to be devastating, most of them being treatable. learning disabilities, speech issues, and psychiatric manifestations respond well if remedial and supportive therapy is begun early. dgs is the most common microdeletion syndrome, with a notoriety of being clinically varied. timely diagnosis aids better management, more holistic counseling, and the opportunity for prenatal diagnosis in a subsequent pregnancy. the authors certify that they have obtained all appropriate patient consent forms. in the form the patient(s) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal. the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity can not be guaranteed. this research received no specific grant from any funding agency in the public, commercial, or not - for - profit sectors. the authors certify that they have obtained all appropriate patient consent forms. in the form the patient(s) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal. the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity can not be guaranteed. this research received no specific grant from any funding agency in the public, commercial, or not - for - profit sectors. | our patient presented with congenital heart disease (chd : tetralogy of fallot), hypocalcemia, hypoparathyroidism, and facial dysmorphisms. suspecting digeorge syndrome (dgs), a fluorescence in situ hybridization (fish) analysis for 22q11.2 deletion was made. the child had a hemizygous deletion in the 22q11.2 region, diagnostic of dgs. unfortunately, the patient succumbed to the heart disease. dgs is the most common microdeletion syndrome, and probably underrecognized due to the varied manifestations. this case stresses the importance of a detailed physical examination and a high index of suspicion for diagnosing this genetic condition. timely diagnosis can help manage and monitor these patients better and also offer prenatal diagnosis in the next pregnancy. |
astrocytic tumors are the most common primary glial tumors of the central nervous system, and are divided according to the degree of anaplasia and presence of necrosis into low grade astrocytomas (diffuse astrocytoma, pilocytic astrocytoma) or high grade astrocytomas (anaplastic astrocytoma, glioblastoma multiforme). glioblastoma multiforme (gbm) represents 50% of all gliomas and 20% of all operated intracranial solid lesions. the median age for diagnosis for all primary brain tumors is 57 years, although gbms are often diagnosed at slightly older ages. symptoms at presentation may be acute or subacute and include seizures, headaches, weakness or sensory disturbances, language dysfunction, changes in behavior or personality, or cognitive impairment. magnetic resonance imaging is the preferred method for radiologic diagnosis, as these tumors are contrast enhancing, often with central necrosis and peritumoral edema. imaging with positron emission tomography and magnetic resonance spectroscopy may allow for non - invasive grading of gliomas, but pathologic confirmation is needed in most cases [13 ]. the last decade has brought many advanced research discoveries such as the discovery of linolenic acid selective activity against tumor cells or the deciphering the role of cysteine protease inhibitors in brain tumor development. despite advances in therapeutic management of gbm, prognosis remains poor and the median overall survival rate is below 1 year. even in the most favorable cases (young patients treated with radical surgery, radiotherapy and chemotherapy), death occurs in most cases within 2 years. several prognostic factors have been established : age, performance status, therapy (quality of surgery, radiotherapy, chemotherapy), and specific tumor characteristics such as location and nature (de novo or secondary to a low grade glioma ; methylation of the mgmt gene promoter) [68 ]. at the time of tumor recurrence, the infiltrative nature of gbms may preclude repeat surgeries because of the risk of damage to adjacent areas. her-2/neu, also called c - erbb2 oncogene, is located on chromosome 17q21 and encodes for a 185 kd transmembrane glycoprotein with intracellular tyrosine kinase activity. the c - erbb-2 receptor belongs to the family of epidermal growth factor receptors that are crucial in the activation of subcellular signal transduction pathways controlling epithelial cell growth and differentiation. overexpression of c - erbb-2 protein is observed in 20% to 40% of breast cancers and other solid tumors and is generally associated with a poor prognosis. it has been reported to have a prognostic value in medulloblastoma and other neoplasms such as breast and lung cancers. recently, there have been some experimental approaches associated with anti c - erbb-2 receptor strategies as a gene therapeutic intervention in glioblastoma [912 ]. the c - erbb-2 protein was originally identified in the brain and is thought to play a critical role in neurogenesis. the overexpression of the c - erb protein appears to be correlated with aggressiveness of various tumors, including malignant gliomas. the aim of the study was to evaluate c - erbb-2 protein expression in astrocytic tumors of various grades. the patients were divided in 3 groups based on histopathologic diagnosis of their tumors : diffuse astrocytomas g2 (group i), anaplastic astrocytomas g3 (group ii), glioblastomas g4 (group iii) based on the criteria accepted by who (based on who classification of tumors of the central nervous system by louis dn lyon, 2007). following deparaffinization and rehydration, epitope retrieval was carried out in the envision flex target retrieval solution (dako) in high ph. endogenous peroxidases were blocked by incubating the sections in methanol and 30% hydrogen peroxidase for 20 minutes. next, slides were incubated with polyclonal antibody against c - erbb-2 protein (novocastra ncl - c - erbb-2316) in 1:50 dilution overnight in 4c. visualization reagent (abc kit, novostain super) was applied for 30 minutes followed by dab solution for 5 minutes. the results were expressed as the percentage of cells with a strong positive membrane staining as follows : 10% positive cells- negative (), between 1150% (+), and 51% positive cells (+ +). negative controls were performed using a nonimmunized igg replacing the primary antibody. known c - erbb-2 protein expressing breast cancer and lymph node with metastatic disease the c - erbb-2 protein expression was evaluated and compared in all of the groups and the intensity of the staining was related to the nonneoplastic cerebral tissue containing neocortex and adjacent white matter (figure 1). chi squared and pearson s correlation tests and statistica 8.0 statsoft software were used for statistical analysis. chi squared and pearson s correlation tests and statistica 8.0 statsoft software were used for statistical analysis. among 65 patients, 17 (26.1%) had diagnosis of diffuse astrocytoma (group i), 25 (38.5%) had anaplastic astrocytoma (group ii) and 23 (35.4%) had a diagnosis of glioblastoma (group iii). in the glioblastoma group, 19/23 patients had previous history of low grade astrocytic tumors. demographic features and immunohistochemical findings of 65 patients are summarized in table 1. in mean ages were 49.19 years in group i, 54.08 in group ii, and 62.39 in group iii. there was statistically significant correlation between age (p=0.0146) and sex (p=0.0426) in all 3 examined groups. diffuse astrocytomas (g2) occurred mainly in younger and in male patients, compared to glioblastoma which more commonly occurred in older patients. in group i, c - erbb-2 protein expression was observed in 15/17 cases (88.3%) ; 7 (41.2%) were (+) positive, and 8 (47.1%) cases were (+ +) positive (figure 2). in 8 cases the immunohistochemical reaction was very intense in 8090% of the neoplastic cells. in group ii, c - erbb-2 protein expression was estimated in 22/25 cases (88%) ; 12 (48%) were (+) positive, and 10 (40%) were (+ +) positive (figure 3) with strong membrane and cytoplasmatic granular reaction. the immunohistochemical staining was seen in 7080% of the neoplastic cells. in group iii, c - erbb-2 protein expression was observed in 19/23 cases (82.6%) ; however, 11 (47.8%) were (+) positive for c - erbb-2 protein and only 8 (34.8%) tumors were (+ +) positive) (figure 4). even among this group only 5060% of the neoplastic cells were positive for c - erbb-2 protein. it is worth to notice that in younger patients (2348 years) with glioblastoma (12/23 cases) all cases were c - erbb-2 positive. comparing the percentage of all glial tumors cases negative for c - erbb-2 protein expression with the percentage of glial tumors with the strongest immunoreaction, it was discovered that the most evident positive staining was observed mainly in diffuse astrocytomas (47.1%), similar to anaplastic astrocytomas (40%), and less intensive in glioblastoma cases (34.8%). only secondary glioblastomas (arising from low grade astrocytoma) were positive for c - erbb-2 protein, and all of the primary (arising de novo) glioblastoma were negative for c - erbb-2 protein. c - erbb-2 expression was the most significant in diffuse astrocytomas (g2) and anaplastic astrocytomas (g3), and less significant in glioblastoma (g4) cases. in the glioblastoma group the intensity of the staining and the percentage of the positive cells were more suggestive in younger patients, although it was not statistically significant (p=0,829). in the glioblastoma group most cases showed weak positive immunostaining, estimated as (+) positive, and 17.4% of glioblastoma cases were negative for c - erbb-2 expression. astrocytic tumors are the most common primary central nervous system tumor classified according to tumor cell type and histological grade. for newly diagnosed patients with glioblastoma recent data suggest that combining temozolomide with rt significantly improves median survival when compared with rt alone (from 12.1 to 14.6 months) ; however, survival still remains poor. recent studies suggest that aberrations of c - erbb-2 may be involved in astrocytic brain tumor development and progression, and its expression might be potentially significant owing to the role of recombinant monoclonal anti - her-2/neu antibody trastuzumab (herceptin) in the treatment of tumors. there is a tremendous amount of recent literature on gene and microarrays on glial (including astrocytic) tumors, and many have presented egfr, vegf, and other markers as therapeutic targets ; nevertheless, there are many inconsistencies, resulting in poor prognosis and failure of treatment in patients with primary brain tumors. similar to the results of the present study, bian. suggested that aberrations of c - erbb-2 might be merely an early event in the initiation and progression of astrocytomas, presenting, similarly to our results, the strongest c - erbb-2 expression in low grade astrocytomas.. observed negative immunoexpression for c - erbb2 protein in all 94 astrocytic neoplasms, similar to the results of haynik., who estimated all 49 glioblastoma tumors as negative for c - erbb-2 protein reaction, torp. who evaluated c - erbb-2 protein expression in 9 of 21 glioblastoma tumors (43%) and duhem - tonnelle., who suggested that only erbb-3 may represent a new potential therapeutic target to gliomas. our results differ from those presented by kristt., who observed increased c - erbb-2 protein expression in glioblastomas, similarly to liu., who estimated c - erbb-2 expression in 76% of glioblastomas the important role of c - erbb-2 expression in gliomas is also confirmed by giles. and gao., who observed that inhibition of c - erbb-2 greatly decreased glioma cell survival after irradiation, concluding that simultaneous inhibition of c - erbb-2 expression can promote potent antitumor activity and radiosensitizing activity in human glioma. the results of our study show c - erbb-2 protein expression as an early phenomenon in astrocytic tumor development, as the strongest c - erbb-2 protein expression was observed in diffuse astrocytomas compared to the other examined glial tumors. secondary glioblastomas, which in our study occurred mostly in younger patients, also showed very strong c - erbb-2 immunoreaction compared to primary glioblastomas. this might be also be evidence for a role of c - erbb-2 disorder in progression of astrocytic tumors. the c - erbb-2 protein expression differences between primary and secondary glioblastoma in our study might suggest that secondary glioblastoma development is associated with c - erbb-2 protein expression, but there are no clear data to confirm this suggestion. our research will be continued with a larger group of the patients and we will attempt to confirm the presented results using ish methods. | summarybackgroundastrocytic tumors are the primary brain tumors, which often progress to glioblastoma, a highly malignant neoplasm of the central nervous system. there is much new data regarding to the formation and progression of these tumors ; however, glioblastoma remains one of the most fatal neoplasms in humans.the aim of the study was to evaluate the role of c - erbb-2 protein expression in various groups of astrocytic tumors.material/methods65 cases of astrocytic tumors were divided into 3 groups : diffuse astrocytoma (group i ; n=17 cases), anaplastic astrocytoma (group ii ; n=23 cases) and glioblastoma (group iii ; n=25 cases). c - erbb-2 protein expression was estimated semiquantitatively on immunohistochemically stained tissue sections using antibodies against c - erbb-2 protein. statistical analysis was performed in all examined groups.resultsthe c - erbb-2 protein expression was observed in 15 out of 17 cases (88.3%) in group i, 22 out of 25 cases (88%) cases in group ii, and in 19 out of 23 cases (82.6%) in group iii. there were no statistically significant differences between the examined groups. the strongest c - erbb-2 immunoexpression was observed in low grade astrocytomas (diffuse astrocytomas g2) ; in the glioblastoma group the c - erbb-2 protein expression was weak and 17.4% of cases were negative.conclusionsc-erbb-2 protooncogene alteration is an early phenomenon in glial tumor development and progression. |
surgical services in many areas of rural africa, where most of the population reside, are mostly provided by government and mission hospitals in contrast to urban areas where private hospitals play a more substantial role in fulfilling surgical needs of the involved population. furthermore for a variety of reasons, the majority of the medical workforce practice in the urban settings further compounding the provision of surgical care to the mostly needy rural populace. hence it is not unusual for the surgical wards to be frequently filled to an overflowing situation and the medical staff stretched to the limit. in many hospitals it is not uncommon to see patients share beds or be placed on extra beds placed in between regular beds, hindering movement in the wards. a complex problem of surgical ward congestion in a low resource economy certainly does not have an easy solution as there are not enough infrastructures, well - trained professionals, and supportive services to tackle the problem head on. paradoxically in many areas, the structural buildings are not the problem but the lack of professionals in combination with lack of basic supplies and appropriate working incentives. day - case surgery has been developed in many countries as an important approach in reducing inpatient admissions. ideally day - case surgery demands a dedicated ward and a dedicated day - case theater or variations of above.1 hence day - case surgery should ideally function as a system demanding initial capital investment but more importantly incorporating : surgeons, anesthesiologists, nurses, and other supporting staff dedicated to make the system function. the shortage of a trained workforce and a functioning health network system, in most of the african countries, remain hindering factors to full development of day - case procedures. hence different options or modifications are needed to help solve the problem of inpatient congestion with an option being for individual specialists to develop lists of very safe procedures that can be done on a day - case basis but without further demand on the limited workforce in their respective services. many minor and major procedures that can be done on a day - case basis abound in the rural areas and many of the patients harboring these pathologies detest the congested wards and more especially the thought of possible bed sharing., several procedures like circumcision, diagnostic cystoscopy, prostate biopsy, suprapubic cystostomy for urine retention, and dorsal slits for phimosis have commonly been done on an outpatient basis at a reduced admission rate of 21.6% over the period of january to november 2009. the urology service since september 2009 has both a theater and outpatient clinic slots separate from general surgery assigned to it. the purpose of this study was to further increase the number of urology procedures done on a day - case basis using the allocated slots. this was a prospective and descriptive study of consecutive series of 71 patients that were treated and followed - up on a day - case basis between december 2009 and february 2011. these patients were treated for urological pathologies that prior to this study were usually admitted to the ward for surgical interventions as well as five cases of unilateral inguinal hernia and three cases of epigastric / umbilical hernia all treated by the lead author on a day - case basis. all other cases that were already commonly done on a outpatient basis ; cases of emergency admissions ; cases with concomitant medical problems that were assessed as needing in hospital observation ; cases whereby patients preferred to be admitted to the hospital ; and cases of long - distance travel to the hospital and patients of asa - ps classes iii, iv, and v were excluded from this series. several factors including preoperative evaluation with detailed history of symptoms that prompted patient 's presentation, medical history with emphasis on patient 's fitness for the outpatient procedure, and information on the distance of travel to the hospital were considered when evaluating patients for outpatient day cases. patients received detailed explanation indicating that they will go back home on the day of surgery and should therefore present with a care giver that will accompany them at home. laboratory and other investigations were tailored to presenting pathology and symptoms but in all cases hemoglobin count was a basic laboratory work - up. patients present to the preoperative holding area the morning of surgery where they are prepared for surgery. there is a nurse assigned to the preoperative holding area and this area is equipped with basic equipment for monitoring vital signs. in our hospital, there is no dedicated day - case ward or theater. after surgery the patients are again observed at this same preoperative holding area or occasionally in the main recovery room with the participation of their caregivers who then gets introduced to the process of patient monitoring. emphasis was placed on using local anesthesia whenever possible. for inguinal hernia, after blind ilioinguinal - iliohypogastric (ii - ih) block at 3 cm medial to the anterior superior iliac spine, the method for local anesthesia as reported by amid.2 was utilized with the difference that 1% lidocaine with epinephrine at 1:100,000 was used at a reduced volume of 2 - 4 ml from the various steps to compensate for the 7 - 10 ml used for the ii - ih block and also for any additional local infiltration along the course of surgery with the aim of not exceeding therapeutic maximum. for scrotal procedures, spermatic cord block using 1% lidocaine was utilized with the additional inclusion of step by step infiltration of the scrotal subdermic, intradermic, and deep subcutaneous layers circumferentially and toward the external inguinal ring and as such copying the step by step local anesthetic approach of amid.2 for patients that were operated on using local anesthesia, adequacy of anesthesia was assessed on the table by continuous communication with the patient. a simplified method of assessing adequacy of intraoperative anesthesia was used ; if the patient was comfortable with no pain or at a point during the surgery, had moderate transient pain (per patients description) that was completely alleviated with additional lidocaine infiltration at the surgical site, then the anesthesia was assessed as good ; if the patient had significant pain but responded to additional local infiltration, the anesthesia was assessed as fair ; and if a patient had any degree of pain that could not be alleviated with local anesthesia but instead needed supplementary intravenous sedation / analgesia, then the anesthesia was assessed as poor. for other procedures where local anesthesia was not possible, the patients scheduled for day - case procedures were given priority start times over inpatient procedures in order to have adequate time for postoperative monitoring before been discharged home. postoperative care consisted of detailed instruction to the patient on not driving himself home, having the care - giver close - by for at least the first 24 hours, removing the wound dressings after 2 days. further instructions were also given to both the patient and care - giver on what should prompt calling a contact person at the hospital or possibly returning to the hospital and included uncontrollable pain, excessive and progressive swelling at area of surgery, large amount of drainage from incision or disruption of an incision and also for any unusual behavior or confusion, otherwise analgesics ; combination of nsaid and acetaminophen was prescribed. further follow - up depended on individual diagnosis and further plan of treatment ; however all patients were to be seen at least 1 month from surgery. in addition patients were encouraged to call the mobile phone number of the lead author or the surgical emergency number in the case of any emergency situation after discharge. the mean age in our patients was 56.3 years (range of 1 - 86 years). over the study period seventy - eight patients needing more invasive surgeries were admitted for their surgical procedures while 71 (47.65%) received surgical intervention on a day - case basis [table 1 ]. on these 71 patients, all the advanced prostate cancer patients on presentation had indwelling catheters inserted for urinary retention. forty - nine of our patients were operated on using local anesthesia and included hydrocelectomy ; bilateral orchiectomy for hormonal treatment of advanced prostate cancer and unilateral orchiectomy for testicular abscesses ; unilateral inguinal herniorrhaphy ; epigastric / umbilical herniorrhaphy ; spematocelectomy ; epidydimal cystectomy ; and orchiopexy for retractile testes. in 16 patients the choice of intravenous sedation depended on the anesthetist assigned to the urology room on the day of surgery and included varying combinations of midazolam / fentanyl / propofol ; propofol / fentanyl ; midazolam / fentanyl / ketamine ; midazolam / pethidine / ketamine. two cases of orchiopexy were done under a combination of local anesthesia and sedation / analgesia while four other cases of orchiopexy were done under general anesthesia with additional local anesthesia for better postoperative pain control. on adequacy of pain control, 44 of the 49 patients that were operated using local anesthesia had good anesthetic effect while 4 and 1 patients respectively had fair and poor anesthetic effect. the one patient that had poor anesthetic effect had a very large hydrocele with adherent subcutaneous tissues for unknown reasons. immediate postoperative recovery for all the patients was uneventful with all the patients leaving recovery area between 15 minutes and 2 hours 30 minutes postsurgery with none needing admission. on further follow - up at the outpatient clinic two of the patients (2.8%) that had intravenous sedation / analgesia reported episode of nausea and vomiting at home. six patients (8.4%) felt that their pain was more significant than they had anticipated but responded to analgesia. there was no mortality. when participants were asked if they would consider and opt for outpatient surgery again ; all the patients felt that they would still prefer outpatient procedure over admission to the hospital if they were to make a choice again. in this series, we treated 71 patients with mostly urological pathologies on a day - case basis thereby decreasing the number of inpatient admissions to the lead author 's service by 47.65% with associated low complication rate. with the allocation of specific theater days to the urology service, we were able to prioritize day - case procedures with these cases done first on the list before in - patient procedures. furthermore an equipped preoperative holding area was adequately utilized to receive, observe and discharge these patients avoiding admission to the main ward. across africa and in institutions where day surgery is practiced, probably all the different models of day - case surgery setup are practiced including separate day - case ward and day - case theater,46 day - case ward but no separate day - case theater,7 day - case theater but no day - case ward.8 many of these authors have demonstrated good outcome measures using these variations with day - case rates of 30.4 - 65% reported.457 our day - case rate of 47.6% compares favorably with these reports but still far from those from north america where day - case rates of nearly 90% are obtainable;9 hence there is still room for further improvement. as the desire to expand further to include more demanding cases may naturally arise, it will be our desire that a more dedicated day - case unit should preferably be in place before such expansion. we attribute our low complication rate to very careful patient selection using careful history taking and adequate physical examination to help ascertain fitness of our patients for surgery. incorporating the asa - ps classification10 and limiting patient inclusion to classes i and ii may have further helped exclude more at risk patients for post - operative complications. an ideal day case surgical service involves establishing a system where there is a dedicated facility and staff to run such a unit.1 as establishing such a unit is capital intensive1 and considering the need to develop and expand day surgery in africa,11 individual specialists can play a significant role in alleviating the problem by using any of the existing variations of day - case surgery or coming up with modifications that can suit their local environments. in our modification, we have adequately utilized our pre - operative holding area that has all the basic facilities for patient monitoring thereby avoiding congestion of the main recovery room. since most of our patients were operated on using local anesthesia, the recovery time was usually very short facilitating quick patient turnover. our modification did succeed in avoiding admitting day - case patients to the general ward and in giving these patients timely access to theatre without significant complication rate and without need for initial capital investment thereby making this approach very practical in a low resource economy like ours. in this report, we have expanded our list of surgical procedures that can be done on a day - case basis encompassing mostly urological and few general surgical procedures that prior to this study were regularly admitted to the ward with orchiectomy, dviu, and hydrocelectomy being the most common procedures [table 1 ]. however, the spectrum of day - case procedures will certainly depend on the prevailing circumstances in the different institutions as demonstrated in other studies.48 in selecting our cases, emphasis was placed on doing as many of the procedures as possible using local anesthesia. use of local anesthesia in outpatient procedures has a number of advantages that include shorter recovery period and as such quicker discharge from the hospital;1213 lower rate of admission into the hospital;12 cost effective;1315 and better postoperative pain control.14 furthermore local anesthesia for the outpatient procedure can be done without need for an anesthesiologist monitoring16 and this can be especially helpful in many low - resource economies where shortage of anesthesiologists is still significant. our emphasis on the use of local anesthesia and careful patient selection for short surgical procedures proved its importance in the development of our day - case practice as it helped prevent unplanned admission to the ward, delayed discharge home, and significant complications. as experience, availability of technology, organizational structures, surgical, and anesthetic techniques among many other factors improve, the spectrum of procedures that can be done on a day - case basis in our environment should potentially not be less than what is already obtained in more developed countries where day - case rates of up to 90% are possible.9 day - case surgery has been reported by many authors to be a safe practice and associated with high patient satisfaction.1722 patients satisfaction and acceptance of day - case procedures in this series were very encouraging. it is then hoped that our experience will encourage more interest in day - case procedures across africa. there is no doubt that for day - case units to flourish, day - case champions who are passionate to the course should be raised and encouraged as mere establishment of day - case units without the needed enthusiastic leadership may fail to bring out the needed results to match the investment. persisting lack of adequate infrastructures, well - trained professionals, and supportive services among many other factors continues to hinder systemic development of day - case surgery in rural africa and other low resource economies. in the absence of a functioning day - case service, individual specialists can and should develop safe lists of cases in their respective services that can be done on an outpatient basis with the goal to reduce demand for inpatient beds as well as offer the many advantages of outpatient surgery to qualified patients. | background : surgical ward congestion continues to be a problem across rural africa. day - case surgery has helped minimize this problem in most developed countries but remains underdeveloped across africa. the objective of this study was to carefully expand day - case services within the framework of already existing hospital infrastructure.materials and methods : seventy - one consecutive patients out of 149 mostly urologic patients that met the study criteria were treated and followed up on a daycase basis over a 15-month period. in the absence of a day surgery unit, these patients were prioritized and operated on urologic theater days while adequately utilizing the equipped preoperative holding area for patient recovery. patients were all nonemergent, of american society of anesthesiologists physical status (asa - ps) classes 1 and 11 and accepting to undergo day - case procedure among other selection criteria. the main outcome measures were to determine the percentage reduction in admission rate and encountered complications.results:forty-nine (69%) of these 71 patients were treated using local anesthesia. the day - case surgery rate for the urology service was increased to 47.65% from a previous rate of 21.6%. six patients (8.4%) felt that their postoperative pain was more significant than they had anticipated. postoperative nausea and vomiting occurred in two patients (2.8%). there was one case of scrotal hematoma that resolved on observation. there was no mortality.conclusions:in the absence of a dedicated day - case service, individual specialists should develop or increase safe lists of cases in their respective fields that can be done on a day - case basis in order to reduce demand for in - patient beds. |
human articular cartilage has limited repair potential. marrow - stimulation procedures as microfracture for cartilage repair rely on the formation of a primitive mesenchymal blood clot that forms fibrous tissue with variable outcome. limitations of osteochondral grafts include limited donor site, morbidity, questionable cell viability, and fibrocartilage formation in between osteochondral plugs. cell - based strategies have explored chondrocytes and mesenchymal stem cells (mscs) with extensive basic science and preclinical studies. however, clinical strategies have focused on autologous chondrocyte implantation, yet the concept itself is not ideal ; it is a 2-step procedure with donor site morbidity, the quantity of harvested chondrocytes is limited, and chondrocytes dedifferentiate into fibroblasts when cultured ex vivo. because of these limitations, the focus in cartilage repair is shifting toward bone marrow mesenchymal stem cells (bm - mscs) and biodegradable scaffolds with or without growth factors. bm - mscs are available in larger quantities, are easier to isolate and expand, and demonstrate strong chondrogenic potential. platelet - rich preparation such as platelet - rich plasma (prp) and platelet - rich fibrin glue (pr - fg) is a novel biological scaffold that has not been widely tested by itself as an msc carrier in clinical chondrogenesis. the -secretory granules of platelets contain transforming growth factor (tgf)-1 and insulin growth factor-1 (igf-1), which stimulate cartilage regeneration. it is expected that the biological effect of multiple growth factors on tissue regeneration is greater than that of a single growth factor. pr - fg has also been shown to provide sustained release and protection against proteolytic degradation of endogenous growth factors. because the average baseline blood platelet count in an individual is 200,000 75,000/l, a platelet concentrate count of 1,000,000/l (5-fold) has been commonly described for therapeutic platelet - rich preparations. concentrations of growth factors in pr - fg are found to be in supraphysiological levels (5- to 27-fold, according to different processing methods). the concentrations of tgf-1 and igf-1 in pr - fg are in the range of 90 to 400 ng / ml and 50 to 200 ng / ml, respectively. this pilot study was conducted for the following aims : first, to test the hypothesis that pr - fg can be used clinically as a scaffold to deliver cultured mscs for cartilage repair. the 2nd aim is to report on the clinical results 1 y after implantation of mscs - pr - fg. the study included 5 patients : 4 men and 1 woman (range = 21 - 37 y ; mean = 25.4 y). inclusion criteria were a full - thickness cartilage lesion in weightbearing areas of the femoral condyle (outerbridge grade iii or iv), size 3 to 12 cm, disabling symptoms not responding to conservative treatment, age between 18 and 50 y, and patients agreeing to comply with strict rehabilitation. exclusion criteria were complex ligamentous knee instability, kissing lesions, axial malalignment (> 10 varus / valgus) assessed clinically, osteochondral neoplasia, inflammatory joint disease, immune suppression, and symptomatic vascular / neurologic disorder of the lower extremities. all of the patients provided written informed consent according to regulations and after approval of the ethical committee and were operated on by the same surgeon (the 1st author). characteristics of the 5 patients with cartilage defects treated with bone marrow mesenchymal stem cells transplanted in platelet - rich fibrin glue note : ocd = osteochondritis dissecans ; oa = osteoarthritis ; mfc = medial femoral condyle ; lfc = lateral femoral condyle ; rt = right ; lt = left ; na = non applicable ; acl = anterior cruciate ligament ; aclr = anterior cruciate ligament reconstruction ; mf = microfracture. for clinical evaluation, we used 2 scores collected by an independent observer : the lysholm score (subjective) and the revised hospital for special surgery knee score (subjective and objective). in addition, plain x - rays (ap, lateral tunnel views) and magnetic resonance images (mris ; t1 weighted, t2-fat - suppressed weighted, proton density imaging) of the involved knee were performed using a 1.5 t mri machine (philips, amsterdam, the netherlands) and evaluated by an independent senior musculoskeletal radiologist. the scores, x - rays, and mris were taken preoperatively and 6 and 12 mo postoperatively. second - look arthroscopy was performed after 12 mo on 2 patients who consented to the procedure. the repair tissue was rated according to the international cartilage repair society (icrs) arthroscopic grading system. bone marrow (20 ml) was aspirated from the iliac crest under aseptic standard operative procedures and placed in heparinized tubes. cell cultures were performed at the molecular biology and tissue engineering unit at the same university. briefly, nucleated cells were isolated with a density gradient (ficoll - paque ; ge healthcare, waukesha, wi) and resuspended in culture medium (delbecco s modified eagle s medium) supplemented with 10% fetal bovine serum (fbs) and 1% penicillin - streptomycin (10,000 g / ml). fbs used in this study was purchased from reliable countries without bovine virus traceability, where it is furthermore subjected to serial filtration and terminal sterilization with gamma irradiation and followed by bacterial and viral testing using validated procedures to ensure pathogen freedom. cells were incubated at 37 c in 5% co2 for ~14 d. culture medium was changed every 2 to 3 d. when cells reached 80% to 90% confluence, cultures were washed twice with phosphate - buffered saline (pbs), and the cells were trypsinized with 0.25% trypsin in 1 mm edta for 5 min at 37 c. after centrifugation, cells were resuspended in medium and subcultured for ~10 d, reaching an average count of 15 10. mscs in culture were characterized by their adhesiveness and fusiform shape, by flow cytometry for msc surface markers (cd34, cd45, and cd73) and for cd29 gene expression by reverse transcriptase polymerase chain reaction. on the day of implantation, cells were trypsinized, collected, and resuspended in 1 ml pbs and transferred to the operating room in sterile tubes. potential immunogenicity from fbs proteins was furthermore minimized by repeated copious irrigation of the msc pellet with pbs before final resuspension and transfer to the operating room. platelet concentrate was prepared in the hospital blood bank (el - shabrawishi hospital blood bank, cairo, egypt) according to standardized protocols previously reported elsewhere. briefly, using a cell saver centrifuge system, 500 ml volume of blood from healthy donors was collected into 70 ml of citrate - phosphate - dextrose anticoagulant. this was delivered into the 125-ml disposable bowl at 60 ml / min, with the centrifuge spinning at 2039 g until the red blood cells reached 1.25 cm from the collar of the bowl. the centrifuge speed was then reduced to 368 g, and a blood flow rate of 60 ml / min was used to continuously collect the prp, from which 1 ml of platelet - rich concentrate was used with fg to form pr - fg. this method has been shown to yield a platelet count of 7.7 1.1 10/ml. in a 2nd - stage implantation procedure, under a tourniquet, the knee joint was exposed via medial or lateral parapatellar arthrotomy. multiple 3- to 4-mm - deep perforations in the subchondral bone were performed using a 1.2 kirschner wire at 3- to 4-mm intervals. an autologous periosteal flap was harvested from the anteromedial ipsilateral proximal tibia and tailored to the defect size. the flap was sutured to surrounding cartilage with the cambium layer facing toward the subchondral bone using absorbable 4 - 0 sutures, leaving the 12 oclock position open for introduction of the bm - mscs / pr - fg mixture. allogenous pr - fg was supplied as 1 ml platelet concentrate, 1 ml fibrinogen, and 1 ml thrombin in separate syringes from the aforementioned blood bank. bm - mscs were mixed with the pr - fg, and the mixture was left to gel and then introduced through the 12 oclock position and subsequently sutured and sealed by fg (fig. the inoculum density of bm - mscs at the time of implantation was 2 10 cells / cm. autologous mesenchymal stem cell (mscs) platelet - rich fibrin glue (pr - fg) implantation. (a) cartilage defect curetted to a peripheral stable rim of cartilage and subchondral bone drilled. (c) defect covered with periosteal flap, sutured, and then sealed with fibrin glue. (d) mscs mixed with pr - fg intraoperatively to be introduced through the 12 oclock position of the defect. weightbearing was restricted, whereas passive, active - assisted, and active range - of - motion exercises were initiated toward a full range equal to the contralateral knee. after 4 to 6 wk, partial weightbearing was initiated as tolerated. by 8 wk, statistical analyses were done using statistical package for social studies (spss) software, version 11 for windows. the clinical scores were shown as the mean standard deviation at 3 time points : preoperatively and 6 and 12 mo postoperatively. data were analyzed statistically by friedman s test and the wilcoxon signed - rank test. none of the cell cultures had bacterial or fungal infection, nor were there any perioperative complications in any of the patients. details of the clinical scores of the 5 patients preoperatively, 6 and 12 mo postoperatively, as well as the arthroscopic scores of the 2 patients who underwent follow - up arthroscopy at 1 y statistically significant difference. compared with preoperative status, all patients experienced marked improvement in symptoms and knee function, allowing them to return to normal daily activities. all 4 patients without preexisting radiographic signs of advanced degenerative changes returned to their previous sporting levels. clinical scores 6 mo and 12 mo postoperative were statistically different from preoperative scores (p 1-mm wide, and a fibrillated surface all through. the patient had described the outcome as good. patients 3, 4, and 5 described their outcomes as excellent, and all showed complete defect fill with no overgrowth or periosteal hypertrophy and congruity of the repair tissue with the native cartilage on 6 to 12-mo follow - up mri (fig. x - rays showed complete filling of the defects with restoration of the congruity of the femoral condyles and no evidence of degenerative changes. in patient 4, 2nd - look arthroscopy after 12 mo showed the graft surface to be level with the surrounding cartilage, a demarcating border 4 cm). we have demonstrated that pr - fg successfully fixed the cultured mscs within the defects and provided them with a suitable environment to synthesize a hyaline - like grossly appearing cartilaginous matrix. the positive 1-y clinical outcomes support further randomized controlled clinical trials of this treatment modality with larger numbers of patients and longer follow - up periods, as previously stated. | objective : to test the hypothesis that platelet - rich fibrin glue (pr - fg) can be used clinically as a scaffold to deliver autologous culture - expanded bone marrow mesenchymal stem cells (bm - mscs) for cartilage repair and to report clinical results 1 y after implantation of mscs pr-fg.patients and methods : autologous bm - mscs were culture expanded, placed on pr - fg intraoperatively, and then transplanted into 5 full - thickness cartilage defects of femoral condyles of 5 patients and covered with an autologous periosteal flap. patients were evaluated clinically at 6 and 12 mo by the lysholm and revised hospital for special surgery knee (rhssk) scores and radiographically by x - rays and magnetic resonance imaging (mri) at the same time points. repair tissue in 2 patients was rated arthroscopically after 12 mo using the international cartilage repair society (icrs) arthroscopic score.study design : case series ; level of evidence 4.results:all patients symptoms improved over the follow - up period of 12 mo. average lysholm and rhssk scores for all patients showed statistically significant improvement at 6 and 12 mo postoperatively (p < 0.05). there was no statistically significant difference between the 6 and 12 mo postoperative clinical scores (p = 0.18). icrs arthroscopic scores were 8/12 and 11/12 (nearly normal) for the 2 patients who consented to arthroscopy. mri of 3 patients at 12 mo postoperatively revealed complete defect fill and complete surface congruity with native cartilage, whereas that of 2 patients showed incomplete congruity.conclusion:autologous bm - msc transplantation on pr - fg as a cell scaffold may be an effective approach to promote the repair of articular cartilage defects of the knee in human patients. |
in the gastrointestinal (gi) tract, 5-hydroxytryptamine (serotonin, 5-ht) is a major signaling molecule, which mediates gi functions (motility and secretion), emesis, nausea and pain [1, 2 ]. most of colonic 5-ht is synthesized and released from mucosal enterochromaffin (ec) cells ; therefore, alterations in the release of 5-ht from ec cells affect both physiological and pathophysiological colonic functions. irritable bowel syndrome (ibs) is a gi chronic disorder characterized by abdominal discomfort or pain associated with altered bowel habits. abnormal levels of chromogranin a (a marker for all endocrine cells) in the colon of ibs patients have been demonstrated, thus indicating a role of the colonic endocrine cells (namely, 5-ht - containg ec cells or peptide yy (pyy)-containing l cells), in the regulation of colonic function in ibs patients [4 - 6 ]. indeed, a recent human study suggests that the increased 5-ht release from colonic ec cells contributes to the development of abdominal pain in ibs patients. therefore, an improved understanding of endogenous modulator system for 5-ht release from ec cells may provide novel insights into the etiology and pharmacotherapy of functional bowel disorders such as ibs. our in vitro studies have demonstrated that isolated guinea - pig colon is a helpful in vitro - preparation for studying the mechanism modulating the release of 5-ht from ec cells [8 - 10 ]. experimental data from the in vitro - preparations indicate that melatonin, endogenous tachykinins (tks ; neuropeptides such as substance p and neurokinin a) and pyy play important roles in the modulation of the release of 5-ht from ec cells via a unique endogenous tachykinergic nk2/nk3 receptor cascade system [8 - 11 ]. this review focuses on the evidence establishing a potential role of the endogenous nk2/nk3 receptors cascade system controlling the release of 5-ht from ec cells, with special attention being paid to the pathophysiology of gut disorders including ibs. a selective tachykinin nk2 receptor agonist ala - nka-(4 - 10) (0.01 - 1 m) evoked a long - lasting 5-ht release from isolated guinea - pig colon in a concentration - dependent manner [8 - 11 ]. the 5-ht - releasing action of ala - nka-(4 - 10) was unaffected by tetrodotoxin (ttx), and was also seen in myenteric plexus - free mucosal preparations, thus suggesting that the nk2 agonist - evoked 5-ht release is mediated by activation of nk2 receptors located on non - neuronal cells in the mucosal layer. this agrees with a previous report which showed tachykinin nk2 receptor expression on the surfaces of enterocytes of guinea - pig colon. the l - type calcium channel blocker nicardipine or the syntaxin inhibitor botulinum toxin type c also reduced the long - lasting 5-ht release evoked by ala - nka-(4 - 10), thus suggesting that the nk2 receptor - triggered 5-ht release is mediated via syntaxin - related exocytosis mechanisms. however, an involvement of tachykinin nk1 receptors in the evoked 5-ht release is unlikely because a selective tachykinin nk1 receptor agonist, [sar, met (o2)]-substance p (100 nm) failed to affect basal 5-ht release. a selective tachykinin nk3 receptor agonist senktide also evoked a transient 5-ht release from the isolated guinea - pig colon implicating tachykinin nk3 receptors, in addition to nk2 receptors in modulating 5-ht release from colonic mucosa. 1, the enhancing effect of senktide on 5-ht release was prevented by both ttx and the nk2 receptor - selective antagonist sr48968, thus suggesting that senktide stimulates the release of neuronal tks, which in turn enhances 5-ht release via mucosal nk2 receptors. this agrees with previous findings in guinea - pig colon where nk3 receptor activation releases tks and 5-ht [13, 14 ]. the 5-ht - releasing action of senktide was also inhibited by hexamethonium, and was not seen in myenteric plexus - free mucosal preparations, thus suggesting that myenteric cholinergic interneurons also participate in the mechanism for nk3 receptor - triggered 5-ht release. thus, these findings support the view that the cascade of neuronal nk3 receptors and mucosal nk2 receptors represents an endogenous modulator system for 5-ht release from ec cells. these results are also in line with previous findings (1) in human colon where nk3 receptors present predominantly on myenteric and submucosal plexus neurons, and nk3 receptors come into a play only in pathological conditions such as rescue responses or inflammation, and (2) in healthy volunteers where the stimulation of nk2 receptors appears to induce ibs - like symptoms. moreover, it has been shown that the 5-ht release from mucosal biopsy specimens of ibs patients is increased by 10-fold over controls, and the increased 5-ht release contributes to development of abdominal pain in ibs patients. therefore, the view that the endogenous tachykininergic nk2/nk3 receptor cascade system can regulate the release of 5-ht from colonic ec cells, has an important implication for the potential role of endogenous tks in the generation of the symptoms associated with ibs ; it can be speculated that endogenous tks acting through the nk2/nk3 receptor cascade system facilitate the release of 5-ht which in turn affects the colonic function of ibs patients through the activation of 5-ht receptor subtypes. a sensory nerve - derived neuropeptide, calcitonin gene - related peptide (cgrp, 1 - 100 nm) has been also shown to produce a ttx - sensitive but atropine - resistant 5-ht release via stimulation of myenteric plexus neurons in the isolated segments of guinea - pig colon. the enhancing effect of cgrp on 5-ht release was also prevented by both the nk2 receptor - selective antagonist sr48968 and the nk3 receptor - selective antagonist sr142801, but not by the nk1 receptor - selective antagonist l703606. thus, these findings support the view that cgrp stimulates the release of neuronal tks, which in turn enhances 5-ht release via the endogenous nk2/nk3 receptor cascade system. thus, it appears that cgrp - sensitive tks - releasing neurons play a role in activating the endogenous nk2/nk3 receptor cascade system. this conjecture is also supported by a previous report which showed that cgrp can stimulate intramural tachykinergic neurons in the guinea - pig colon. furthermore, the evidence for the elevation of the levels of endogenous cgrp and tks in human diseased colonic mucosa has also been demonstrated [18, 19 ] ; irritants, immunological and inflammatory mediators can release tks and cgrp within the intestinal wall where these peptides may facilitate the release of 5-ht via the endogenous nk2/nk3 receptor cascade system. thus, these findings suggest a possible role of the endogenous nk2/nk3 receptor cascade system controlling the release of 5-ht from ec cells in pathophysiological conditions that are associated with an upregulation of cgrp or tks release. the 36 amino - acid long peptide, peptide yy (pyy) is known as a major peptide hormone, which regulates colonic functions (motility and secretion) and appetite by acting via different neuropeptide y receptor subtypes [20, 21 ]. in the colon, pyy the blood pyy levels have been shown to increase rapidly in response to food ingestion, and the postprandial blood pyy levels remain elevated for several hours. abnormal levels of pyy in large intestinal endocrine cells have also been reported in patients with ibs [6, 22 ], indicating a possible role of pyy in the generation of the symptoms associated with ibs. the nk2 receptor agonist, ala - nka-(4 - 10) (10 - 100 nm) also evoked a long - lasting and ttx - resistant pyy release from isolated guinea - pig colonic mucosa, thus suggesting that activation of nk2 receptors on the mucosal layer induces a long - lasting pyy release from l cells. 2, pretreatment with the neuropeptide y1 receptor - selective antagonist bibo3304 attenuated the ala - nka-(4 - 10)-evoked 5-ht release. these findings support the hypothesis that activation of nk2 receptors on pyy - containing l cells provokes the release of pyy, which in turn activates mainly y1 receptors located on ec cells to induce 5-ht release (fig. this hypothesis is further corroborated by findings that exogenously applied pyy (10 nm) evoked a long - lasting and bibo3304-sensitive 5-ht release with a time course similar to that found for the nk2 agonist - evoked 5-ht release. overall, the above observations indicate that endogenously released pyy play a fundamental role in the nk2 receptor - triggered 5-ht release from ec cells ; pyy - containing l cells appears to control 5-ht release from ec cells (fig. 3). given that food intake is a major and physiologically significant factor stimulating the release of pyy from mucosal l cells and both pyy and 5-ht are well - known mediators of nausea and vomiting, it is hypothesized that the cascade of nk2 receptors on pyy - containing l cells and y1 receptors on ec cells (fig. 3) might play a role in the generation of postprandial symptoms (nausea and vomiting) in patients with ibs ; in fact, a postprandial increase in plasma 5-ht levels have been shown to be related to the generation of postprandial symptoms in diarrhea - predominant ibs [24, 25 ]. therefore, the nk2 receptor - triggered pyy release from colonic mucosa may be expected as a pharmacological target for postprandial symptoms in functional bowel disorders including ibs. activation of tachykinin nk3 receptors on enteric cholinergic neurons has also been shown to provoke the release of pyy from colonic mucosa. thus, to elucidate whether the endogenous tachykinergic nk2/nk3 receptor cascade system participates in the mechanism for food intake - induced pyy release, is an interesting topic for future study. the 5-ht - derived neurohormone, melatonin (mt) is mainly synthesized in the pineal gland as well as in 5-ht - rich ec cells of the intestinal mucosa. it has been shown that exogenous mt modulates colonic motility and pain in ibs patients [28, 29 ] ; therefore, mt is considered as a possible therapeutic agent for ibs [29, 30 ]. in the isolated guinea - pig colonic mucosa, mt has been shown to inhibit the nk2 receptor - triggered 5-ht release by acting through mt3 melatonin receptors located on the mucosal layer, thus suggesting a possible role of mt as a negative - regulator for the nk2 receptor - triggered 5-ht release from ec cells (fig. it is not yet known whether or not endogenous mt participates in the pathogenesis of ibs, but oral mt has been shown to reduce abdominal pain and the sensation of bowel urgency in ibs patients. it is therefore to be expected that if low mt concentrations in the colonic mucosa facilitate 5-ht release from ec cells, the enhanced 5-ht release leads to abdominal pain in ibs patients. in fact, it has been shown that ibs patients have lower mt levels compared with healthy controls. thus, to elucidate whether the endogenous nk2/nk3 receptor cascade system participates in the modulatory effect of mt on colonic motility or pain, is an interesting topic for future study. the present review shows that the cascade pathway of nk3 receptors on cholinergic interneurons and nk2 receptors on pyy - containing endocrine l cells represents an endogenous modulator system for 5-ht release from colonic ec cells, and that melatonin, endogenous tks and pyy play important roles in the modulation of the release of 5-ht from ec cells via the endogenous nk2/nk3 receptor cascade system (fig. 3). given that tks, 5-ht - containing ec cells, pyy - containing l cells and melatonin play important roles in the pathophysiological regulation of colonic functions in the diseased gut including ibs, the potential role of the endogenous nk2/nk3 receptor cascade system controlling the release of 5-ht from ec cells, will undoubtedly be a focus of future investigations. | 5-hydroxytryptamine (5-ht) released from colonic mucosal enterochromaffin (ec) cells is a major signaling molecule, which participates in the pathophysiological regulation of colonic functions in gut disorder including irritable bowel syndrome (ibs), but the endogenous modulator system for the 5-ht release is not yet well elucidated. our in vitro studies in guinea - pig colon have indicated that the cascade pathway of neuronal tachykinergic nk3 receptors and nk2 receptors on peptide yy (pyy)-containing endocrine l cells represents an endogenous modulator system for 5-ht release from ec cells and that melatonin, endogenous tachykinins and pyy play important roles in modulation of the release of 5-ht from ec cells via the endogenous nk2/nk3 receptor cascade system. this review aims at examining the potential role of the endogenous tachykinergic nk2/nk3 receptor cascade system controlling the release of 5-ht from ec cells, with special attention being paid to the pathophysiology of gut disorders including ibs. |
undernutrition in childhood, causing wasting, fetal growth restriction, stunting, and micronutrient deficiencies, is one of the main burdens of the health system and also affects the economic and sociocultural status of society. poverty and malnutrition play a crucial role in increasing morbidity and mortality, impairing cognitive development in children, and increasing common childhood infections. acute malnutrition is the clinical term for undernutrition, a major concern of the health sector. it is defined as a weight - for - height z - score (whz) 11.5 cm) severe acute malnutrition (muac 25 years. early marriage of both sexes before they finish their higher education has been a tradition in afghanistan. this may suggest that because of early marriage, they might still be dependent on their parents, and may not have a steady income to support their family financially. it also draws doubts about their knowledge of dietary management and child health in the family. present study indicates that the mother s education is also an important predictor for child wasting. it further suggests that providing education for mothers at the primary or higher level may bring more than a two - fold decrease in the incidence of wasting among u5 children. educated mothers might be more nutritionally literate and might have more knowledge of child feeding practices. a study conducted in cambodia suggested similar findings. more than one - fourth of mothers in this study had been suffering from moderate acute malnutrition. no significant association was found between nutrition status of mothers and u5 acute malnutrition in our study. however, it has been reported as a risk factor for u5 acute and chronic malnutrition in previous studies. diarrhea has been a major cause of malnutrition in u5 children in resource - poor countries like afghanistan. our study showed that children who had a history of diarrhea in the last two weeks were about two times more prone to be acutely malnourished than their counterparts with no such history. the synergistic relationship between under - nutrition and diarrheal episodes might be exacerbated, as children tend to eat less and their ability to absorb nutrition and physical activity might be decreased during the episodes. previous similar studies have suggested that the combination of fever and pneumonia with diarrhea might increase the risk of u5 wasting and mortality, and it was strongly associated with household sanitation and hygiene practices. the current study showed that u5 children whose frequency of food intake was three times or less in one 24-hour period were more than three times as likely to be acutely malnourished. the mother s knowledge and attitude regarding child feeding might play an important role in child s eating frequency and behavior. analysis of this study showed that wasting was more than two times as pronounced among children who were aged < 24 months. this may be because of early weaning and obstetric factors of the mothers, as 57.3% of them were found to be either pregnant or lactating, and they might not be able to feed the u5 children properly and sufficiently. the who estimates that 50% of child undernutrition cases are the result of repeated diarrheal and intestinal infections due to poor sanitation and hygiene, and a lack of safe drinking water. this study indicated that u5 children of households that had been using unprotected water sources were two times more likely to be acutely malnourished than their counterparts. the main reason for acute malnutrition in these children might be repeated diarrheal disease and intestinal parasites because of unsafe drinking water. in previous studies it was declared that the source and distance of water collection in rural areas of developing countries have a significant role in the quality and quantity of the water, as in most rural settings, the farther the distance to the water source is, the more residents decrease their water use. these practices may have a direct impact on u5 children nutritional status. this study also showed that the u5 children in the households that had been using kitchen salt with the proper amount of iodine were two times less likely to be acutely malnourished. the majority (76.9%) of households this might be one of the underlying causes of undernutrition among the children due to micronutrient deficiency. first, the data may not reflect each cluster, because data collection of households started from the center of the cluster, although the sampling method has been commonly used elsewhere. second, in assessing knowledge of household heads and caregivers, the respondents might guess desirable answers, which may show good scores, but not reflect reality. third, the sample size was relatively small, so that the acute malnutrition frequency was not estimated among subgroups, because the 95% ci of the percentage would be wider for the subgroups. there has been remarkable progress toward reducing u5 malnutrition and mortality in last decade ; however, the health sector still could not achieve the mdg 2015 guidelines. to improve results from all national and international efforts toward managing malnutrition, first, nutrition programs should be horizontally implemented to the body of the bphs to allow community management of acute malnutrition. third, funding the wash sector should be done at a level that reflects its impact on undernutrition. fourth, strategies and programs for fighting undernutrition must incorporate a long - term multi - sectorial component. fifth, food fortification should be considered as one of the key strategies for improving the nutrition quality of the nation. sixth and perhaps the most important, health knowledge skills should be included in school curricula. this may lead to substantial improvement in child nutritional status by directly enabling the girls and boys who will be future mothers and household heads to have improved health knowledge, practice, and health - seeking behavior. in conclusion, the present study indicated that education of parents, household income and age of household head had a strong relation with the nutritional status of children ; a further increase in these factors may result in a remarkable decrease in wasting of u5 children. in addition, wash had a significant association with the nutritional status of u5 children. moreover, low water quality might be a major risk factor for wasting among children. naim musammem, and the cooperative staff of the provincial office of solidarity for afghan families in faryab province for their technical support and assistance with the data collection process. the authors declare that they have no competing interests that might affect the results of this study. | abstractacute malnutrition affects more than 50 million under - five (u5) children, causing 8.0% of global child deaths annually. the prevalence of acute malnutrition (wasting) among u5 children in afghanistan was 9.5% nationally and 3.7% in faryab province in 2013. a cross - sectional study was conducted for 600 households in faryab to find the prevalence and causes of acute malnutrition. odds ratios (ors) and 95% confidence intervals (cis) were estimated using a logistic model. demographic results of this study showed that 54.0% of the household heads and 92.3% of the mothers had no education. three - fourths of households had a monthly income 250 usd. according to the measurement of weight for height z - score (whz), 35.0% (210/600) of the children had acute malnutrition (wasting, whz < 2). in more than half of the households, water, sanitation, and hygiene (wash) conditions were poor. when adjusted, a significant association of acute malnutrition among u5 children was found with the education level of household heads (or=1.49 ; 95% ci, 1.022.17), age of household heads (or=2.01 ; 95% ci, 1.213.35), income (or=1.66 ; 95% ci, 1.042.27), education level of mothers (or=2.21 ; 95% ci, 1.004.88), age of children (or=1.99 ; 95% ci, 1.322.93), history of children with diarrhea in the last two weeks of data collection (or=1.57 ; 95% ci, 1.102.27), feeding frequency (or=3.01 ; 95% ci, 1.217.46), water sources (or=1.89 ; 95% ci, 1.262.83), and iodized salt (or=0.59 ; 95% ci, 0.390.88). the present study indicated that an increase in education level of parents, household income, and quality of wash would result in a significant decrease in prevalence of wasting among u5 children. |
night - guard bleaching is an effective and simple method for regaining the esthetic appearance of intrinsically discolored or stained teeth. there is concern about the adverse effect of bleaching materials on existing restorations in the oral cavity. many studies have reported that there is little evidence of bleaching agents causing significant changes in dental materials, including glass - ionomer cements, ceramics and gold. however, many in vitro studies have reported a significant increase in mercury release from dental amalgam after treatment with peroxides [13 ]. bleaching agents such as carbamide peroxide decompose into free radicals, which theoretically have the potential to corrode metal alloys such as dental amalgam existing in or near the teeth being whitened. mercury is contained in dental amalgam from which it can be released into the oral cavity and distributed throughout the body as a result of being inhaled or swallowed. so, ionic mercury leached out from amalgam restorations may present a risk to the dental patient [5, 6 ]. treatment duration, the ph and concentration of the bleaching agent, aging and surface polishing of amalgam restorations are some of the factors that control mercury release from dental amalgam exposed to carbamide peroxide bleaching agent. therefore, it is of clinical importance to find ways to reduce mercury release from amalgam restorations following a routine procedure such as home bleaching. this study assessed the effect of surface polish of dental amalgam on the amount of mercury release after treatment with 16% carbamide peroxide bleaching gel. the null hypothesis was that polishing dental amalgam does not decrease the amount of mercury release after treatment with 16% carbamide peroxide gel. two commercially available dental amalgams were selected for this in vitro experimental study. the characteristics and chemical compositions of these materials forty - eight samples from two amalgam brands (totally 96 samples) [(oralloy magicap s, coltene co. 235 ascot parkway, cayahoga falls, ohio. 44223 /usa) and (sdi - gs80, southern dental industries limited, bayswater, victoria 3153, australia) ] were selected for the purpose of the study. the amalgam capsules were mixed according to the manufacturers instructions with a dental amalgamator (doumat 2, degussa, frankfurt, germany). each amalgam disc was prepared in a truncated cone - shaped pvc polymer mold with a diameter of 9 mm at the base and 8 mm at the top and a height of 3 mm. the specimens were removed from the molds after 60 minutes and stored in sealed glass tubes containing distilled water for 24 hours at room temperature. subsequently, half of the specimens were polished with green and red rubber (dtz geozalee 307 - 14167 berlin, germany), a brush and tin oxide paste mounted on a slow - speed contra - angle hand piece without water coolant. the specimens were again immersed in distilled water at room temperature (24c) for 1 month. then, the amalgam discs were removed from the tubes and dried with cotton wool. twenty - four samples from each group were polished and the other 24 were not polished. all of the samples were treated with 16% carbamide peroxide gel (nite white, discus dental, inc. culver city, usa) in tubes containing 3 ml of carbamide peroxide gel and 0.1 ml of distilled water in a manner that the entire amalgam disc surfaces were coated with the gel. according to the manufacturer s instructions, this gel should be used 12 hours per day and we chose 2 hours per day for our study. half of the samples in each group (12) were kept in these tubes for 14 and 28 hours. after these periods, the amalgam samples were removed from the assay tubes, and carbamide peroxide gel on the sample surfaces was carefully rinsed with 7.0 ml of distilled water until the volume of each tube was 10 ml. mercury levels of each solution were measured using the vav440 mercury analyzer system (thermo jarrell ash co. sh-22 model, franklin, massachusetts, usa). the chemical reaction in this system was based on the cold - vapor atomic absorption method. the solution was treated with nitric acid and sulfuric acid in the presence of potassium permanganate and potassium per sulfate to oxidize all the mercury to mercuric ions (hg). mercury concentration in each solution was determined by comparing it with a standard curve of known mercury levels. considering the surface area of each amalgam disc, mercury levels were calculated in g / mm. two commercially available dental amalgams were selected for this in vitro experimental study. the characteristics and chemical compositions of these materials forty - eight samples from two amalgam brands (totally 96 samples) [(oralloy magicap s, coltene co. 235 ascot parkway, cayahoga falls, ohio. 44223 /usa) and (sdi - gs80, southern dental industries limited, bayswater, victoria 3153, australia) ] were selected for the purpose of the study. the amalgam capsules were mixed according to the manufacturers instructions with a dental amalgamator (doumat 2, degussa, frankfurt, germany). each amalgam disc was prepared in a truncated cone - shaped pvc polymer mold with a diameter of 9 mm at the base and 8 mm at the top and a height of 3 mm. the specimens were removed from the molds after 60 minutes and stored in sealed glass tubes containing distilled water for 24 hours at room temperature. subsequently, half of the specimens were polished with green and red rubber (dtz geozalee 307 - 14167 berlin, germany), a brush and tin oxide paste mounted on a slow - speed contra - angle hand piece without water coolant. the specimens were again immersed in distilled water at room temperature (24c) for 1 month. then, the amalgam discs were removed from the tubes and dried with cotton wool. twenty - four samples from each group were polished and the other 24 were not polished. all of the samples were treated with 16% carbamide peroxide gel (nite white, discus dental, inc. culver city, usa) in tubes containing 3 ml of carbamide peroxide gel and 0.1 ml of distilled water in a manner that the entire amalgam disc surfaces were coated with the gel. according to the manufacturer s instructions, this gel should be used 12 hours per day and we chose 2 hours per day for our study. half of the samples in each group (12) were kept in these tubes for 14 and 28 hours. after these periods, the amalgam samples were removed from the assay tubes, and carbamide peroxide gel on the sample surfaces was carefully rinsed with 7.0 ml of distilled water until the volume of each tube was 10 ml. mercury levels of each solution were measured using the vav440 mercury analyzer system (thermo jarrell ash co. sh-22 model, franklin, massachusetts, usa). the chemical reaction in this system was based on the cold - vapor atomic absorption method. the solution was treated with nitric acid and sulfuric acid in the presence of potassium permanganate and potassium per sulfate to oxidize all the mercury to mercuric ions (hg). mercury concentration in each solution was determined by comparing it with a standard curve of known mercury levels. considering the surface area of each amalgam disc, mercury levels were calculated in g / mm. the mean levels of mercury released from amalgam samples after 14- and 28-hour treatments are summarized in table 2 and graph 1. two - way anova showed significant differences between the mean levels of mercury release from polished vs. unpolished amalgam surfaces after treatment with 16% carbamide peroxide gel (p=0.015). two - way anova indicated that increasing the storage time from 14 to 28 hours did not cause significant changes in the amount of mercury release (p=0.385). in addition, there was no statistically significant interaction effect between amalgam surface polish and storage time (p=0.768). the results of the present study showed that less mercury is released from polished amalgam after treatment with 16% carbamide peroxide bleaching agent as compared to unpolished amalgam ; therefore, the study s null hypothesis was rejected. according to literature, polishing high - copper amalgam surfaces has no influence on the longevity of the restoration. it is suggested that carefully placed and carved high - copper amalgam restorations may have the same longevity as polished restorations. reavis - scruggs indicated that burnishing silver amalgam restorations alone creates surface smoothness, improved marginal seal, decreased corrodibility and decreased dissipation of mercury vapor. therefore, dental practitioners may prefer to omit the polishing procedure to save time in the office. however, after pos - carve burnishing of amalgam restorations it is likely that excess amalgam remains beyond the margin, which necessitates finishing / polishing to remove the excess amalgam. reaction of the surface area of amalgam samples with carbamide peroxide decreases after polishing ; therefore, the amount of mercury release diminishes. additionally, scanning electron microscopy and x - ray energy dispersive microanalysis in a study carried out by ferracane suggested that unpolished amalgam surfaces have a slightly greater surface concentration of gamma-1 phase than polished surfaces, which is a potential source for mercury release. the results of a study carried out by canay. showed that unpolished amalgam has a higher corrosion rate in comparison with the polished amalgam. the corrosion current density is rather low for polished surfaces, and burnishing increases resistance of dental amalgam to corrosion. in addition, the chemical dissolution of mercury phase and its diffusion to the outer surface is easier on unpolished amalgam surfaces. as a result, canay suggested that the existing amalgam restorations should be polished prior to bleaching procedures. although it has been reported that the greatest amount of mercury release occurs during polishing and removal of amalgam without high volume evacuation, according to a study carried out by sweeney, if polishing dental amalgam would be carried out under moist conditions and high - volume evacuation, the mercury exposure is minimized to levels not exceeding the threshold level value (tlv), which is 50 g hg / m of air. the world health organization (who) guideline for maximum intake of mercury is 40 g / day. in the present study, the average amounts of mercury released from each unpolished and polished amalgam disc were about 1.2 g / mm and 0.8 g / mm, respectively. therefore, the average mercury release from unpolished samples was 234 g and 468 g over 14- and 28-hour periods, respectively. in addition, the average mercury release from polished samples was 156 g and 312 g over 14- and 28-hour periods, respectively. according to the white nite gel instructions, the product should be used two hours per day. as a result, the maximum release of mercury using 16% carbamide peroxide in this study was 33.4 g / day for unpolished samples and 22.3 g / day for polished samples, both of which are in the safe range of mercury intake. in the present study, mercury release is expressed as the amount released per unit surface area of specimen as a standard form, to be comparable with other studies, which is the same method used by al - salehy. rotstein reported that mercury released from amalgams immersed in 10% cp solutions after 48 hours was between 23 and 161 g / ml. al - salehi recalculated the data reported by rotstein and reported that considering the 1.9 cm surface area of the samples the amount of mercury release would be 0.64.24 g / mm, which is consistent with the results obtained in our study. similarly, the data reported for mercury release by hummert and mackert and berglund after recalculation were 0.0140.020 and 0.016 g / mm, respectively. it has been reported in some studies that mercury release from dental amalgam after bleaching is time - dependent. in the present study, the increase in mercury release from 14 to 28 hours was not statistically significant, which might be attributed to the fact that the diffusion rate of mercury is inversely proportional to the film thickness of the oxide layer covering the amalgam restoration because mercury atoms must pass through the oxide film to reach the environment. the layer formed by corrosion, which covers the amalgam samples, inhibits further corrosion in vitro. in the oral cavity, this layer dislodges as a result of mastication and occlusion ; therefore, mercury release may be time - dependent in vivo. according to a study carried out by rotstein, the size of amalgam samples in the current study was approximately the same as a class i or class ii amalgam restorations in a molar tooth. patients may have more than one posterior amalgam restoration in their mouth, so these patients may be in danger of higher - than - maximum mercury intake after treatment with 16% carbamide peroxide gel in the home bleaching technique. in addition, patients who abuse over - the - counter products by prolonged contact periods of the bleaching agent with amalgam restorations may expose themselves to the risk of increased total mercury intake. therefore, it is prudent to minimize mercury release from amalgam restorations as much as possible, and one of the most effective ways is to polish the existing amalgam restorations in the patients mouth, which is the same conclusion as reached by canay. within the limitations of this study, it was concluded that polished amalgam restorations release less mercury after treatment with carbamide peroxide bleaching gel in comparison with unpolished amalgam restorations. | objectives : this study evaluated the effect of surface polishing on mercury release from dental amalgam after treatment with 16% carbamide peroxide gel.materials and methods : ninety - six samples from two different amalgam brands were prepared in truncated cone - shaped pvc polymer molds with an external surface area of 195 mm2. half of the specimens were polished with green and red rubber, a brush and tin oxide paste at low speed. samples were treated with 16% carbamide peroxide gel in tubes containing 3 ml of carbamide peroxide gel and 0.1 ml of distilled water for 14 and 28 hours. subsequently, carbamide peroxide gel on the sample surfaces was rinsed away with 7.0 ml of distilled water until the volume of each tube increased to 10 ml. the mercury level of each solution was measured using the vav440 mercury analyzer system. considering the surface area of each amalgam disc, mercury amounts were calculated in g / mm2. data were analyzed using two - way anova.results:there were significant differences between the mean levels of mercury release from polished vs. unpolished amalgam surfaces after treatment with 16% carbamide peroxide. increasing the storage time from 14 to 28 hours did not result in significant changes in the amount of mercury release. there was no significant interaction effect between amalgam surface polish and storage time statistically.conclusion:polished amalgam restorations release less mercury after treatment with carbamide peroxide bleaching gel in comparison with unpolished amalgam restorations. |
the genomes of higher organisms are littered with repetitive sequence elements, and evidence for how this came to be is accumulating through a combination of bench work and bioinformatics. long interspersed nuclear elements (lines or l1s) are retrotransposons that have generated integrated dna copies of themselves through rna intermediates. intact l1 rnas encode two proteins, orf1p and orf2p, which together associate with l1 rna and direct its nuclear importation, reverse transcription to dna, and integration at a random site within the host genome [1 - 3 ]. sequence analyses show a succession of l1 families in mammalian genomes, each family expanding over time before being relegated to the status of fixed dna by host - defense mechanisms and perhaps by direct competition from newer, more competent l1s. in humans, this history is traced in order through the inactive l1pa5, l1pa4, l1pa3b and l1pa2 families and the currently active l1pa1 family. in the mouse, the a, gf and tf families are currently active. although typically regarded as genetic baggage, martin vingron and his colleagues (zemojtel.) estimate in a recent paper in bmc genomics, that the mouse genome has approaching 5,000 intact active l1s in the diploid nucleus this is a timely reminder of both the harmful and potentially beneficial effects of l1 elements to cause functional changes in the genome. catalog examples of mouse transcripts that have incorporated portions of line sequences embedded in their genomic loci. although such incorporation into exons ' exonization ' and the related phenomenon of the co - option of transposon sequences for functional purposes ' exaptation ' have been described previously (reviewed in), the new work from vingron 's team provides an update on the prevalence of l1 exonization within the mouse transcriptome. in most examples of exonization described by the authors, the l1 sequence provides a new start site of transcription (figure 1) and provides exonic sequence followed by a gu splice donor site. in other examples, however, lines provide entire exons or ag splice acceptors with downstream transcript end points. contributions of single l1-derived splice sites creating chimeric l1/non - l1 exons were found less frequently. a hypothetical gene locus is diagrammed (a) before and (b, c) after insertion of an l1 in antisense orientation. (b) in one of these, the l1 sequence provides a novel transcription start site, an alternative first exon (ex1 ') and a splice donor site. (c) in the second, an entire new exon (ex3.1) with splice sites on either side is derived from the inserted l1., the l1 sequence is oriented in antisense with respect to the mrna (figure 1). first is the apparent potential of the antisense promoter in the orf1p sequence to initiate transcription, as was observed for 17 cdnas in this analysis. second is the presence of a greater number of functional donor and acceptor splice sites on the antisense strand compared with the sense strand, totaling 18 (a - type l1s) and 22 (f - type l1s) functional antisense splice sites versus four functional splice sites in the sense orientation. third is the repeated use of particular splice sites : sa -154 in the antisense 5 ' untranslated region and sd + 52 in antisense orf1p were used in 13 and 16 cdnas, respectively (sa (splice acceptor) and sd (splice donor) numbers refer to the distance in nucleotides from the beginning of the orf1p coding sequence in sense - oriented l1mda2). finally, there is a relative prevalence of antisense intragenic l1 insertions, which are found 1.7 times more frequently than sense insertions. it has been proposed that this last observation is the result of rapid selection against sense - oriented l1s that interrupt gene loci. disruption of gene function by premature polyadenylation may be one reason an intragenic l1 insertion is selected against, and aauaaa sites in the sense direction of murine l1s outnumber those in the antisense orientation by a ratio of 9:2 using the m13002 l1mda2 sequence as a reference. perhaps more importantly, sense - oriented orf2 sequence interferes with transcriptional elongation, resulting in profoundly compromised transcript levels. disrupted transcription in the absence of true exonization can create hypomorphic and null alleles, but these are only two of many potential sequelae of an intragenic l1 insertion. allelic changes resulting in new functions by creating a novel transcription start or end site, or in the case of antisense l1s, both a premature polyadenylation site and a new transcript initiation site (a scenario described as ' gene breaking '). exonized repetitive sequences can also contribute to protein - coding sequence. give the example of guanylate - binding protein 5a (encoded by gbp5), which is predicted to have a 174-amino - acid carboxy - terminal extension derived from an exonized l1. because of the activity of murine l1s, comparison across inbred mouse strains reveals many polymorphic l1 insertions (david symer, personal communication). this observation, coupled with the genetic tractability of the mouse, will make it instrumental in our understanding of the functional aspects of endogenous l1s. interestingly, cell context is emerging as an important factor in the exonization of transposon sequences, with tissue- and tumor - specific transposon - derived cdnas being identified. as yet, the functional consequences of such exonizations are not well understood, but recognition that exonization might depend on cellular context lends special significance to the annotations of predicted intronic l1 splice sites by zemojtel. as similar projects are undertaken in the human genome, experimentalists with an interest in a particular locus in a given cell type or tumor context will have a new tool for predicting alternative l1-containing transcripts, even in the absence of expressed sequence tags indicating splice - site usage. in addition to changes to an mrna that result from transposon insertion, pressure created by acquisition of a novel function (neofunctionalization) may act on transposon sequences over evolutionary time. it has been proposed that transposon exonization is a prelude to sequence exaptation, and that alternative splicing of the exon initially allows either its loss or functional co - option [16 - 18 ]. selective divergence of exonized sequence from the l1 consensus is not appreciable in the examples given by zemojtel. : that is, an exonized l1 sequence segment is equally similar to a consensus l1 as is the entirety of the l1 containing it (our unpublished analysis). thus, the status of these relatively young murine l1 sequences may be similar to that of recently exonized alu sequences in primates in that there is no appreciable sequence co - option. neofunctionalization of more ancient transposon sequences is, however, being increasingly recognized from examinations of mammalian gene structure. examples include an alternatively spliced exon in poly(rc)-binding protein 2 (pcbp2) that is borrowed from a silurian period sine (short interspersed nuclear element) transposon ; the recombination - activating gene 1 (rag1), which may be derived from a transib dna transposon ; the primate set domain and mariner transposase fusion gene (setmar) related to the mariner - like hsmar1 transposon ; and the derivatives of tf1/sushi ltr retrotransposons, the mar gene family, which includes an essential gene for mammalian development, paternally expressed gene 10 (peg10). we expect that, along with identifying already neofunctionalized sequences, studies of recent exonization events will enhance our understanding of mobile element exaptation. in summary, the recent paper by vingron and colleagues provides an updated view of the mouse genome and transcriptome with respect to already exonized l1 sequences and intronic l1s with the potential to become part of processed transcripts. this work furthers our understanding of the complex relationships between mammalian genes and the retroelements within them. | long interspersed nuclear elements (lines) are among the most successful parasitic genetic sequences in higher organisms. recent work has discovered many instances of line incorporation into exons, reminding us of the hazards they pose to genes in their vicinity as well as their potential to be co - opted for the host 's purposes. |
within clinical trials (cts) there are three dimensions where activities, their duration, and the conditions on which these time - bound activities unfold decide the fate of the study. these are : (1) assessment of safety prediction if a potential medicinal product will have any safety concerns through the evaluation of its potential adverse effects, (2) proof of concept and large studies efficacy, (3) postmarketing surveillance, pharmacovigilance, and periodic safety update reports. the coordination of an individual ct project and the general management of the projects running simultaneously have a strategic importance to ensure timely marketing authorization of the promising candidates in the portfolio. in all phases of cts, the common management items tend to remain the same, i.e., the scope, resources, and the timelines. the productivity of the pharmaceutical industry has decreased over time, and the costs of producing new medicines have been rising sharply. critical path analysis (cpa) is a method which is intended to improve drug development and reduce uncertainty and cost by applying scientific tools to the clinical development process. when activity durations are deterministically or experientially known, the cpa can be applied to manage time and resources for a given trial. the motivation for cpa and overall project management for cts come from the fact that there are many dependent and independent (parallel) activities involved, and also there are many uncertainties. to meet the objective of systematic planning, cpa is one of the many network techniques which have been widely used for planning, scheduling and controlling the large and complex projects. cpa formally identifies tasks which must be completed on time for the timely completion of the whole project. it also identifies which tasks can be delayed if the resource needs to be reallocated to catch up on missed or overrunning tasks. a further benefit of cpa is that it helps us to identify the minimum length of time needed to complete a project. where we need to run an accelerated project, it helps us to identify which project steps we should accelerate to complete the project within the available time. the disadvantage of cpa, if we use it as the technique by which our project plans are communicated and managed against, is that the relation of tasks to time is not as immediately obvious as with gantt charts. it is not always possible to sort out completely identifiable activities and their start and finish times., we tried to design and develop an innovative ct management approach for a project - specific operating plan during the conduct of the study. this ct management approach does not replace an understanding of or adherence to the requirements contained in all applicable regulations, guidelines, or standard operating procedures governing these studies but ensures optimization of ct design, analysis, trial management, and cost. initially, the activity list of a model ct was listed in different 78 activities, which was further merged into the major 35 activities. after further consideration and dependence analysis, it was finalized in the 25 activities which were taken in activity predecessor table for the purpose of network diagram and cpa considering patients, conduct and outcome [table 1 ]. activities are inclusive ; describe the trial entirely with accuracy, in chronological and logical sequences. dependency nature is both qualitative and quantitative [table 2 ] and also validated by experts. primary and secondary outcomes were appropriate, ethically approved, approved by the concerned regulatory agency, scientifically standardized as well as patients were well cared [figure 1 ]. these 25 activities were taken to create a network diagram and perform cpa considering patients, conduct, and outcome activity - predecessor relationship to describe how each activity is dependent or independent from another activity which precedes this given activity network diagram based on the activity - predecessor table including activities from start to finish. in this figure, all 25 activities mentioned in tables 1 and 2 were taken to design the network a y. the activities which were linked to another activity (ies) are connected with the direct line to show inter - dependent relationships to determine the flow of these activities, we need to create a precedence diagram. activities which must be completed before a particular activity starts are called predecessor activities, and those which must follow a particular activity are called successor activities. after deciding the precedence order, the activities are put in a logical sequence using the graphical notations. while constructing the network, to ensure that the activities fall in a logical sequence, the following questions need to be answered : (1) what activities must be completed before a particular activity starts ? (2) what activities follow this ? (3) what activities must be performed concurrently with this ? a delay in any of the critical path activities will delay the entire project, regardless of whether the other project activities are completed on or before time. the precedence diagram shown in figure 2 has multiple paths highlighted as the green line, red line. the red line of this diagram describes the critical path of the activities from the starting point to completion of a clinical trial y slack and float both refer to the amount of time by which a particular event or activity can be delayed without affecting the time schedule of the network. the total float of an activity is the difference of its latest start time and its earliest start time. the activities which have zero floats are critical activities and are on the path of critical activities, the critical path. total float represents the time by which an activity can be delayed without affecting the project completion time. in this network diagram, critical activities such as development of protocol and investigator brochure, informed consent form translations, review, approval, translation certificate, study insurance certificate, site initiation visit (siv) preparation, dispatch of investigational product and other site requirements, siv, data entry, data validation, data clarification form generation and tracking, reconcile final records, and close study plays vital role. some of the noncritical activities of this project include development of drug supply management plan, clinical trial registry of india registration, preparation of ethics committee submission packets, and allocation of sites to the clinical research associate [figure 3 ]. the diagram shown above describes the multiple paths highlighted with different colors with multiple activities and longest path that is, red lined path is the critical path of the project the longest path without any slack is the critical path. from the above diagram, we can see that path red lined is the longest as compared to green and blue lined path. therefore, if activity on this path is delayed, then the project will be delayed. after identifying the critical path, we can deduce the activities that when delayed will not impact the project. calculating the float of each activity using the cpa to determine the amount of time an activity may be late without delaying the project. a delay in any of the critical path activities will delay the entire project, regardless of whether the other project activities are completed on or before time. the precedence diagram shown in figure 2 has multiple paths highlighted as the green line, red line. the red line of this diagram describes the critical path of the activities from the starting point to completion of a clinical trial y slack and float both refer to the amount of time by which a particular event or activity can be delayed without affecting the time schedule of the network. the total float of an activity is the difference of its latest start time and its earliest start time. the activities which have zero floats are critical activities and are on the path of critical activities, the critical path. total float represents the time by which an activity can be delayed without affecting the project completion time. in this network diagram, critical activities such as development of protocol and investigator brochure, informed consent form translations, review, approval, translation certificate, study insurance certificate, site initiation visit (siv) preparation, dispatch of investigational product and other site requirements, siv, data entry, data validation, data clarification form generation and tracking, reconcile final records, and close study plays vital role. some of the noncritical activities of this project include development of drug supply management plan, clinical trial registry of india registration, preparation of ethics committee submission packets, and allocation of sites to the clinical research associate [figure 3 ]. the diagram shown above describes the multiple paths highlighted with different colors with multiple activities and longest path that is, red lined path is the critical path of the project the longest path without any slack is the critical path. from the above diagram, we can see that path red lined is the longest as compared to green and blue lined path. therefore, if activity on this path is delayed, then the project will be delayed. after identifying the critical path, we can deduce the activities that when delayed will not impact the project. calculating the float of each activity using the cpa to determine the amount of time an activity may be late without delaying the project. by executing this approach repeatedly, an arbitrarily large number of realizations of project completion time may be generated allowing inference of its uncertainty. in addition, cost uncertainty with time - dependent effects can also be quantified by secondary calculations in the project network. some believe that a project plan is just a list of specific tasks and deliverables, organized across time and into larger groupings that begin and end with major project milestones. however, a project plan is not just a timeline management but also achieving the objectives clearly and being answerable to stakeholders by managing well all sorts of resources. major factors in determining the success or failure of clinical development projects are how well the planning process is carried out, whether the clinical project managers are involved early enough to understand the product development goals, executive sponsorship, and stakeholder involvement. senior level leadership in the planning phase is common in established pharmaceutical companies, but we recommend that the attention is given to developing strategic development plans also extend to individual clinical study plans. in smaller biopharmaceutical companies without dedicated project management offices, strategic product development planning conducted to satisfy investors usually lacks a rigorous operational component. in the absence of mid to senior management leadership, this planning is almost nonexistent, increasing the risks for each individual clinical study. in the clinical research the project team works within a matrix reporting environment to ensure that the tasks required for completing the project are done within the project timeline and with the quality that meets or exceeds the client 's expectations. within a clinical research organization, project management can include satisfying the client, supporting the project team, and senior management. most of the time, the project manager ends up sitting on a fence between what the client wants and what senior management wants. at the end of each day, the project manager must ensure that both sides are happy with the project 's performance. because the planning gap for clinical projects is persistent and widespread, managing each section of a typical project plan during the definition and planning stages is key to the success of the study. as the need to meet deadlines becomes more important, producing good and stable project plans become imperative. we can see that the critical path method or cpa is very useful for determining activities that can lead to project delay. based on actual results, resource reallocation can be considered for further better management of complex cts. the management of ct resources can only be carried out effectively if a series of accurate resource models can be produced that can provide a range of project execution alternatives. in this way | clinical research operates in a strictly regulated environment under various management models, but a distinct management model of clinical trial (ct) still needs exploration and research. critical path analysis (cpa) is a management approach can be used for monitoring, analysis, and prediction of success of its time - bound operational activities. a model ct was compiled with 78 activities, which were further merged into 35 major activities. after performing dependence analysis, the list was finalized with 25 activities which were taken in activity predecessor to create a network diagram and perform cpa considering patients, conduct, and outcome. activities were inclusive, described the trial entirely with accuracy, and were in chronological and logical sequences. this approach does not replace an understanding of or adherence to the requirements contained in all applicable regulations, guidelines or standard operating procedures governing clinical studies but ensures the proper use of operational and decisional approaches including optimal resource management. as the need to meet deadlines becomes more important and the need to produce good, stable project plans, cpa is very useful for determining activities that can lead to project delay. with this approach, project may be effectively monitored, and realistic schedules can be maintained. |
from may 2004 to may 2006, we conducted face - to - face interviews with diabetic patients aged 18 years attending clinics affiliated with an academic medical center (university of chicago, chicago, il) or physician offices affiliated with a suburban hospital (macneal hospital, berwyn, il). before conducting the study, we knew that patients attending the university of chicago were predominately african american, whereas patients attending macneal hospital were mainly latino and caucasian. to achieve an ethnically diverse study population, we planned for balanced recruitment from the two sites. we identified patients using electronic billing data provided by the clinics. patients were included if they had an icd-9 billing code of 250.xx in the past year and were 18 years of age. we excluded patients with type 1 diabetes, as well as those who had dementia during telephone recruitment. additional patients were excluded if they scored 90% of patients having a prescription drug plan. there was no significant difference in the mean duration of diabetes across ethnic groups (910 years). however, there were differences in the prevalence of comorbid conditions or complications across ethnic groups. latinos had less self - reported hypertension than african americans and caucasians, but there was no difference in prevalence of hypercholesterolemia. african americans had higher rates of self - reported diabetes complications such as eye disease, heart disease, and stroke, whereas latinos had the lowest rates. for overall health status, latinos had higher mean physical component summary scores than the other ethnic groups but lower mental component summary scores. mean a1c levels were higher for african americans (7.54%) and latinos (7.69%) than for caucasians (7.18%) (p < 0.01) ; consistent with these differences, lower proportions of african americans (41%) and latinos (47%) had a1c levels < 7% than of caucasians (55%) (p < 0.01). with regard to cholesterol control, latinos also had the highest mean ldl cholesterol levels (102 vs. 95 mg / dl) and the lowest proportion of patients with ldl cholesterol levels < 100 mg / dl (56 vs. 63%) of the three racial / ethnic groups (p < 0.01). the picture was reversed in the case of blood pressure control in that latinos had the lowest mean systolic blood pressure levels (126 mg / dl) of the three groups, whereas african americans had the highest (136 mg / dl) (p < 0.01). consistent with our findings related to risk factor levels, latinos had the lowest use of medications among the three racial / ethnic groups : the lowest mean number of both total medications and diabetes - related medications (three vs. four medications for african americans and caucasians ; p < 0.01). in terms of intensity of glucose control regimen, latinos also had the lowest percentage of insulin use (18%), whereas african americans had the highest use of insulin (27%) (p < 0.01). similarly, latinos had the lowest percentage of aspirin prophylaxis use (24 vs. 47% for caucasians) and cholesterol - lowering drug use (51 vs. 68% for caucasians ; both p < 0.01). frequently used nondiabetes medications included multivitamins, proton - pump inhibitors, calcium supplementation, and thyroid replacement therapy. in direct questions regarding concerns for medications, both african americans and latinos had significantly more concerns about various elements of medication - taking than caucasians (table 2). they were more likely to say that they worried about side effects (african americans 49% and latinos 66% vs. caucasians 39%), development of dependency on medications (52% and 65% vs. 39%, respectively), and the potential harms of generic substitutes (35 and 26% vs. 13%, respectively ; all p < 0.01). three - quarters of latinos had concerns about the costs of medications compared with one - half of african americans and caucasians (p < 0.01). with regard to the implications of future changes to medication regimens, african americans and latinos were also more likely to report that changes in their medication regimen would represent a disruption in their daily routine and would raise concerns about their health. when we directly queried patients about their willingness to adopt more medications, we found significant differences across racial / ethnic groups. more african americans (18%) and latinos (12%) than caucasians would be opposed to the addition of more medications if recommended by their physician (7%) (p < 0.01), although the majority of patients in all groups would accept such changes in their medications. when asked about the possibility of adding insulin (among those not using insulin), the proportion of patients opposed to such a change was larger than the proportion opposed to the general addition of more medications. as observed with the prior question, larger proportions of african americans (26%) and latinos (22%) than caucasians (17%) were opposed to the addition of insulin to their regimens (p = 0.09), although this difference did not reach statistical significance. the majority of these unadjusted racial / ethnic differences in medication concerns were attenuated by adjustment for socioeconomic, demographic, and clinical covariates but remained significant (table 3). for example, the association between race / ethnicity and side effect concerns became less pronounced in adjusted analysis (for latinos, unadjusted odds ratio (or) 3.00 [95% ci 1.984.55 ] adjusted or 2.92 [1.834.64 ]). with regard to responses to questions regarding the addition of more medications, african americans remained significantly more likely to express a reluctance to add new medications than caucasians (unadjusted or 3.05 [1.665.61 ] adjusted or 2.53 [1.354.72 ]) even after adjustment for socioeconomic, demographic, and clinical covariates. latino - caucasian differences in opinion were borderline in significance in unadjusted analysis (1.95 [0.973.93 ]) and became clearly nonsignificant (1.48 [0.693.15 ]) when accounting for covariates. in our comprehensive analysis of predictors of a reluctance to add medications, we found that concerns about growing dependent on medications, report of unpleasant or painful experience with medications, anticipated disruption of daily routine, concerns regarding health if faced with medication changes, and concerns over switches from brand - name to generic drugs were all significantly associated with a reluctance to add more medications (all p < 0.01). the patient 's physical health status, current number of medications, and current number of diabetes - related medications were not significantly associated with a reluctance to add more medications. higher mental health status was associated with a lower likelihood of opposing additional medications (p < 0.01). after identification and exclusion of collinear variables, the final model of a reluctance to add more medications included report of unpleasant or painful experience with medications (or 2.43 [95% ci 1.394.27 ] ; p < 0.01) and anticipated disruption of daily routine (1.97 [1.143.42 ] ; p = 0.02). african american race remained a significant predictor of reluctance to add more medications even within the fully adjusted model (2.48 [1.324.69 ] ; p < 0.01). our study provides valuable insights into how concerns regarding quality of life with medications and the willingness to adopt more medications vary across type 2 diabetic patients of various racial / ethnic groups. over one - half of racial / ethnic minority patients in our sample expressed concerns regarding side effects of medications and the development of possible dependency on medications, in comparison with over one - third of caucasian respondents. similarly, approximately one - half of minority patients would be concerned about their health as a result of changes in medication regimens in comparison with 22% of caucasian patients. latinos and african americans were also more likely to voice reluctance to adding medications. in adjusted analyses, patients who expressed a concern regarding the use of medications were more likely to express reluctance to adding medications. the basic finding of ethnic differences in concerns regarding life with medications suggests that the everyday experiences of living with diabetes treatments are, on average, experienced differently by ethnic groups. these differences in medication concerns may contribute to differences in perceived quality of life related to diabetes (23). ethnic differences in perception will require additional study to determine their origins and to clarify their implications for long - term treatment adoption and disparities in care. it is important to note that many of the concerns and attitudes related to medications were shared among all patients. patients of ethnic minorities were more likely to express concerns regarding current and future medications, but many caucasian patients also shared these concerns. concerns regarding the physical and logistical burdens of managing multiple medications were quite prevalent throughout the population and are clearly important to acknowledge with patients before intensifying or altering medication regimens. we found that the experience of pain or discomfort with the use of current oral medications was an independent predictor of reluctance to add more medications. it should be noted that the majority of patients within each racial / ethnic subgroup were willing to take additional medication, including insulin, if advised to do so by their health care providers. these results are important reminders that it is important for providers to approach patients as individuals and elicit concerns about current and future medications from patients irrespective of race / ethnicity. it may be possible to acknowledge medication concerns while also intensifying diabetes care through a shared decision - making approach to treatment decisions (24). our recruitment rate was low, which is likely related to our attempt to recruit patients irrespective of frequency of prior visits to a physician. we speculate that nonrespondents were less frequent attendees of clinic and potentially less adherent to medical therapies than respondents. if that is the case, our findings likely underestimate the level of concern about life with medications among patients with diabetes. a related limitation of our study is that the views of the patients whom we surveyed may not be representative of all patients with diabetes. the surveyed patients all had physicians that granted us permission to recruit them ; the majority of subjects had health insurance and prescription drug coverage. in addition, our spanish - speaking latino patients all saw physicians who spoke spanish. given known disparities in insurance coverage and access to providers, it is likely that the degree of health disparities in this sample and the extent of racial / ethnic differences in responses to questions might be less pronounced than in a population - based study. nonetheless, we still found important disparities in risk factor control and important differences in medication concerns. it is also important to recognize that the ethnic group assignment in our sample reflects the specific ethnic mix of chicago neighborhoods that composed our clinical sites. the makeup of the same ethnic group categories may differ in other parts of the city, state, and country. our study findings also reflect the views of patients before the publication of reports regarding the potential risks associated with very intensive glucose control. current concerns about quality of life with diabetes - related medications may be even more widespread than we have found. our study provides insights into how reported concerns regarding diabetes - related medications vary across racial / ethnic groups. however, it remains unclear whether these concerns and perceptions regarding medications are at the root of observed disparities in diabetes care and outcomes (25). longitudinal studies that help to test the association between medication concerns with intermediate outcomes after adjusting for diabetes severity will be valuable to addressing this critical question in diabetes health disparities research. | objective to evaluate ethnic differences in medication concerns (e.g., side effects and costs) that may contribute to ethnic differences in the adoption of and adherence to type 2 diabetes treatments.research design and methods we conducted face - to - face interviews from may 2004 to may 2006 with type 2 diabetic patients 18 years of age (n = 676 ; 25% latino, 34% non - hispanic caucasian, and 41% non - hispanic african american) attending chicago - area clinics. primary outcomes of interest were concerns regarding medications and willingness to take additional medications.resultslatinos and african americans had higher a1c levels than caucasians (7.69 and 7.54% vs. 7.18%, respectively ; p < 0.01). latinos and african americans were more likely than caucasians to worry about drug side effects (66 and 49% vs. 39%, respectively) and medication dependency (65 and 52% vs. 39%, respectively ; both p < 0.01). ethnic minorities were also more likely to report reluctance to adding medications to their regimen (latino 12%, african american 18%, and caucasian 7% ; p < 0.01). in analyses adjusted for demographics, income, education, and diabetes duration, current report of pain / discomfort with pills (odds ratio 2.43 [95% ci 1.394.27 ]), concern regarding disruption of daily routine (1.97 [1.143.42 ]), and african american ethnicity (2.48 [1.324.69 ]) emerged as major predictors of expressed reluctance to adding medications.conclusionslatinos and african americans had significantly more concerns regarding the quality - of - life effects of diabetes - related medications than caucasians. whether these medication concerns contribute significantly to differences in treatment adoption and disparities in care deserves further exploration. |
metaiodobenzylguanidine (mibg) is an agent that is specifically taken up by sympathetic nervous system tissues, including neuroblastoma tumors. i - mibg plays an essential role in the diagnostic evaluation of patients with neuroblastoma. in addition, high - dose i - mibg therapy is an important part of the treatment of patients with relapsed or refractory neuroblastoma. the norepinephrine transporter (net ; encoded by slc6a2 gene) is thought to be the primary transporter responsible for specific active cellular uptake of mibg. several studies have demonstrated that neuroblastoma cell lines that lack net mrna expression fail to accumulate mibg [46 ]. net mrna levels appear to correlate in vitro with extent of mibg uptake [68 ]. moreover, a range of cells that do not typically accumulate mibg can be engineered to do so by transfection of the net gene [917 ]. additional studies in neuroblastoma and other neuroendocrine tumors have suggested that vesicular monoamine transporters (vmats) and organic cation transporters (octs) may also play a role in mediating uptake of mibg [1820 ]. approximately 10% of patients with neuroblastoma have tumors that do not accumulate mibg on the basis of negative diagnostic i - mibg scans. one small study utilized rt - pcr to evaluate net gene expression in 6 neuroblastoma tumors from patients with negative baseline mibg diagnostic scans. none of these tumors had detectable net mrna, while 90% of the 48 mibg - avid tumors had detectable net transcripts. the correlation between clinical mibg uptake with net protein expression and mrna expression of other putative mibg transporters has not been studied. the primary aim of the current study was to determine the association between tumor net mrna and protein expression with mibg avidity in patients with neuroblastoma. secondary aims included evaluation of the association of slc6a2 gene polymorphisms with mibg avidity, association of mrna expression for other membrane transporters with mibg avidity, as well as correlation of net protein expression levels with clinical features. patients were selected from children 's oncology group (cog) protocols a3961 and a3973 for the treatment of intermediate - risk or high - risk neuroblastoma, respectively [22, 23 ]. for the primary analyses focused on net expression, all patients with institutional report of mibg nonavid tumors and available tumor mrna were included (n = 29). an unselected group of patients with institutional report of mibg avid tumors and available tumor mrna was also included (n = 54) to achieve the desired sample size for the primary analyses. for evaluation of net genotype, patients from a3961 and a3973 who had been included in a previous large - scale neuroblastoma genome - wide association project were included [24, 25 ]. patients were treated according to protocol therapy, as previously described [22, 23 ]. all laboratories performing rt - pcr, immunohistochemistry, and genotyping were blinded to patient characteristics, including mibg avidity. all participants or legal guardians provided consent for use of submitted tissue at the time of initial enrollment onto a cog or legacy group neuroblastoma biology study. the cog neuroblastoma biology committee and the ucsf committee on human research approved the study. patients underwent diagnostic i- or i - mibg whole body scans at the time of study entry onto either clinical trial a3973 or a3961. scans were obtained according to local institutional practice and coded as mibg avid or nonavid by the treating investigator. baseline diagnostic mibg scans were available for central review for 27 patients with high - risk disease treated on protocol a3973 who also had tumor mrna available for analysis. the primary clinical endpoint for the current study, mibg avidity, is based upon the results of central review in these 27 patients. central review was independent of the current study and preceded determination of tumor transporter expression levels for the current study. investigator assessment of mibg avidity was used only for evaluation of correlation of net gene polymorphisms with mibg avidity. neuroblastoma tumor material from patients participating in the cog neuroblastoma biology study was submitted frozen to the neuroblastoma tumor bank at the cog biopathology center (columbus, oh). for tumors with a minimum of 60% neuroblastoma tumor in the submitted material, rna was extracted using invitrogen life technologies trizol reagent (total rna isolation reagent ; grand island, ny). mrna quality was assessed using either rna integrity number (rin) or ratio of absorbance at 260 and 280 nm (a260/a280). reverse transcriptase pcr (rt - pcr) was performed with applied biosystem 's (abi) high capacity reverse transcription kit using 2 g of total rna as per manufacturer instructions. quantitative real - time pcr (q - pcr) reactions were conducted with taqman fast universal master mix (abi) in 384-well reaction plates using 10 ng of cdna per reaction. commercial taqman probe and primer sets for net (assay i d : hs01567441_m1), oct-1 (assay i d : hs00427552_m1), oct-2 (assay i d : hs00533907_m1), oct-3 (assay i d : hs01009568_m1), vmat-1 (assay i d : hs00915193_m1), vmat-2 (assay i d : hs00161858_m1), mate-1 (assay i d : hs00217320_m1), gapdh (assay i d : hs99999905_m1), and pgk1 (assay i d : hs99999906_m1) were purchased from abi. reactions were run on an abi prism 7900ht, and the thermal cycling conditions were 95c for 20 seconds followed by 60 cycles of 95c for 3 seconds and 60c for 30 seconds. results using either control were well correlated (r = 0.91) such that only the results normalized to pgk1 are presented. expression vector containing net cdna was used as a positive control and demonstrated that net expression levels in the tumor tissue were within the linear range of the standard curve. all samples were tested in triplicate, and the result reported is the mean of three separate experiments. paraffin - embedded tumor material was prepared using standard methods at each treating institution and submitted to the cog neuroblastoma tumor bank. each of the formalin fixed paraffin embedded glass slides was stained with a commercially available net antibody (net17 - 1 ; mab technologies ; stone mountain, ga) at a 1 : 1000 dilution. antigen retrieval was performed in a pressure cooker with 0.01 m - citrate buffer at a ph of 7.6. slides were blocked using the avidin biotin blocking kit (sp-2001 ; vector laboratories ; burlingame, ca). the secondary antibody used was a biotinylated anti - mouse igg 1 : 200 dilution (vector). slides were then incubated per manufacturer protocol with an avidin biotin complex (vector). lastly, the slides were incubated with diaminobenzidine using a high - sensitivity substrate chromogen system (dako ; carpinteria, ca) and counterstained with hematoxylin. staining results were assessed by one pathologist (bp) using a four point scoring system : 0 + = tumor cells display complete lack of staining ; 1 + = tumor cells show faint cytoplasmic staining ; 2 + = tumor cells show intermediate cytoplasmic staining ; 3 + = tumor cells display intense cytoplasmic staining (see supplemental figure 1 for representative sections in suplementary material available online at doi:10.1155/2012/250834.) the percentage of cells positive for net by ihc was also determined (supplemental figure 1). a net protein expression composite score was derived by multiplying the percentage of tumor cells expressing net by the intensity score. germline dna was isolated and genotyped as previously described. in brief, the illumina humanhap550 beadchip was used to genotype samples at over 550,000 single nucleotide polymorphisms (snps). from this larger dataset, all snps in the net gene (slc6a2) locus were chosen for analysis in the current study. while no snps were excluded due to low minor allele frequencies, only 4 snps had minor allele frequencies 0.10 for all comparisons ; table 4). we report for the first time a positive association between clinical mibg tumor avidity and net protein expression by neuroblastoma cells. this finding supports the critical role of net in mediating specific active uptake of mibg into neuroblastoma cells. our results are also consistent with previous preclinical studies demonstrating a correlation between net protein levels and mibg uptake into myocardial cells. while we observed a significant association between mibg avidity and net protein expression, we also noted overlap in net protein expression between patients with mibg avid and mibg nonavid tumors. specifically, our cohort includes both patients with high net protein expression and mibg nonavid tumors as well as patients with low net protein expression and mibg avid tumors. given that mibg avidity was centrally assessed by independent review, misclassification of mibg avidity is unlikely to account for these findings. our genotyping results suggest that individual variations in the structure of the slc6a2 gene are also unlikely to account for these results. instead, our findings raise the possibility that mibg uptake may be influenced by factors in addition to net protein. it is possible that tumors with low net protein expression may accumulate mibg via other transporters. candidate transporters include octs and vmats as these transporters have been implicated in mibg uptake in other neuroendocrine tumors [1820 ]. our evaluation of oct and vmat mrna expression did not reveal any statistically significant associations with mibg avidity, including subset analysis focusing only on patients with low net protein expression and mibg avid tumors (data not shown). given the trend showing higher vmat1 and vmat2 mrna median levels in mibg avid tumors, future studies will focus on tumor vmat protein levels. likewise, it is possible that tumors with high net protein expression that fail to accumulate mibg may have increased expression of mibg efflux transporters that account for low net uptake of mibg. while mate-1 mrna expression did not correlate with mibg avidity, evaluation of protein levels of mate-1 and other efflux transporters may be informative. alternatively, other physiologic parameters, such as tumor vascularity and ph, may impede distribution of mibg to the tumor cell membrane. we were unable to replicate previous findings demonstrating an association between net mrna expression and clinical mibg avidity. first, our study relied on archived tumor mrna obtained within the context of cooperative group trials. as such, degradation of mrna may have resulted in a false negative result, though our mrna quality data may argue against this point. our findings suggest that immunohistochemistry for net protein may be a more practical approach for evaluating net expression in future cooperative group studies and in clinical practice. second, as discussed above, it is possible that the association between mibg avidity and either net protein or mrna expression is imperfect. we note that in previous studies of net mrna levels as predictors of clinical mibg uptake, cases of clinical mibg avidity in the setting of low net mrna expression have been reported [4, 30 ]. in one previous study, 5 of 11 patients with negative pcr for net mrna nevertheless had positive mibg scans. third, it is possible that, as we observed, net mrna expression does not correlate with net protein expression in human neuroblastoma tissue, perhaps through posttranslational modification of net protein. one of our secondary analyses yielded the previously unreported association between mycn amplification and low net protein expression. other statistically significant clinical correlations with lower net protein expression (patients with high - risk disease and patients with metastatic disease) may be driven by this association with mycn amplification. as an exploratory secondary analysis unadjusted for multiple statistical testing, it is also possible that this association is a chance finding and therefore requires replication by other groups. we note that one previous report did not detect a difference in clinical mibg avidity according to tumor mycn status. in addition, response rates after high - dose i - mibg therapy do not appear to differ between patients with mycn amplified and mycn nonamplified tumors, though all patients were required to have mibg avid tumors to receive i - mibg therapy. the association between mycn status and clinical mibg avidity will be investigated further in a future analysis by our group. we have demonstrated that neuroblastoma tumor cell net protein expression correlates with clinical mibg avidity and also with tumor mycn status. additional work, including gene and protein expression profiling efforts, will need to investigate other mechanisms of mibg uptake in cases with low net protein expression. future studies may also focus on the correlation between tumor net protein expression and response to targeted radiotherapy with high - dose i - mibg. | purpose. 123i - metaiodobenzylguanidine (mibg) is used for the diagnostic evaluation of neuroblastoma. we evaluated the relationship between norepinephrine transporter (net) expression and clinical mibg uptake. methods. quantitative reverse transcription pcr (n = 82) and immunohistochemistry (ihc ; n = 61) were performed for neuroblastoma net mrna and protein expression and correlated with mibg avidity on diagnostic scans. the correlation of net expression with clinical features was also performed. results. median net mrna expression level for the 19 mibg avid patients was 12.9% (range 1.673.7%) versus 5.9% (range 0.6110.0%) for the 8 nonavid patients (p = 0.31). median percent net protein expression was 50% (range 0100%) in mibg avid patients compared to 10% (range 080%) in nonavid patients (p = 0.027). mycn amplified tumors had lower net protein expression compared to nonamplified tumors (10% versus 50% ; p = 0.0002). conclusions. net protein expression in neuroblastoma correlates with mibg avidity. mycn amplified tumors have lower net protein expression. |
erosion is defined as the loss of tooth structure due to the function of acids and irrespective of bacterial activity. by changing lifestyle during the recent decades, consumption of acidic foods and beverages role of food acids as the main cause of erosion has been documented in numerous studies. dentin hypersensitivity is usually associated with cervical erosion and has been suggested as a direct clinical outcome of erosion. clinical pattern of erosion includes porosities in dentin surrounded by a prominent enamel margin. various studies have evaluated the role of fluoride - containing solutions and varnishes and casein, calcium and phosphate - containing compounds (casein phosphopeptide - amorphous calcium phosphate [cpp - acp ]) in the prevention and reduction of erosive wear. application of ionized fluoride, i.e., sodium fluoride, amine fluoride or stannous fluoride on tooth surfaces results in deposition of calcium fluoride (caf2) on the enamel surface. under neutral conditions, caf2 deposition is facilitated by increasing the concentration of fluoride ion, longer exposure time, and lowering the ph of the solution. the fluoride ions released from caf2 can incorporate into the dental hard tissue and result in its further stability, hardness and increased abrasion resistance. when enamel and dentin are exposed to fluoride ions, the calcium and phosphate present in tooth structure form fluorapatite crystals with these ions. cpp - acp complex provides optimal concentrations of calcium and phosphate ions for enhancement of enamel remineralization. tooth mousse (tm) is a water - based sugar - free cream that contains cpp - acp. when applied, it maintains optimal concentrations of calcium and phosphate ions on enamel surfaces to enhance remineralization. in vitro studies have demonstrated that cpp - acp can be absorbed by the salivary pellicle and dental plaque. thus, a calcium - rich reservoir is formed that can facilitate remineralization. until date, no study has compared the efficacy of cpp - acp paste and sodium fluoride mouthwash for prevention of erosive wear. considering the popularity of sodium fluoride mouthwash among the iranian population and the recent introduction of tm to the iranian market, the present study was conducted aiming at comparing the efficacy of 0.2% sodium fluoride mouthwash and cpp - acp paste for prevention of dentin erosion in human teeth. in this study, various researchers have shown that profilometry is a highly accurate method for the measurement of surface loss (> 0.4 m). this in vitro experimental study was conducted on 36 sound human premolar teeth (with no caries or fracture) that had been extracted as the result of periodontal disease or for orthodontic treatment. calculation of sample size in each group was done using ncss software (ncss 2007. the surfaces were then rinsed with deionized water and placed in ethylenediaminetetraacetic acid 17% solution for 1 min for smear layer removal. afterwards, half the tooth surfaces were covered with adhesive tape and samples were then randomly divided into three groups of 12 each. for higher precision and comfort in the next steps (pretreatment and erosion phases), three clean and transparent glasses were prepared and a circle was drawn on their internal surfaces in a height higher than the mid - height of the longest tooth. the drawn circle was covered with adhesive tape to prevent wiping off or fading when in contact with acid and mouthwash. in the next step, the samples were glued to the external surface of glasses (each group on one glass) in a way that the covered halves of the teeth were placed above the drawn circle and their remaining halves were located below it. in the next steps, these glasses were placed in dishes containing acid or mouthwash. the amount of acid and mouthwash was regulated in a way that the surface of acid or mouthwash was in alignment with the drawn circle. by doing so, we made sure that the upper halves of teeth were not exposed to acid or mouthwash and were safely protected from the effects of these solutions. in the first group, samples received pretreatment with cpp - acp paste (tm, gc, japan) according to the manufacturer 's instructions. tm paste was applied on samples 4 times a day (5 min each time) for 5 days. after each phase of pretreatment, the teeth were irrigated with deionized water and stored in artificial saliva (ph = 7.3) until the next phase. deionized water, carboxymethyl cellulose, mgcl2, kcl, cacl2, nacl, k2hpo4, and sorbitol were the constituents of artificial saliva. this solution was prepared by the iran polymer and petrochemical research center upon the author 's request. in the second group, samples were pretreated with 0.2% sodium fluoride mouthwash (behsa, iran). samples were placed in the mouthwash 4 times a day (1 min each time) for 5 days according to the manufacturer 's instructions. after each phase of pretreatment, the teeth were irrigated with deionized water and stored in artificial saliva. after completion of pretreatment phase, the teeth in all three groups were exposed to acid under similar conditions. the samples were placed in a glass of coca - cola 4 times a day (2 min each time) for 3 days. as mentioned earlier, the surface of drink was in alignment with the drawn circle to prevent penetration of drink under the adhesive tape. after each phase, samples were rinsed with deionized water and stored in artificial saliva until the next phase of the test. furthermore, in order to have a stable rest for profilometry each group of teeth were glued on a piece of cardboard. in the next phase, the amount of surface loss in samples was measured by a profilometer (emd-1500 - 311, mahr federal inc. the output of profilometer is the greatest difference in surface area scanned by the stylus which is shown as ymax. for each tooth, profilometry was performed 3 times (for the protected surface, for the eroded surface and for a total surface area) and three output data were obtained. finally, the protected and eroded surfaces in each tooth were compared with each other. values measured by the profilometer data were analyzed using spss version 16 software (spss inc, chicago, il, usa). one - way anova was used for comparison of erosion rate and surface changes in the three understudy groups. the surfaces were then rinsed with deionized water and placed in ethylenediaminetetraacetic acid 17% solution for 1 min for smear layer removal. afterwards, half the tooth surfaces were covered with adhesive tape and samples were then randomly divided into three groups of 12 each. for higher precision and comfort in the next steps (pretreatment and erosion phases), three clean and transparent glasses were prepared and a circle was drawn on their internal surfaces in a height higher than the mid - height of the longest tooth. the drawn circle was covered with adhesive tape to prevent wiping off or fading when in contact with acid and mouthwash. in the next step, the samples were glued to the external surface of glasses (each group on one glass) in a way that the covered halves of the teeth were placed above the drawn circle and their remaining halves were located below it. in the next steps, these glasses were placed in dishes containing acid or mouthwash. the amount of acid and mouthwash was regulated in a way that the surface of acid or mouthwash was in alignment with the drawn circle. by doing so, we made sure that the upper halves of teeth were not exposed to acid or mouthwash and were safely protected from the effects of these solutions. two groups received pretreatment before placing in acid solution. in the first group, samples received pretreatment with cpp - acp paste (tm, gc, japan) according to the manufacturer 's instructions. tm paste was applied on samples 4 times a day (5 min each time) for 5 days. after each phase of pretreatment, the teeth were irrigated with deionized water and stored in artificial saliva (ph = 7.3) until the next phase. deionized water, carboxymethyl cellulose, mgcl2, kcl, cacl2, nacl, k2hpo4, and sorbitol were the constituents of artificial saliva. this solution was prepared by the iran polymer and petrochemical research center upon the author 's request. in the second group, samples were pretreated with 0.2% sodium fluoride mouthwash (behsa, iran). samples were placed in the mouthwash 4 times a day (1 min each time) for 5 days according to the manufacturer 's instructions. after each phase of pretreatment, the teeth were irrigated with deionized water and stored in artificial saliva. after completion of pretreatment phase, the teeth in all three groups were exposed to acid under similar conditions. the samples were placed in a glass of coca - cola 4 times a day (2 min each time) for 3 days. as mentioned earlier, the surface of drink was in alignment with the drawn circle to prevent penetration of drink under the adhesive tape. after each phase, samples were rinsed with deionized water and stored in artificial saliva until the next phase of the test. furthermore, in order to have a stable rest for profilometry each group of teeth were glued on a piece of cardboard. in the next phase, the amount of surface loss in samples was measured by a profilometer (emd-1500 - 311, mahr federal inc., germany). the output of profilometer is the greatest difference in surface area scanned by the stylus which is shown as ymax. for each tooth, profilometry was performed 3 times (for the protected surface, for the eroded surface and for a total surface area) and three output data were obtained. finally, the protected and eroded surfaces in each tooth were compared with each other. data were analyzed using spss version 16 software (spss inc, chicago, il, usa). one - way anova was used for comparison of erosion rate and surface changes in the three understudy groups. table 1 shows the number of samples and statistical data regarding ymax for the protected and eroded surfaces, the total ymax and the surface loss. since, the whole tooth had similar surface roughness before exposure to acid, the surface loss or amount of erosion was calculated by subtracting ymax of the protected area from the total ymax. this rate was 2.24 2.4 m in the paste pretreatment and 1.14 0.81 m in the mouthwash group, respectively. test results of the three understudy groups anova failed to find a significant difference in mean surface roughness of protected surfaces between the three groups (p = 0.051). furthermore, no significant difference was detected in mean surface roughness of eroded surfaces after exposure to acid between the three groups (p = 0.052). comparison of mean total ymax and mean surface loss revealed a significant difference between the three groups (p < 0.05). comparison of mean total ymax between the three groups showed no significant difference between the paste and the mouthwash groups (p = 0.098). the paste and the control groups did not have a statistically significant difference in this respect either (p = 0.853). however, the difference between the mouthwash and the control group in this regard was statistically significant (p < 0.05). comparison of mean surface loss between the three groups revealed no statistically significant difference between the paste and the mouthwash groups (p = 0.284). the paste and the control groups did not have a statistically significant difference in this regard either (p = 0.094). however, the difference between the mouthwash and the control group was statistically significant (p < 0.05). figure 2 shows ymax values for the protected surfaces of the three groups. error bar of mean and 95% confidence interval of ymax for protected surfaces in the three groups of tooth mousse, mouthwash, and control figure 3 shows ymax values for surfaces exposed to acid. error bar of mean and 95% confidence interval of ymax for eroded surfaces in the three groups of tooth mousse, mouthwash, and control total ymax values are demonstrated in figure 4. the ymax values are obtained when diamond stylus is moved vertically in contact with the protected and eroded tooth surfaces and then moved laterally across them for a specified distance and specified contact force. error bar of mean and 95% confidence interval of total ymax for the three groups error bar of mean and 95% confidence interval of surface loss in the three groups dentin hypersensitivity is among the direct outcomes of erosion that may occur in clean tooth surfaces. acid reflux and acidic foods and beverages can dissolve the smear layer and expose dentinal tubules to the oral cavity resulting in aggravation of dentin hypersensitivity. consumption of acidic foods and beverages play a significant role in occurrence of acid erosion in teeth. considering the growing consumption of soft drinks and increased prevalence of dentin hypersensitivity among patients, the present study was conducted aiming at evaluating the role of preventive factors in prevention of erosion due to the consumption of coca - cola which is a popular drink worldwide. the effectiveness of fluoride in toothpastes and mouthwashes is mainly because of strengthening the tooth surface against dissolution. studies conducted on caries have shown that fluoride supplementation in the form of mouthwash increases the concentration of fluoride ion in the mouth which subsequently results in strengthening of teeth surfaces. topical application of fluoride compounds leave behind a considerable amount of fluoride on tooth surfaces due to the porosities and water content of dentin. in deeper dentin layers, high fluoride concentrations act as a fluoride reservoir. various studies have compared different fluoride - containing solutions and varnishes, fluoride mouthwashes with the different compositions and fluoride products in combination with other preventive materials. others have evaluated the effect of increased concentration of sodium fluoride and the efficacy of fluoride compounds at various phs for prevention of erosion. furthermore, it has been shown that calcium - containing compounds are capable of preventing erosive wear. cpp - acp complex provides super - saturated concentrations of calcium and phosphate ions that prevent demineralization by providing a rich source of neutral ion pair (capo4) and enhance the formation of hydroxyapatite crystals in demineralized lesions. however, it has been revealed that cpp - acp remineralizes the enamel surface eroded as the result of acid exposure and increases its hardness. many compounds other than cpp - acp are involved including glycerol, xylitol, propylene glycol, water, metal oxides, and hydroxybenzoates. glycerol has been suggested to play a major role in the efficacy of tm for reduction of erosive wear of enamel and dentin due to its lubricating effect. rees., in their study compared the efficacy of tm (containing cpp - acp) and pronamel toothpaste for erosion prevention taleb., compared cpp - acp and fluoride gel in remineralization of demineralized human enamel surfaces. some other studies assessed the effect of cpp - acp on prevention of erosive enamel and dentin wear and demineralization. ymax value for protected surfaces from the total ymax was calculated, since ymax of protected surfaces was not significantly different between the three groups. the results of this study revealed that both cpp - acp paste and sodium fluoride mouthwash were capable of reducing erosion which is in accord with the findings of wiegand., magalhes., rees., and ranjitkar., comparison of means showed that the rate of erosion (surface loss) in the control group was higher than the tm and mouthwash groups. furthermore, the level of erosion in the tooth mouse group was greater than the mouthwash group. thus, the efficacy of mouthwash for prevention of erosion was greater than that of tm. paired comparison of groups revealed that although the rate of erosion (surface loss) in the cpp - acp group was lower than the control and higher than the mouthwash groups, none of these differences were statistically meaningful. in contrast, rate of erosion (surface loss) in the mouthwash group was significantly lower than the control group which was in agreement with the findings of hamba., they showed that sodium fluoride and cpp - acpf paste were more effective than cpp - acp for inhibition of enamel demineralization. more recently many valuable articles are published regarding effect of cpp - acp on enamel by shetty. in 2014, somasundaram. in 2013, vashisht. in 2013, and patil. in 2013, and different results have been obtained, which all show that cpp with fluoride is a promising material for remineralization of enamel subsurface lesions and it means that still more studies have to be done to evaluate exact protective effect of cpp - acp. this study showed that using mouthwash is an effective method for prevention of erosion in at - risk patients. sodium fluoride mouthwash provides protection against caries and prevents erosive tooth wear by strengthening the enamel surface. | aim : the purpose was to compare the effect of 0.2% sodium fluoride mouthwash and casein phosphopeptide - amorphous calcium phosphate paste on prevention of dentin erosion.materials and methods : buccal surfaces of 36 sound premolar teeth were ground flat and polished with abrasive discs. half the polished surfaces were covered with tape to maintain a reference surface. samples were randomly allocated into three groups. group a was pretreated with tooth mousse (tm) 4 times a day for 5 days. group b was pretreated with 0.2% sodium fluoride mouthwash 4 times a day for 5 days. group c was considered as the control group with no pretreatment. in the next step, the samples were exposed to coca - cola 4 times a day for 3 days. after each erosive cycle, the samples were rinsed with deionized water and stored in artificial saliva. the surface loss was determined using profilometry.results:the erosion in both groups a and b was less than the control group. the surface loss in mouthwash group was significantly lower than in the control group. erosion in tm group was more than the mouthwash group and less than the control group.conclusion:sodium fluoride mouthwash is more effective for prevention of dentin erosion. |
coarctation of the aorta is a localised stenosis of the aorta, most commonly located just distal to the origin of the left subclavian artery and closely related to the attachment of the ductus arteriosus with the aorta. frequently associated congenital defects are a bicuspid aortic valve, valvular and subvalvular aortic stenosis, mitral stenosis, ventricular septal defect and persistent ductus arteriosus. coarctation causes an increased afterload for the left ventricle, resulting in an increased systolic pressure in the left ventricle and upper part of the body. treatment of aortic coarctation can be performed surgically or percutaneously. even after successful correction hypertension often persists due to abnormal vascular physiology. uncorrected or unnoticed coarctation results in a high morbidity and mortality, mainly due to hypertension - associated problems. however, patients with isolated coarctation (coarctation without additional congenital defects) often remain asymptomatic for years and the defect may be unnoticed even until the fifth or sixth decade of life. although a late detected coarctation almost always includes irreversible cardiovascular damage, adequate treatment can result in a substantial improvement in both quality of life and prognosis [47 ]. in the present article, we describe two patients with late detected coarctation to illustrate the clinical consequences, diagnostic clues for earlier detection and current therapeutic options to achieve optimal treatment in these patients. a 40-year - old woman was referred to our hospital in 2004 because of a recently discovered difference in blood pressure between the right and left arm and a cardiac murmur. hypertension had already been diagnosed 20 years before and she was treated with an angiotensin receptor blocker. every now and then she suffered from chest pain, especially in bed at night, sporadically during exertion. she had no pregnancy wish and besides hypertension and migraine she had no further medical history. the arterial blood pressure at her right arm was 155/85 mmhg and on the left arm 135/70 mmhg, arterial blood pressure was unobtainable in the lower extremities, and there was a marked delay in the femoral pulses compared with the brachial arteries (femoral brachial delay). auscultation of the chest revealed an early systolic ejection murmur and a soft diastolic murmur. the electrocardiogram showed normal sinus rhythm and conduction times, but also signs of left ventricular hypertrophy (high r - waves in the left precordial leads with associated st - segment changes). at transthoracic echocardiography, left and right ventricular dimensions and function appeared within normal ranges. however, a functional bicuspid aortic valve was identified, with a grade i / iii regurgitation. suprasternal view revealed poor acoustic images, but in the abdominal aorta a clear diastolic forward flow was demonstrated. aortic coarctation was suspected and confirmed by magnetic resonance (mr) imaging showing a wide aortic arch and a significant narrowing, with minimal diameter of 6 mm, just distal of the left subclavian artery and poststenotic dilatation of the descending aorta. the systolic blood pressure gradient between upper and lower extremities appeared to be 3040 mmhg. an uncomplicated balloon angioplasty was performed (using a 30 14 and 40 18 mm balloon, respectively) resulting in a coarctation diameter of 16 mm (fig. a stent was not placed because of the existence of wide pre- and post - coarctation diameters. given the elastic aortic properties, solid stent fixation was not expected. during follow - up of 5 years, no recoarctation occurred, the systolic blood pressure gradient between upper and lower extremities remained 10 mmhg and blood pressure control was satisfactory using low doses of atenolol and losartan. 1a aortic coarctation (red line : 6 mm) distal to the left subclavian artery, before balloon angioplasty ; b aortic coarctation (red line : 16 mm), after balloon angioplasty a aortic coarctation (red line : 6 mm) distal to the left subclavian artery, before balloon angioplasty ; b aortic coarctation (red line : 16 mm), after balloon angioplasty a 55-year - old woman was seen at the outpatient clinic in 2007 because of progressive complaints of angina. her medical history included hypertension and a cardiac murmur, which had already been diagnosed during a medical examination when she was 18 years old. in 1991, besides systolic hypertension, no structural heart disease was found except for a mild aortic regurgitation and a peak systolic pressure gradient of 17 mmhg (heart rate 68 beats / min) over the aortic valve. she was treated with antihypertensive drugs and visited her cardiologist on a regular, yearly basis. because of therapy refractory hypertension she was also referred to the internist to exclude underlying causes of her hypertension, but no underlying cause was diagnosed. when she was 50 years old, she stopped smoking because mild chronic obstructive pulmonary disease was diagnosed. at that time echocardiography showed left ventricular hypertrophy (septum en posterior wall diameter both 12 mm) with normal systolic function, still a mild aortic regurgitation and a peak systolic pressure gradient across the aortic valve of 28 mmhg (heart rate 64 beats / min). in the meantime, she was treated with four different antihypertensive drugs, but she remained hypertensive. to evaluate her complaints of angina in 2007 a nuclear exercise stress test was performed, which showed signs of reversible perfusion defects in the inferior, posterior and lateral walls suggestive of myocardial ischaemia. during following cardiac catheterisation, via the right femoral artery, the catheter could not be advanced up the ascending aorta because of a discrete narrowing of the aorta (fig. 2). catheterisation performed via the radial artery showed a significant stenosis in the circumflex artery which was subsequently dilated and stented. additional computed tomography (ct) scan of the aorta confirmed the diagnosis of coarctation with a pinpoint stenosis in the aorta just distal to the left subclavian artery. moreover, an extensive collateral network, mainly consisting of bronchial and lumbar arteries, was seen (fig. it was decided by our congenital team to accomplish an extra - anatomical bypass 1 year after coronary stenting (fig. an extra - anatomical bypass was constructed between the aortic isthmus and descending aorta by the use of a 16-mm prosthetic graft. no perioperative complications occurred and postoperatively, for the first time in her life, her blood pressure was better under control with antihypertensive drugs even with dose reduction of the medication. after more than 2 years of follow - up she was still free of angina, experienced a better exercise tolerance and her blood pressure levels were under control. 2pinpoint aortic coarctation (a) and collateral vessel (b)fig. 3ct image of severe aortic coarctation (red arrow) just distal to the left subclavian artery, and an extensive collateral networkfig. 4extra - anatomical bypass of aortic coarctation pinpoint aortic coarctation (a) and collateral vessel (b) ct image of severe aortic coarctation (red arrow) just distal to the left subclavian artery, and an extensive collateral network extra - anatomical bypass of aortic coarctation we have described two women with a late diagnosed isolated coarctation, although hypertension had been diagnosed decades before. in retrospect, in both cases, hypertension in combination with a cardiac murmur was already noticed during early adulthood which should have raised the physicians suspicion. femoral delay (delayed femoral pulses), weak femoral pulsations, arterial blood pressure difference between the right and left arm as well as between upper and lower extremities and finally auscultation on the posterior thorax (continuous murmur caused by collateral vessels or systolic murmurs due to coarctation), see also table 1. from a clinician s standpoint it is important to realise that the absence of a difference in blood pressure between the upper extremities does not exclude the presence of aortic coarctation, because in the majority of cases the coarctation is localised distal to the origin of the left subclavian artery. the key sign of an aortic coarctation is therefore a difference in arterial blood pressure measured between the upper and lower extremities. this examination should always be performed in case of unexplained hypertension in (young) adults. specific signs on a chest x - ray are inferior rib notching due to enlarged and tortuous intercostal arteries (in the posterior anterior view), and aortic contour abnormalities (on the lateral view). in addition, directed (colour) doppler echocardiography can confirm the diagnosis or make it less likely, and is important to screen for additional cardiac defects. in the suprasternal long - axis view discrete narrowing of the thoracic aorta, diastolic forward flow can be identified in the descending thoracic aorta and/or in the abdominal aorta which is specific for an aortic coarctation (fig. the severity of the stenosis can be estimated by measuring maximal flow velocity in the descending aorta as compared with prestenosis flow velocity. calculation of the gradient by the simplified bernoulli s equation : pressure = 4 (velocity) should be used with care because the formula is not applicable in non - localised, elongated stenosis. when a coarctation is diagnosed or suspected, imaging by mr of ct can clearly define the localisation of the coarctation, gives a complete view of the aorta including collateral vessels, and can be used to exclude cerebral aneurysms. additional cardiac catheterisation can help to determine the severity of the stenosis and is indicated when associated coronary artery disease is suspected. table 1physical diagnostic clues for aortic coarctationhypertension in combination with a cardiac murmurdifference in arterial blood pressure between upper and lower extremities (and between right and left arm)brachial femoral delay (delayed femoral pulses)weak femoral pulsationscontinuous murmur during auscultation on the posterior thoraxfig. sawtooth with diastolic forward flow in descending aorta, in a patient with severe aortic coarctation physical diagnostic clues for aortic coarctation echo doppler image of typical sawtooth with diastolic forward flow in descending aorta, in a patient with severe aortic coarctation a severe aortic coarctation detected during (early) childhood or in young adults needs to be repaired [10, 11 ]. until recently, however, there was no consensus regarding the optimal treatment strategy for late detected aortic coarctation, because the benefits of surgery were unclear. in addition, expected surgical risks in these patients are higher as compared with surgery during childhood [12, 13 ]. yet, the main concern was that a late detected coarctation has already resulted in substantial degenerative changes in the aorta and the presence of an extensive network of collateral vessels resulting in an increased perioperative risk including bleeding, stroke and, although rare, paraplegia especially in case of a poor collateral circulation. moreover, preoperative coexisting coronary artery disease, left ventricular dysfunction and cerebral vascular disease increase the risk of perioperative complications. however, in the last 10 years several studies have showed the feasibility and benefits of surgical repair in patients older than 50 years [47, 14, 15 ]. the reported perioperative and early mortality in these studies was 0% and the late mortality 04% (mean follow - up 0.14.3 years). perioperative complications occurred in 025% of patients including postperfusion lung oedema, significant pleural effusion, re - exploration for control of bleeding and persistent left vocal cord paralysis. however, the majority of studies have also demonstrated a substantial improvement in the control of systolic hypertension and symptoms during follow - up, even in patients with mild preoperative hypertension [57, 14, 16 ]. these results are of overriding importance in the decision to repair late detected aortic coarctation, preventing hypertension - related complications in the further course of the patient s life. the most applied surgical method in patients with a late detected coarctation perioperative surgical morbidity or mortality may occur but is rare. in recent years, a less invasive alternative for surgery, balloon angioplasty with or without stenting of the aorta, has increasingly been used and shows promising results [17, 18 ]. the technique proved to be feasible, and the rate of recoarctation appeared to be low, similar to that of surgical methods [18, 19 ]. disadvantages of this technique include a per procedure risk of aortic dissection, immediate residual pressure gradients, stent migration and the risk (comparable with surgical repair) of aortic aneurysms during follow - up. the final choice of treatment for late detected aortic coarctation depends upon several factors including the morphology and severity of the coarctation, vascular condition including collateral vessels and comorbidity, and should always be determined in a specialised centre by a team of cardiologists and surgeons with experience in adult congenital heart disease. in patients with unexplained hypertension, especially in combination with a cardiac murmur, the presence of an aortic coarctation should always be ruled out given the high morbidity and mortality. the key sign of an aortic coarctation, a difference in arterial blood pressure measured between the upper and lower extremities, should always be examined, followed by echocardiography. even in case of a late detected severe coarctation, surgical or percutaneous repair has proven to be feasible and substantially effective, improving quality of life and lowering the risk of further problems associated with hypertension. finally, aortic coarctation should be regarded as a diffuse arteriopathy and therefore evaluation and treatment in a specialised centre remains essential aiming for an optimal treatment strategy. | in patients with unexplained hypertension, especially in combination with a cardiac murmur, the presence of an aortic coarctation should always be ruled out given the high morbidity and mortality. however, particularly patients with an isolated coarctation often remain asymptomatic for years and the defect may be unnoticed even until the fifth or sixth decade of life. in the present article, we describe two patients with late detected coarctation to illustrate the clinical consequences, diagnostic clues for earlier detection and current therapeutic options to achieve optimal treatment. the key sign of an aortic coarctation, a difference in arterial blood pressure measured between the upper and lower extremities, should always be examined, followed by echocardiography. we conclude that even in case of a late detected severe coarctation, surgical or percutaneous repair has proven to be feasible and substantially effective, improving quality of life and lowering the risk of further hypertension - associated problems. |
onchocerciasis is a vector - borne parasitic disease caused by infection with the filarial nematode onchocerca volvulus and can result in eye or skin lesions. although adult worms can live for years under the skin in fibrous nodules that are often palpable, morbidity is caused by the body 's immune reaction to the microfilariae (mf) that leave the nodules and migrate through the skin and sometimes enter the eye. in 2007, the world health organization (who) estimated 37 million persons were infected with onchocerciasis in 37 endemic countries (30 in africa, six in the americas, and one in the arabian peninsula). in the americas there are 13 geographically isolated endemic foci found within mexico, guatemala, colombia, venezuela, brazil, and ecuador [3, 4 ] where 470,222 persons were at risk of infection in 2011. the onchocerciasis elimination program of the americas (oepa) was established in 1992 in response to a resolution of the directing council of the pan american health organization (paho) calling for the elimination of onchocerciasis ocular morbidity in the americas by 2007. a new resolution in 2008 by paho called for the interruption of transmission throughout the region by 2012. a subsequent 2009 paho resolution (cd49.r19), calling for the elimination or drastic reduction of 12 neglected infectious diseases of poverty in the americas by 2015, includes onchocerciasis as an elimination target. the oepa strategy is to support national programs in the six endemic countries to provide twice - yearly mass drug administration (mda) of ivermectin (mectizan, donated by merck & co.) to 85% of the eligible population at risk [6, 9, 10 ]. the guatemala ministry of public health (moph), with the assistance of oepa, began mda with ivermectin in 1996 and has achieved 85% coverage of the eligible population at risk in twice - yearly mda rounds since 2002. interruption of transmission was demonstrated in two of the four foci (santa rosa in 2007 and escuintla - guatemala in 2008) [11, 12 ] ; in 2010 both foci completed the posttreatment surveillance, and the evaluations showed elimination of transmission. the huehuetenango focus has participated in 22 rounds of mda with ivermectin over the past 13 years, with 85% coverage in 17 consecutive rounds of twice - yearly ivermectin treatment. e. w. cupp and m. s. cupp calculated that the death of adult worms is accelerated in the presence of ivermectin and that the adult population should die (in the absence of ongoing transmission) after 6.5 years (13 rounds) of twice - yearly treatment. thus, the treatment period appears more than adequate to have eliminated the parasite in the focus of huehuetenango. we, therefore, report the assessment of the status of 2007 - 2008 pretreatment and 2010 - 2011 posttreatment onchocerciasis transmission evaluations in the huehuetenango focus. the huehuetenango focus (15.35n, 91.90e) is situated in the western highlands of guatemala along the border with mexico and consists of eight historically endemic municipios (similar to counties) (figure 2). historical data from this area showed a gradual reduction of infection prevalence well before the initiation of mda. yamagata., in an analysis of nodule prevalence data gathered since 1940 by moph nodulectomy teams working in the area, showed that nodule prevalence decreased from 41% (in 1940) to 21% in 1969. with the launching of ivermectin mda, surveys conducted by the moph in 1987 and 1992 showed that the prevalence of nodules was 1.1% and 2.2%, respectively (f. o. richards, jr., unpublished data). the first surveys to measure the prevalence of infection with o. volvulus mf using skin snips in huehuetenango demonstrated a prevalence of 0% in any survey conducted between 1987 and 1992. due to access problems, distance between communities, availability of the project ophthalmologist, and the number of persons who could be examined in one day, it was considered feasible to sample 40 residents from each of nine communities, which were selected from among the 19 eligible communities by probability proportional to size sampling. all nine selected communities were mapped and a census conducted on all households using a personal digital assistant (pda) to identify all eligible persons (residents 7 years of age with at least five years living in the community) for the evaluation. we selected 13 houses (estimating three eligible individuals per household) at random from each of the selected communities after mapping, and all eligible individuals from these households were invited to participate in the evaluation. an ophthalmologist with extensive experience in onchocerciasis - related ocular morbidity examined participants using a method described previously [11, 14 ]. briefly, residents were examined with a slit lamp for the presence of microfilaria in the anterior chamber or the cornea of either eye. the cumulative incidence of infection with o. volvulus was determined serologically by measuring the prevalence of igg4 antibodies to the recombinant antigen ov16 in school age children (6 to 12 years old) using methods previously described [11, 15 ]. briefly, a sample size of 3000 children was required ; assuming a 30% non - response rate, our final sample size was 4286 children. all schools in the pec were identified, and the number of enrolled children 6 to 12 years old determined. the schools were ordered at random and then selected in order until the required sample size was achieved. forty - three schools from 37 pecs (seven municipios) were selected by this procedure. all children aged 6 to 12 from each selected school were asked to participate in the evaluation, and children that were not present at school during the evaluation were followed up at their homes and asked to participate. finger - prick, filter paper blood samples were collected from all participating children as previously described. igg4 antibodies to the recombinant antigen ov16 were determined using the enzyme - linked immunosorbent assay (elisa) method described previously [11, 15 ]. black flies were collected twice per month from november 2007 to april 2008 (the peak black fly biting season), in four coffee plantations located in the same geographical areas of the pecs. plantations were purposively selected to find those with high densities of s. ochraceum and owners willing to participate in the evaluation. we used a standard black fly collection method described in previous studies [11, 16 ]. briefly, in each plantation, four collection sites, two near the residences and two in the coffee plantation, were identified. a team, comprised of a collector and a paid human attractant (male resident from 18 to 50 years of age), was employed at each collection site and worked two days per month per site. all human attractants received a dose of ivermectin before the evaluation and were assessed for the presence of antibodies to o. volvulus using the ov16 elisa test before and after the evaluation. flies were pooled and evaluated by a standard polymerase chain reaction (pcr) method to identify o. volvulus dna [1719 ]. the evaluation of transmission elimination was conducted between the second and third year after treatment suspension, during peak black fly biting season. this evaluation consisted only of the entomological assessment since o. volvulus recrudescence should first be detected in the vectors. the criteria to determine elimination of transmission is the absence or near absence of infective blackflies (< 1 infective fly per 2000 tested). collection of backflies was carried out from december 2010 to april 2011 in the same four coffee plantations as the pretreatment evaluation and following the same procedures. the exact one - sided 95% ci for the prevalence of mfas and igg4 antibodies to ov16 was obtained as previously described using sas software (version 9.0, sas institute, cary, nc, usa). the proportion of infective flies and the 95% ci were calculated using poolscreen 2.0 [17, 18, 21 ]. standard procedures were used to obtain geometric mean of the biting rate, arithmetic mean of the biting rate, biting density, and the seasonal transmission potential. the seasonal transmission potential (stp) was calculated as the product of the seasonal biting density (sbd ; number of bites per person during the transmission season), the proportion of flies with infective - stage o. volvulus larvae (pcr positive), and the mean number of infective larvae per infective fly (assumed to be one in an area of low transmission). the protocol for these evaluations was reviewed and approved by the centers for disease control and prevention (atlanta, ga, usa), the universidad del valle de guatemala (guatemala city, guatemala), and the guatemala moph. written informed consent was obtained from participants 18 years of age and older ; parents or guardians of children less than 18 years provided written, informed consent for their participation. ophthalmological, serological, and entomological evaluations were conducted from june 2007 to april 2008. the results from these evaluations showed the transmission status of o. volvulus in the huehuetenango focus, after 14 rounds of ivermectin treatment with coverages higher than 85% (figure 3). table 1 shows the nine pecs with the eligible population and the number of individuals evaluated in each pec (figure 2). of these individuals, 82% reported having lived in the community throughout their life, 55% were female, and 64% were between the age of seven and 29. ophthalmological evaluation showed that 97% of the participants had a visual acuity between 20/20 and 20/70. we identified 3910 children aged from 6 to 12 years old enrolled in the 43 selected schools (table 2), of which 3118 (80%) participated in the evaluation. the mean age was 9.1 years (sd 2.1 years), 52% were male, and the average of years of living in the community was 9.3 years (sd 1.8 years). none of the children tested were found to be seropositive, resulting in a cumulative incidence of 0% (95% ci 00.1%). entomological evaluation was carried out in four coffee plantations (two located in agua dulce, cuilco and two in marilandia, san pedro necta) which presented high densities of s. ochraceum. thirty nine collections days, equivalent 1220 hours of collection, were completed between november 2007 and april 2008 (table 3). a total of 8252 s. ochraceum and 11473 s. metallicum were collected as reported before, the biting density of s. ochraceum is higher in december and starts to decrease in february (table 3). overall, the geometric mean biting rate was 3.2 (95% ci 2.93.5) bites / person / hour, with the highest biting rate in november (16.7 bites / person / hour) and the lowest in april (0.7 bites / person / hour). the sbd indicating the total number of bites per person per season was 5765 (95% ci 52636300). the paid human attractants were tested at the beginning and end of the evaluation for antibodies to ov-16, but none was found positive. the s. ochraceum collected were grouped in 357 pools, and all were negative for o. volvulus infection. the prevalence of infection was 0% (95% ci 00.02%), and the maximum prevalence of infection was estimated to be 0.4/2000 flies. the stp was 0 infective larvae / person / season, but the maximum potential stp (using the upper boundary on the prevalence of infection in s. ochraceum) was 1.3 infective larvae / person / season. the results from the previous evaluations showed that transmission of o. volvulus through s. ochraceum was interrupted, thus in 2009 ivermectin mda was suspended from the huehuetenango focus. following who guidelines, from december 2010 to april 2011, we conducted the entomological evaluation to evaluate the transmission of o. volvulus after treatment suspension. the entomological evaluation was conducted in the same four coffee plantations as the previous entomological evaluation. a total of 4587 s. ochraceum and 4912 s. metallicum were collected (table 4), and a significant reduction in the density of both species was observed when compared to the 2007 - 2008 collection. however the biting density pattern of s. ochraceum was similar as that observed in 2007 - 2008. the overall geometric mean biting rate was 1.8 (95% ci 1.62.0) bites / person / hour, and the sbd was 2693 bites / person (95% ci 24542945). the paid human attractants did not present antibodies to ov16 before or after the evaluation. the s. ochraceum collected were grouped in 147 pools, and all were negative for o. volvulus infection. the prevalence of infection was 0% (95% ci 00.04%), and the maximum prevalence of infection was estimated to be 0.8/2000 flies. the stp was 0 infective larvae / person / season, with the maximum potential stp (using the upper boundary on the prevalence of infection in s. ochraceum) being 1.1 infective larvae / person / season. as a result of low number of s. ochraceum collected, all s. metallicum were grouped in pools in the same manner as described for s. ochraceum and processed by pcr to determine if they were infected with o. volvulus. all s. metallicum were negative for o. volvulus ; thus, there are no parasites circulating in these black flies. in 2001, the who established a set of guidelines to assist onchocerciasis programs to determine whether interruption of transmission had occurred and mda with ivermectin could be stopped. the process outlined by who involves four phases : (1) suppression of transmission, where new infective stage larvae are no longer introduced into the human population by the vectors, but the parasite population maintains the ability to recover if interventions are withdrawn ; (2) interruption of transmission, when the parasite population is thought to be unable to recover and interventions (in this case, twice - yearly ivermectin treatment) can be halted ; (3) precertification, during which time posttreatment surveillance is needed for three years with an in depth evaluation to take place during the second or third year, depending on the peak transmission season ; (4) if this evaluation was negative, a declaration of elimination. when all foci within a country reach the final phase, the country may request certification of elimination of onchocerciasis from who. we conducted an assessment of the status of o. volvulus transmission in the third onchocerciasis focus of guatemala to be so evaluated by this process. data from evaluations conducted by the moph in huehuetenango in the 1990s indicated that the parasite had, at best, a tenuous hold when mda was launched in 1996. our results in 2007 - 2008 confirmed that transmission of onchocerciasis had been successfully interrupted in huehuetenango after 22 rounds of mda over 13 years. similarly, we found no serological evidence of recent exposure to the parasite among 612-year - old children residing in the endemic area, nor any entomological evidence of infected or infective black fly vectors. the maximum stp in this area was conservatively calculated at 1.2 infective larvae / person / season, which is not sufficient to sustain transmission in s. ochraceum areas. the expert steering committee of oepa (the program coordinating committee (pcc)) reviewed the results of this evaluation and formally recommended to the guatemala moph that mda with ivermectin be suspended in huehuetenango, beginning in 2009. this recommendation was accepted, and huehuetenango joined the santa rosa and escuintla - guatemala foci in the posttreatment surveillance period. one challenge to maintaining the huehuetenango focus free of onchocerciasis is its proximity to the border with mexico (figure 1) and, in particular, to the south chiapas onchocerciasis focus. there has been conjecture that the two foci were linked through migrant coffee workers moving between the two countries and acquiring onchocerciasis in one or both foci. the unlikelihood of migrant workers being able to maintain onchocerciasis transmission in guatemala and mexico has been studied and discussed elsewhere [25, 26 ]. of the two foci, south chiapas historically had the highest levels of active onchocerciasis transmission in all of mexico whereas the evidence from huehuetenango during the same time period showed significant decreases in onchocerciasis prevalence even before the mda program, suggesting that south chiapas had little effect on transmission in huehuetenango. an mda program requiring treatment four times per year was required to interrupt transmission in the south chiapas focus ; at the beginning of 2012 mda was suspended and post treatment surveillance initiated there, thus it is unlikely that onchocerciasis could be reintroduced into huehuetenango from mexico. following pts guidelines, from december 2010 to april 2011 an entomological evaluation was conducted in huehuetenango to verify that o. volvulus transmission had not recrudesced. the results from this evaluation showed that o. volvulus l3 are not circulating in the primary vector s. ochraceum or in potentially a secondary vector, s. metallicum. a conservative calculation of stp using upper 95% confidence interval values for s. ochraceum was 1.1 infective larva / person / season, which is insufficient to maintain the parasite population. currently in guatemala the three hypoendemic foci (santa rosa, escuintla - guatemala and huehuetenango) have demonstrated, through completion of pts, elimination of transmission. the only focus that is currently under post treatment surveillance is the hyperendemic central endemic focus, which only stopped mda at the beginning of 2012. when the central endemic focus successfully completes pts and attains elimination status, then the entire country of guatemala will be able to request who certification of elimination in 2015. in may 2009, the us president barack obama announced a new global health initiative (ghi) to improve health outcomes in partner countries. neglected tropical diseases feature prominently in the ghi, and a key target is elimination of onchocerciasis from the americas (http://www.america.gov/st/texttrans-english/2010/august/20100817134101su0.3731152.html, accessed on september 1, 2010). as the elimination of onchocerciasis from the americas by 2015 looks to be on target, this could be one of the early successes of the ghi and a significant public health success story that can help motivate and inform other elimination efforts. | in latin america, onchocerciasis is targeted for elimination by 2012 through twice - yearly mass treatment of the eligible population with ivermectin. in guatemala, two of the four historical endemic foci have demonstrated elimination of transmission, following world health organization guidelines. using established guidelines ophthalmological, serological, and entomological evaluations were conducted in 2007 - 8 to determine the transmission status of onchocerciasis in the huehuetenango focus. the prevalence of onchocerca volvulus microfilariae in the anterior segment of the eye in 365 residents was 0% (95% confidence interval [ci ] 00.8%), the prevalence of infection of o. volvulus in simulium ochraceum among 8252 flies collected between november 2007 and april 2008 was 0% (95% ci 00.02%), and the prevalence of antibodies to a recombinant o. volvulus antigen in 3118 school age children was 0% (95% ci 00.1%). these results showed transmission interruption ; thus, in 2009 mass treatment was halted and posttreatment surveillance began. to verify for potential recrudescence an entomological evaluation (from december 2010 to april 2011) was conducted during the 2nd and 3rd year of posttreatment surveillance. a total of 4587 s. ochraceum were collected, and the prevalence of infection of o. volvulus was 0% (95% ci 00.04%). transmission of onchocerciasis in the huehuetenango focus has been eliminated. |
uterine granuloma is a rare entity and may be either focal or diffuse in nature. diffuse granulomas represent a local reaction without an obvious cause or may be associated with infection or systemic granulomatous disorders. infective conditions associated with uterine granulomas could be tuberculosis, atypical mycobacteria, endemic mycosis, cytomegalovirus infection and parasites. we report two cases of uterine granuloma, the first in which there was diffuse non - necrotizing granuloma. the second case showed diffuse granulomas with caseation in the myometrium and was positive for acid fast bacillus (afb) stain. a 50-year - old patient presented in august 2010 with heavy and irregular bleeding per vaginum for last 10 months. she had undergone dilatation and curettage (d and c) and cervical biopsy in december 2009 for menorrhagia. she had a second episode of menometrorrhagia in march 2010 followed by a repeat d and c. the endometrial curettings was reported as simple hyperplasia without atypia. her general, local examination and routine investigations were normal. a total abdominal hysterectomy with bilateral salpingo - oophorectomy was performed and the patient was discharged in satisfactory condition on eighth post - operative day. histology revealed endometrium in proliferative phase and myometrium showed multiple non - necrotizing epithelioid cell granulomas. photomicrograph showing non - caseating epithelioid cell granulomas with multinucleated giant cells present in the myometrium (h and e, 200) a 40-year - old presented with pain and mass lower abdomen of five months duration with no menstrual irregularity. general physical examination was normal, local examination was suggestive of fibroid corresponding to size of 18 weeks pregnant uterus. ultrasonography revealed a large single intramural fibroid 10 9.7 cm with thin endometrium and normal ovaries. histopathological examination revealed endometrium in secretory phase, uterine tumor as leiomyoma and in addition myometrium showed caseating epithelioid granulomas along with langerhans and foreign body giant cells [figure 2 ]. stain for afb was positive. patient was started on att despite hysterectomy and absence of tuberculosis elsewhere because the adnexa was left behind where the possibility of a tubercular focus could not be ruled out. a 50-year - old patient presented in august 2010 with heavy and irregular bleeding per vaginum for last 10 months. she had undergone dilatation and curettage (d and c) and cervical biopsy in december 2009 for menorrhagia. she had a second episode of menometrorrhagia in march 2010 followed by a repeat d and c. the endometrial curettings was reported as simple hyperplasia without atypia. her general, local examination and routine investigations were normal. a total abdominal hysterectomy with bilateral salpingo - oophorectomy was performed and the patient was discharged in satisfactory condition on eighth post - operative day. histology revealed endometrium in proliferative phase and myometrium showed multiple non - necrotizing epithelioid cell granulomas. photomicrograph showing non - caseating epithelioid cell granulomas with multinucleated giant cells present in the myometrium (h and e, 200) a 40-year - old presented with pain and mass lower abdomen of five months duration with no menstrual irregularity. general physical examination was normal, local examination was suggestive of fibroid corresponding to size of 18 weeks pregnant uterus. ultrasonography revealed a large single intramural fibroid 10 9.7 cm with thin endometrium and normal ovaries. histopathological examination revealed endometrium in secretory phase, uterine tumor as leiomyoma and in addition myometrium showed caseating epithelioid granulomas along with langerhans and foreign body giant cells [figure 2 ]. stain for afb was positive. patient was started on att despite hysterectomy and absence of tuberculosis elsewhere because the adnexa was left behind where the possibility of a tubercular focus could not be ruled out. when clinicians encounter patients with granulomatous inflammation, they are faced with a diagnostic dilemma to determine its cause. non infections conditions include granulomatous inflammation following surgical instrumentation, foreign body reaction, systemic granulomatous disorders like sarcoidosis, systemic vasculitides like giant cell arteritis or may be idiopathic i.e., without an obvious underlying cause. the first case in our study showed diffuse granulomatous lesions in myometrium of non - necrotizing variety. there was history of surgical intervention for uterine bleeding in the form of dilatation and curettage done twice at an interval of three months., showed that 8 of 11 patients with incidental granulomas of the uterus had a known history of instrumentation, which consisted of dilatation and curettage (n = 4), endometrial biopsy (n = 2), hysterosalpingogram (n = 1) and hysteroscopy (n = 1). none of the patients had clinical evidence of sarcoidosis or systemic infection and stains for microorganisms were negative. the clinical scenario of the first case correlates with this study where well formed, non - necrotizing granulomas of the uterus were found with a history of instrumentation. the patient had endometrial defects as shown on histopathology which can explain all the clinical features. similarly there was no evidence of systemic granulomatous disease or tuberculosis based on clinical and laboratory evidence. hence a history of instrumentation may explain the presence of uterine granulomas in the first case. despite its contiguity with endometrium, sinha., reported a case of myometrial tuberculosis detected on sonography as multiple anechoic areas of variable size with well defined borders scattered throughout the myometrium. after receiving antitubercular treatment patient was relieved of menorrhagia and a repeat sonogram showed a homogenous myometrium with absence of anechoic areas. the second case presented with a pelvic mass suggestive of leiomyoma, confirmed on fnac and had no menstrual complaint. there was incidental detection of tubercular granulomatous inflammation of myometrium along with leiomyoma in this case., in a 31 year study on pathology of female genital tuberculosis reported that microscopic involvement of myometrium was observed in 20% of the cases. in the indian context, tuberculosis remains the most common cause of diffuse granulomas since two thirds of tuberculosis cases in south east asia are found here., reviewed 5,085 biopsy specimens (3,510 endometrial and 1,575 cervical) of these 28 endometrial and 2 cervical biopsy specimens were reported as granulomatous inflammation, consistent with tuberculosis. myometrial involvement was not observed in any of the endometrial biopsy specimens in which myometrium was included. this supports the infrequent involvement of myometrium in genital tuberculosis. to conclude, uterine granuloma involving myometrium is rare. there is need for awareness about instrumentation causing granuloma formation, and also to look for an additional pathology such as tubercular granuloma which may present as an incidental finding warranting therapy. | uterine granulomas confined to the myometrium are uncommon. granulomas maybe infectious, non - infectious or idiopathic in origin. they may be diffuse or focal in nature. in this report, we describe two cases of granulomas of diffuse variety involving the myometrium. the first case shows non - necrotizing granuloma probably related to previous instrumentation. the second case depicts caseating granulomas along with leiomyoma of uterus. |
at 23 years of age, our patient presented with a palpable neck mass with no other underlying disease. sonographic findings revealed an enlarged isoechoic mass (42.52.5 cm) with peripheral rim - like calcification in the right lobe of the thyroid. a preoperative diagnosis of papillary thyroid carcinoma was determined and a bilateral total thyroidectomy with ccnd and right internal jugular neck dissection was performed. the pathologic diagnosis determined that the mass was a diffuse sclerosing variant of papillary thyroid carcinoma confined to the right thyroid parenchyma (fig. the tumor metastasized to several lymph nodes on the right side of the neck and the following regional lymph nodes (8/30) : level ii (2/6), level iii (2/13), level iv (3/10), and perithyroidal (1/1). approximately 30 mci of radioactive iodine (i) was used for postoperative adjuvant treatment. excision of the left surgical site with left modified radical neck dissection and right level ii and iii selective node dissection were performed. the tumor had again metastasized to several lymph nodes on the left side of the neck and the following regional lymph nodes (8/33) : right level ii (0/3) ; left level ii (1/9), level iii (1/4), and level iv (4/15), and perithyroidal (2/2). postoperatively, 200 mci of radioactive iodine (i) was administered and a daily dose of 200 g of levothyroxine was prescribed for adjuvant treatment. even though the patient continued with treatment, the tumor recurred at the left surgical site and the tumor size increased. at 31 years of age, the patient identified another newly developed, palpable small mass at the right postauricular area, which grew rapidly within a month. excision of the trachea with right modified radical neck node dissection and left level vi selective neck dissection was performed. histologic diagnosis of the pretracheal lesion determined that the lesion was an anaplastic thyroid carcinoma with a focal papillary carcinoma component, and the tumor metastasized to the following bilateral lymph nodes (8/18) : right level ii (5/14) and level v (0/1) and left level vi (3/3). after surgery, the patient experienced dysphagia and dyspnea, and despite intensive care for respiratory failure, he died 4 months later. the right thyroid gland contained a 42.5-cm ill - defined, yellow - tan mass. histologically, the tumor showed typical papillary thyroid carcinoma features with lymphocytic thyroiditis, squamous morules, and abundant psammoma bodies in the outside of the tumor.. the histologic findings of the recurred tumor showed a pattern similar to the previous primary thyroid carcinoma. our review of this tumor histology revealed a focal (a) and a low level of the braf v600v (c.1800g > a) mutation were found in the initially diagnosed papillary thyroid carcinoma and poorly differentiated carcinoma component of the recurred papillary thyroid carcinoma, but only the prominent braf v600v (c.1800g > a) mutation was found in the anaplastic carcinoma component. at 23 years of age, our patient presented with a palpable neck mass with no other underlying disease. sonographic findings revealed an enlarged isoechoic mass (42.52.5 cm) with peripheral rim - like calcification in the right lobe of the thyroid. a preoperative diagnosis of papillary thyroid carcinoma was determined and a bilateral total thyroidectomy with ccnd and right internal jugular neck dissection was performed. the pathologic diagnosis determined that the mass was a diffuse sclerosing variant of papillary thyroid carcinoma confined to the right thyroid parenchyma (fig. the tumor metastasized to several lymph nodes on the right side of the neck and the following regional lymph nodes (8/30) : level ii (2/6), level iii (2/13), level iv (3/10), and perithyroidal (1/1). approximately 30 mci of radioactive iodine (i) was used for postoperative adjuvant treatment. excision of the left surgical site with left modified radical neck dissection and right level ii and iii selective node dissection were performed. the tumor had again metastasized to several lymph nodes on the left side of the neck and the following regional lymph nodes (8/33) : right level ii (0/3) ; left level ii (1/9), level iii (1/4), and level iv (4/15), and perithyroidal (2/2). postoperatively, 200 mci of radioactive iodine (i) was administered and a daily dose of 200 g of levothyroxine was prescribed for adjuvant treatment. even though the patient continued with treatment, the tumor recurred at the left surgical site and the tumor size increased. at 31 years of age, the patient identified another newly developed, palpable small mass at the right postauricular area, which grew rapidly within a month. excision of the trachea with right modified radical neck node dissection and left level vi selective neck dissection was performed. histologic diagnosis of the pretracheal lesion determined that the lesion was an anaplastic thyroid carcinoma with a focal papillary carcinoma component, and the tumor metastasized to the following bilateral lymph nodes (8/18) : right level ii (5/14) and level v (0/1) and left level vi (3/3). after surgery, the patient experienced dysphagia and dyspnea, and despite intensive care for respiratory failure, he died 4 months later. the first surgical specimen was comprised of bilateral thyroid glands and lymph nodes. the right thyroid gland contained a 42.5-cm ill - defined, yellow - tan mass. histologically, the tumor showed typical papillary thyroid carcinoma features with lymphocytic thyroiditis, squamous morules, and abundant psammoma bodies in the outside of the tumor. the histologic findings of the recurred tumor showed a pattern similar to the previous primary thyroid carcinoma. our review of this tumor histology revealed a focal (a) and a low level of the braf v600v (c.1800g > a) mutation were found in the initially diagnosed papillary thyroid carcinoma and poorly differentiated carcinoma component of the recurred papillary thyroid carcinoma, but only the prominent braf v600v (c.1800g > a) mutation was found in the anaplastic carcinoma component. we report a rare case of anaplastic carcinoma in a young patient, analyze the follow - up pathology data related to anaplastic transformation, and discuss the mechanism of anaplastic transformation. the mean age of diagnosis is 55 to 65 years, with the peak incidence in the sixth to seventh decade of life. the age of most reported cases was more than 30 years, and only a few reported cases were younger than 30 years.1 most reported cases of anaplastic transformation by dedifferentiation from a wdtc were in the elderly.5 therefore, thorough clinical history of previous thyroid diseases should be determined and the possibility of an anaplastic transformation should be considered when a diagnosis of anaplastic carcinoma is confirmed in an elderly patient. the mortality rate is > 90%, with a mean survival of 6 months after diagnosis.6 almost all patients complain of a rapidly growing neck mass and symptoms associated with a large mass such as hoarseness, dysphagia, vocal cord paralysis, cervical pain, and dyspnea are the most frequent and important. the overall 5-year survival ranges from 0% to 14% and the median survival is 4 to 12 months. the enlarged tumor mass often causes death by obstruction or invasion of a vital structure, similar to the occurrence in our patient.6 anaplastic thyroid carcinoma may arise de novo or, more commonly, through anaplastic transformation (or dedifferentiation) of a preexisting papillary or follicular thyroid carcinoma. the mechanism of this transformation, however, is not well understood.5 in this study, we performed immunohistochemical staining and braf mutation analysis to determine the mechanism of anaplastic transformation in the wdtc, and we found that the well - expressed markers in the wdtc showed loss of expression in the anaplastic carcinoma. we also confirmed that the markers known to participate in transformation including p53, ki-67, and others were not initially expressed but were expressed at the full anaplastic transformation. todd and wenig7 reported that thyroglobulin and ttf-1 immunoreactivity was completely reduced in recurrent or metastatic thyroid carcinoma and anaplastic thyroid carcinoma, which showed less differentiated histologic features, and these results were identical to the immunostaining results of our case. ozaki.8 reported that high ki-67 expression was found in more malignant tumors, which was also similar to the findings in our case. many studies have examined the role of the p53 gene in the thyroid as well as other organs, and most of these studies have shown that a loss in p53 gene expression is related to malignant transformation. furthermore, studies of the thyroid have reported to be related to anaplastic transformation in wdtc.9 the p53-positive staining that appears in the full anaplastic transformation of our case is similar to other reports. however, additional research is necessary to determine the stage of anaplastic transformation in which the change in p53 and ki-67 expression appears. mutation of the braf gene represents the most common genetic alteration in papillary thyroid cancer and is found in 45% of these tumors in western countries and up to 90% of these tumors in korea.10 the vast majority of braf alterations in papillary thyroid cancer are braf v600e mutations,10 but braf k601e mutations have been reported in both follicular and solid variants.11 in multifocal thyroid cancer, different tumor nodules have distinct genetic alterations, such as different types of ret / ptc rearrangement or variation in the presence of the braf mutation.12 recently, in korea, kim.11 reported braf mutations that appeared in different forms according to the histologic subtype in a multifocal papillary carcinoma. because the conventional papillary carcinoma component in their patient revealed the braf v600e mutation, but the follicular variant component revealed the braf k601e mutation, they determined that the multifocal papillary carcinoma occurred de novo rather than as a result of intrathyroid metastasis, although no clear classification can be determined yet. in our case, the braf v600e and braf v600v mutations were found in the initially diagnosed papillary thyroid carcinoma and the poorly differentiated carcinoma component of the recurred papillary thyroid carcinoma, but only the braf v600v mutation was found in the anaplastic carcinoma component. however, there has not been any report of a braf v600v (c.1800g > a) mutation in thyroid carcinoma. we found two reports of a braf v600v mutation in malignant melanoma13 and colorectal cancer,14 but the reports did not attempt to explain the relevance of the braf v600v mutation expression. we found that the braf mutation type changed during the tumor relapse process, but additional research is necessary to determine whether the alteration of the braf mutation is just an incidental finding or a phenotypic change relevant to the anaplastic transformation. in conclusion, we evaluated the rare occurrence of anaplastic carcinoma in young patients and did not find p53 or ki-67 expression or decreased thyroglobulin, ttf-1, and galectin-3 expression in the poorly differentiated tumor cells. therefore, we considered the possibility that overexpression of p53 and ki-67 contributed to the anaplastic transformation. additional studies are necessary to ascertain the intermediate steps of anaplastic transformation. if the markers that are well manifested in wdtc are lost even though p53 and ki-67 are not expressed, then we can consider those changes in expression as precursors to anaplastic transformation. therefore, future studies with thorough and careful histologic examination may allow us to predict the anaplastic transformation of the tumor. | this study reports a case of anaplastic transformation from a well - differentiated thyroid carcinoma in a young patient. the first recurrent tissue contained poorly differentiated foci that revealed lower thyroglobulin, thyroid transcription factor 1 (ttf-1), and galectin-3 expression than the well - differentiated area. however there was no increased p53 or ki-67 expression in the poorly differentiated foci, nor in the well - differentiated area. the tissue subsequently relapsed and revealed only anaplastic features, complete loss of thyroglobulin, ttf-1, and galectin-3 expression and revealed an increase in p53 and ki-67 expression. the braf v600e and braf v600v mutation were found in the initially diagnosed papillary thyroid carcinoma and the poorly differentiated foci of the recurring papillary thyroid carcinoma ; however, only the braf v600v mutation was found in the anaplastic carcinoma. these results suggest that overexpression of p53 and ki-67 contributed to the anaplastic transformation. we also found that the braf type changed during the tumor relapse. |
spontaneous intracranial hypotension (sih) is an increasingly recognised syndrome with the hallmark of orthostatic headache (oh). pathophysiology suggests spontaneous leakage of cerebrospinal fluid (csf) through a spinal meningeal fistula.1 diagnosis of sih remains challenging and the criteria of the international headache society are evolving.2 3 a simple, cost - effective, non - invasive, and repeatable diagnostic tool that would aid in diagnosis, follow - up, and understanding of the pathophysiology of sih would be useful. high resolution transorbital sonography of the optic nerve sheath diameter (onsd) is increasingly used for the assessment of raised intracranial pressure.4 sonographic measurements showing increased onsd in patients with raised intracerebral pressure (icp) were confirmed using high - resolution mri.5 6 literature on ultrasound diagnostics in patients with sih is very sparse and consists only of a case study and an investigation in 10 patients on the effect of epidural blood patch on onsd and ophthalmic venous flow.79 up to now, all measurements reported in the literature were only performed with patients lying supine, and no measurements were made after change to the upright position. based on pathophysiological considerations, we hypothesised that there is a change in onsd from supine to upright position and that this change should be more pronounced in patients with acute ohs. in the current study we evaluated the diagnostic value of onsd by performing two high - resolution orbital ultrasound assessments, in supine and upright positions, in three groups : symptomatic and asymptomatic sih patients, and controls. ultrasound of the onsd is part of our routine diagnostic workup in all patients presenting with sih in our departments. we included all patients (18 years or older) with confirmed sih from january 2013 to december 2014. sih was diagnosed according to criteria of the international headache society.2 these criteria are : (a) any headache fulfilling criterion c ; (b) low csf pressure (< 60 mm h2o) and/or evidence of csf leakage on imaging ; (c) headache has developed in temporal relation to the low csf pressure or csf leakage, or has led to its discovery ; (d) not better accounted for by another international classification of headache disorders 3rd edition (ichd-3) diagnosis. all patients with confirmed sih had evidence of csf leakage on imaging as required by criterion (b) in the international headache classification.2 imaging findings included subdural fluid collections, enhancement of the pachymeninges, sagging of the brain, or a fluid collection circumscribing the spinal dural sac in t2-weighted mri (see online supplementary tables s1a, b). exclusion criteria included any pathology (ie, erosions) of the upper eyelid, any eye surgery within the previous 6 months, and recent head or neck trauma or conditions not allowing positional changes. patients with postdural puncture headache and patients who had recent spinal surgery potentially affecting csf dynamics were excluded. sih patients were dichotomised into : group a, those with current severe or immediate oh (ie, within 15 min after assuming an upright position) ; or group b, those with no oh after assuming an upright position, but who had a history (< 4 weeks) of oh that developed in less than 15 min on shifting to upright position. all consecutive patients received dynamic ultrasound assessment of the onsd. additionally, we examined 20 patients without headache or any orthostatic symptoms (group c1) and 19 healthy controls (c2). group c consisted of similarly aged patients who were admitted for back surgery due to spinal stenosis or disc herniation ; all their data are based on presurgical examinations. group c2, which served as an external control, comprised healthy volunteers from regensburg, germany. a joint meeting between the groups from bern and regensburg was held prior to the start of the study to ensure consistency of examinations at the two study centres. ultrasound was performed using the same protocol in the two centres. in bern, the change in onsd was measured with a 715 mhz linear array transducer in transorbital b - mode ultrasound (iu 22 equipped with a 15l8 transducer, philips, amsterdam, the netherlands). in regensburg, the ultrasound machine was toshiba xario xg equipped with a high frequency linear array transducer (plt-1204bt, tokyo, japan). the examination was conducted first with the patient in supine position and then in upright position with the patient standing for 2 min (figure 1). the probe was placed on the temporal part of the upper eyelid using a thick layer of gel. left : illustration of change in diameter of the ons in a patient with spontaneous intracranial hypotension. illustration of the changes in the diameter of the ons in a symptomatic patient that occur with the change from supine to upright body position. the upper left picture shows the diameter of the ons in a symptomatic patient while supine. the upper right picture shows a decreased ons diameter when this patient assumes upright position. right - side image : onsd was assessed 3 mm behind the papilla in axial and sagittal planes on both sides using a perpendicular axis. (sas / t = subarachnoid space / trabeculae ; ons, optic nerve sheath). to comply with maximum patient safety, mechanical index as an on - screen parameter was reduced to 0.3, which is concordant with the guidelines of the british medical ultrasound society and us federal drug administration, which recommends a mechanical index of 00.3 and less than or equal to 0.23 for ultrasound of the eye.10 11 the field of view was adjusted to a depth of 40 mm and a single focal zone placed behind the orbit. the b - mode gain was reduced for optimisation of the optic nerve sheath (ons). onsd was measured 3 mm behind the echogenic lamina cribrosa in axial and sagittal planes to the cranium on both sides using a perpendicular axis as previously described.8 11 the documented onsd was computed from the mean of four values for each patient, that is, two on each eye (figure 1). in both study centres, for this exploratory study, the primary analysis population consisted of subjects who had an onsd assessment in supine and in upright position. the primary analysis variable was the change in the onsd from supine to upright position. the comparison of onsd among groups was performed using the wilcoxon rank - sum test (two groups) or the kruskal - wallis test (more than two groups). analysis of covariance was employed for supportive evaluations and was adjusted for age and gender. for evaluation of the onsd change, analysis of covariance was adjusted for the value in supine position. an analysis of receiver operating characteristics (roc) curves was used to assess the diagnostic test for the presence of ohs based on onsd. the analysis was carried out in accordance with good clinical practice guidelines, swiss academy of medical sciences guidelines, and the declaration of helsinki. the ethics board of the bern university hospital approved the study (bern, switzerland, kek - nr. the prospective study of healthy volunteers in regensburg was approved by the local ethics committee (regensburg, germany, 14 - 101 - 0076). ultrasound of the onsd is part of our routine diagnostic workup in all patients presenting with sih in our departments. we included all patients (18 years or older) with confirmed sih from january 2013 to december 2014. sih was diagnosed according to criteria of the international headache society.2 these criteria are : (a) any headache fulfilling criterion c ; (b) low csf pressure (< 60 mm h2o) and/or evidence of csf leakage on imaging ; (c) headache has developed in temporal relation to the low csf pressure or csf leakage, or has led to its discovery ; (d) not better accounted for by another international classification of headache disorders 3rd edition (ichd-3) diagnosis. all patients with confirmed sih had evidence of csf leakage on imaging as required by criterion (b) in the international headache classification.2 imaging findings included subdural fluid collections, enhancement of the pachymeninges, sagging of the brain, or a fluid collection circumscribing the spinal dural sac in t2-weighted mri (see online supplementary tables s1a, b). exclusion criteria included any pathology (ie, erosions) of the upper eyelid, any eye surgery within the previous 6 months, and recent head or neck trauma or conditions not allowing positional changes. patients with postdural puncture headache and patients who had recent spinal surgery potentially affecting csf dynamics were excluded. sih patients were dichotomised into : group a, those with current severe or immediate oh (ie, within 15 min after assuming an upright position) ; or group b, those with no oh after assuming an upright position, but who had a history (< 4 weeks) of oh that developed in less than 15 min on shifting to upright position. all consecutive patients received dynamic ultrasound assessment of the onsd. additionally, we examined 20 patients without headache or any orthostatic symptoms (group c1) and 19 healthy controls (c2). group c consisted of similarly aged patients who were admitted for back surgery due to spinal stenosis or disc herniation ; all their data are based on presurgical examinations. group c2, which served as an external control, comprised healthy volunteers from regensburg, germany. a joint meeting between the groups from bern and regensburg was held prior to the start of the study to ensure consistency of examinations at the two study centres. in bern, the change in onsd was measured with a 715 mhz linear array transducer in transorbital b - mode ultrasound (iu 22 equipped with a 15l8 transducer, philips, amsterdam, the netherlands). in regensburg, the ultrasound machine was toshiba xario xg equipped with a high frequency linear array transducer (plt-1204bt, tokyo, japan). the examination was conducted first with the patient in supine position and then in upright position with the patient standing for 2 min (figure 1). the probe was placed on the temporal part of the upper eyelid using a thick layer of gel. left : illustration of change in diameter of the ons in a patient with spontaneous intracranial hypotension. illustration of the changes in the diameter of the ons in a symptomatic patient that occur with the change from supine to upright body position. the upper left picture shows the diameter of the ons in a symptomatic patient while supine. the upper right picture shows a decreased ons diameter when this patient assumes upright position. right - side image : onsd was assessed 3 mm behind the papilla in axial and sagittal planes on both sides using a perpendicular axis. (sas / t = subarachnoid space / trabeculae ; ons, optic nerve sheath). to comply with maximum patient safety, mechanical index as an on - screen parameter was reduced to 0.3, which is concordant with the guidelines of the british medical ultrasound society and us federal drug administration, which recommends a mechanical index of 00.3 and less than or equal to 0.23 for ultrasound of the eye.10 11 the field of view was adjusted to a depth of 40 mm and a single focal zone placed behind the orbit. the b - mode gain was reduced for optimisation of the optic nerve sheath (ons). onsd was measured 3 mm behind the echogenic lamina cribrosa in axial and sagittal planes to the cranium on both sides using a perpendicular axis as previously described.8 11 the documented onsd was computed from the mean of four values for each patient, that is, two on each eye (figure 1). in both study centres, an examiner trained in ultrasound diagnostics of the ons performed all ultrasound examinations. for this exploratory study, the primary analysis population consisted of subjects who had an onsd assessment in supine and in upright position. the primary analysis variable was the change in the onsd from supine to upright position. the comparison of onsd among groups was performed using the wilcoxon rank - sum test (two groups) or the kruskal - wallis test (more than two groups). analysis of covariance was employed for supportive evaluations and was adjusted for age and gender. for evaluation of the onsd change, analysis of covariance an analysis of receiver operating characteristics (roc) curves was used to assess the diagnostic test for the presence of ohs based on onsd. the analysis was carried out in accordance with good clinical practice guidelines, swiss academy of medical sciences guidelines, and the declaration of helsinki. the ethics board of the bern university hospital approved the study (bern, switzerland, kek - nr. the prospective study of healthy volunteers in regensburg was approved by the local ethics committee (regensburg, germany, 14 - 101 - 0076). nine patients had to be excluded from further analysis because of recent surgery with a possible effect on csf dynamics (posterior fossa surgery, n=2 ; recent spinal surgery, n=1 ; postdural puncture headaches, n=6). thus, 39 patients with newly confirmed sih as well as 39 controls were included for analysis (figure 2). the demographic and clinical characteristics of the included patients are shown in online supplementary tables s1a, b. subject disposition diagram. all patients in groups a and b were selected according to the international headache society criteria for sih and had confirmed diagnosis of sih.2 at the time of the examination, 18 patients of group a (10 men, 8 women ; mean age 52 (range 2775) years) had ohs when assuming the upright position. the 21 patients comprising group b (11 men, 10 women ; mean age 63 (range 3983) years) did not have immediate (ie, within < 15 min) severe ohs. the control group (c1+c2) comprised 39 patients : 20 patients (14 men, 6 women ; mean age 51 (range 1873) years) from one study centre (c1), plus 19 healthy controls (10 male, 9 female ; mean age 58 (range 5067) years) from the other study centre (c2). in the primary analysis population no missing ultrasound assessments were reported. no adverse events were reported. in the supine position onsd was similar among patients with and without oh (p=0.921). the mean onsd in supine position in patients with active oh (group a) was 5.380.91 mm, which was similar to values for patients with confirmed sih but without active oh (group b), 5.480.89 mm. in the upright position there was a statistically significant difference between patients with and without oh (p=0.044). oh (group a) had a mean onsd of 4.840.95 mm, whereas patients without active oh (group b) had a mean of 5.531.02 mm. the change () in onsd between supine and upright position was statistically significantly different between groups a and b (p<0.001). the mean difference () between supine and upright position in patients with active oh (group a) was 0.53sd 0.34 mm. patients without active ohs (group b) had a mean difference () of 0.050.41 mm. comparison of patients with oh and sih (group a) to patients without oh (group b), versus the controls (groups c1 and c2 combined : 0.010.38 mm) showed a statistically significant difference () (p<0.001 ; figure 3). bee swarm box - plot data with means and sds showing the differences between groups. bee swarm box - plot data with means and sds illustrate the differences () in patients with orthostatic headaches (group a, green), without (group b, red) and patients without an spontaneous intracranial hypotension (sih) diagnosis (c1, grey, internal control and c2, black, external control). only patients with confirmed sih and oh had a significant decrease (10%) of onsd (group a supine : 5.380.91 mm vs group a upright : 4.840.99 ; p<0.001). analysis of covariance was used to adjust estimates for the potentially confounding effects of age and sex. results in a model comparing groups a, b and c confirmed that the only group with a significant change in onsd was group a (sih with oh ; p<0.001). we evaluated the diagnostic accuracy of the difference in onsd with the operator : presence of ohs (group a) against groups b, c1 and c2. roc analysis for difference of onsd between supine and upright position revealed an area under the curve of 0.874 (95% ci 0.776 to 0.972 ; p<0.001). in the supine position onsd was similar among patients with and without oh (p=0.921). the mean onsd in supine position in patients with active oh (group a) was 5.380.91 mm, which was similar to values for patients with confirmed sih but without active oh (group b), 5.480.89 mm. in the upright position there was a statistically significant difference between patients with and without oh (p=0.044). patients with active oh (group a) had a mean onsd of 4.840.95 mm, whereas patients without active oh (group b) had a mean of 5.531.02 mm. the change () in onsd between supine and upright position was statistically significantly different between groups a and b (p<0.001). the mean difference () between supine and upright position in patients with active oh (group a) was 0.53sd 0.34 mm. patients without active ohs (group b) had a mean difference () of 0.050.41 mm. comparison of patients with oh and sih (group a) to patients without oh (group b), versus the controls (groups c1 and c2 combined : 0.010.38 mm) showed a statistically significant difference () (p<0.001 ; figure 3). bee swarm box - plot data with means and sds showing the differences between groups. bee swarm box - plot data with means and sds illustrate the differences () in patients with orthostatic headaches (group a, green), without (group b, red) and patients without an spontaneous intracranial hypotension (sih) diagnosis (c1, grey, internal control and c2, black, external control). only patients with confirmed sih and oh had a significant decrease (10%) of onsd (group a supine : 5.380.91 mm vs group a upright : 4.840.99 ; p<0.001). analysis of covariance was used to adjust estimates for the potentially confounding effects of age and sex. results in a model comparing groups a, b and c confirmed that the only group with a significant change in onsd was group a (sih with oh ; p<0.001). we evaluated the diagnostic accuracy of the difference in onsd with the operator : presence of ohs (group a) against groups b, c1 and c2. roc analysis for difference of onsd between supine and upright position revealed an area under the curve of 0.874 (95% ci 0.776 to 0.972 ; p<0.001). using ultrasound measurements, we showed a significant reduction of 10.0% in the onsd in the standing position compared to the supine position in patients diagnosed with sih and current ohs. unlike patients with ohs, patients diagnosed with sih but without active ohs at the time of examination, as well as controls, did not show a significant collapse of their onss when moving from a supine to an upright position. the demonstration of a collapse of the onsd in sih patients when changing from supine to upright position is a novel finding and assessment of the onsd using ultrasound might become a useful diagnostic tool for diagnosis and follow - up of patients with sih. furthermore, our findings give intriguing insights into the pathophysiology of sih. the current results of the diameter of the ons are in line with the reported ultrasound values in the literature of 5.4 and 5.75 mm in healthy adults, as well as mri of 5.1 and 5.3 mm.1216 a study by takeuchi showed significantly smaller onsd using mri techniques and measured the onsd in two slices (just behind the eyeball and 3.5 mm behind the first slice, mean 3.34 and 4.85 mm, respectively). these differences may be due to the different depth as we, and the previously mentioned groups, performed the onsd measurements 3 mm behind the ocular bulb, as suggested by helmke and hansen.17 two other studies using coronal short tau inversion recovery sequences behind the optic bulb suggested a loss of csf signal around the optic nerve in supine position although these were not quantitative studies and did not include control groups.14 18 the width of the ons is related to the intracranial pressure ; numerous reports and prospective studies have shown an increase of the onsd in patients with intracranial hypertension.12 1921 measurements of the ons response to increased csf pressure by lumbar intrathecal infusion tests showed an immediate increase. however, to date, significant differences in onsd between patients with oh, patients without oh, or healthy controls have not been found. therefore, and based on the pathophysiology and mri findings of sih as summarised by schievink and mokri,22 we hypothesised that changes of the onsd should be detectable when the body position is changed from supine to upright, which causes an orthostatic decrease of intracranial pressure.1 2226 the dynamic paradigm of performing two measurements first supine, then upright showed a significant change in onsd in patients with sih. however, the change in onsd was significant only in sih patients when they were symptomatic, indicating a higher compliance capacity, or an ongoing loss of csf.27 currently the sensitivity of transorbital ultrasound for sih seems to be rather low and would, thus, not be a suitable screening tool. however, to provide robust numbers of sensitivity and specificity, clearly a larger sih is mainly a clinical diagnosis with the hallmark of orthostatic symptoms and mri findings. however, sih has a wide clinical spectrum and some patients do not show typical orthostatic symptoms or mri findings. for these patients transorbital ultrasound may be of diagnostic help and the current data suggests that it has the potential for stratifying patients for more invasive tests and as a prognostic marker. furthermore, dynamic ultrasound may assess the efficacy of treatment, may be used for follow - up examinations, and may provide insight into the pathophysiology of sih. one explanation for the collapse of the ons could be that patients who are symptomatic with headache suffer from an this in turn leads to a more pronounced loss of intracranial csf and decrease of icp while in the upright position as compared to control patients or sih patients without headaches, eventually leading to a measurable and significant change of the subarachnoid space around the optic nerve (figure 1). assessing onsd both in supine and upright positions might enhance the usefulness of ultrasound examinations of the ons and could be a useful bedside tool for patients with oh. it has several advantages : (1) it is not affected by the normal variability of the ons diameter between patients, (2) it ameliorates the unknown relationship between ons diameter and csf pressure in individual cases, and (3) it eliminates the interobserver variability of ultrasound measurements of the absolute onsd because the same observer can perform both examinations. the stratification of our patients is based on the hypothesis that the amount of csf moving from the intracranial to the spinal compartment when patients shift from a supine to standing position is larger in patients with oh. unlike patients without oh or controls, there is not enough csf in the intracranial space to support the weight of the brain at its normal intracranial position.28 the csf leakage causes sagging of the brain and consequent tension to pain - sensitive structures like veins and the dura mater. thus ongoing oh serves as a surrogate marker for active disease. we classified our patients according to the current criteria for sih of the international headache society.2 the difficulty in making a sound diagnosis is reflected by the recent evolution of the diagnostic criteria. we recently proposed a classification system for the likelihood of a spinal csf - fistula according to imaging findings.29 inclusion of imaging findings in the diagnosis of sih might improve understanding of sih. one group comprised 20 patients hospitalised prior to elective back surgery ; the other comprised 19 healthy controls in a different hospital. each of the two control groups was examined by a different ultrasound unit with a different examiner. evaluation of control subgroups (c1 and c2), showed slightly higher ons diameters in the c2 subgroup in both supine and upright positions (see online supplementary table s2), which may result from the different clinical units or equipment used for these subgroups. however, the mean individual difference between supine and upright position () was not notably different between the c1 and c2 subgroups (see online supplementary table s3). a 715 mhz linear array transducer has an axial and lateral resolution of 0.1 mm, while the mean change of the onsd was only 0.53 mm.30 prospective studies in patients with oh following spinal tap or epidural anaesthesia may be appropriate to confirm the results of this study. we classified our patients according to the current criteria for sih of the international headache society.2 the difficulty in making a sound diagnosis is reflected by the recent evolution of the diagnostic criteria. we recently proposed a classification system for the likelihood of a spinal csf - fistula according to imaging findings.29 inclusion of imaging findings in the diagnosis of sih might improve understanding of sih. one group comprised 20 patients hospitalised prior to elective back surgery ; the other comprised 19 healthy controls in a different hospital. each of the two control groups was examined by a different ultrasound unit with a different examiner. evaluation of control subgroups (c1 and c2), showed slightly higher ons diameters in the c2 subgroup in both supine and upright positions (see online supplementary table s2), which may result from the different clinical units or equipment used for these subgroups. however, the mean individual difference between supine and upright position () was not notably different between the c1 and c2 subgroups (see online supplementary table s3). a 715 mhz linear array transducer has an axial and lateral resolution of 0.1 mm, while the mean change of the onsd was only 0.53 mm.30 prospective studies in patients with oh following spinal tap or epidural anaesthesia may be appropriate to confirm the results of this study. in summary, this report describes a difference in the diameter of the ons on postural changes from supine to upright positions. therefore, transorbital ultrasound in supine and upright positions may provide a novel method for the diagnostic workup of patients suspected to have sih. results of the current analysis are promising and suggest the need for a larger prospective series to assess sensitivity and specificity of this novel diagnostic tool for sih. | objectivespontaneous intracranial hypotension (sih) is most commonly caused by cerebrospinal fluid (csf) leakage. therefore, we hypothesised that patients with orthostatic headache (oh) would show decreased optic nerve sheath diameter (onsd) during changes from supine to upright position.methodstransorbital b - mode ultrasound was performed employing a high - frequency transducer for onsd measurements in the supine and upright positions. absolute values and changes of onsd from supine to upright were assessed. ultrasound was performed in 39 sih patients, 18 with oh and 21 without oh, and in 39 age - matched control subjects. the control group comprised 20 patients admitted for back surgery without headache or any orthostatic symptoms, and 19 healthy controls.resultsin supine position, mean onsd (sd) was similar in patients with (5.380.91 mm) or without oh (5.480.89 mm ; p=0.921). however, in upright position, mean onsd was different between patients with (4.840.99 mm) and without oh (5.530.99 mm ; p=0.044). furthermore, the change in onsd from supine to upright position was significantly greater in sih patients with oh (0.530.34 mm) than in sih patients without oh (0.050.41 mm ; p0.001) or in control subjects (0.010.38 mm ; p0.001 ; area under the curve : 0.874 in receiver operating characteristics analysis).conclusionssymptomatic patients with sih showed a significant decrease of onsd, as assessed by ultrasound, when changing from the supine to the upright position. ultrasound assessment of the onsd in two positions may be a novel, non - invasive tool for the diagnosis and follow - up of sih and for elucidating the pathophysiology of sih. |
the fluid from young unopened pitchers is used in cleaning wounds or treating incontinence, distress and pain. aerial parts are used in the treatment of kidney stones, hypertension, fever and cough (http://www.forestry.gov.my/). the earlier studies have shown that endophytic microorganisms isolated from medicinal plants produce the same metabolites as their hosts. therefore, there is a great potential in exploring endophytes as a source of therapeutic natural products. however, despite several traditional medicinal applications of nepenthes spp. the objective of this study was to isolate and to identify the endophytic bacteria (eb) from nepenthes spp. namely, nepenthes ampullaria, n. gracilis, n. macfarlanei, n. mirabilis, n. rafflesiana and n. sanguinea were collected from frim, selangor, malaysia. however, leaves and twigs from one to three individual plants of nepenthes spp., namely, n. alba, n. albomarginata, n. gracillima and n. sanguinea were collected from the gunung jerai (gj), gurun, kedah, malaysia. leaves with petioles were thoroughly washed under running tap - water and the surface - sterilization of plant material samples was carried out as reported elsewhere. the stem pieces were soaked in 70% ethanol and flamed to make their surface sterile. aseptically, the leaf and stem tissue pieces were inoculated in the petri plates containing luria - bertani (lb) agar medium. the plates were incubated in an incubator at 37c (3c) for 18 - 20 h in the dark. the isolation, cultivation of endophytes, amplification of 16s rdna, sequencing of 16s rdna, identification of endophytes and rooted phylogenetic tree construction was carried out as reported by bhore. incubation of the inoculated leaf discs and stems pieces on lb agar medium enabled cultivable eb to grow, and the colonies of grown eb were visible on the margins of the leaf and stem tissues. all 96 isolates were identified based on 16s rdna sequence blast (megablast) hits analysis. the annotated 16s rrna gene fragment (16s rdna) nucleotide sequences of all isolates have been submitted to the international dna database (genbank / ddbj / embl) under accession numbers : jf819686-jf819713 and jf938974-jf939041. were from the bacilli (59.4%) class, followed by gammaproteobacteria (35.4%) and betaproteobacteria (5.2%) [table 1 ]. the genera of endophytic bacterial isolates (ebis) isolated from 9 nepenthes plant species the 16s rdna multiple sequence alignment output from clustalw was used in the construction of a rooted dendrogram. rooted dendrogram showing clustering of 44 diverse types of endophytic bacteria (eb) isolated from nine nepenthes spp. the accession number of 16s rdna (rrna gene) sequence of the respective isolate is given in front of the species name in this short and snappy study, we isolated and identified 96 isolates from nine nepenthes spp. eb have been reported from several medicinal plants, for instance glycyrrhiza spp. however, to the best of our knowledge, our study is the first to illustrate diversity and types of eb in nine stated nepenthes spp. comparison between the annotated 16s rdna fragment sequences from isolates and the sequences from genbank / ddbj / embl database using the blastn program revealed the identity of the respective isolates. the rooted dendrogram clearly showed the clustering of the species from the bacilli, betaproteobacteria and gammaproteobacteria groups. endophytes are found abundantly in various plant species studied to date, and soil bacteria such as bacillus spp., pseudomonas spp. and azospirillum spp. however, we did not find any pseudomonas spp. and azospirillum spp. in our isolates. during isolation of eb, the growth medium used might be directly affecting the number and type of endophytic microorganisms that can be isolated from the plant tissues. the tissue samples used in the isolation of eb were from a single or few plants of each nepenthes spp. the location and the conditions in which plant species are grown also determine the types of endophytes in it. we have used plant samples from nepenthes spp. that were collected from their wild habitat in gj and diverse collection available at frim. this could be the reason for the wide diversity of eb in the studied nepenthes spp. it is important to note that from 96 isolates, 22 isolates (representing 15 species) belonged to the enterobacteriaceae family, which contains human enteric pathogens. a number of species from the enterobacteriaceae family have been reported as endophytes, viz entrobacter cloacae and klebsiella pneumonia in maize, entrobacter asburiae in cotton, and klebsiella spp. and entrobacter cloacae in banana. of the 44 species of eb isolated from nepenthes spp. however, we have not found any published record that reported acinetobacter soli, bacillus cibi, b. horneckiae, b. indicus, b. koreensis, b. stratosphericus, citrobacter gillenii, c. youngae, kluyvera ascorbata, providencia alcalifaciens and serratia liquefaciens as endophytes. perhaps, this is the first study that reports these bacterial species as endophytes. however, the benefits derived by nepenthes spp. from these bacterial endophytes and its quantum are not clearly understood yet. from this study contains diverse types of cultivable eb, and that the majority of bacterial endophytes (59.4%) were from the bacilli class. nonetheless, these research findings could serve as a foundation in further research on the therapeutic properties of nepenthes spp. in correlation with their bacterial endophytes. we hypothesize that in nepenthes spp., these eb might be involved in producing bioactive compounds of pharmaceutical importance, and further research is required to ascertain the same. | background : nepenthes species are used in traditional medicines to treat various health ailments. however, we do not know which types of endophytic bacteria (eb) are associated with nepenthes spp.objective:the objective of this study was to isolate and to identify eb associated with nepenthes spp.materials and methods : surface - sterilized leaf and stem tissues from nine nepenthes spp. collected from peninsular malaysia were used to isolate eb. isolates were identified using the polymerase chain reaction - amplified 16s ribosomal dna (rdna) sequence similarity based method.results:cultivable, 96 isolates were analyzed ; and the 16s rdna sequences analysis suggest that diverse bacterial species are associated with nepenthes spp. majority (55.2%) of the isolates were from bacillus genus, and bacillus cereus was the most dominant (14.6%) among isolates.conclusion:nepenthes spp. do harbor a wide array of cultivable endophytic bacteria. |
a 41-year - old male injection drug user was brought to the emergency department unresponsive, febrile and hypotensive. initial investigations revealed a white blood cell count of 19.310/l ; urinalysis, chest x - ray and abdominal computed tomography were all unremarkable. fluid resuscitation, vasopressor support and empirical antibiotic therapy with intravenous (iv) piperacillin / tazobactam, vancomycin and clindamycin were initiated. following resuscitation, the patient provided a vague history : he was well until the day of admission, at which time he developed a headache, severe fatigue and generalized malaise. his medical history was significant for hepatitis c and previous episodes of bacteremia related to injection drug use. blood cultures collected on admission grew b cereus (four of four bottles) and serratia marcescens. blood cultures were persistently positive for b cereus until day 14 of admission. on day 29, the patient developed a small antecubital abscess that was drained and cultured b cereus. the patient received a total of six weeks of iv vancomycin and was discharged home. nine days following the admission of case 1, a 49-year - old man with a history of daily heroin and crystal methamphetamine injection drug use presented to hospital with confusion and agitation. he was afebrile, with no obvious focus of infection on history or physical examination. his medical history was significant for recently diagnosed hiv infection and hepatitis c. initial laboratory results showed a white blood cell count of 13.110/l. blood cultures collected on admission grew b cereus and lactobacillus, and the patient was switched to iv vancomycin and ciprofloxacin. three subsequent blood cultures were positive for b cereus ; blood cultures eventually cleared on day 14 of admission. iv vancomycin was continued for a total duration of eight weeks and the patient was discharged. twenty days after case 1 was admitted to hospital, a 33-year - old man with a history of heroin and cocaine injection drug use presented to the emergency department with confusion. his medical history included hepatitis c and previous episodes of methicillin - resistant staphylococcus aureus infective endocarditis, one of which necessitated a tricuspid valve replacement. the patient left hospital against medical advice, but was called to return the following day when blood cultures returned positive for gram - positive bacilli, subsequently identified as b cereus. repeat blood cultures were drawn and iv vancomycin therapy was initiated. only the initial blood culture (one of two bottles) grew b cereus, and all subsequent blood cultures were negative. the patient received a total of two weeks of iv vancomycin and was discharged home. the b cereus isolates from the present cluster of three cases were submitted to the british columbia public health microbiology and reference laboratory for pulsed - field gel electrophoresis (pfge) analysis to determine genetic relatedness. pfge was performed for b cereus using smai restriction endonuclease for digestion and based on the parameters described by liu (5). additionally, the vancouver police department was able to provide five samples of confiscated heroin (dry powder) for microbiological analysis. the samples of confiscated heroin were randomly chosen and were not related to the patients who presented to hospital. of the five samples of heroin provided, three grew b cereus from microbiological culture. coagulase - negative staphylococci (three of five) and escherichia vulneris (one of five) were also recovered in culture. given the preliminary findings from the pfge performed on the clinical isolates of b cereus, the organisms recovered from the heroin samples were not submitted for further genetic analysis. a common source for the cluster was not confirmed. a 41-year - old male injection drug user was brought to the emergency department unresponsive, febrile and hypotensive. initial investigations revealed a white blood cell count of 19.310/l ; urinalysis, chest x - ray and abdominal computed tomography were all unremarkable. fluid resuscitation, vasopressor support and empirical antibiotic therapy with intravenous (iv) piperacillin / tazobactam, vancomycin and clindamycin were initiated. following resuscitation, the patient provided a vague history : he was well until the day of admission, at which time he developed a headache, severe fatigue and generalized malaise. his medical history was significant for hepatitis c and previous episodes of bacteremia related to injection drug use. blood cultures collected on admission grew b cereus (four of four bottles) and serratia marcescens. blood cultures were persistently positive for b cereus until day 14 of admission. on day 29, the patient developed a small antecubital abscess that was drained and cultured b cereus. the patient received a total of six weeks of iv vancomycin and was discharged home. nine days following the admission of case 1, a 49-year - old man with a history of daily heroin and crystal methamphetamine injection drug use presented to hospital with confusion and agitation. he was afebrile, with no obvious focus of infection on history or physical examination. his medical history was significant for recently diagnosed hiv infection and hepatitis c. initial laboratory results showed a white blood cell count of 13.110/l. blood cultures collected on admission grew b cereus and lactobacillus, and the patient was switched to iv vancomycin and ciprofloxacin. three subsequent blood cultures were positive for b cereus ; blood cultures eventually cleared on day 14 of admission. iv vancomycin was continued for a total duration of eight weeks and the patient was discharged. twenty days after case 1 was admitted to hospital, a 33-year - old man with a history of heroin and cocaine injection drug use presented to the emergency department with confusion. his medical history included hepatitis c and previous episodes of methicillin - resistant staphylococcus aureus infective endocarditis, one of which necessitated a tricuspid valve replacement. the patient left hospital against medical advice, but was called to return the following day when blood cultures returned positive for gram - positive bacilli, subsequently identified as b cereus. repeat blood cultures were drawn and iv vancomycin therapy was initiated. only the initial blood culture (one of two bottles) grew b cereus, and all subsequent blood cultures were negative. the patient received a total of two weeks of iv vancomycin and was discharged home. the b cereus isolates from the present cluster of three cases were submitted to the british columbia public health microbiology and reference laboratory for pulsed - field gel electrophoresis (pfge) analysis to determine genetic relatedness. pfge was performed for b cereus using smai restriction endonuclease for digestion and based on the parameters described by liu (5). additionally, the vancouver police department was able to provide five samples of confiscated heroin (dry powder) for microbiological analysis. the samples of confiscated heroin were randomly chosen and were not related to the patients who presented to hospital. of the five samples of heroin provided, three grew b cereus from microbiological culture. coagulase - negative staphylococci (three of five) and escherichia vulneris (one of five) were also recovered in culture. given the preliminary findings from the pfge performed on the clinical isolates of b cereus, the organisms recovered from the heroin samples were not submitted for further genetic analysis. a common source for the cluster was not confirmed. b cereus has been reported as a cause of extraintestinal infections, including those of the musculoskeletal, ocular, respiratory, cardiovascular and central nervous systems (6). injection drug use is often attributed as an etiological factor, with reports of cellulitis (4), endophthalmitis and panophthalmitis (3,7,8), and endocarditis (1,6,9,10). the prognosis of bacteremia and native valve endocarditis with b cereus is typically good, with prompt antibiotic therapy of adequate duration being suitable for recovery (2). conversely, high morbidity and mortality have been observed with prosthetic valve endocarditis, and authors of a review on the subject recommend prompt valve replacement in conjunction with iv antibiotics (11). no guidelines exist for treatment of invasive b cereus infections, and reports in the literature describe clinical recovery with two to four weeks of iv antibiotics for bacteremia, and four to six weeks for endocarditis (2). published susceptibility reports describe complete susceptibility of b cereus to vancomycin, quinolones, gentamicin, carbapenems and tigecycline ; intermediate susceptibility to clindamycin, tetracycline and erythromycin ; and high - level resistance to trimethoprim / sulfamethoxazole, penicillins and cephalosporins (12). in the present study, case 1 was treated with a prolonged six - week course of iv vancomycin for b cereus bacteremia because of persistently positive blood cultures for the first 14 days. case 2 was treated for mitral valve endocarditis with an eight - week course of iv vancomycin and his blood cultures cleared over a two - week period. although case 3 had a prosthetic tricuspid valve, there was no evidence of endocarditis and only a single initial positive blood culture. he received a two - week course of iv vancomycin, despite the possibility that this was a culture contaminant. all patients in the cluster shared the risk factor of injection drug use, having recently injected heroin. b cereus infections have been sporadically associated with injection drug use, particularly with heroin. these rare occurrences were first reported (9) and reviewed (1) in the 1970s. b cereus, a known contaminant related to drug use, is resistant to heat and capable of surviving in harsh environments. a report from 1983 conducted in washington, dc, found that nearly one - half of injection paraphernalia and 32% of heroin samples were contaminated with bacillus species (13) the microbial burden was significantly higher on the injection paraphernalia than the heroin itself, and brown heroin (mainly from mexico) was found to have a higher burden compared with white heroin (mainly from overseas). the authors speculated that heroin users experience frequent transient episodes of bacillus species bacteremia, which are rarely of clinical significance. in the literature, one case exists with a largely conclusive link of a b cereus infection to contaminated heroin (4). a patient with cellulitis provided a heroin sample, and both his wound aspirate and heroin cultured b cereus, which was found to be indistinguishable through pfge. in our cluster possible explanations for our cluster not having a common source include increasing microbial burden of heroin in general, supported by the burden found on the randomly acquired heroin or, particularly, contaminated injection paraphernalia among users. b cereus infections are an underappreciated cause of bloodstream infections in injection drug users, for which the treatment requires prompt identification and antibiotic coverage. our observation of three unrelated but temporally associated cases of b cereus infection in heroin users suggests that suspicion of b cereus infection in this patient population may become increasingly warranted. | bacillus cereus is a bacteria commonly found in nature that is most frequently implicated as the cause of gastrointestinal illness caused by ingestion of contaminated food ; however, there are also reports of extraintestinal infections, although rare. this article describes three cases in which b cereus bacteremia occurred among injection drug users living in vancouver, british columbia, within a short period of time. pulsed - field gel electrophoresis was performed to determine whether these three isolates were related. |
a 38-year - old man came to the netherlands in january 2009 to work as a rigger in the harbor of rotterdam. since october 2008, he had lived in kapit, sarawak, in borneo and hunted wild animals in the surrounding jungles. one week after arriving in the netherlands, he came to our hospital with a 5-day history of fever, myalgia, headache, and low back pain. his medical history was uneventful, and he had not experienced any previous malaria attacks. laboratory investigations showed a moderate anemia (hemoglobin 7.8 mmol / l [reference range 8.511.0 mmol / l ]), a normal leukocyte count (5.8 10/l [reference range 4.310 10/l ]), thrombocytopenia (platelet count 22 10/l [reference range 150400 10/l ]), an increased level of c - reactive protein (158 mg / l [reference range < 10 mg / l ]), and liver function abnormalities (serum alanine aminotransferase 199 u / l [reference range < 41 u / l ] ; aspartate aminotransferase 128 u / l [reference range < 37 u / l ] ; lactate dehydrogenase [ldh ] 1,059 u / l [reference range < 450 u / l ] ; gamma - glutamyltransferase 183 u / l [reference range < 50 u / l ] ; alkaline phosphatase 285 u / l [reference range < 120 u / l ] ; and total bilirubin 99 mol / l [reference range < 17 mol / l ]). plasma lactate level was within normal limits. in a rapid diagnostic test for malaria (binaxnow malaria test ; binax, scarborough, me, usa), his blood sample was negative for p. falciparum histidine - rich protein 2 but showed a positive reaction with pan - malarial aldolase antigen, which suggested a non p. results of quantitative buffy coat analysis were positive for malaria trophozoites, schizonts, and gametocytes. a thin blood film showed parasite density of 2% infected erythrocytes (84,000 trophozoites/l), schizonts, and gametocytes with an inconclusive morphologic appearance (figure 1). a p. knowlesi infection was suspected because of his recent stay in kapit, malaysian borneo. the patient was treated orally with chloroquine, 10 mg / kg, followed by 5 mg / kg after 6, 24, and 48 hours, which resulted in a rapid relief of symptoms and fever. results of quantitative buffy coat analysis, pan - malarial aldolase antigen reactivity, and thick and thin blood smears were negative within 40 hours after administration of chloroquine. infected erythrocytes were not enlarged, lacked schuffner stippling, and contained much pigment. shown are examples of trophozoites (a f), a schizont (g), and a gametocyte (h). subsequently, pcr analysis of blood samples taken at admission was performed to determine the plasmodium species. human plasmodium species were excluded by using a conventional nested pcr and real - time pcr (8,9). in addition, pcr analysis was performed on a blood sample by using diagnostic primers for plasmodium small subunit (ssu) rrna as described (3), including genus - specific and species - specific primers. (3), nested pcr was not necessary because of high parasitemia and availability of fresh material. instead, pcrs were performed directly on 2 l of blood in 25-l volumes by using the phusion blood pcr kit (finnzymes, espoo, finland). specific primers generated pcr products, providing evidence that the patient had p. knowlesi malaria. to confirm the pcr result, we sequenced the cloned amplification product generated with primers rplu1 and rplu5. sequences were compared with known plasmodium a - type ssu rrna sequences by using the neighbor - joining method (figure 2). fj804768) clustered strongly with p. knowlesi a - type ssu rna sequences, confirming that the patient was infected with the p. knowlesi parasite. phylogenetic tree constructed according to the neighbor - joining method based on a - type small subunit rna sequences of several plasmodium species (the sequence of the clinical isolate pkhhr - bprc1 (in boldface) (genbank accession no. fj804768) clusters with all other p. knowlesi strains (indicated by pk isolate numbers). pfrag, p. fragile ; pinui, p. inui ; pcyn, p. cynomolgi ; pfalc, p. falciparum ; pmal, p. malariae. obtaining a correct diagnosis of malaria may be troublesome in centers where laboratory staff are less skilled in the proper identification and quantification of causative plasmodium species, as may occur in countries in which malaria is not endemic. in contrast to our case, bronner. reported that the binaxnow malaria test did not detect a p. knowlesi infection in a traveler from sweden who had a p. knowlesi infection acquired in malaysian borneo (2). low parasitemia (0.1%) in this patient may have caused the lack of reactivity with the pan - malarial antigen aldolase (2). we evaluated the binaxnow malaria and the optimal rapid malaria (diamed, cressier, switzerland) tests for detection of p. knowlesi in human blood by analysis of consecutive blood samples taken after admission. these samples were stored at 20c for 2 weeks until tests were performed (table). the blood sample taken on admission (2% infected erythrocytes) did not react with p. falciparum specific antibody against histidine - rich protein 2, but reacted with the pan - malarial antigen aldolase in the binaxnow malaria test. specific ldh and pan - malarial ldh in the optimal rapid malaria test, confirming the cross - reactivity of p. knowlesi ldh with monoclonal antibody 17e4 against p. falciparum ldh, as shown by mccutchan. this antibody is also used in the optimal rapid malaria test (diamed, pers. therefore, a positive test result for the p. falciparum ldh in the optimal rapid malaria test is not specific for p. falciparum because it can also be caused by a p. knowlesi infection. the positive result for ldh and aldolase in either test became negative after treatment (table), which indicates rapid clearance of parasites after treatment. results of our comparative study suggest that the optimal rapid malaria test may be able to detect lower levels of p. knowlesi parasitemia than the binaxnow malaria test. hrp-2, histidine - rich protein 2 ; ldh, lactate dehydrogenase ; nd, not detectable. all tests were performed on blood samples collected in edta and frozen at 20c for 2 weeks. binax, inc., scarborough, me, usa. diamed, cressler, switzerland. our results indicate that commercially available rapid diagnostic antigen tests for human plasmodium species can detect p. knowlesi infections in humans, although infections with a low parasitemia will not be detected. a negative test result does not exclude a p. knowlesi infection, as it does not exclude infections by other human plasmodium species (11). | we describe a pcr - confirmed case of plasmodium knowlesi infection with a high parasitemia level and clinical signs of severe malaria in a migrant worker from malaysian borneo in the netherlands. investigations showed that commercially available rapid antigen tests for detection of human plasmodium infections can detect p. knowlesi infections in humans. |
fibroblasts are one of the most commonly used somatic cells to produce induced pluripotent stem (ips) cells [1, 2 ]. therefore, it is essential to determine the genetic and epigenetic stability of fibroblasts in long - term culture. it has been shown that long - term in vitro culture of somatic cells increases the risk of cell senescence, chromosomal aneuploidy, as well as multiploidy karyotypes [3, 4 ]. however, the potential changes in epigenetic gene regulation in long - term cultured human fibroblasts are not well investigated. dosage compensation of the x chromosome in mammalian female cells is one of classic examples of epigenetic gene regulation. it is achieved through an event referred to as x chromosome inactivation (xci) in which one of the two x chromosomes is silenced during development [57 ]. xci is initiated in the x - inactivation center of the x chromosome, within which a gene encoding x inactive - specific transcript (xist) is expressed solely from the inactivated x chromosome. xist gene is crucial for xci because its noncoding rna directly interacts with and coats the inactive x chromosome. after the completion of xci, the two x chromosomes in female cells are distinguished by differential transcription of the xist gene, dna methylation, and histone modifications such as histone h3 lysine 27 trimethylation (h3k27me3) [8, 9 ]. in this study, human dermal fibroblasts (hdfs) we then examined the cell - growth rate, the stability of xci in short- and long - term passages. we found that early passages of female hdfs retained a normal karyotype with the expected xci pattern while high - passage cells exhibited a higher risk of carrying more than one copy of the inactive x chromosomes due to the presence of three or four copies of x - chromosomes in one nucleus. this research project was approved and monitored by the ethical committee in the first affiliated hospital of nanjing medical university. written informed consent was obtained from all 8 female patients before collecting their dermal tissue specimens. tissue specimens were washed with phosphate buffered saline (pbs) solution with 100 u / ml penicillin and 100 mg / ml streptomycin. fat and connective tissues were removed, and the epidermis was scraped off by an operating knife blade. the remnant tissues were sheared into clumps of approximately 1 mm, which were then planted uniformly onto the bottom of 25 cm flasks. the flasks were then inverted and placed in a 5% co2 incubator at 37c. culture medium consists of dulbecco 's modified eagle 's medium (dmem, pyruvate - free, high - glucose formulation ; invitrogen) supplemented with 20% fetal bovine serum (fbs ; invitrogen). when the cells reach 8090% confluence, the culture was split at a ratio of 1 : 2. cells from passage 5 through 10 were cultured in a 6-well plate with a density of 4 10 per well. six days later, the growth curves of 8 hdf cell lines were obtained by counting the number of cells per well every 24 hours. karyotype analysis on each hdf cell line was carried out via giemsa - banding (g - banding) between passages 510. when 8090% confluence was reached in the 25 cm flask, culture medium was changed and supplemented with 0.1 g / ml colcemid solution (diluted from 10 g / ml stock, cat., cells were collected and suspended in 5 ml of 0.075 m kcl solution.. 2 ml of fixation fluid (methanol : glacial acetic acid at 1 : 3) was added and mixed together, and the cells were then collected. after two rounds of fixations, the cell suspension was mounted onto slides and baked at 75c for 3 hours. cells were plated on either slides or coverslips after being rinsed three times with 1x pbs for 5 minutes and then fixed with 4% paraformaldehyde for 20 minutes at room temperature. then the cells were washed again with 1x pbs three times and stored in 70% etoh at 20c for subsequent analysis. all reagents used for rna fish were prepared with diethylpyrocarbonate- (depc-, sigma) treated h2o. cells were washed three times with 1x pbs and dehydrated with 70% ethanol for 2 minutes and 90% ethanol for 2 minutes. then the cells were hybridized overnight at 37c in hybridization buffer (50% formamide/2x standard saline citrate (ssc) using a cy3-hxd1 - 3 probe as previously described in. the next day, coverslips were rinsed twice with hybridization buffer for 30 minutes each at 37c and then once with 1x pbs. the cells prepared as above were rinsed and incubated sequentially by the following procedures : cells were rinsed with 1x pbs three times, incubated in 1x pbs plus 0.4% triton x-100 (sigma) for 20 minutes and rinsed again with 1x pbs three times at room temperature. blocking of nonspecific staining was conducted with 10% normalgoat serum in 1x pbs for 1 hour at room temperature, after which the cells were incubated overnight with a rabbit anti - h3k27me3 antibody (1 : 1000, from update biotechnology, usa) at 4c in a humidified chamber. the cells were subsequently rinsed in 1x pbs three times and incubated for 1 hour in the dark with fitc - conjugated secondary antibody (1 : 200, beijing zhong shan golden bridge biotechnology co. ltd, china). the cells were then rinsed with 1x pbs three times, counterstained with 4,6-diamidino-2-phenylindole (dapi, vysis, inc., downers grove, il, usa), and mounted onto slides after 5 minutes. slides were immersed in 2x ssc solution jar, shook for 5 seconds, and removed after 10 minutes. hybridization areas were made, and the temperature of the denaturing solution (70% formamide/2x ssc) was maintained at 73c. after about 5 minutes, slides were dehydrated for 1 minute in 70% etoh, followed by 1 minute in 85% etoh, and then 1 minute in 100% etoh. concurrently, the probe mixture was prepared and placed in a 73c water bath for 5 minutes. 10 l of probe mixture was then added to one target area after which the coverslip was immediately applied. the next day, the coverslip was removed from one slide and the slides were immediately immersed in the 0.4x ssc solution for 2 minutes at 70c. finally, the slides were air - dried in the dark and 10 l of counterstains were applied to the target area of the slides and the coverslips were added. a commercially available probe (vysis, inc., downers grove, il, usa) targeting the centromeric region of x chromosome (green fluorescence) was used to evaluate the number of x chromosomes of the cells. two hundred cells were examined randomly from each culture, and the numbers of signals in each cell nucleus were counted. after the first 24-hour inoculation, dermal tissues were completely adhered to the bottom of the flasks and hdfs began growing out in the following days. when hdfs reached 8090% confluence, they were passaged as described in materials and methods. within 10 passages, few of the cells exhibited senescence including intumescences, arborescence, and cell death. the growth curves showed that the number of hdf slowly increased in the first 24 hours after passage. from 24 to 96 hours, hdf proliferated exponentially. hdf cells entered stationary phase from 96 to 144 hours after passage (figure 1). g - banding was conducted to determine the karyotypes of 8 hdf cell lines, and the results demonstrated that each culture exhibited normal karyotype in twenty metaphase spreads examined within the first 10 passages (data not shown). one intense punctate signal of h3k27me3 was found in most hdf nuclei within passages 1 and 5 (figure 2). occasionally, we also found cells that exhibit either zero or two punctuate staining signals. after randomly selecting and counting the number of nuclei with 0, 1, and 2 h3k27me3 signals, we found that the number of cells without h3k27me3 punctate signals appeared to increase at a higher passage number (table 1). the xist rna fish analysis yielded the same results as h3k27me3 immunostaining (figure 3). interestingly, in four out of total eight cell lines (# 1, 2, 3, and 8), our counting data showed that two positive signals of xist rna coating or h3k27me3 staining of inactive x chromosomes were present in approximately 8.5 ~ 18.5% hdf cells at passage 10 (figures 2 and 4). consistent with the increased number of inactive x - chromosome, a few cells showed three or four copies of x chromosome signals from dna fish analysis of total number of x chromosomes. our counting data showed that approximately 6% of the cells exhibited three copies of x chromosomes and 16% with four copies of x - chromosomes (figure 4). pluripotent cells are considered a major resource for regenerative medicine, and fibroblasts are the most commonly used cell type to produce ips cells by reprogramming [1, 2 ]. in order to produce quality ips cells, the biological properties of fibroblasts must first be characterized prior to reprogramming. these include cell growth state, stability of cytogenetic and epigenetic status such as x chromosome inactivation. as a pioneer on cell senescence study, hayflick was the first to establish a reliable protocol to maintain fibroblast growth in vitro. early studies showed that hdfs can reach over 50 population doublings before senescence sets in, but its proliferative capacity depends on the age of the donor and the biopsy site. in our cultures of eight female hdf lines, cells with fusiform shape and senescence phenotype nevertheless, in vitro cultures may still affect genetic and epigenetic stability of the cultured hdfs. by counting the cell numbers after passaging, we plotted the cell growth curves which demonstrated that cells were in delitescence in the first 24 hours after cell passage. the cells then proliferated rapidly in exponential growth phase from 24 to 96 hours and entered stationary phase from 96 to 144 hours (figure 1). previous in vitro culture studies report spontaneously mutated clones in most species, especially in rodents, usually after 57 passages. these mutated clones have abnormal karyotypes including chromosomal aneuploidy and multi - ploidy karyotypes. in our results, we did not find any definitively abnormal karyotypes through the standard g - banding karyotyping. however, in a detailed analysis of x - inactivation status, we found that a portion of cells (8.518.5%) in half of our cultures (four out of eight hdf lines) exhibited more than two copies of x - chromosomes. our data suggest that identification of the number of inactive x chromosomes by xist rna fish or the total number of x chromosomes by dna fish analysis is a more sensitive method to detect chromosomal variations than the standard g - banding karyotyping. xci is a fundamental epigenetic mechanism in female somatic cells, involving the dosage compensation of x - linkage genes. any improper changes in the dosage of x - linkage gene would impair the normal function of somatic cells. research on human female embryonic stem cell (esc) lines indicated that xci patterns of these lines could change during passaging. xist gene expression in cultured hesc lines was unstable and subjected to epigenetic silencing by dna methylation. in this study, xist gene rna fish and h3k27me3 immunofluorescence were performed to detect the xci status in passages 1, 5, and 10 of hdf cells. the results indicated that in most cell nucleus only one xci signal (green spot for h3k27me3 or red spot for xist rna) was observed, and few of the cells had no xci markers (figures 2 and 3). cell numbers without xci signals tended to increase along with hdf population doublings (table), which could be dna methylation on the xist promoter to silence its expression. additionally, two xist rna signals in coupling with 3 - 4 signals of x chromosome can be observed in the same nucleus in a few cells at passage 10 (figure 4). the presence of two xist rna signals indicate two copies of inactive x chromosomes, consistent with the increased number of total x - chromosomes. in summary, our present study demonstrates that early passages of female hdf cultures are genetically more stable than cultures after 10 passages. by monitoring xci status and the number of inactive x chromosome, we can effectively determine whether the chromosome ploidy of human female somatic cells changes over the course of cell culture. the information of genome stability is important for evaluating the suitability of these cells for other applications such as the derivation of ips cells in regenerative medicine. | human primary fibroblasts are a popular type of somatic cells for the production of induced pluripotent stem (ips) cells. here we characterized biological properties of primary fibroblasts in terms of cell - growth rate, cytogenetic stability, and the number of inactive x chromosomes during long - term passaging. we produced eight lines of female human dermal fibroblasts (hdfs) and found normal karyotype and expected pattern of x chromosome inactivation (xci) at low passages (passage p1 - 5). however, four out of the eight hdf lines at high passage numbers (p10) exhibited duplicated hallmarks of inactive x chromosome including two punctuate signals of histone h3 lysine 27 trimethylation (h3k27me3) and x inactive - specific transcript (xist) rna signals in approximately 8.518.5% of the cells. our data suggest that the copy number of inactive x chromosomes in a subset of female hdf is increased by a two - fold. consistently, dna fluorescent in situ hybridization (fish) identified 3 - 4 copies of x chromosomes in one nucleus in this subset of cells with two inactive xs. we conclude that female hdf cultures exhibit a higher risk of genetic anomalies such as carrying an increased number of x chromosomes including both active and inactive x chromosomes at a high passage (p10). |
the most recent introduction to the dental ceramics family is zirconia, which in its pure form is a polymorphic material. fabrication of fixed prosthesis involves layering of metallic substructure or all ceramic core structure with an aesthetic ceramic veneering material. the underlying substructure provides the required strength and the overlying ceramic gives the required esthetics. yttrium oxide partially stabilized tetragonal zirconia polycrystal (y - tzp) was introduced in dentistry as a core material in the 1990s. many studies on the use of zirconia have been conducted, but some features still remain unclear. there is a lack of information on how temperature change and water treatments affect the material properties and clinical durability of veneered zirconia cores. unlike other prostheses in the body, oral prosthesis recent studies have shown an indication that water molecules have an influence on the bond between veneered and veneering material. slow surface transformation to the stable monoclinic phase occurs through environmental stresses, usually in the presence of water molecules, hot water vapor, or body fluids such as saliva [47 ]. studies have shown the bond between the zirconium core and the veneering ceramic to be influenced by water molecules which eventually affects the clinical durability of the restoration in function [8, 9 ]. aging of zirconia may have detrimental effects on its bonding with veneering ceramics ; mechanical stresses and wetness exposure accelerate this process. presently, different companies are providing milled zirconia cores from presintered zirconia blocks and are offering long time warranty (15 yrs) for their products. on observing the growing trends and future possibilities, it is very important to understand the clinical durability, limitation, and function of the veneered zirconia as a restorative material. this study was undertaken to investigate and compare the bond strengths of commercial zirconia framework materials with veneering material as influenced by (steam and thermal treatment) artificial aging. in this study, sbs testing and fracture surface analysis was carried out to microscopically characterize the failure modes and to evaluate and compare the phase transformation (m - phase fraction) and surface grain size (m) using x - ray diffractometer and sem (scanning electron microscope), respectively, between aged and nonaged specimens. the objective of this study was to simulate the clinical environment so the durability, clinical function, and nature of the bond between zirconia and veneering material, as in a clinical trial of 15 years, can be evaluated. the null hypothesis tested was that the bond strength between different zirconia framework and veneering ceramic will not be affected by artificial aging. three types of zirconia were selected for this study : cercon (degudent, hanau, germany), ziecon (jyoti ceramic industries pvt., ltd., nashik, india), and upcera (shenzhen upcera co., ltd., china) (table 1). a total of 90 samples in the form of disc with each zirconia system having 30 samples were fabricated from their respective zirconia blocks of 98 mm diameter 10 mm height by cad / cam procedure. a cad model of the sample is designed for the purpose of the study and converted into stereolithographic (stl) format in the computer. the cad design was used by milling machine and samples of 7 mm diameter 3 mm height were milled out from the standard blanks of partially sintered zirconia. as per the manufacturer though thermocycling has been advocated in the literature as a conventional aging test, autoclaving induced low - temperature degradation is an established method for accelerated aging of y - tzp materials. autoclaving at 134c for 5 hours is the standard aging protocol according to iso 13356 valid for y - tzp implants for surgery. 20 samples from each group were autoclaved at 134c for 5 hrs to simulate oral conditions for 15 years. ninety samples of different zirconia core material were layered using vita vm9 (vita zahnfabrik, bad sckingen, germany) veneering ceramic material (table 3). first, the liner material, which was a single, thin, continuous layer supplied by the manufacturers, was applied and fired independently according to the manufacturer 's instructions (table 4). the veneering procedure was done using the manual layering technique. for the application of base dentin, dentin powder and modelling liquid were mixed according to the standard procedure and a customized metallic jig was used to achieve a uniform thickness of 1 mm. finally, the samples were finished to achieve the uniform thickness of 2 mm. the prepared core - veneer disks were fired in a programmable vacuum porcelain furnace (vita vacumat 4000 premium t, vita zahnfabrik, bad sckingen, germany) according to the firing programs provided by the manufacturer (table 4). a total thickness of each sample including the zirconia substructure of approximately 5 mm was obtained. a metal jig was used for holding the samples to determine the shear bond strength. the samples were held in such a manner that the junction of zirconia substructure / veneering ceramic interface was faced towards the chisel load applicator. universal testing machine (model 3345, instron corp., norwood, ma, usa) with a 10 kn load cell and crosshead speed of 0.5 mm / min was used. a chisel load applicator was used to direct a parallel shearing force to the substructure / veneer ceramic interface. the fracture load was obtained with the help of the graph on the digital monitor attached to the machine. the drop in the graph determined the point of debonding (fracture load) of each sample. fracture load (kg) was converted to the shear bond strength (mpa) by use of the cross - sectional area of disc. the following formula was used to calculate the shear bond strength (mpa) : (1)shear bond strengthmpa = fracture loadkgarea of discmm2. after shear bond strength analysis, all the debonded samples were analysed under a stereomicroscope (20x) to evaluate the nature of bond failure. each specimen was placed on carbon coated flat platform in such a manner that the surface between zirconia substructure and veneering ceramic faces the pointer of stereomicroscope, so as to identify whether there was cohesive failure, adhesive failure, or a combination of both, that is, mixed failure. the crystalline phases on the surfaces of the specimens were analysed by a philips x'pert 1 x - ray diffractometer. scans were performed in the 2 range of 25 to 35 with a step size of 0.01 and 0.5 s / step interval. the m - phase fraction (xm) was calculated by equation given by garvie and nicholson. the m - phase fraction (xm) was calculated by the following equation : (2)xm = im111+im111im111+im111+it111100,where im(111) is intensity of the peak (areas under the 31.5 peak) that represents the m - phase, im111- is intensity of the peak (areas under the 28.2 peak) that shows the m - phase, and it(111) is intensity of the peak (areas under the 30.3 peak) that represents the t - phase. after phase analysis, to observe the surface microstructure for the surface particle size evaluation, specimens were sectioned and polished with a diamond wheel and grinding machine and coated with gold using a sputter - coating technique. electron imaging was performed at an accelerating voltage of 15 kv to investigate surface geometry with zeiss evo 40 scanning electron microscope at the magnification of 30,000x. 10 areas were randomly selected and analysed for each specimen (figures 1(a) and 1(b)). the data was entered into ms excel spreadsheet and analysed, using spss version 11 statistical software. techniques applied were student 's t - test and one - way analysis of variance (anova) followed by post hoc comparison by bonferroni method. for each group and subgroup p table 5 summarizes the mean values and standard deviations of sbs for all the tested zirconia and veneering ceramics, that is, upcera, ziecon, and cercon. among control groups (without artificial aging), cercon showed the highest mean value (27.9 6.54 mpa) followed by ziecon (24.70 6.76 mpa) and the lowest mean value was shown by upcera (24.43 7.13 mpa). the statistical difference was found to be nonsignificant among them (p = 0.66). among the test groups (with artificial aging) cercon showed the highest mean value (24.20 8.87 mpa) followed by upcera (24.0 7.53 mpa) and the lowest mean value was shown by ziecon (23.9 8.03 mpa). the statistical difference was found to be nonsignificant among the subgroups (p = 0.99). with vita vm9 veneer, ziecon and cercon in control group demonstrated 90% cohesive failure. as for the zirconia of test group, upcera and cercon showed 30% combined failure. table 7 summarizes the mean of phase transformation (m - phase fraction) vol% values and their respective standard deviation of all the three groups, that is, upcera, ziecon, and cercon. among control groups (without artificial aging), cercon showed the highest mean value (12.6 1.15%) followed by ziecon (6.49 0.77%) and the lowest mean value was shown by upcera (6.08 1.16%). the statistical difference was found to be significant among the subgroups (p = 0.00). among the test groups (with artificial aging) cercon showed the highest mean value (14.3 0.70%) followed by upcera (12.4 1.9%) and the lowest mean value was shown by ziecon (11.0 0.18%). the statistical difference was found to be significant among the subgroups (p = 0.04). table 8 summarizes the mean of surface grain size and their respective standard deviation of all the three groups, that is, upcera, ziecon, and cercon. among control groups (without artificial aging), upcera showed the highest mean value (0.43 0.039 m) followed by ziecon (0.41 0.064 m) and the lowest mean value was shown by cercon (0.39 0.060 m). the statistical difference was found to be nonsignificant among the subgroups (p = 0.532). among the test groups (with artificial aging) upcera showed the highest mean value (0.42 0.034 m) followed by ziecon (0.40 0.05 m) and the lowest mean value was shown by cercon (0.40 0.032 m). the statistical difference was found to be nonsignificant among the subgroups (p = 0.781). there is no significant difference in the mean values of shear bond strength among all samples for both aging and nonaging groups. hence the proposed hypothesis was accepted and probably multicentric trials could be taken to get the explicable results. zirconia is classified as a high strength ceramic material ; it was introduced for use in dentistry as a biomaterial with unsurpassed mechanical properties. its clinical application expanded from single crowns, short - span fixed partial denture, and multiunit full - arch zirconia frameworks to implant abutments and complex implant superstructures [1315 ]. there is no doubt that if we compare the strength of zirconia framework in relation to shear bond strength between veneered ceramic and zirconia, the weakest link on comparative note is the bonding between zirconia and veneering zirconia. in the presence of water molecules this leads to a cascade of events as the transformation of one grain results in local volume expansion and causes stress to neighboring grains. t~m transformation induced by aging in the humid environment of the oral cavity is commonly referred to as low - temperature degradation (ltd). at oral temperatures the results showed that there is no significant difference in the mean values of shear bond strength among all samples (table 5). (2004), wherein they obtained sbs values of zirconia veneered porcelain ceramic in the range of 27 mpa-28 mpa. however the results of our study are in disagreement with the low bond strength values (9.4 mpa26 mpa) obtained by guess. this difference in bond strength values could be attributed to the type of veneering ceramic used with the zirconia framework. in the present study between the aged and nonaged samples, aging did not make any clinically significant difference in the shear bond strength ; that is, in clinical restorations the bond between the zirconia core and veneered ceramic would not be affected by time. another factor that may affect the shear bond strength is the multiple firing of veneer ceramic over zirconia core ; due to multiple firing there is relaxation of residual stresses and phase changes from tetragonal to monoclinic transformation. this not only affects the strength of the structure, but also may affect the core - veneer bond strength. surface lifts that occur during tetragonal monolithic transformation can reduce the core - veneer bond strength. zirconia veneered samples of all groups (aged as well as nonaged) after sbs test showed combined and cohesive (within veneer) bond failure as seen under stereomicroscope, with the predominance of cohesive failure in the veneer layer with no adhesive failure (table 6). (2008), and aboushelib (2006) who investigated the type of bond failure after sbs test. the failure mode observed for ceramic systems was mainly combined (adhesive at the interface and cohesive in the veneering ceramic) and rest of them were cohesive, that is, chip off in veneer material. in contrast, ozkurt. (2010) in their study on type of bond failure in zirconia veneered specimens have found that, in all the test groups, both adhesive and combined failures occur between the zirconia cores and their veneering ceramics. the clinical implication of these findings is that in zirconia ceramic systems the interface of the bonded restorations came out to be stronger as there are more of chip - off fractures of the veneering ceramic and delamination rather than catastrophic failure of the core structure. the bond strength between zirconia and the veneering ceramic was higher than the cohesive strength of the veneering ceramic. in other words, the weakest link was not the interface but the veneering ceramic itself. adhesive failure does not occur in the presence of a good bond between compatible ceramic core and veneering materials. hence, to realize the benefit of the high strength of zirconia frameworks, the strength of veneering ceramics needs to be improved. in the present study different analytical methods are applied, namely, xrd and sem, to analyse the aging characteristics of the zirconia ceramics. iso standards allow a maximum of 25% of m - phase to be present, post accelerated aging test conducted at 134c with 2-bar pressure for 5 hrs for any commercial zirconia ceramics to be used in dentistry. xrd analysis results of our study among all the three zirconia samples, namely, upcera, ziecon, and cercon, revealed that, after aging treatment, the m - phase content on the zirconia surfaces in all samples was found to be increased after aging relative to the tetragonal phase peak (table 7). cercon samples were observed to be most sensitive to the aging treatment and this was reflected as a large % increase of m - phase content on the surface of the sample (14.3%). this was followed by upcera (11.4%), and ziecon (11.0%) was observed to be least affected by aging treatment. the present study on simulated aging has shown that ltd is observed to be insignificant over a simulated period of 15 years of clinical usage. the literature is well supplemented with research on phase transformation and its effect on physical properties such as flexural strength and fracture toughness. however, very few studies have evaluated the quantitative phase transformation within zirconia frameworks due to ltd and the correlation of phase transformation with the bond strength between zirconia substructure and veneering ceramic. in a study xiao. observed the change in the monoclinic phase and compared the low - temperature resistance aging performances of the three clinical frequently used zirconia core materials lava frame, cercon smart, and upcera before and after aging. xrd analysis showed that the m - phase contents of the three zirconia materials increased by prolonging the aging time, where in upcera zirconia was the most sensitive to the aging treatment. the differences in ltd observed in the three different brands of yttria - stabilized zirconia ceramics may be attributed to differences in grain sizes, distributions of the grains, and additives (e.g., binder for the pressing step). other factors like technique of compacting the powdered zirconia into block may also influence the homogeneity and the density distribution of the material, hence the strength of the restorations. difference in the sintering techniques (presintering and final sintering) may also cause the variation in material properties. control of grain size in zirconia ceramics is important to maximize the mechanical properties and minimize the possibility of ltd. grain size may also affect the bond strength ; a sem analysis among zirconia groups of upcera, ziecon, and cercon revealed that, after postaging treatment, the grain sizes in all the three zirconia ceramics were found to be insignificantly the same as that of samples without aging (table 8). the results were in support of kim. that the bond strength is not affected if the percentage of monoclinic content is restricted to up to 14% and a good bond between ceramic and veneering material was present at this % age of monoclinic content ; also this % age of phase transformation showed no influence on the surface grain size in zirconia within all samples of the groups. however zirconia has been shown to behave unpredictably in response to stress due to the phase transformation ; it is very important to understand that the above results may be totally in reverse due to the effect of stress generated by clinical function such as masticatory forces, surface cracks, premature contact, or any other mechanism that can lead to stress formation within the prosthesis. the strength and other physical properties of zirconia are related to the quality of the zirconia block, that is, its composition, method of compaction and converting into a blank, and homogeneity within the blank. sintering mechanism may have a determinant influence on the phase transformation, grain size, and the physical and mechanical properties. a wide scope of research is open to investigate further the challenges in improving the properties of zirconia restorations. therefore, the results of this study may not directly transpolate into serviceability of the prosthesis in clinical setting. however, the results do indicate the acceptable values of bond strength upon aging of all commercially available zirconia used in the study as compared to samples without any aging procedure. from the present study it can be concluded that 15 yrs of clinical usage as induced by artificial aging has no effect on the bond strength of zirconia and the veneering ceramic. the effect of stress generated by clinical usage such as masticatory forces, surface cracks, premature contact, or any other mechanism leads to stress formation within prosthesis which could not be simulated in the study samples. this concentration of stress with the passage of time (aging) could affect the performance of zirconia based restorations clinically. within the limitations of the study, following conclusions were drawn:(1)bond strength between zirconia and veneering ceramic in all samples was comparatively same (24.43 mpa to 27.9 mpa) and aging of 15 years had no effect on bond strength (23.9 mpa to 24.20 mpa).(2)the zirconia veneer interface came out to be stronger than the strength of the veneering ceramic ; on average 70% of the samples have shown combined failure ; hence to improve the longevity of zirconia restorations the strength of veneering ceramic has to be improved.(3)bonding of zirconia veneered restoration is not influenced if the phase transformation (% of monoclinic content) is up to 14%. iso standards allow a maximum of 25 wt% of m - phase to be present, post accelerated aging test conducted at 134c with 2-bar pressure for 5 hrs for any commercial zirconia ceramics to be used in dentistry.(4)surface grain size (0.40 0.032 m0.42 0.034 m) may not be affected by the phase transformation (% of monoclinic content) if the zirconia veneered restorations are placed in oral cavity for 15 years (as simulated by artificial aging). bond strength between zirconia and veneering ceramic in all samples was comparatively same (24.43 mpa to 27.9 mpa) and aging of 15 years had no effect on bond strength (23.9 mpa to 24.20 mpa). the zirconia veneer interface came out to be stronger than the strength of the veneering ceramic ; on average 70% of the samples have shown combined failure ; hence to improve the longevity of zirconia restorations the strength of veneering ceramic has to be improved. bonding of zirconia veneered restoration is not influenced if the phase transformation (% of monoclinic content) is up to 14%. iso standards allow a maximum of 25 wt% of m - phase to be present, post accelerated aging test conducted at 134c with 2-bar pressure for 5 hrs for any commercial zirconia ceramics to be used in dentistry. surface grain size (0.40 0.032 m0.42 0.034 m) may not be affected by the phase transformation (% of monoclinic content) if the zirconia veneered restorations are placed in oral cavity for 15 years (as simulated by artificial aging). | a study was undertaken to evaluate the effect of artificial aging through steam and thermal treatment as influencing the shear bond strength between three different commercially available zirconia core materials, namely, upcera, ziecon, and cercon, layered with vita vm9 veneering ceramic using universal testing machine. the mode of failure between zirconia and ceramic was further analyzed as adhesive, cohesive, or mixed using stereomicroscope. x - ray diffraction and sem (scanning electron microscope) analysis were done to estimate the phase transformation (m - phase fraction) and surface grain size of zirconia particles, respectively. the purpose of this study was to simulate the clinical environment by artificial aging through steam and thermal treatment so as the clinical function and nature of the bond between zirconia and veneering material as in a clinical trial of 15 years could be evaluated. |
hodgkin s lymphoma was first described in 1832, but the nature of the pathognomic reed - sternberg cells, on which diagnosis of the disease is based has only been cleared in the past few years. radiotherapy has been employed for treatment of localized disease from the 1940s and combination chemotherapy was presented for anatomically progressive diseases in the 1960s. there has been great improvement in the outcome in the last three decades concluing to the fact that hodgkin s lymphoma is mentioned as one of the most curable non - cutaneous malignancies. combined therapy (chemotherapy and radiotherapy) can lead to the cure of more than 90% of children and adolescents with hodgkin s disease, but the intensive treatment may cause early and late complications. damage of soft tissues, respiratory, cardiovascular, skeletal and endocrine systems, dental development disturbance and second cancers are considered to be late complications of such treatment. these complications may have adverse effects on the patient s quality of life after discontinuation of treatment. hypodontia (partial anodontia), microdontia, altered eruption patterns and root stunting are some of the stated complications. developmental abnormalities resulting after malignant chemotherapy occur when the patient is treated prior to six years of age. diminished root surface area due to radiation exposure is the reason for early tooth loss. combined radio chemotherapy which is commonly used for the treatment of childhood neoplasia causes periodontium effects.there are just few reports of the complications of the dental system caused by radiotherapy in childhood in the literature. we report a girl who received chemotherapy and radiation of the head and neck area at five years of age and demonstrated developmental disturbances following chemoradiotherapy. this case report describes a 26-year - old girl, who at the age of 5 years, received chemotherapy and radiotherapy for hodgkin s disease in the neck region. the patient consulted the department of oral medicine, school of dentistry, tehran university of medical sciences because of dental changes and tooth loss despite adequate dental care and oral hygiene. clinical examination of the oral cavity revealed loose teeth (grade 3) (fig 1) and in flamed mandibular gingivae, but the maxillary teeth were not mobile and oral hygiene was fairly good (fig 2). panoramic view x - ray revealed that the roots of the mandibular teeth showed resorption, had become v - shaped and shortened together with alveolar bone loss and no remarkable changes in the enamel and dentin of the crowns was detected (fig 3). medical work - up did not show cervical lymphadenopathy. considering the patient s age, her height (149 cm) and weight (50 kg) were lower than normal, while her parents were nearly tall. altogether, she was alert and healthy, did not show any relapse of disease and was not taking any medicine. in the medical history, the patient had stage 1 nodular sclerosis type classical hodgkin s lymphoma in the lymph nodes of the neck at the age of 5 years. she was treated by chemotherapy (bleomicin+vinblastin+dacarbazine+prednisone for 6 periods) plus 2400-cgy radiation therapy. the patient had radiotherapy for hodgkin in the neck area to include the cervical nodes. she had two brothers, aged 24 and 15 years, who were alive and healthy. after examination, the patient was referred for extracting mandibular teeth (fig 4) and was subsequently sent to the prosthetics department in order to restore the missing teeth with a lower jaw of complete denture (figs 5,6). chemotherapy and radiotherapy may have serious effects on developing teeth such as delayed dental development, microdontia, hypoplasia, agenesis and v - shaped and shortened roots [810 ]. in some studies they evaluated the effects of therapy on dentofacial development in 27 acute lymphoblastic leukemia pediatric patients before 10 years of age who were treated with radiotherapy (rt) alone or chemotherapy together with 18002400-cgy cranial radiotherapy. the severity of these abnormalities was greater in children who received rt treatment before 5 years of age. minicucci observed in 76 children who were treated with high - dose chemoradiotherapy that 82.9% of them showed at least one dental abnormality including tooth agenesis, arrested root development, microdontia and enamel dysplasia. holtta studied dental development in young children after myeloablative therapy which was either chemotherapy and fractionated total body irradiation (tbi) or chemotherapy alone (non - tbi). in the tbi group, 9 out of 10 patients had very severe root defects in contrast to none in the non - tbi group. takinam reported hypoplasia of the mandible and teeth in a 4-year - old boy who had cystic hygroma. at the age of 7 months he had been treated with 2400-cgy to the head and neck, followed by surgery. panoramic view showed hypoplasia of the roots of the canines, molars and permanent teeth. futhermore, moller and carl like takinam, presented dentomandibular defects after chemo - radiation therapy in boys aged 9 years and 4 years with hodgkin s lymphoma. clinical and radiographic evaluation showed dental abnormalities such as root blunting, mild to severe root shortening, premature closure of the root apices and severe radiation caries with mandibular and maxillary hypoplasia. they observed that new megavoltage radiation machines have reduced the chances of bone complications in adults. in this study, we reported a girl who was treated with chemotherapy plus 2400-cgy radiation therapy at the age of 5 for a lesion of hodgkin s disease in the neck area. she had disturbances in the root development of the mandibular teeth including short roots, arrested root development, v - shaped root and loose teeth. in contrast, roots of the maxillary teeth were normal and the root apexes were closed. our report was similar to the results of other studies on the effects of radiotherapy on dentition and showed the long - term effects of cancer therapy on teeth. although chemoradiation therapy cures more than 90% of children with lymphoma or leukemia, it often causes dental abnormalities that may affect their quality of life these abnormalities are probably caused by the type, intensity and frequency of treatment and the age of the patients at the time of radiotherapy which might have important consequences on the children s dental development. finally, the improved outcome of patients with hodgkin s disease makes it imperative to consider the long - term side effects of treatment and to ameliorate them as soon as possible. in this way, dental evaluation after diagnosis and frequent follow - ups may help to ensure appropriate preventive measures and minimize dental and periodontal diseases. cases similar to this report are rare in the literature and the importance of such cases lies in the fact that dentists and other health care professionals should be aware of the oral and facial manifestations related to treatment of malignances, especially those related to the effects of radiation on bony structures. another important aspect of this case was inadequate attention to the oral condition of the patient for a long time and consequences of such neglect on her general health status such as the impact on height and weight. treatment regimes for childhood cancer are known to affect the root development in the long - term survivors but the available data is subjective in nature with very few quantitative data in the literature. the report of this patient introduces a new case in order to minimize the dental treatment sequel by supportive measures and also to initiate timely dental and prosthetic management. unfortunately, for this case dental treatment was not adequate and she could not eat and brush her teeth well because of loose teeth for many years. therefore, the health status of children and adolescents cured from hodgkin s disease and other childhood cancers should be regularly evaluated. | the improvement in survival and local control measures in children with neoplasm in the head and neck region may lead to increased iatrogenic adverse effects of treatment. the aim of this study was to report a new case of the long - term effects of chemoradiotherapy on oral health and dental development in a patient treated for hodgkin s disease at an early age. in this case report, a 26-year - old female is presented, who at the age of 5 years received chemotherapy and radiotherapy for hodgkin s disease in the neck region. the patient consulted the department of oral medicine because of dental changes and tooth loss despite adequate dental care and oral hygiene. clinical examination revealed loose teeth and inflamed gingiva of the mandible, x - ray showed premature root resorption, v - shaped and shortened roots and alveolar bone loss. after examination, the patient was referred for extracting the mandibular teeth and then wassent to the prosthetics department. therefore, in order to decrease dental treatment sequelae in patients who have had cured malignant disease, these cases should have life - long dental care and follow - up. |
internal limiting membrane (ilm) free autografts have been used to repair refractory macular holes instead of using conventional surgical techniques like vitrectomy and peeling off the ilm, with encouraging results according to several authors. furthermore, to treat retinal tears in retinal detachment surgery, laser therapy or cryotherapy are used with excellent results, achieving appropriate sealing of the tears and thus contributing to retinal reattachment. laser therapy is also applied for the transoperative treatment of retinotomies that are occasionally performed to drain the subretinal fluid. not having those means to achieve tear sealing poses a complication that in some hospital sites, mainly in third world countries like mexico, is not entirely uncommon. in this case report we describe the use of an ilm autograft to seal a transoperative retinotomy in a patient who underwent primary vitrectomy for rhegmatogenous retinal detachment and proliferative vitreoretinopathy. a 55-year - old female patient came for consultation with sudden loss of vision and also reporting a rapidly progressive superior altitudinal scotoma of her right eye. on examination, the patient had a visual acuity of hand motion on her right eye, and fundus examination with a goldman 3-mirror lens revealed total retinal detachment with peripheral horseshoe tears in the 1, 2, 7, and 9 o'clock positions, with presence of an inferior and temporal fixed fold. vitrectomy surgery was then performed with membrane peeling under perfluorocarbon fluids, placing a # 287 exoplant with a # 240 band and silicone oil as intraocular tamponade. the tears were all treated with laser therapy. while the air - fluid exchange was being completed, a retinotomy in the raphe was inadvertently made. laser treatment of the retinotomy site was no longer possible at that moment due to malfunction of the laser device. 0.02 ml of brilliant blue g (fluoron gmbh, ulm, germany) were injected into the vitreous cavity to stain the ilm. afterwards, a tano scratcher and an ilm forceps were used to peel off a small graft of the ilm adjacent to the retinotomy site. once obtained, it was displaced under a perfluorocarbon fluid bubble and placed inside the retinotomy (fig 1, fig 2). finally, a perfluorocarbon fluid - air exchange was performed, and 5 ml of silicone oil were injected into the vitreous cavity. three weeks later the retina was completely reattached and the retinotomy sealed with the ilm graft (fig 3). it was not possible to take an optical coherence tomography (oct) image due to the presence of a lens opacity. peeling of the ilm is nowadays considered one of the main steps that contribute to a higher success rate in macular hole surgery. this has been achieved more efficiently with the use of dyes such as brilliant blue g that provide better visualization of such membrane [3, 4 ]. furthermore, some techniques have been reported using an ilm flap as well as free ilm autografts over and inside the macular hole, with good anatomical and functional results according to several authors [1, 5, 6 ]. conventionally, to treat rhegmatogenous retinal tears, methods such as diathermy, cryotherapy or laser therapy have been used. in the latter, for example, an inflammation area this effect induces the formation of a chorioretinal scar around the retinal tear that seals it and prevents vitreous fluid from entering into the subretinal space and detaching the retina again. in this case report we describe the use of an ilm autograft to block the retinotomy, since we did not have these conventional methods available anymore at that moment of the surgery. the apparent sealing of the tear at the 3-week postoperative follow - up could be observed clinically, although it was not possible to confirm this finding by an oct image. in different histological preparations, mller cell remains were found in the ilm once it had been removed from the rest of the retina. these cell remains might be involved in the migration and repositioning of photoreceptors as well as in the formation of fibroglial tissue. hence, the ilm autograft might work as a potential source of mller cell remains and as a scaffold where fibroglial tissue can probably grow, contributing to the sealing of the retinal defect [1, 5, 8 ]. in their study of a modified technique of autologous transplantation of ilm for macular holes, hernndez - da mota and bjar - cornejo reported the presence of oct hyperreflective images where the ilm autograft was placed, which might suggest the presence of this type of tissue filling the macular hole defect. the same phenomenon might occur in rhegmatogenous retinal tears and retinotomies such as the one presented in this case report. animal studies as well as larger case series of patients and eventually comparative studies with conventional techniques are required to determine the real impact of the use of ilm autografts for the treatment of rhegmatogenous retinal detachment and retinal tears. other possible applications of this surgical technique combined with a retinal pigment epithelium autograft might be in diseases such as albinism and degenerative myopia that have little pigmentation of the retinal pigment epithelium. | during a pars plana vitrectomy, an unplanned retinotomy in the raphe was performed in a 55-year - old female patient with rhegmatogenous retinal detachment and proliferative vitreoretinopathy. since diathermy and laser therapy were not available at that moment, it was decided to peel off a small graft of the internal limiting membrane adjacent to the retinotomy site which had been previously stained with brilliant blue g. the graft was displaced under perfluorocarbon fluids and placed inside the retinotomy. three weeks after surgery, the apparent closure of the retinotomy was observed clinically. |
laser in situ keratomileusis (lasik) has a high rate of improving uncorrected snellen visual acuity (1), however it can degrade the quality of vision, resulting in reports of reduced night vision clarity, glare, and halos (2). the contrast sensitivity test more effectively evaluates the visual quality over a range of sizes and daytime contrast levels (3) and is necessary to assess the visual performance in refractive surgery patients (4). the increased higher order aberrations induced after lasik is one reason for the reduced contrast sensitivity (5, 6). the contrast sensitivity after wavefront - guided lasik using the ablation depth based on the individual higher order aberrations compared with standard lasik after surgery showed a significantly improvement (7). the reduction in the contrast sensitivity was greater for higher amounts of myopia in standard lasik (8). however, previous studies for contrast sensitivity after wavefront - guided lasik did not include high myopia patients. the aim of this study was to evaluate the contrast sensitivity, glare sensitivity and total high order aberrations after wavefront - guided lasik in both moderate and high myopia patients. the high myopia group included 25 eyes of 14 patients with -7.78~-6.17d (table 1). the uncorrected visual acuity, spherical equivalent, contrast sensitivity and glare sensitivity were measured preoperatively, and at 1 week, 1 month and 2 months postoperatively on patients undergoing wavefront - guided lasik. the ocular total higher order aberration (hoa) was measured at 2 months postoperatively. were performed using a hartmann - shack aberrometer (wavescan, visx, sunnyvale, ca, u.s.a.) and visx star s4 (visx) excimer laser, respectively. in all patients, the individual aberrations gained including the higher order aberrations by wavescan was programmed into the star s4 laser to create a customized treatment. a flap thickness of 130 m was created using a moria m2 microkeratome (moria, france). after surgery, ofloxacin 0.3% (samil pharm, seoul, korea) and fluorometholone 0.1% (samil pharm) were prescribed 4 times daily beginning one day after surgery for a one week. the contrast sensitivity and glare were examined using a visual capacity analyzer (vca, l2 informatique, france) with landolt rings as the optotypes in a darkened room with the monitor as the only light source. a standard 15-inch computer monitor was used, and the horizontal distance between the monitor and the eyes was 1 m. there were five spatial frequencies, each with 20 levels of 0.1 - 100% contrast : 3, 4.8, 7.5, 12, 19 (cycle / degree ; cpd). the monitor illumination for the day and nighttime contrast sensitivity testing were 100 cd / m and 30 cd / m, respectively. the measured levels were calculated as the log units, and the minimum contrast levels were recorded. the nighttime glare test used a vca attaching light source (sb99, l2 informatique, france) of 500,000 cd / m during 60 sec. if the patients did not read the landolt ring in the monitor as a result of a glare disturbance, the observer run the decrease button of vca, which was automatically was recorded. the pupil diameter was measured at the daytime (220 lux) and nighttime condition (5 lux) using a pupillometer (colvard pupillometer, oasis medical, u.s.a.). the hartmann - shack wavefront analyzer (wavescan, visx) was used to measure the ocular wavefront aberrations for a 4 mm and 6 mm entrance pupil. the magnitudes of the coefficients of the zernike polynomials are represented as the root mean square (rms ; in microns) and were used to show any ocular wavefront aberrations. from the contrast sensitivity data obtained using this system, the area under the log contrast sensitivity function (aulcsf) was calculated according to the method reported by applegate. (9). the log of the contrast sensitivity was plotted as a function of the log of the spatial frequency, and the third - order polynomials were fitted to the log spatial frequency limits of 0.48 (corresponding to 3 cpd) and 1.28 (19 cpd). the resulting value was defined as the aulscf, which is a single quantity used to characterize the overall visual performance of the eye. an independent t - test, analysis of variances (anova), chi - square test and pearson correlation analysis using spss software (spss inc., chicago, il, u.s.a.) were used. age, gender and preoperative pupil diameter in the daytime and nighttime did not differ between the two groups (p>0.05) (table 1). table 2 shows time course of the changes in the uncorrected visual acuity (ucva). a ucva of 20/20 or better was achieved by 96.0% in those with moderate myopia and by 94.1% in those with high myopia 2 months after surgery. 1 shows the daytime contrast sensitivity at all spatial frequencies after wavefront - guided lasik. the contrast sensitivity increased at 7.5 cpd at 1 month after wavefront - guided lasik in the moderate myopia group (p=0.018). 2 shows the nighttime contrast sensitivity at all spatial frequencies after wavefront - guided lasik. the contrast sensitivity increased at 7.5 cpd 2 month after wavefront - guided lasik in the moderate myopia group (p=0.005). all other frequencies were not significantly different after wavefront - guided lasik both in the daytime and nighttime (p>0.05). the contrast sensitivity at all spatial frequencies did not differ in both groups (p>0.05, fig. 3, 4). table 3 shows correlation analysis between the amounts of myopia and postoperative 2 months contrast sensitivity at all spatial frequencies in the daytime and nighttime. no significant correlation was found between the ablation depth by wavefront - guided lasik and the contrast sensitivity (p>0.05, table 3). in all patients with moderate myopia and high myopia, no correlation was found between the photopic pupil diameter and the daytime contrast sensitivity. the scotopic pupil diameter showed no correlation with the nighttime contrast sensitivity (p>0.05, table 4). the reduction in glare sensitivity was observed at 1 week and 1 month after surgery. however, this was recovered at 2 months postoperatively and the difference between the groups was not significantly different in the glare test (table 5). for all patients, the aulcsf in the daytime did correlate with the total hoa in the 4 mm entrance pupil (r=-0.071, p=0.612). the correlation between the aulcsf in the nighttime and the total hoa in the 6 mm entrance pupil was not significant (r=-0.176, p=0.260) (fig. the visual acuity had improved after laser refractive surgery but most patients have reported blurring and glare symptoms (8). their vision is also susceptible to the changes in illumination and contrast (2, 10). therefore, a contrast sensitivity test is needed to more accurately and objectively evaluate the visual function (5, 11). this study was designed to evaluate the visual quality with a contrast sensitivity and glare test after wavefront - guided lasik. the visual acuity test determines the ability to resolve small details at a high contrast (12). it does not mean that an individual performs normally on all visual tasks, and the acuity is a poor predictor of the visual performance in certain daily perceptual tasks such as face recognition (12). on the other hand, contrast sensitivity discriminates the luminance differences between a material or an area in a space (13). as a sine - wave grating system, the contrast sensitivity is 3 or 5 times more sensitive than the letter acuity (13). this study used the vca, which assesses overall visual function with difference modes (8). (8) reported that the reliability coefficient ranged from 89.1% to 99.8% under maximum background luminance. under 3 cd / m background luminance, some studies have reported decreased low - contrast sensitivity after lasik (10, 13 - 22). chan. (4) related the contrast sensitivity reduction after lasik to some optical factors (high order aberration) (5, 6). eighty eight percent of the contrast sensitivity measurements improved 1 month after the wavefront - guided lasik based on the individual high order aberration ablation pattern (7). however, previous studies did not include the high myopia patients. in standard lasik, the reduction in the contrast sensitivity was greater for correction of higher amounts of myopia (1). in this study pop and payette (23) reported that the aulcsf did not correlate with the total hoa (r=-0.11) after lasik. using a cutoff point of a total hoa of either 0.5, 0.6, or 0.7 m, the independent t - test showed that the aulcsf did not differ between the lower and higher total hoa groups. these supports the hypothesis proposed in earlier studies in that a low wavefront aberration does not completely fit the entire visual performance (11, 24). in this study, the total hoa after wavefront - guided lasik in most patients was < 0.5 m, and there was no correlation between the aulcsf and the total hoa. a larger pupil causes a spurious resolution in an optically aberrant system and the pupil size is theoretically important in determining the optical quality of the retinal image and the visual performance (25, 26). however, previous studies reported that a low correlation between the contrast sensitivity and the glare symptoms after lasik and the pupil size (27). this study also found no significant correlation between the contrast sensitivity and the pupil size in both the daytime and nighttime conditions. however, the number of cases in our study limited scotopic pupil was not larger than 7.0 mm. a large sample size with a wider range of pupil sizes will be necessary to confirm previous reports showing that the visual performance may demonstrate a decline in function related to the clearance zone compromised by a large pupil diameter (27). el danasoury (28) found, using a questionnaire, that 49% of eyes reported glare after lasik with an optical zone of 5.5 mm. several studies reported night - driving difficulties and glare ranging from 2% to 55.6% (29). the score of the nighttime glare symptoms with 75 lux after standard lasik was 1.481.16 and 2.161.11 in those with moderate and high myopia, respectively (27). in all cases, the glare symptom was recovered 2 months after lasik and there was no significant difference between the groups. in conclusion, the reduction in the contrast sensitivity and glare was not caused after the wavefront - guided lasik based on the individual higher order aberration in both moderate myopia and high myopia groups. | this study evaluated the visual quality after wavefront - guided laser in situ keratomileusis (lasik) for treating myopia. thirty - two eyes with moderate myopia (-5.78~-2.17d) and 25 eyes with high myopia (-7.78~-6.17d) were prospectively reviewed. the contrast sensitivity (cs), glare and the total higher order aberrations (hoa) were measured before and 1 week, 1 month and 2 months after lasik. the pupil diameter was measured at day- and night - time illumination. the cs and glare at all spatial frequencies were not reduced after wavefront - guided lasik (p0.05). the area under the log contrast sensitivity function (aulcsf) showed no correlation with the total hoa (r2=-0.071, p=0.612, between the daytime aulcsf and the total hoa with a 4 mm entrance pupil, r2=-0.176, p=0.260, between the nighttime aulcsf and the total hoa with a 6 mm entrance pupil). there was no decrease in cs and glare after wavefront - guided lasik for myopia. in conclusion, wavefront - guided lasik based on the individual ablation patterns is a good option for refractive surgery to improve the visual quality in both moderate and high myopia cases. |
introduction of imaging modalities, especially magnetic resonance (mr), and of modern methods of neurosurgery and pharmacotherapy revolutionized diagnosis and therapy of pituitary tumors. currently, mr is the method of choice for imaging of the pituitary gland and the perisellar area. advanced mr techniques mr diffusion, mr spectroscopy and mr perfusion have been increasingly applied [112 ]. the proper diagnosis and management of patients with pituitary tumors are of high importance in clinical practice [1417 ]. mr imaging protocol of pituitary and sellar region including postoperative studies usually consists of unenhanced and enhanced t1-weighted images in coronal and sagittal planes. t2-weighted unenhanced images are also performed sometimes, but most authors have reported postoperative studies of the sellar region using only t1-weighted images [1820 ]. there are only a few papers concerning applications of t2-weighted sequences in imaging of the postoperative sella [2123 ]. nakasu found that t2-weighted imaging could be a reliable and sufficient method to assess the sella in patients after pituitary surgery. in the literature there is still disagreement whether t2-weighted images are useful in assessment of the sellar region after resection of pituitary tumors. the purpose of this study was to investigate the magnetic resonance features of the postoperative sella with fast spin echo (fse) t2-weighted imaging (t2) and to evaluate the benefits of this sequence compared to the classically performed contrast - enhanced t1-weighted imaging (t1+c) on a 1.5 t unit. the aim of our study was also to assess whether t2 sequence could replace t1-weighted images after contrast administration.. this could be especially useful in patients with high risk of nephrogenic systemic fibrosis (nsf). the study group consisted of 101 patients who underwent resection of pituitary tumors with the following diagnoses : pituitary adenoma (89 cases), pituitary abscess (1 case), rathke s cleft cyst (1 case), pituitary cyst (1 case), germinoma (1 case), craniopharyngioma (6 cases) and meningioma (2 cases). among 89 pituitary adenomas there were : 37 hormonally inactive (nf - nonfunctioning) adenomas, 31 growth hormone- (gh) secreting adenomas, 11 prolactin- (prl) secreting adenomas, 7 adrenocorticotropin- (acth) secreting adenomas, 2 luteotropin- (lh) secreting adenomas and 1 case of gh- and prl - secreting adenoma. in the study group there were 58 women (57.4%) aged 22 to 75 years (mean 52.67) and 43 men (42.6%) aged 21 to 79 years (mean 49). overall age of the analysed patients ranged from 21 to 79 years (mean 51.1). because the patients were observed prospectively, patient s age at the time of surgery was considered. types of pituitary tumors in the analysed material with regard to patient s sex are presented in figure 1. in all patients preoperative and multiple postoperative mr studies were performed. the number of postoperative examinations ranged from 1 to 8 (mean 3). the duration of the follow - up from the moment of the operation until the last mri study ranged from 3 to 348 months (mean 70.8). seventy - three patients were operated only once ; in these cases we used follow - up studies and endocrinological assessment to confirm our diagnosis. twenty - eight patients underwent more than 1 surgery ; in these cases we had histopathological confirmation of the mr findings performed after the first operation. each study began with a single pilot image and 3 localization images, followed by t1-weighetd images in coronal and sagittal planes, using the fast sequence (fourier acquired steady - state sequence) and fast spin echo (fse) t2-weighted coronal images. paramagnetic contrast medium was administered intravenously in each patient, at a dose of 0.1 ml / kg bw, and post - contrast t1-weighted images were taken in coronal and sagittal planes. post - contrast t1 and pre - contrast t2 images were interpreted by 2 independent readers (neuroradiologists) using the following criteria for assessing particular features : 0 non - visible, 1 visible, but not clearly ; 2 clearly visible ; 3 excellently visible. the following features of the postoperative sella were evaluated : presence of pituitary, contours of pituitary (i.e. visible border between the pituitary gland and the cavernous sinus or residual mass if present), infundibulum, optic chiasm, presence of residual tumor, contours of residual tumor (visible border between the residual tumor and pituitary gland or the cavernous sinus). appearance of the postoperative sphenoid sinus (implanted materials, fluid collections) was also described. implanted materials (muscle with fascia) were used only in 3 patients ; therefore this feature was excluded from the statistical analysis. the pituitary gland persistent after operation was recognised by homogeneous low signal intensity on t2-weighted images and homogeneous contrast enhancement on t1-weighted images, as well as by its anatomic relationship to the infundibulum on t1-weighted images. residual tumor was differentiated from postoperative changes by means of location, pattern of signal intensity and enhancement presented on preoperative images. fluid collection was diagnosed when a cyst - like area was found in the surgical cavity and underwent resorption during the follow - up period. as was known from the surgical reports results of visual assessment by 2 independent readers were analyzed using wilcoxon signed ranks test for statistical analysis using the pasw statistics 18.0 program. the study group consisted of 101 patients who underwent resection of pituitary tumors with the following diagnoses : pituitary adenoma (89 cases), pituitary abscess (1 case), rathke s cleft cyst (1 case), pituitary cyst (1 case), germinoma (1 case), craniopharyngioma (6 cases) and meningioma (2 cases). among 89 pituitary adenomas there were : 37 hormonally inactive (nf - nonfunctioning) adenomas, 31 growth hormone- (gh) secreting adenomas, 11 prolactin- (prl) secreting adenomas, 7 adrenocorticotropin- (acth) secreting adenomas, 2 luteotropin- (lh) secreting adenomas and 1 case of gh- and prl - secreting adenoma. in the study group there were 58 women (57.4%) aged 22 to 75 years (mean 52.67) and 43 men (42.6%) aged 21 to 79 years (mean 49). overall age of the analysed patients ranged from 21 to 79 years (mean 51.1). because the patients were observed prospectively, patient s age at the time of surgery was considered. types of pituitary tumors in the analysed material with regard to patient s sex are presented in figure 1. in all patients preoperative and multiple postoperative mr studies were performed. the number of postoperative examinations ranged from 1 to 8 (mean 3). the duration of the follow - up from the moment of the operation until the last mri study ranged from 3 to 348 months (mean 70.8). seventy - three patients were operated only once ; in these cases we used follow - up studies and endocrinological assessment to confirm our diagnosis. twenty - eight patients underwent more than 1 surgery ; in these cases we had histopathological confirmation of the mr findings performed after the first operation. each study began with a single pilot image and 3 localization images, followed by t1-weighetd images in coronal and sagittal planes, using the fast sequence (fourier acquired steady - state sequence) and fast spin echo (fse) t2-weighted coronal images. paramagnetic contrast medium was administered intravenously in each patient, at a dose of 0.1 ml / kg bw, and post - contrast t1-weighted images were taken in coronal and sagittal planes. post - contrast t1 and pre - contrast t2 images were interpreted by 2 independent readers (neuroradiologists) using the following criteria for assessing particular features : 0 non - visible, 1 visible, but not clearly ; 2 clearly visible ; 3 excellently visible. the following features of the postoperative sella were evaluated : presence of pituitary, contours of pituitary (i.e. visible border between the pituitary gland and the cavernous sinus or residual mass if present), infundibulum, optic chiasm, presence of residual tumor, contours of residual tumor (visible border between the residual tumor and pituitary gland or the cavernous sinus). appearance of the postoperative sphenoid sinus (implanted materials, fluid collections) was also described. implanted materials (muscle with fascia) were used only in 3 patients ; therefore this feature was excluded from the statistical analysis. the pituitary gland persistent after operation was recognised by homogeneous low signal intensity on t2-weighted images and homogeneous contrast enhancement on t1-weighted images, as well as by its anatomic relationship to the infundibulum on t1-weighted images. residual tumor was differentiated from postoperative changes by means of location, pattern of signal intensity and enhancement presented on preoperative images. fluid collection was diagnosed when a cyst - like area was found in the surgical cavity and underwent resorption during the follow - up period. as was known from the surgical reports, 3 patients had muscle with fascia implanted into sphenoid sinus. results of visual assessment by 2 independent readers were analyzed using wilcoxon signed ranks test for statistical analysis using the pasw statistics 18.0 program there were no statistically significant differences in visual assessment of the postoperative sella by the 2 independent readers. the descriptive statistics with the results of the wilcoxon signed ranks test made by each neuroradiologist are summarized in tables 2 and 3. contrast - enhanced t1-weighted imaging was significantly superior to t2-weighted imaging in assessment of infundibulum (p<0.05) for both radiologists (figure 2). there was no statistically significant difference for each of the readers between t1- and t2-weighted images regarding to the following features : visualization of residual pituitary gland (p=0.062 and p=0.368), contours of pituitary (p=0.959 and p=0.265), optic chiasm (p=0.294 and p=0.843), and visualization of presence of residual tumor (p=0.204 and p=0.169). we present the spectrum of other results measured by 2 observers in figures 3 to 6 to demonstrate features without statistically significant difference between t1- and t2-weighted images. on the other hand, results of each reader indicated that t2-weighted images were significantly superior to contrast - enhanced t1-weighted imaging with regard to visualization of contours of residual tumor (p<0.05) (figure 7). muscle with fascia appeared on t1-weighted images as a round hypointense structure, located in the lumen of the sphenoid sinus. after contrast administration, the peripheral part of that material demonstrated slight enhancement. on t2-weighted images, the implanted muscle produced a high signal intensity (increased signal intensity due to degenerative processes of the denervated muscle), while fascia presented as a line of low signal intensity. in 3 cases with muscle and fascia implanted within the sphenoid sinus, the follow - up examinations showed a decreasing intensity of contrast enhancement on t1-weighted images. in contrast, t2-weighted images showed consistent appearance and did not reveal any significant changes in signal intensity within the implanted muscle and fascia for up to 31 months following the operation no further mr examinations were performed in those cases. in 1 case there was fluid collection inside the sphenoid sinus what was excellently visible on t2-weighted images. post - surgical follow - up of the sellar region is very important, especially in cases of hormonally inactive tumors. detecting recurrence of a proliferative process in subsequent follow - up examinations plays a crucial role for further therapy. however, while presurgical pituitary tumor presentation in mr was described in detail and usually does not present any diagnostic difficulties, post - surgical evaluation of the pituitary gland often becomes a serious problem. interpretation of mr images performed after surgical therapy of pituitary tumors is difficult because of a major change in anatomical conditions in this area. the interpretation depends also on numerous other factors, including : size and expansion of a tumor before surgery, type of surgical access, quality and volume of implanted material, and time of its resorption. proper evaluation of post - surgical mr images is crucial to determination of the completeness of surgical resection. neuroradiologists therefore face the important and difficult task of evaluation of structures present in the surgical area, differentiating residual tumor from residual normal gland, the filling materials and from other post - surgical changes (such as fibrous and cicatricial). due to the problems mentioned above, as pre- and postcontrast t1-weighted images are considered as main sequences for assessing the postoperative sella, the role of t2-weighted images has not been established yet. we therefore decided to assess the value of t2-weighted images in comparison to contrast - enhanced t1-weighted images. it is not a surprise that contrast - enhanced t1-weighted imaging was significantly superior to t2-weighted imaging in assessment of infundibulum (p<0.05), which corresponds with data from the literature. nakasu also indicated that the pituitary stalk was less frequently visible on t2-weighted images compared to post - contrast t1. however, in some of our cases the infundibulum was also well delineated on t2-weighted images (figure 8). moreover nakasu claim that contrast - enhanced t1-weighted images had no advantages in demonstration of the optic chiasm, and that post - contrast t1 did not improve the detection of the pituitary gland or its contours. our results demonstrate that t2-weighted imaging is significantly superior to t1-weighted contrast - enhanced imaging regarding visualization of the border between the residual tumor and pituitary gland or the cavernous sinus (p=0.015 for the first reader and p= 0.001 for the second reader). moreover, in 3 cases it was only possible to recognize the residual tumor on t2-weighted images, not on t1-weighted contrast - enhanced images, because the residual mass demonstrated signal intensity and enhancement pattern identical to normal pituitary gland. in these 3 cases, on the basis of t1-weighted contrast - enhanced images, persistent pituitary gland in the postoperative sellar cavity was diagnosed. as indicated by bladowska, mr imaging of postsurgical pituitary gland does not necessarily correlate with its hormonal function, thus the endocrinological assessment is not always helpful in differential diagnosis of masses persistent in the postoperative sellar region. some patients with small residual pituitary gland may show normal hormonal function, while in other cases the pituitary seems normal on mr but the patients have hormonal disorders. because of inconclusive endocrinological assessment in cases of postsurgical pituitary gland, correct diagnosis based on imaging is of great importance. in our opinion, t2 images can significantly improve this diagnosis and in some cases are even superior to post - contrast t1 images. to our knowledge, t2-weighted images revealed the intrasellar mass, which demonstrated high signal intensity that indicated the residual tumor and excluded persistent pituitary gland (figure 9). the careful assessment of preoperative t2-weighted images in these 3 cases showed that the pituitary adenomas demonstrated exactly the same high intensity signal. sometimes the residual tumor may present on post - contrast t1-weighted images with the same signal intensity as postoperative changes in the sphenoid sinus ; this may cause misdiagnosis as showed the case in figure 10. in this patient there was no border line between the tumor infiltrating the right cavernous sinus and fluid collection inside the right sphenoid sinus on post - contrast t1-weighted images. furthermore, t2-weighted imaging may play an important role in assessment of implanted muscle with fascia. in our material in 3 individuals, an implanted muscle with fascia was identified. it was represented by a round, isointense structure, filling nearly the whole sphenoidal sinus in t1-weighted mri images. after intravenous contrast administration, the structure was enhanced slightly in its peripheral part, with a central round area of lower signal intensity. such an image of the implanted muscle was found in 2 patients after 4 months following the operation and in 1 patient 5 months after surgery (figure 11). the follow - up mris (beginning approximately from the 12 postoperative month) revealed a gradual change in the appearance of that material. after contrast administration, the previously distinct border between the enhanced peripheral part and the central hypointense part of the implanted muscle became ill - defined. in further mri examinations, performed approximately 25 months after surgery or later, the structure inside the sphenoidal sinus remained hypointense after contrast administration. without the analysis of previous images and the knowledge of the patient s history, the correct diagnosis of that mass on t1-weighted images would have been impossible (especially its differentiation from fluid cistern). on the other hand, the implanted muscle with fascia produced a very characteristic and almost stable image in t2-weighted sequence for at least 31 months following the operation. in the t2-weighted sequence, the material is represented by a hyperintense mass with a linear structure of very low signal intensity, consistent with fascia (figure 12). kakite indicated that 3d - gre is a more suitable sequence for evaluating sellar lesions on contrast - enhanced t1-weghted imaging at 3.0 t mr units. however, in most radiology departments all over the world, pituitary mr imaging is performed using 1.5 t equipment, so the state of the art in everyday clinical practice, should be based on 1.5 t units. our results demonstrate that a careful review of t2-weighted images appears to offer more precise information regarding many anatomical and pathological details that are not visible on post - contrast t1-wieghted images. the advantages of our study, compared to the studies mentioned above, are the compatible results of 2 independent readers, as well as the greater number of analyzed patients. in addition, our study supports the findings of nakasu, but adds further information to the literature regarding the changes in the sphenoid sinus, including the characteristic mr appearance of implanted muscle with fascia. reduction of contrast material administration could be especially useful in patients with high risk of nephrogenic systemic fibrosis (nsf). t2-weighted images may help to discriminate between tumorous and non - tumorous involvement of the postoperative sella and the sphenoid sinus, especially in cases in which the signal intensity and enhancement pattern of pituitary gland and tumor are the same on t1-weighted images. t2-weighted images are also very useful in the postoperative evaluation of the implanted muscle with fascia, especially during long - term follow - up. the best protocol for the postoperative imaging after pituitary tumor resections includes both t1- and t2-weighted imaging, because t1- and t2-weighted images compliment each other in the postoperative examination of the sella and sellar region. however, in some cases t2 could replace post - contrast t1, especially in patients with high risk of nsf. | summarybackgroundthe proper diagnosis and management of patients after surgery for pituitary tumors are of great importance in clinical practice. the purpose of this study was to investigate the magnetic resonance features of the postoperative sella with fast spin echo t2-weighted imaging and to evaluate the benefits of this sequence compared to the classically performed contrast - enhanced t1-weighted imaging at 1.5 t unit.material/methodsthe study group consisted of 101 patients who underwent resection of pituitary tumors. there were 58 women (57.4%), aged 22 to 75 (mean age, 52.67 years) and 43 men (42.6%), aged 21 to 79 (mean age, 49 years). in all patients preoperative and multiple postoperative mr studies were performed. post - contrast t1 and pre - contrast t2 images were interpreted by 2 independent readers (neuroradiologists).resultscontrast - enhanced t1-weighted imaging was significantly superior to t2-weighted imaging in assessment of infundibulum (p<0.05). there was no statistically significant difference for each of readers between t1- and t2-weighted images regarding to the following features : visualization of residual pituitary gland (p=0.062 and p=0.368), contours of pituitary (p=0.959 and p=0.265), optic chiasm (p=0.294 and p=0.843), and visualization of presence of residual tumor (p=0.204 and p=0.169). t2-weighted images were significantly superior to contrast - enhanced t1-weighted imaging with regard to visualization of contours of residual tumors (p<0.05).conclusionst2-weighted images may help to discriminate tumorous from non - tumorous involvement of the postoperative sella and the sphenoid sinus. t2-weighted images are also very useful for a long time after the resection in the postoperative evaluation of the implanted muscle with fascia. |
mosquitoes, the most important group of nuisance pest insects, due to their diversity and abundance, demonstrated vector competence and frequent infection in nature, they are regarded as one of the most important vectors of diseases (sardelis. 2008a, b). tracking the movement of mosquitoes in their natural habitat is critically important for understanding their basic biology, demography, ethology and vector - borne disease control as well as prevention. animal marking have been used since 218 bc when fisher and peterson used banding to distinguish ownership of birds (fisher and peterson 1964). unfortunately, for marking insects, most marking techniques for vertebrate, such as bands, brands, tattoos, tags, notches, paints are not practical because they are tedious, time - consuming, heavy and costly (southwood 1978, basavaraju. insect marking for scientific studies dates back to 1920, since then, a variety of marking techniques, paints, dyes etc. were used to in studies of insect population dynamics (geiger. 1919, dudley and searles 1923). 2007), paints (trpis and hausermann 1986, niebylski and meek 1989, service 1993, bellini. 2007), and radioactive isotopes (jenkins 1949, abdel - malek 1966, lindquist. 1967, hood - nowotny. however, for study on the behavioral characteristics of post bloodmeal, existing techniques tend to be labor intensive, as they require rearing mosquitoes, marking them in large quantities, and then inspecting large numbers of individuals to detect recaptures (walker. furthermore, compared with natural populations, rearing mosquitoes, marking them in large quantities by using artificial methods, and releasing them may change their behavior (reisen. 2003, silver 2008). for study the behavior characteristics after blood meal, these methods are not ideal however, the problem is how to mark breeding mosquitoes without inhibiting their normal biology, and with long - term retention after blood meal, it is still bothering most biological scientists. (2014) marked adult mosquitoes by feeding them on cow injected with isotope p and subsequent ecological investigations. in some studies, successfully labeled mosquitoes by feeding them on radioactive animal blood (hassett and jenkins 1951), use of large bait animals for marking wild population of adult anophels aquasalis injected with a dose of 1.7 curies of p (bruce - chwatt 1956). however, such high dosage would be dangerous to the animal (winteringham london meeting, 1953). the aim of the current study was to develop a marking technique as an internal marker to mark post blood meal mosquitoes by using stable phosphate isotope p and determine the optimal concentration of it. before experimenting, 6 healthy rabbits (2 kg) were selected and physically examined by veterinarian. the injection method of normal saline solution to the rabbits was according to smith. (1951), i e. an isotonic buffered saline solution, containing different concentration of na2hpo4, was injected intravenously of rabbits. the dosage for no. 1, 2 rabbits, no. 3, 4 rabbits, no. 5 rabbit and no. 6 were 0.2 mci, 0.4 mci, 0.8 mci and 1.7 mci, respectively. for the negative control, 1 healthy rabbits was injected with isotonic buffered saline solution without na2hpo4. then 20 to 50 emerged female cx. pipiens fed on these rabbits at 6, 12, 24, 48, 72 h and 120 h after injected with na2hpo4. at the same time, 0.2 ml of blood was extracted from rabbits, as well as at 16 and 32 days for radioactivity level measure. pipiens radioactivity levels were measured at 2 h, 6 h, 12 h, 24 h, 48 h, 72 h and 120 h after blood meal. the measurement of radioactivity was conducted using a liquid scintillation counters (model ysj-76). before injection of p to rabbits, to normalize background radiation, radioactivity level of 30 emerged adult cx. mosquitoes tested were anesthesia with ether, placed in a bell counter tube of the vitriol chambers for 1 minute, as for the rabbits blood test, 0.2 ml of blood was dipped on the paper in the tinfoil sample plates, counts that exceeding 50% of the background was as the standard that were labeled. at the 2 day after no. 6 rabbit was injected with na2hpo4, up to 5 g feces was placed into a container with 300 ml of tap water and cx. pipiens larvae and pupae inside. as the negative control, placed 5 g feces of the rabbit without injected na2hpo4 into the water. the experimental project was reviewed and approved by the ethical committee of shandong academy of medical sciences (jinan, shandong). urine and feces of the rabbits were collected and sent to the institute of radiation medicine, shandong academy of medical sciences for appropriate processing to prevent spread of the isotope. before experimenting, 6 healthy rabbits (2 kg) were selected and physically examined by veterinarian. the injection method of normal saline solution to the rabbits was according to smith. (1951), i e. an isotonic buffered saline solution, containing different concentration of na2hpo4, was injected intravenously of rabbits. the dosage for no. 1, 2 rabbits, no. 3, 4 rabbits, no. 6 were 0.2 mci, 0.4 mci, 0.8 mci and 1.7 mci, respectively. for the negative control, 1 healthy rabbits was injected with isotonic buffered saline solution without na2hpo4. then 20 to 50 emerged female cx. pipiens fed on these rabbits at 6, 12, 24, 48, 72 h and 120 h after injected with na2hpo4. at the same time, 0.2 ml of blood was extracted from rabbits, as well as at 16 and 32 days for radioactivity level measure. pipiens radioactivity levels were measured at 2 h, 6 h, 12 h, 24 h, 48 h, 72 h and 120 h after blood meal. the measurement of radioactivity was conducted using a liquid scintillation counters (model ysj-76). before injection of p to rabbits, to normalize background radiation, radioactivity level of 30 emerged adult cx. mosquitoes tested were anesthesia with ether, placed in a bell counter tube of the vitriol chambers for 1 minute, as for the rabbits blood test, 0.2 ml of blood was dipped on the paper in the tinfoil sample plates, counts that exceeding 50% of the background was as the standard that were labeled. at the 2 day after no. 6 rabbit was injected with na2hpo4, up to 5 g feces was placed into a container with 300 ml of tap water and cx. pipiens larvae and pupae inside. as the negative control, placed 5 g feces of the rabbit without injected na2hpo4 into the water. the experimental project was reviewed and approved by the ethical committee of shandong academy of medical sciences (jinan, shandong). urine and feces of the rabbits were collected and sent to the institute of radiation medicine, shandong academy of medical sciences for appropriate processing to prevent spread of the isotope. pipiens and rabbits blood was measured, the background was determined as was 1213 counts per minute (cpm), i e counts that exceeded 50% of background (> 20 cpm) were considered positive. within 5 days after no pipiens fed on the rabbits, among the 222 mosquitoes blood feeding, except one mosquito was fewer than 20 cpm, the others 221 mosquitoes were labeled no matter how much dosage of na2hpo4 was injected. the more dosage the rabbits injected with na2hpo4, the higher radioactive levels of cx. the radioactive levels started to decrease from the 48 hours (2 day) after injection. compared with radioactive level at 48 h, though the radioactive level of labeled mosquitoes was higher than background at 72 h, 96h and 120 h, radioactive level decreased to a lower level (figs. 1, 2, 3). levels of radioactivity of culex pipiens after fed on no. 1, 2 rabbits after injection of p (0.2 mci) legend 6, 12, 24, 48, 72, 120 : mosquito fed time after injection p into rabbit (hours) levels of radioactivity of culex pipiens after fed on no. 3, 4 rabbits after injection of p (0.4 mci) legend 6, 12, 24, 48, 72, 120 : mosquito fed time after injection p into rabbit (hours) levels of radioactivity of culex pipiens after fed on no. 5 rabbits after injection of p (0.8 mci) legend 6, 24, 48, 72, 120 : mosquito fed time after injection p into rabbit (hours) the radioactive level decreased very fast in the blood of rabbits. at 6 hour the radioactive level was 100%, the radioactive level decreased 2648%, 6075% and 9597% at the 2, the 5 and the 32 day, respectively (table 1). radioactivity levels of rabbit blood after injection of p (1.7 mci) at different time cpm = pulse per minute, - do not measured no. 1 to 4 rabbits showed good health, have no change on body temperature and body weight. on the 2 day, no. 5 rabbit abortion, and gave birth to 2 died bunnies, the radioactive level was 15 mr / h at 5 cm away from these died bunnies. the no. 5 rabbit presented no abnormalities and was dissected at the 143 day after injected with na2hpo4, no pathological changes were found in the internal organs. the radioactivity level of liver was measured, which decreased to similar level of background. 6 rabbit showed diarrhea, appetite loss, weight loss and other symptoms. at the 31 days, and no special lesions it still presented a higher radioactivity level in liver, spleen, intestine, heart, lung, kidney, muscle etc (table 2). 6 rabbit tissue after injection of p (1.7 mci) 31 days background radiation= 22 cpm at the 2 day after no. 6 rabbit was placed into a container, at the 2 and 7 day, there were total 39 adult cx. pipiens mosquitoes emerged, among them, 22 (56.5%) were marked, their cpm was 21875, the average was 323. pipiens and rabbits blood was measured, the background was determined as was 1213 counts per minute (cpm), i e counts that exceeded 50% of background (> 20 cpm) were considered positive. within 5 days after no pipiens fed on the rabbits, among the 222 mosquitoes blood feeding, except one mosquito was fewer than 20 cpm, the others 221 mosquitoes were labeled no matter how much dosage of na2hpo4 was injected. the more dosage the rabbits injected with na2hpo4, the higher radioactive levels of cx. the radioactive levels started to decrease from the 48 hours (2 day) after injection. compared with radioactive level at 48 h, though the radioactive level of labeled mosquitoes was higher than background at 72 h, 96h and 120 h, radioactive level decreased to a lower level (figs. 1, 2, 3). levels of radioactivity of culex pipiens after fed on no. 1, 2 rabbits after injection of p (0.2 mci) legend 6, 12, 24, 48, 72, 120 : mosquito fed time after injection p into rabbit (hours) levels of radioactivity of culex pipiens after fed on no. 3, 4 rabbits after injection of p (0.4 mci) legend 6, 12, 24, 48, 72, 120 : mosquito fed time after injection p into rabbit (hours) levels of radioactivity of culex pipiens after fed on no. 5 rabbits after injection of p (0.8 mci) legend 6, 24, 48, 72, 120 : mosquito fed time after injection p into rabbit (hours) the radioactive level was 100%, the radioactive level decreased 2648%, 6075% and 9597% at the 2, the 5 and the 32 day, respectively (table 1). radioactivity levels of rabbit blood after injection of p (1.7 mci) at different time cpm = pulse per minute, - do not measured 1 to 4 rabbits showed good health, have no change on body temperature and body weight. on the 2 day, 5 rabbit abortion, and gave birth to 2 died bunnies, the radioactive level was 15 mr / h at 5 cm away from these died bunnies. the no. 5 rabbit presented no abnormalities and was dissected at the 143 day after injected with na2hpo4, no pathological changes were found in the internal organs. the radioactivity level of liver was measured, which decreased to similar level of background. however, no. 6 rabbit showed diarrhea, appetite loss, weight loss and other symptoms. at the 31 days, and no special lesions were found in internal organs after dissection. it still presented a higher radioactivity level in liver, spleen, intestine, heart, lung, kidney, muscle etc (table 2). 6 rabbit tissue after injection of p (1.7 mci) 31 days background radiation= 22 cpm at the 2 day after no. 6 rabbit was injected with na2hpo4. after 5 g feces of no. 6 rabbit was placed into a container, at the 2 and 7 day, there were total 39 adult cx. pipiens mosquitoes emerged, among them, 22 (56.5%) were marked, their cpm was 21875, the average was 323. we could see that the more dosage the rabbits injected with na2hpo4, the higher radioactive levels of cx. the radioactive levels started to decrease from the 48 hours (2 day) after injection. compared with radioactive level at 48 h, though the radioactive level of labeled mosquitoes was higher than background at 72 h, 96 h and 120 h, radioactive level decreased to a lower level. the more injection of na2hpo4 into rabbits, the higher radioactivity level in the blood and the slower decrease, was also consistent with the labeling condition of cx. 1, 2, 3). no. 1 to 4 rabbits showed good health, but as for the no. 5 rabbit injected with 0.8 mci of na2hpo4, on the 2 day, abortion, and gave birth to 2 died bunnies. at the 143 day, the no. 5 rabbit presented no abnormalities, no pathological changes were found in the internal organs. 6 rabbit injected with 1.7 mci of na2hpo4 showed diarrhea, appetite loss, weight loss and other symptoms. at the 31 days, it still presented a higher radioactivity level in liver, spleen, intestine, heart, lung, kidney, muscle etc. therefore, the appropriate concentration for not only marking mosquitoes but also no harm to rabbits was not more than 0.4 mci. stable isotopes occur naturally in the environment, are safe and non - invasive, pose no health or environmental risks (hood - nowotny and knols 2007). in medical research, most stable isotopes are non - toxic and are routinely used for mosquito feeding trials, in which human adults are labelled up through supplementary feeding with stable isotopes, may be useful for host seeking behavior and repellent testing, etc. several tracers were studied, such as co, sr, zn, i, ca and p, whereas p was the most applied radioisotope for tagging due to its short half - life, safety, activity and easy of detection (obrien and wolfe 1964). hasset and jenkins (1951) also performed a detailed study of the conditions affecting mosquitoes labelled with p and compared stages, p concentrations and age. the filarial larvae, setaria digitata linstow was marked after adult mosquitoes (armigerea obturbans walker) fed on cows or men infected with microfilaria, that larvae of mosquitoes were reared in water containing 1 c of p / ml (dissanaike. 1957). toxicity to the insect was also a serious problem to be considered in many studies (quarterman. the radioisotopes ca and i were very toxic when fed to adult houseflies at 1 c / ml of milk, whereas p was satisfactory (quarterman. 1954). by using n - labeled potassium nitrate and c - labeled glucose to mark larval mosquitoes, there were no consistent effects of isotopic enrichment on immature mosquito survival or adult mosquito body size (hamer. although fluorescent dyes or powders are also suitable for marking mosquitoes (takken. 1998, mccall. 2001, pates 2002, lapointe 2008, baber. 2010), and no effect of these dyes and powders on performance of ant (pogonomyrmex owyheei), mountain pine beetle (dendroctonus ponderosae), grasshopper (melanoplus spp.), ae. aegypti, anopheles sinensis in some studies (porter and jorgenson 1980, linton. others have found a reduced longevity, behavioural response or survival of opine parasitoids (diachasmimorpha spp.), weevils, ae. aegypti, asian citrus psyllid (diaphorina citri) and codling moths (laspeyresia pomonella) (sheppard. be caused of the restrictions of the retention, most studies researches primarily focus on dispersal of nulliparous female mosquitoes during the initial host - seeking event and sometimes a second host - seeking event (hamer., mosquitoes were successfully marked after feeding on hosts injected with trace element rb and cs (anderson. they are not radioactive, safe for workers and for the environment. as for insects marked with an element, no tags, paints, dyes, dusts, or visible marks were found left to alter insects behavior or interactions with other insects (hagler and jackson 2001). however, a limitation to use of trace elements as insect markers in large fields is that the detection of elements can be difficult, expensive, and time - consuming, requires technical expertise and expensive detection equipment (akey and burns 1991). some trace elements are not retained very well in certain insect species, for example, rb could be detected for only 26 days after marking aphids (guillebeau. high concentrations of trace elements can adversely affect development, survival, increased adult deformity, reduced pupation, eclosion, egg production and fecundity of certain insects (stimmann. naturally, occurring stable isotope markers are useful, as they do not require the pre - marking of individuals (hood - nowotny and knols 2007). labelling a distinct portion of an ecosystem with stable isotopes is a useful, minimally invasive method to study insect dispersal from an ecophysiological perspective (macneale. stable isotopes occur naturally in the environment, unlike painting, dusting, etc., stable isotope methods are non - invasive and samples require only minimal preparation following collection, which makes the cost of the process as a completely comparable to methods such as polymerase chain reaction (hood - nowotny and knols 2007). other advantages are the analysis costs (depending on the isotope and the matrix, the cost per sample may range from us$ 5100.00), shipping stable isotope samples is simple, safe and inexpensive (iaea 2009). it would cost between $ 150250 to label 1 000 000 anopheles mosquitoes with c - labelled glucose in the larval stages (hood - nowotny. it is possible to trace the fate of labelled sperm into female spermatheca in studies of male mosquitoes labelled with c (helinski. these stable isotope markers meet the usual criteria for use in insect studies : retention, no effect on behavior, durability, easily applied, clearly identifiable, and not expensive (hagler and jackson 2001). a labelled blood source also provides an easily identifiable point source for post feeding dispersal studies. tracing of labelled blood to determine resource allocation to the eggs or other tissues could also provide useful physiological information. it was possible to mark large numbers of mosquitoes by providing blood meals from a host fed injected with a stable isotope substance. na2hpo4 was injected via the jugular of rabbit vein ; these rabbits were bitten by emerged female cx. pipiens, so mosquitoes were successfully marked. the appropriate dosage (not only can label mosquito but also have no ill effects on rabbits) of na2hpo4 was 0.1 mci / kg. pipiens mosquitoes were also can be marked by placing rabbit feces or na2hpo4 into container. the technique was less time - consuming, more sensitive, and safer, can be used to study post - blood meal dispersal of mosquitoes and other blood - sucking insects. the short half - life of 14 days of p, however, was a disadvantage in studies where prolonged observations were necessary. | background : the aim of the current study was to develop a marking technique as an internal marker to mark post blood meal mosquitoes by using stable phosphate isotope 32p and determine the optimal concentration of it.methods:an isotonic physiological saline solution, containing different concentration of radioactive isotope 32p - labeled disodium phosphate (na2h32po4) was injected into rabbits via the jugular vein in the laboratory. emerged cx. pipiens were marked after feeding on rabbit. at the same time, the labeled conditions of emerged cx. pipiens were also measured by placing feces of no. 6 rabbit into containers with mosquito larvae and pupae inside.results:according to the label condition of cx. pipiens after taking blood and the effect of different dosage na2h32po4 on rabbit health, the optimal concentration of radioactive isotope was determined, that is, 0.1211 mci / kg. by placing feces of no. 6 rabbit into containers with mosquito larvae and pupae inside, the emerged mosquitoes were also labeled. therefore, feeding mosquitoes on the animal injected with radioactive na2h32po4 was more practical for detecting and tracing mosquitoes.conclusion:the method was less time - consuming, more sensitive and safer. this marking method will facilitate post - bloodmeal studies of mosquitoes and other blood - sucking insects. |
crimean - congo hemorrhagic fever (cchf) is a fatal infectious disease with an average death rate of 50%. the disease is caused by the nairovirus group from the bunyaviridae family (1) and is transmitted through the saliva of blood - sucking arthropods (ixodidae ticks), or by contact with the blood and tissues of infected livestock. moreover, transmission of cchf virus from person to person may occur by close contact with infected patients or via nosocomial transmission (1 - 6). a number of tick genera are capable of becoming infected with the cchf virus, including hyalomma spp. and rhipicephalus spp., but the most common vectors appear to be members of the hyalomma genus (2, 6, 7). crimean - congo hemorrhagic fever has been reported in 30 countries in africa, the middle east, europe, and asia (4, 8). it is prevalent in most of iran s neighboring countries, including pakistan, afghanistan, russia, turkey, iraq, kazakhstan, kuwait, oman, saudi arabia, tajikistan, and the united arab emirates (4). although there had been sporadic reported cases of the disease in livestock and humans since 1970 in iran, the first reported outbreak occurred in 1999. since that time, cchf has been considered one of the country s major public health challenges (3, 4). currently, cchf is prevalent in 23 of 30 provinces of iran ; sistan - baluchestan, isfahan, fars, and khuzestan are respectively the most heavily - infected provinces (4). due to high fatality rates and many challenges in its treatment, prevention, and control, cchf is a disease that should be dealt with immediately by public health authorities, so the disease is subject to immediate reporting (9). health center walk - in patients with symptoms of severe headache, sudden onset of high fever, muscular pain, and bleeding should be immediately suspected for cchf (9, 10). in probable cases, thrombocytopenia, leukopenia, and leukocytosis are detected in the hematological work - up, and are ultimately diagnosed and confirmed by a seropositive test for cchf. the incubation period of this disease is 1 - 7 days, and onset of disease is usually sudden (11). in 2002, khuzestan province was ranked as having the lowest rate of infection, with 10 or fewer cases annually (9). furthermore, khuzestan is also included as one of the highest - ranking infected areas on this list, with seven probable cases and five confirmed cases (4, 9). this retrospective study was conducted to investigate the epidemiological aspects of cchf and the determine the disease trend in khuzestan province in 1999 - 2015. this survey is a retrospective descriptive study based on statistics from the khuzestan health center during 1999 - 2015. various epidemiological parameters, including the annual prevalence of the cchf and the victims age group, occupation, sex, clinical symptoms, living area (urban or rural), and fatality ratio, were collected from the patient - history documents. the recorded data were analyzed with spss software, and the case - fatality rates were determined using the formula below (5). fatality rate = (the number of deaths due to cchf during the specific time / total cases of disease during specific time) 100. eighty - one patients with cchf symptoms were referred to various health centers in khuzestan province during 1999 - 2015. among these patients, 42 were diagnosed as probably cchf - positive with hematological findings positive for thrombocytopenia, of which 17 were confirmed as definite cases with seropositive testing for the cchf virus. the highest numbers of cases were reported in 2003 and 2010, with seven cases during each of those years ; there were five death cases in 2003 compared to two in 2010 (table 1). however, no cchf was reported in 2007, 2008, and 2013 (figures 1 and 2). the annual variations of probable, confirmed, and death cases of cchf in khuzestan province during 1999 - 2015 are illustrated in figures 1 and 2. the most confirmed cases occurred in 2010, whereas the most death cases occurred in 2003, when five out of seven patients died with a probable or confirmed diagnosed. c, confirmed case ; p, probable case. in 95% of the patients, symptoms of fever, bleeding, and thrombocytopenia were detected. bleeding was not observed in 5% of the patients, and the nose was the most - bleeding organ (table 2). the prevalence of the disease was 45.2%, 26.2%, 14.3%, and 14.3% in the spring, summer, autumn, and winter, respectively. of all cases, 57.1% of patients were males and 42.9% were females, and 35.7% of patients lived in rural dwellings compared to 64.3% who were from urban areas. the age groups of 10 - 19 and 20 - 29 years were the highest - risk groups, each with a frequency of 26.2% (table 3). the occupational groups most often exposed to cchf were farmers and housewives, with frequencies of 28.6% and 26.2%, respectively (table 4). there have been several reports regarding the prevalence of cchf in iran (1 - 4). however, it is necessary to calculate the disease incidence and trends in provinces with high rates of infection. this information provides a better idea and awareness regarding this disease s prevalence, and will better illustrate the efficiency of the organizations involved in disease prevention and surveillance programs. khuzestan province has been ranked as one of the most heavily - infected provinces (4) with seven cases in 2003, of which five led to death. following this outbreak, the disease demonstrated a downward trend from 2004 until 2008, as there were no reported cases in 2007 and 2008. however, an upward trend was noted again in 2009 and 2010, with seven probable cases reported in 2010 ; four cases were serologically confirmed and two of them eventually died. the disease followed the same downward trend in iran from 2000 to 2009 (4), but it followed an upward trend in turkey during 2002 - 2007 (150 cases in 2002 - 2003 compared to 717 in 2007). in iran, the highest disease prevalence occurred in 2002 and 2008, with 111 and 120 confirmed cases, respectively ; of these, 14 and 19 patients died (3). there were no reported cases in khuzestan province in 2008. in this epidemiological survey of khuzestan province, 42 probable cchf cases were reported from 1999 to 2015, of which 64.3% were from urban areas and 35.7% were from rural areas. the male : female ratio was 1.3:1.0 and the case - fatality ratio was calculated to be 28.6% (12/42) during this time period. in a similar epidemiological survey of cchf in turkey during 2002 - 2007, 1,820 cchf cases were reported and the fatality ratio was calculated to be 5% (92/1820), of which 69.4% of patients were from rural areas and the male : female ratio was 1.13:1.00 (8). in iran, 1,638 probable cchf cases were collected from different provinces from june 2000 until december 2009, of which 635 were confirmed and 89 died (4). therefore, the fatality ratio was calculated to be 5.4% in iran during this time period. this ratio has a wide range, 2% - 80%, in different countries (11). the case - fatality ratio was 28.6% among 38 persons in mauritania infected with cchf virus from february to august 2003. the first patient was a young woman who became ill shortly after butchering a goat ; she transmitted the infection to 15 hospital health workers and four members of her family (12). in an assessment of patients with cchf symptoms referred to the amir - almoemenin hospital of zabol county, sistan- baluchestan province, southeast iran, during 2003 - 2005, a total of 65 patients were enrolled. of them, 52 (80%) were male and 13 (20%) were female. the signs and symptoms included fever (98.4%), headache (80%), myalgia (72.3%), nausea and vomiting (60%), abdominal pain (49.2%), skin rash (13.8%), and splenomegaly (4%). laboratory findings included thrombocytopenia (100%), leukopenia (70.7%), and anemia (20%) (13). during the first half of 2009 in khorasan razavi province, 13 patients suffered from cchf, which confirmed the upward trend of the disease compared to the same time in previous years. four patients were health workers, four were butchers, and the others were farmers (10). in another epidemiological survey from birjand county, khorasan province, iran, a total of 32 probable cchf patients were hospitalized during 2011 and 2012. forty percent of the patients were men and 60% were women. in terms of occupation, 48% of the patients had direct contact with livestock and 70% were from rural areas. in 85% of the cases, fever and bleeding were the most common clinical symptoms seen in all of the patients (14). eighty - two probable cases were reported in an epidemiological study of cchf in fars province during 1999 - 2011, leading to the deaths of 18 patients. of all patients, men had the most cases, and the highest - risk occupational groups were farmers, butchers, and slaughterhouse workers. the most cases were reported in summer and spring, which is the ticks growing season (15). two peaks of cchf were observed in khuzestan province during 1999 - 2015 (in 2003 and 2010), confirming the probability of re - emergence of the disease. the increased numbers of disease cases in khuzestan in 2010 were attributed to smuggling of cattle and bufflehead, followed by drought in pakistan, afghanistan, and sistan- baluchestan. in the last few decades, cchf has emerged or re - emerged in various countries due to climate and anthropogenic factors, such as land - use changes, agricultural practices, and movement of livestock, which may influence host - tick - virus dynamics (16). climate changes, unhealthy and illegal slaughter, and smuggling of livestock could also lead to the emergence or re - emergence of the disease, as occurred in many provinces of iran, including khuzestan, in 2010. finally, considering that the highest - risk occupational groups for cchf infections are farmers and housewives, and the riskiest age group is 10 - 39 years, these individuals should be instructed and trained with regard to transmission modes, prevention, symptoms, and treatment of cchf. particular inspections of veterinary organizations with regard to livestock slaughter and trade, treatment of tick - infested livestock, and training of high - risk groups can help reduce the prevalence of cchf in iran. | backgroundcrimean - congo hemorrhagic fever (cchf) is an arboviral zoonotic disease transmitted to humans mainly through the bite of blood - sucking ixodidae ticks and also via contact with the blood and tissues of infected livestock.objectivesthis study is a retrospective descriptive survey based on data collected from the health center of khuzestan province, iran, during 1999 - 2015.patients and methodspatients with symptoms of severe headache, high fever, and bleeding were evaluated. laboratory tests and serological or molecular assays were used to detect probable and confirmed cases, respectively. the epidemiological parameters of this study were analyzed on the basis of probable cases.resultsa total of 42 patients were diagnosed as probable cases, and 17 of these (42.5%) were confirmed serologically. two peaks of the disease occurred in khuzestan province, in 2003 and 2010, with seven cases each of those years, leading to the deaths of five and two patients, respectively. men and women comprised 57.1% and 42.9% of the patients, respectively. of all probable cases, 64.3% were from urban areas and 35.7% were from rural areas. the age groups of 10 - 19 and 20 - 29 years, with a frequency of 26.2% in each group, were exposed to the most infections. farmers and housewives were the highest at - risk occupational groups with a frequency of 28.6% and 26%, respectively. fever, bleeding, and thrombocytopenia were reported in 95% of the patients, and the case - fatality ratio was calculated to be 28.6% (12 of 42 cases).conclusionscontinuous training is necessary to improve the knowledge and awareness of the highest - risk groups with regard to the transmission modes, prevention, symptoms, and treatment of this disease. |
dogs : four adult male and 4 adult female beagle dogs were used in this study. it was carried out in accordance with hokkaido university guidelines for the care and use of experimental animals, which basically conform to those of the association for assessment and accreditation of laboratory animal care (aaalac) international. the study was approved by the committee for laboratory animals, graduate school of veterinary medicine, hokkaido university (approval number 08 - 0473). the ultracentrifugation - precipitation method for canine lipoprotein analysis : the ultracentrifugation - precipitation (u - p) method for canine lipoprotein analysis briefly, blood samples were collected from dogs that had been fasted for 24 hr. peripheral blood was taken into tubes with edta, and the plasma was separated by centrifugation at 1,500 g for 10 min at 4c. four milliliters of the plasma was placed in a thermoplastic ultracentrifuge tube and overlayered with 6 ml of 154 mm nacl. the tube was capped and centrifuged at 164,000 g for 18 hr at 4c in an ultracentrifuge (beckman l7 - 55 ultracentrifuge, beckman coulter inc., brea, ca, u.s.a.). three milliliters of the upper layer, which included lipoproteins with a density of less than 1.006 g / ml(vldl), were removed as the vldl fraction. seven milliliters of the lower layer containing lipoproteins with a density greater than 1.006 g / ml(hdl and ldl) were collected as the hdl+ldl fraction. the ldl in the hdl+ldl fraction was precipitated by incubation with 400 l of 1 u/l heparin for 10 min and 5 ml of 101 mm mncl2 for 30 min on ice. after centrifugation at 10,800 g for 30 min at 4c, the resultant supernatant was collected as the hdl fraction, and the sediment was collected as the ldl fraction. measurement of cholesterol concentration in fractions separated by the ultracentrifugation - precipitation method : the cholesterol concentrations of intact plasma (total cholesterol), the hdl+ldl fraction and the hdl fraction (hdl - cholesterol, hdl - c) were measured using an enzymatic assay (chorestest, sekisui medical co., ltd., tokyo, japan) with a spectrophotometer (u-3210, hitachi, tokyo, japan). the ldl - cholesterol (ldl - c) concentration was calculated as the difference between the cholesterol concentration of the hdl+ldl fraction and that of the hdl fraction. for the u - p method, the ldl / hdl ratio was calculated from the ldl - c and hdl - c concentrations calculated above. analysis of canine lipoprotein using gp - hplc : lipoprotein analyses of intact plasma and the hdl+ldl and hdl fractions were performed using commercial gp - hplc service (lipotest, spectrum lab. the concentrations of hdl - c and ldl - c determined by the u - p method were compared with those determined by gp - hplc. lc - ms / ms sample preparation by tryptic digestion : lc - ms / ms sample preparation was performed using the methods of rosenfeld. and wilm. with some modifications. briefly, 7 microliters of the hdl+ldl, hdl and ldl fractions, separated using the u - p method, were subjected to sds polyacrylamide gel electrophoresis (sds - page) with 420% gradient acrylamide gels (ready gel j, bio - rad laboratories, inc., the stained protein s bands were excised with a scalpel, washed with 100 l of 50% acetonitrile in 25 mm ammonium carbonate and left to dry at rt for 10 min with shaking in an eppendorf thermomixer. the gel slices were dehydrated with 100 l acetonitrile and dried in a vacuum centrifugal dryer (thermo savant spd speedvac, with a savant uvs 400 universal vacuum system, thermo fisher scientific inc., then, the gel slices were alkylated with 55 mm iodoacetamide and 25 mm ammonium carbonate in light interception with aluminum foil for 45 min., madison, wi, u.s.a.) was added to the gel slices, and the gel slices were added to 50 mm ammonium carbonate and incubated on ice for 30 min. after absorption of the protease solution, the gel slices were incubated at 37c for 12 hr. to extract hydrophilic and hydrophobic peptides from the gel slices, we performed a three - step extraction. the first extracted solution (50% acetonitrile and 5% trifluoroacetic acid [tfa ]) was added to the gel slices and then incubated in an ultrasonic bath (usc-6, osada, inc. next, the second extracted solution (99% acetonitrile and 0.1% tfa) was added to the gel slices and incubated in an ultrasonic bath for 10 min. finally, the third extraction (99.9% ultrapure water and 0.1% tfa) was added to the gel slices and incubated in an ultrasonic bath. these samples produced by the three - step extraction were concentrated to ~20 l in the vacuum centrifugal dryer. lc - ms / ms analysis : the samples prepared above were analyzed by fourier transform mass spectrometry (ltq orbitrap, thermo fisher scientific inc., waltham, ma, u.s.a.) interfaced with the ultimate 3000 nano lc systems (thermo fisher scientific inc.) equipped with a trap column (zorbax 300 sb - c18, 0.3 5 mm, agilent technology, santa clara, ca, u.s.a.) and analytical column (acclaim pepmap100 3 m 100, dionex corp., sunnyvale, ca, u.s.a.) in acetonitrile gradient (acetonitrile : water=70:30, v / v). peak lists were generated from raw lc - ms / ms spectra using xcalibur 2.0.7 (thermo fisher scientific inc.). the raw data acquired by the mass spectrometry experiment were searched against the national center for biological information (ncbi) databases (www.ncbi.nlm.nih.gov) using proteome discoverer 1.0 (thermo fisher scientific inc.). statistical analysis : the correlation of the hdl - c and ldl - c concentrations between the u - p method and gp - hplc was investigated by pearson s correlation coefficient test. results were considered as correlated when r was significantly different from 0 (p<0.05) and over 0.7. analysis of canine lipoproteins in fractions separated using the ultracentrifugation - precipitation method : first, intact canine plasma, the hdl plus ldl fractions and hdl fractions separated by the u - p method were analyzed using gp - hplc. the peaks of vldl, ldl and hdl cholesterol were identified in intact plasma, but no peak was observed for cm cholesterol, because of the 24 hr fast (fig. 1.gp-hplc of canine lipoprotein in intact dog plasma (a) and of the hdl+ldl fraction (b) and hdl fraction (c) separated by the u - p method. the hdl+ldl fraction showed peaks for ldl and hdl cholesterol, but not for vldl cholesterol (fig. 1c). next, the concentrations of hdl and ldl cholesterol determined using the u - p method were compared with those determined by gp - hplc. the canine hdl - c concentration determined by the u - p method correlated well (r=0.94), but was not the same as that determined by gp - hplc (fig. 2.correlation of hdl - c (a) and ldl - c (b) concentrations and ldl / hdl ratio (c) between the u - p method and gp - hplc. the concentrations of canine hdl - c determined by the u - p method were lower than those determined by gp - hplc. the canine ldl - c concentration determined by the u - p method correlated well with that determined by gp - hplc (r=0.93) (fig. however, the concentrations of canine ldl - c determined by the u - p method were higher than those determined by gp - hplc. the canine ldl / hdl ratio determined by the u - p method correlated with that determined by gp - hplc (r=0.75), but was not the same as that determined by gp - hplc (fig. 2c). gp - hplc of canine lipoprotein in intact dog plasma (a) and of the hdl+ldl fraction (b) and hdl fraction (c) separated by the u - p method. correlation of hdl - c (a) and ldl - c (b) concentrations and ldl / hdl ratio (c) between the u - p method and gp - hplc. sds - page of the canine hdl+ldl, hdl and ldl fractions : the canine hdl+ldl, hdl and ldl fractions separated by the u - p method were subjected to sds - page. it was found that the canine hdl+ldl, hdl and ldl fractions from 3 healthy dogs included several proteins, and the amounts of these proteins were different in each dog (fig. 3fig. 3.sds-page analysis of the hdl+ldl (a), hdl (b) and ldl (c) fractions. the hdl+ldl, hdl and ldl fractions separated from plasma of three healthy dogs by the u - p method were analyzed using sds - page with the gradient gels. lanes 1, 2 and 3, respectively, indicate dogs 1, 2 and 3. proteins with estimated molecular weights of approximately 25 (a), 35 (b) and 60 kda (c) were detected in both the hdl and the ldl fractions. the protein with a molecular weight of more than 250 kda (d) proteins with an estimated molecular weight of greater than 250 kda and of approximately 25 kda were detected in all dogs (fig., proteins with an estimated molecular weight of approximately 25 kda were detected in all dogs (fig. proteins with an estimated molecular weight of approximately 35 and 60 kda were also detected (fig. sds - page analysis of the hdl+ldl (a), hdl (b) and ldl (c) fractions. the hdl+ldl, hdl and ldl fractions separated from plasma of three healthy dogs by the u - p method were analyzed using sds - page with the gradient gels. lanes 1, 2 and 3, respectively, indicate dogs 1, 2 and 3. proteins with estimated molecular weights of approximately 25 (a), 35 (b) and 60 kda (c) were detected in both the hdl and the ldl fractions. the protein with a molecular weight of more than 250 kda (d) was detected only in the ldl fraction. lc - ms / ms analysis of the canine apolipoprotein a - i and apolipoprotein b-100 : the proteins with a molecular weight of 25 kda in the hdl fraction were analyzed using lc - ms / ms. (a) sequence coverage (coverage), molecular weight (mw [da ]) and description of the analyzed protein are shown as a table. accession #, ncbi accession number ; # peptides, number of peptides ; # aas, number of amino acids. (b) the coincident amino acid sequence between the analyzed protein and the canine apo a - i (gray highlighted sequence). the proteins were identified significantly as apo a - i (fig. 4). the proteins with a molecular weight of more than 250 kda in the ldl fraction were also analyzed using the lc - ms / ms and identified significantly as canine apo b-100 (fig. 5fig. (a) sequence coverage (coverage), molecular weight (mw [da ]) and description of the analyzed protein are shown as a table. accession #, ncbi accession number ; # peptides, number of peptides ; # aas, number of amino acids. (b) the coincident amino acid sequence between the analyzed protein and the apo b-100 (gray highlighted sequence).). the proteins with a molecular weight of 60 kda were identified as canine albumin (data not shown). the proteins with a molecular weight of 35 kda were identified as haptoglobin (data not shown). (a) sequence coverage (coverage), molecular weight (mw [da ]) and description of the analyzed protein are shown as a table. accession #, ncbi accession number ; # peptides, number of peptides ; # aas, number of amino acids. (b) the coincident amino acid sequence between the analyzed protein and the canine apo a - i (gray highlighted sequence). (a) sequence coverage (coverage), molecular weight (mw [da ]) and description of the analyzed protein are shown as a table. accession #, ncbi accession number ; # peptides, number of peptides ; # aas, number of amino acids. (b) the coincident amino acid sequence between the analyzed protein and the apo b-100 (gray highlighted sequence). the present study examined the u - p method for its suitability as a gold standard method for canine lipoprotein analysis. since it was reported that gp - hplc was an efficient method in small animals, the u - p method was compared with gp - hplc first. in the u - p method, an ultracentrifuge separates vldl particles by specific gravity, and a heparin manganese chloride solution precipitates ldl particles. gp - hplc separates the particles based on size by using a specialized column. it was confirmed that vldl particles were removed from canine plasma by ultracentrifugation and that ldl was removed from the hdl+ldl fraction by precipitation. moreover, the hdl - c and ldl - c concentrations determined by the u - p method correlated well with those determined by gp - hplc, although the results of the u - p method were not consistent with those of gp - hplc. it was suggested that some of the canine hdl could be precipitated with heparin manganese chloride solution, resulting in a higher ldl - c concentration and lower hdl - c concentration in the u - p method than in gp - hplc. furthermore, the presence in canine plasma of an apolipoprotein e (apo e)-rich subfraction of hdl with a similar density to ldl and apo e, which may co - precipitate with ldl under certain conditions, may invalidate some of the methodologies used for the measurement of canine ldl - c and hdl - c concentrations. therefore, the hdl and ldl fractions separated by the u - p method were analyzed further by lc - ms / ms. in the present study, analysis of the enzymatic fragment by lc - ms / ms was obviously an important way of detecting canine apo a - i and apo b-100. in the hdl fraction, this result shows that the hdl fraction contained only lipoprotein having apo a - i. in the ldl fraction, this result shows that the ldl fraction contains lipoproteins having apo a - i and apo b-100. it is possible that a portion of the canine hdl was precipitated with the heparin manganese chloride solution. the canine hdl that was precipitated with canine ldl might include apo e, as barrie. suggested. it is known that an apo e - rich subfraction of hdl is present in canine plasma and has a similar density and behavior to ldl, although this indication is not studied. accordingly, this canine hdl could be a different lipoprotein from true canine hdl. since the characteristics of this canine lipoprotein remain unclear, so does its role. furthermore, it is also possible that there would be lipoproteins having both apo b-100 and apo a - i in dogs. quantitative analysis of lipoprotein fractions shows that human ldl particles having apo b-100 have certain amounts of apo a - i and apo e [5, 11 ]. similarly, certain canine particles might have both apo b-100 and apo a - i, although this speculation has yet to be validated. based on the present results, it is difficult to suggest the u - p method as an accurate method for analysis of canine lipoprotein. the u - p method can not be a gold standard method for analyzing canine lipoprotein. lc - ms / ms might be an efficient method for detecting canine apo e. however, analysis by sds - page showed that dogs 13 had different protein amounts in the hdl+ldl, hdl and ldl fractions, indicating that there are individual differences among these dogs. because the hdl - c and ldl - c concentrations determined by the u - p method correlated well with those determined by gp - hplc, this method may be able to roughly separate canine hdl and ldl, meaning that the u - p method could be useful for detecting alterations in the balance of canine lipoproteins and for clarifying the clinical significance of canine lipoprotein profiles. however, the ldl / hdl ratio determined by the u - p method was quite different from that determined by gp - hplc. since it is possible that gp - hplc was unable to accurately separate hdl from ldl, it is unclear which of the 2 methods is more accurate at separating canine hdl from ldl. in conclusion, the present study indicated that the u - p method would be hard to use as a gold standard method for analysis of canine lipoprotein, because it precipitates a certain lipoprotein, which includes apo a - i, with canine ldl. | abstractdue to the lack of a gold standard method in canine lipoprotein analysis, it is unclear whether canine high - density lipoprotein (hdl) and low - density lipoprotein (ldl) can be accurately evaluated by the lipoprotein analysis methods used for dogs. this study investigated whether the ultracentrifugation - precipitation (u - p) method was suitable as a gold standard method for analyzing canine lipoprotein. first, the u - p method was compared with a gel permeation high - performance liquid chromatography system (gp - hplc). the concentrations of canine hdl cholesterol (hdl - c) and ldl cholesterol (ldl - c) determined by the u - p method correlated closely with those determined by gp - hplc. however, the canine hdl - c concentration determined by the u - p method was lower than that determined by gp - hplc, and the canine ldl - c concentration determined by the u - p method was higher than that determined by gp - hplc. this study showed that some canine hdl could be precipitated with heparin manganese chloride solution. second, the hdl and ldl fractions separated by the u - p method were analyzed by lc - ms / ms. the hdl fraction was found to contain only apolipoprotein a - i, which is an apolipoprotein of hdl, whereas the ldl fraction contained both apolipoprotein a - i and apolipoprotein b-100, which is an apolipoprotein of ldl. this data showed that a certain lipoprotein that includes apolipoprotein a - i might precipitate with canine ldl when using heparin manganese chloride solution. these results indicated that the u - p method is not currently a gold standard method for analyzing canine lipoproteins. |
extensive research has shown that some of our fastest motor reactions express a degree of sophistication which rivals voluntary actions but little is known about the neural substrates which underlie this sophistication. the present monkey and human studies test whether primary motor cortex (m1) provides a neural substrate for integrating shoulder and elbow motion information for fast feedback control, a key ability for generating fast and accurate corrections. m1 is a prime candidate to mediate this ability because : 1) m1 forms part of a transcortical feedback pathway, giving it access to the required afferent information ; 2) m1 is a key node for voluntary control, which appropriately incorporates shoulder and elbow information when generating commands for voluntary actions ; and 3) influential theories posit that voluntary movement involves the sophisticated manipulation of sensory information, suggesting substantial functional and anatomical overlap between voluntary and feedback control. we first determined whether individual neurons in monkey m1 exhibit a pattern of activity consistent with multi - joint motion integration during fast feedback control. two rhesus monkeys were trained to counter unpredictable step - torque perturbations applied at the shoulder and/or elbow which displaced their hand from a central target. to receive water reward, the monkeys needed to return their hand to the target within 750 ms and remain within it for an additional 3 s, allowing us to analyze both fast feedback responses (50 ms following the perturbation, suggesting that the two stimuli interact through a common cortical circuit and that feedback control at latencies > 50 ms reflects processing in m1. we established the validity of this technique for shoulder muscles since a supra - linear response occurred when tms was delivered ~65 ms after the shoulder muscle was stretched (i.e. during the long - latency reflex ; t - test ; extensor : t9 = 6.7, p 0.5 ; flexor : t8 = 0.5, p > 0.5) when only spinal processes could contribute (supplementary fig. the critical question is whether m1 causally contributes to multi - joint integration for fast feedback control. we tested this hypothesis by applying tms in conjunction with the torque perturbation that causes pure elbow displacement (fig. any supra - linearity of the shoulder muscle response in this condition must reflect afferent information from the elbow joint onto cortical circuits controlling shoulder muscles since local shoulder afferents are not physically affected by pure elbow motion. the predicted supra - linear effect was observed for both shoulder flexors and extensors (fig. 4, supplementary fig. 3, right column) with tms delivered at 65 ms (extensor : t9 = 3.8, p 0.5 ; flexor : t8 = 0.1, p > 0.5). the observed supra - linearity likely reflects latency - specific engagement of m1 rather than a general change in motor neuron excitability since we found no correlation between the magnitude of perturbation - evoked activity and the amount of supra - linearity at either latency (p > 0.1, supplementary fig. these results provide strong evidence that m1 causally underlies multi - joint integration for fast feedback control. previous studies have demonstrated that fast feedback responses in m1 are scaled by task - constraints such as movement amplitude, surface texture and intended vigor. our results show that m1 also integrates locally - ambiguous motion information into a global response that accounts for the limb s mechanical properties, a more complex capability that is central to successfully guiding whole - arm movements. it is well established that the voluntary motor system accounts for the mechanical properties of the limb and that this capability is expressed in the activity of m1 neurons. we have previously argued that the functional similarity of voluntary and feedback control is not an accident and likely arises because of a common neural implementation that includes m1. this expectation is consistent with recent theories of sensorimotor control which posit that voluntary behavior involves the sophisticated manipulation of sensory information. if our suggestion is true then feedback processing in m1 should possess all the capabilities of voluntary processing in m1 and, likewise, studying feedback processing may provide a useful window into voluntary control. the studies presented in this paper were approved by the queen s university research ethics board. all monkey (n = 2, macaca mulatta, ~10 kg, male) procedures were approved by the queen s university animal care committee. human subjects (6 females, 4 males, median age = 27) were neurologically unimpaired, had normal / corrected vision and provided informed consent. human and monkey experiments were performed using different versions of the same robotic exoskeleton (kinarm, bkin technologies, kingston, on) which allows combined flexion and extension movements of the shoulder and elbow in the horizontal plane and can independently apply mechanical loads to the shoulder and/or elbow. target lights and simulated hand feedback were presented in the horizontal plane of the task via a virtual reality display and direct vision of the hand was limited either by a physical barrier (humans) or by a lack of ambient light (monkeys). recording chambers were surgically implanted under inhalation anesthetic and neural recordings were performed according to standard techniques. single tungsten microelectrodes (fhc, bowdoin, me) were advanced until neural activity was observed. individual neurons were then isolated and neural activity was recorded from those neurons with clear responses to either passive or active movements of the shoulder and/or elbow. neurons recorded in the task (n = 356) were located in the rostral bank of the central sulcus as well as more superficial sites where previous mapping efforts showed that trains of electrical stimulation (11 pulses, 333hz, 0.2ms pulse width, < 50ua) could elicit shoulder and/or elbow movement. post - mortem histology confirmed that recording sites from monkey p were located in m1. monkey muscle activity was acquired from mono - articular shoulder muscles (anterior deltoid, middle deltoid, posterior deltoid, pectoralis major ; n = 34) using fine - wire electrodes. electrodes consisted of two single - strand wires and were individually inserted into the muscle belly spaced ~5 mm apart. human experiments used surface electrodes (de-2.1, delsys, boston, ma) and focused on those mono - articular muscles which could be readily recorded from the surface (posterior deltoid, pectoralis major ; n = 19). muscle activity was recorded at either 4 khz (monkey) of 1 khz (human), aligned on perturbation onset and full - wave rectified prior to analysis. only those muscles with clear phasic responses to the mechanical perturbation were analyzed. kinematic data and applied torques were acquired directly from the kinarm device and were sampled at the same rate as the muscle activity. single pulses of tms (mes-10, cadwell, kennewick, wa) were applied over left m1 with a posterior orientation of 3045. placement and orientation of the double coil was chosen to evoke the largest response from the muscle of interest, ~4.5 cm lateral from vertex. stimulation magnitude was selected to deliver the smallest - possible consistent response (evoked response on seven consecutive stimulations, average of 40% and 51% of the stimulator s maximum output for the posterior deltoid and pectoral major, respectively) when the muscle of interest actively countered a 3 nm background load (i.e. active motor threshold). the major difference in the human portion of this study was the parallel implementation of tms. briefly, subjects stabilized their hand in a small central target while countering a steady state shoulder torque (3 nm) which activated either the shoulder flexor or extensor muscles. after a random hold time (14 s), an unpredictable torque pulse (100 ms duration) was introduced and the trial ended when the subjects re - stabilized in the target for 500 ms. in total, four torque perturbations were used in the human study. two single - joint torque perturbations (3 nm shoulder - flexion for shoulder flexor muscles and 3 nm shoulder - extension for shoulder extensor muscles) made up experiment 1 and two multi - joint torque perturbations (3 nm shoulder - flexion / 3 nm elbow - flexion for shoulder flexor muscles ; 3 nm shoulder - extension / 3 nm elbow - extension for shoulder extensor muscles) made up experiment 2. perturbation only, tms only and combined tms and perturbation trials were randomly interleaved. in combined tms and perturbation trials, the tms was timed to evoke shoulder muscle activity either ~25 ms or ~65 ms after perturbation onset. the perturbation, tms placement and tms intensity were chosen for the shoulder flexor muscle and shoulder extensor muscle in two successive blocks. half the subjects began with the conditions for the shoulder flexor muscle and half the subjects began with the conditions for the shoulder extensor muscle. thirty repeats of the 14 conditions were collected for a total of 420 trials in a session that lasted about 2.5 hours. unlike humans, the monkeys did not counter a pre - perturbation background load and they were exposed to eight randomly - interleaved step - torque perturbations ([shoulder torque, elbow torque ], applied flexion / extension = positive / negative : 1. [0.28 nm, 0 nm ], 2. [0.24, 0.24 ], 3. [0, 0.24 ], 4. four of these perturbations (1,3,5,7) formed experiment 1 and two (2,6) formed experiment 2. all eight conditions were used to calculate the steady - state tuning of each neuron by performing a planar regression on the neural activity when the monkey had re - stabilized their hand at the central target. to receive water reward, the monkeys needed to return their hand to the target within 750 ms and remain within it for an additional 3 s, allowing us to analyze both fast feedback responses (< 100 ms post - perturbation) and steady - state motor outputs (last 1s of stabilization) to the applied torque within the same trial. population responses for both muscles and neurons were calculated by collapsing across shoulder flexion and extension conditions according to their predicted excitatory and inhibitory effects. that is, applied shoulder - flexion torque perturbations were excitatory for shoulder extensor muscles / neurons and inhibitory for shoulder flexor muscles / neurons. applied shoulder - extension torque perturbations were excitatory for shoulder flexor muscles / neurons and inhibitory for shoulder extensor muscles / neurons. the studies presented in this paper were approved by the queen s university research ethics board. all monkey (n = 2, macaca mulatta, ~10 kg, male) procedures were approved by the queen s university animal care committee. human subjects (6 females, 4 males, median age = 27) were neurologically unimpaired, had normal / corrected vision and provided informed consent. human and monkey experiments were performed using different versions of the same robotic exoskeleton (kinarm, bkin technologies, kingston, on) which allows combined flexion and extension movements of the shoulder and elbow in the horizontal plane and can independently apply mechanical loads to the shoulder and/or elbow. target lights and simulated hand feedback were presented in the horizontal plane of the task via a virtual reality display and direct vision of the hand was limited either by a physical barrier (humans) or by a lack of ambient light (monkeys). recording chambers were surgically implanted under inhalation anesthetic and neural recordings were performed according to standard techniques. single tungsten microelectrodes (fhc, bowdoin, me) were advanced until neural activity was observed. individual neurons were then isolated and neural activity was recorded from those neurons with clear responses to either passive or active movements of the shoulder and/or elbow. neurons which primarily responded to motion of the wrist or fingers were not recorded. neurons recorded in the task (n = 356) were located in the rostral bank of the central sulcus as well as more superficial sites where previous mapping efforts showed that trains of electrical stimulation (11 pulses, 333hz, 0.2ms pulse width, < 50ua) could elicit shoulder and/or elbow movement. post - mortem histology confirmed that recording sites from monkey p were located in m1. monkey muscle activity was acquired from mono - articular shoulder muscles (anterior deltoid, middle deltoid, posterior deltoid, pectoralis major ; n = 34) using fine - wire electrodes. electrodes consisted of two single - strand wires and were individually inserted into the muscle belly spaced ~5 mm apart. human experiments used surface electrodes (de-2.1, delsys, boston, ma) and focused on those mono - articular muscles which could be readily recorded from the surface (posterior deltoid, pectoralis major ; n = 19). muscle activity was recorded at either 4 khz (monkey) of 1 khz (human), aligned on perturbation onset and full - wave rectified prior to analysis kinematic data and applied torques were acquired directly from the kinarm device and were sampled at the same rate as the muscle activity. single pulses of tms (mes-10, cadwell, kennewick, wa) were applied over left m1 with a posterior orientation of 3045. placement and orientation of the double coil was chosen to evoke the largest response from the muscle of interest, ~4.5 cm lateral from vertex. stimulation magnitude was selected to deliver the smallest - possible consistent response (evoked response on seven consecutive stimulations, average of 40% and 51% of the stimulator s maximum output for the posterior deltoid and pectoral major, respectively) when the muscle of interest actively countered a 3 nm background load (i.e. active motor threshold). the major difference in the human portion of this study was the parallel implementation of tms. briefly, subjects stabilized their hand in a small central target while countering a steady state shoulder torque (3 nm) which activated either the shoulder flexor or extensor muscles. after a random hold time (14 s), an unpredictable torque pulse (100 ms duration) was introduced and the trial ended when the subjects re - stabilized in the target for 500 ms. in total, four torque perturbations were used in the human study. two single - joint torque perturbations (3 nm shoulder - flexion for shoulder flexor muscles and 3 nm shoulder - extension for shoulder extensor muscles) made up experiment 1 and two multi - joint torque perturbations (3 nm shoulder - flexion / 3 nm elbow - flexion for shoulder flexor muscles ; 3 nm shoulder - extension / 3 nm elbow - extension for shoulder extensor muscles) made up experiment 2. perturbation only, tms only and combined tms and perturbation trials were randomly interleaved. in combined tms and perturbation trials, the tms was timed to evoke shoulder muscle activity either ~25 ms or ~65 ms after perturbation onset. the perturbation, tms placement and tms intensity were chosen for the shoulder flexor muscle and shoulder extensor muscle in two successive blocks. half the subjects began with the conditions for the shoulder flexor muscle and half the subjects began with the conditions for the shoulder extensor muscle. thirty repeats of the 14 conditions were collected for a total of 420 trials in a session that lasted about 2.5 hours. the monkeys did not counter a pre - perturbation background load and they were exposed to eight randomly - interleaved step - torque perturbations ([shoulder torque, elbow torque ], applied flexion / extension = positive / negative : 1. [0.2, 0.2 ]) and catch trials where no perturbations occurred. four of these perturbations (1,3,5,7) formed experiment 1 and two (2,6) formed experiment 2. all eight conditions were used to calculate the steady - state tuning of each neuron by performing a planar regression on the neural activity when the monkey had re - stabilized their hand at the central target. to receive water reward, the monkeys needed to return their hand to the target within 750 ms and remain within it for an additional 3 s, allowing us to analyze both fast feedback responses (< 100 ms post - perturbation) and steady - state motor outputs (last 1s of stabilization) to the applied torque within the same trial. population responses for both muscles and neurons were calculated by collapsing across shoulder flexion and extension conditions according to their predicted excitatory and inhibitory effects. that is, applied shoulder - flexion torque perturbations were excitatory for shoulder extensor muscles / neurons and inhibitory for shoulder flexor muscles / neurons. applied shoulder - extension torque perturbations were excitatory for shoulder flexor muscles / neurons and inhibitory for shoulder extensor muscles / neurons. | a basic difficulty for the nervous system is integrating locally ambiguous sensory information to form accurate perceptions about the outside world14. this local - to - global problem is also fundamental to motor control of the arm since complex mechanical interactions between the shoulder and elbow allow a particular amount of motion at one joint to arise from an infinite combination of shoulder and elbow torques5 (fig. 1a). here we show that a transcortical pathway through primary motor cortex (m1) resolves this ambiguity during fast feedback control. we demonstrate that single m1 neurons of behaving monkeys can integrate shoulder and elbow motion information into motor commands which appropriately counter the underlying torque within ~50 ms of a mechanical perturbation. moreover, we reveal a causal link between m1 processing and multi - joint integration in humans by showing that shoulder muscle responses occurring ~50 ms after pure elbow displacement can be potentiated by transcranial magnetic stimulation. our results show that m1 underlies multi - joint integration during fast feedback control, demonstrating that transcortical processing permits feedback responses to express a level of sophistication previously reserved for voluntary control and providing neurophysiological support for influential theories positing that voluntary movement is generated by the intelligent manipulation of sensory feedback6,7. |
interest in vertebrate head segments first arose because of a transcendental concept of morphology [goethe 1790 (cited in gaupp 1898), 1820 ] and was subsequently revived in the search for a common embryonic design reflecting the morphology of a common ancestor (goodrich 1930). current studies of vertebrate head segments involve the discipline of evolutionary developmental biology, in which regulatory gene networks and their functions are compared between vertebrates and nonvertebrate chordates to elucidate evolutionary changes in developmental programs (gene regulatory networks) that result in the vertebrate body plan (see olsson. several types of segments are recognized or assumed present in the vertebrate head (see jefferies 1986 for a classification of segmental theories). for example, pharyngeal arches and cranial (branchiomeric) nerves innervating the arches are iterated in the ventral part of the head. in the vertebrate embryo, these nerves arise on even - numbered rhombomeres (r2, r4, and r6), the segmental bulges in the hindbrain (lumsden and keynes 1989). consequently, the neuromeres in the neuraxis were thought by some morphologists to reflect the segmental design of the vertebrate axis (reviewed by jarvik 1980). however, the most intriguing issue in the history of comparative embryology has concerned the hypothesis that the mesodermal segments in the head are equivalent (serially homologous) to the somites in the trunk. in a general sense, goethe 's vertebral theory of the skull (goethe 1790, 1820) is based on this theory, as are many of the concepts that are based on mesodermal segmentation (fig. 1a) includes the peripheral nervous system and a differentiated skeletal system, if these anatomical structures are removed, it is apparent that the scheme assumes that the paraxial mesoderm is segmented along the entire axis as somites (a) goodrich 's (1930) formulation of the vertebrate head is based on the morphology of the mid - pharyngula of elasmobranchs. (b) when the skeletal and peripheral nervous systems are removed from the scheme, it is evident that it was based on the segmentation of the paraxial mesoderm. note that each of the head somites (pm, mm, hm, s03) is associated ventrally with the pharyngeal arch mesoderm. the vertebrate head is assumed to contain only one type of segmentation that involves metamerism of paraxial mesodermal segments and pharyngeal arches. (d) an example of a nonsegmentalist theory that assumes independent patterns of metamerisms for somites and pharynegal arches but questions the presence of segments in head mesoderm. abbreviations : hm, hyoid somite ; mm, mandibular somite ; ot, otic vesicle ; pm, premandibular somite ; pv, platt 's vesicle ; s07, somites. (a) goodrich 's (1930) formulation of the vertebrate head is based on the morphology of the mid - pharyngula of elasmobranchs. (b) when the skeletal and peripheral nervous systems are removed from the scheme, it is evident that it was based on the segmentation of the paraxial mesoderm. note that each of the head somites (pm, mm, hm, s03) is associated ventrally with the pharyngeal arch mesoderm. the vertebrate head is assumed to contain only one type of segmentation that involves metamerism of paraxial mesodermal segments and pharyngeal arches. (d) an example of a nonsegmentalist theory that assumes independent patterns of metamerisms for somites and pharynegal arches but questions the presence of segments in head mesoderm. abbreviations : hm, hyoid somite ; mm, mandibular somite ; ot, otic vesicle ; pm, premandibular somite ; pv, platt 's vesicle ; s07, somites. classical segmental theories assumed that the iterating intervals of pharyngeal arches are identical to those of head segments. therefore, the typical segmentalist theory assumed that each head somite was associated with a single pharyngeal arch, as is evident in the theories of balfour (1878), van wijhe (1882), goodrich (1930), bjerring (1977), and jarvik (1980) (fig. 1c). the dual segmental theory of romer (1972), in which the segmental pattern of somites is considered independent of that of the gills (fig. 1d), is typical of theories that arose in opposition to the aforementioned segmentalist theories. the series of reports by froriep and kuratani (froriep 1882, 1885, 1902 ; kuratani and eichele 1993 ; kuratani 1997, 2003) make assumptions similar to those of romer (1972). in addition, moderate segmentalists recognized head mesodermal segments but denied (or questioned) their association with the pharyngeal arches (de beer 1922, 1937 ; damas 1944). in the modern context of evolutionary developmental biology, the primary areas of interest involve the absence or presence of paraxial mesodermal segmentation in the vertebrate embryonic head and whether the segments are related to branchiomeric elements. thus, the issues in this field are similar to those considered by the moderate segmentalists. unlike comparative embryology that attempted to formulate an archetype of vertebrate head until the beginning of the 20th century the issue of head segmentation can now be rephrased as did the embryonic head of the vertebrate ancestor have somitomeric segmentation ? as is evident from the search for plesiomorphic features of the patterning program of the vertebrate head. developmental biology, on the other hand, searches for generalized developmental programs of the vertebrate head (reviewed by hunt. 1991 ; graham 2001), which may or may not be equivalent to the ancestral pattern because such a generalized scheme may include vertebrate synapomorphies that are absent from the ancestor. the aims of this review are to refute the theory of head mesodermal segments, to examine the validity of goodrich 's scheme (and, indirectly, most segmentalist theories published prior to goodrich 's scheme), and to consider how to deal with this classical problem in the context of evolutionary development. unlike the clearly segmented paraxial mesoderm in the trunk, there are no clear segments in the embryonic head mesoderm of amniotes. however, meier and his colleagues (meier 1979, 1981 ; anderson and meier 1981 ; meier and tam 1982 ; jacobson and meier 1984 ; meier and packard 1984 ; reviewed by jacobson 1988, 1993) claim to have observed incomplete segments called somitomeres in various gnathostome embryos by using scanning electron microscopy (sem). the presence of these segments has never been confirmed by other scientists (see freund. 1996 ; jouve. 2002 ; also see s. kuratani and t. schilling in this issue). as the somitomeres in gnathostomes were thought to reflect their ancestral morphology, the embryos of basal species were expected to exhibit segments with even greater clarity. koltzoff (1901) and damas (1944) illustrated the location of segmented head mesoderm in lamprey embryos and larvae based on histological observations of embryonic petromyzon marinus and lampetra fluviatilis (fig. these segments are depicted as a rostral continuation of somites and are delineated by clear boundaries. however, veit (1947), who described petromyzon planeri embryos, and kuratani. (1999), who conducted an sem study of lethenteron japonicum, did not observe segmental boundaries in the head mesoderm other than the posterior boundary of the premandibular mesoderm (kuratani. 2is the head mesoderm of the lamprey segmented ? (a) a parasagittal section of a young lamprey embryo published by koltzoff (1901) (modified from de beer 1937). the head mesoderm is colored light gray and real somites in the trunk are colored dark gray. in this section, segments starts from the yet undeveloped premandibular mesoderm (labeled s1) followed by the mandibular mesoderm (labeled s2). thus, the first (real postotic) somite in the generic terminology is labeled s4, and s3 in this figure corresponds to the sum of hyoid mesoderm and somite 0 (see kuratani., the head mesoderm appears to be segmented only between s2 and s3. the unsegmented mesoderm is simply regionalized by surrounding structures and no real segmentation is evident. (d) a scheme showing that parasagittal sections can not discriminate between segmentation and regionalization. is the head mesoderm of the lamprey segmented ? (a) a parasagittal section of a young lamprey embryo published by koltzoff (1901) (modified from de beer 1937). the head mesoderm is colored light gray and real somites in the trunk are colored dark gray. in this section, segments starts from the yet undeveloped premandibular mesoderm (labeled s1) followed by the mandibular mesoderm (labeled s2). thus, the first (real postotic) somite in the generic terminology is labeled s4, and s3 in this figure corresponds to the sum of hyoid mesoderm and somite 0 (see kuratani., the head mesoderm appears to be segmented only between s2 and s3.. the unsegmented mesoderm is simply regionalized by surrounding structures and no real segmentation is evident. (d) a scheme showing that parasagittal sections can not discriminate between segmentation and regionalization. in the early development of lampreys, segmental boundaries appear only in the postotic paraxial mesoderm, and the initially unsegmented head mesoderm is only regionalized into domains by the protruding pharyngeal pouches and otic vesicle (kuratani. the premandibular mesoderm arises in later development from the prechordal plate, and for this reason the premandibular and mandibular mesoderm are separated by a clear boundary such as that between the prechordal plate and the mandibular mesoderm. it is noteworthy that the latter boundary is not formed in the initially continuous sheet of mesodermal cells, as is the case with somitogenesis, and thus is not identical to the somitic boundaries. therefore, the head somites described by koltzoff (1901) and damas (1944) are most likely to have been histological exaggerations of a divided enterocoel and sulci on the surface of the head mesoderm (fig. embryos at stages 19.5, 20, and 20.5 (tahara 1988) are schematically illustrated. as in other vertebrate embryos, the head mesoderm of the lamprey is primarily regionalized by pharyngeal pouches (pp1 and pp2) and the otic vesicle (ot) ; real somitic boundaries arise only in the postotic paraxial mesoderm. embryos at stages 19.5, 20, and 20.5 (tahara 1988) are schematically illustrated. as in other vertebrate embryos, the head mesoderm of the lamprey is primarily regionalized by pharyngeal pouches (pp1 and pp2) and the otic vesicle (ot) ; real somitic boundaries arise only in the postotic paraxial mesoderm. mesoderm of the lamprey described by koltzoff (1901) does not constitute valid evidence for presence of segments. the term enterocoely refers to a mode of mesodermal development, not to a morphological pattern or process of segmentation in the mesoderm (the presence or absence of head cavities is discussed in a subsequent section). although one of koltzoff 's parasagittal sections appears to show segmental organization of the paraxial mesoderm, only two mesodermal domains are depicted in the head (fig. as discussed previously, the division of these two regions corresponds to a sulcus between the mandibular and hyoid mesodermal domains in l. japonicum (kuratani. koltzoff 's paper was strongly biased by the so - called elasmobranch worship that prevailed at the time (gee 1996), and the mesodermal domains he called head somites are similar to those present in early elasmobranch pharyngula, in which head cavities and pharyngeal arch mesoderm are not well dissociated. koltzoff 's second somite (s2) corresponds to the mandibular arch mesoderm and mandibular cavity in well - formed elasmobranch pharyngula, and the third somite (s3) corresponds to the more posterior head mesodermal domain (hyoid mesoderm + somite 0 in the lamprey ; fig. 3) rostral to the real first somite that koltzoff called the fourth somite (s4 in fig. 2a is also produced by regionalization in embryos of l. japonicum (kuratani. it is clear that it has never been proven that the head mesoderm of p. marinus is segmented and that it is merely regionalized by the growth of surrounding embryonic structures, as occurs in l. japonicum. although the head mesoderm of the lamprey develops as an enterocoel, it never becomes segmented nor does it persist as head cavities surrounded by thin epithelium (many classical descriptions have confused enterocoelic head mesodermal domains with head cavities). the head segmental theories were aimed at formulating the morphological patterns of the vertebrate head and referred not only to mesodermal segments but also indirectly to mechanisms responsible for the developmental patterns of the skeletal, nervous, and vascular systems. in developmental biology, for example, many of the segments in the neural tube (neuromeres) represent developmental compartments whose cell lineages are separated from those in other compartments (fraser. 1990 ; figdor and stern 1993). they are often centers of cell proliferation and impose domain restrictions on regulatory gene expression, as observed in the hindbrain and forebrain. they represent typical epithelial segments that develop autonomously (kllen 1956). the spinal cord, on the other hand, contains a different type of neuromeres (myelomeres) whose development is largely dependent on the presence of somites (lim. although neal (1918) did not realize the morphogenetic (segmental) significance of neuromeres, he was aware that neuromeres arise for different reasons at each level of the neural tube (neal 1898). neal (1918) recognized the primary role of somites in the development of myelomeres, which is now termed thus, modern segmental theory should indicate the nature and distribution of generative constraints in vertebrate head development. as has been recognized by several experimental embryologists, the anlage of the spinal cord is not primarily segmented, but the segmental patterns of dorsal root ganglia and motor roots are imposed secondarily by the presence of somites. experimental removal or addition of somites causes a loss or an increase of peripheral nerve elements in a manner similar to changes in mesodermal segments (lehmann 1927 ; detwiler 1934 ; tosney 1982 ; keynes and stern 1984 ; rickmann. 1985 ; lim. 1987, 1991 ; teillet. 1987 ; sechrist. 1993). in amniotes, the segmental pattern of the nerve is ascribed to the fact that neither the motor axons nor the neural crest cells are segmented per se and can only penetrate or migrate into the rostral half of the somites during development. thus, the pattern of spinal nerves is constrained to develop into a somitomeric pattern and the primary factor responsible for their segmental pattern is the presence of somites (the somitomeric constraint). in contrast, it is not the head mesoderm but the rhombomeres that determine the position of nerve root formation in the hindbrain (lumsden. 1991 ; kuratani and eichele 1993 ; kuratani and aizawa 1995 ; niederlnder and lumsden 1996). in the hindbrain, unlike the spinal cord, the segmental pattern of peripheral nerves is constrained to the segmental pattern of the neurectoderm, and selective adhesion of the crest cells to even - numbered rhombomeres prefigures the developmental pattern of the nerves (moody and heaton 1983a, 1983b, 1983c), as was first recognized by froriep (1902). thus, even - numbered and odd - numbered rhombomeres serve as prepatterns for the formation of the roots of cranial nerves (rhombomeric constraint ; kuratani 1991 ; guthrie. 1997), and this is likely to prevail in the lamprey (horigome. 1999). by considering the mechanism responsible for pattern formation, primary segments responsible for generation of secondarily segmented (constrained) structures can be identified. in the ventral part of the head, mesoderm, and crest cells (ectomesenchyme) of the pharyngeal arch and epibranchial placodes may be considered secondarily segmented structures that are constrained or induced by primarily segmented endodermal protrusions, namely, the pharyngeal pouches (branchiomeric constraint ; see begbie. 2005 for induction of epibranchial placodes through signals from endodermal pouches). because comparative embryology and morphology depends solely on the observation of shapes, it is incapable of identifying generative constraints. thus, in the classical segmental theories, cranial nerves (rhombomeric and branchiomeric) were often regarded as highly modified somitomeric spinal nerves. only amphioxus (fig. (a) peripheral nerves of the amphioxus head. as indicated by arrows, rostral peripheral nerves are likely to be patterned segmentally by the presence of somites, as spinal nerves are in vertebrates. (b) developmental logics responsible for the spatial distribution patterns of crest cells (peripheral nerve primordia) in vertebrate embryos. in all the vertebrate embryos examined, the cephalic crest cell streams adhere to the even - numbered rhombomeres (r2, r4, r6, and posterior) of the hindbrain, whereas in the trunk, crest cell streams are subdivided into a somitomeric pattern by the presence of somites (s). abbreviations : mb, midbrain ; ot, otic vesicle ; sc, spinal cord ; sp, spinal nerve primordia ; v, vii + viii, ix, and x, cranial nerve primordia. modified from kuratani (2004). (a) peripheral nerves of the amphioxus head. as indicated by arrows, rostral peripheral nerves are likely to be patterned segmentally by the presence of somites, as spinal nerves are in vertebrates. (b) developmental logics responsible for the spatial distribution patterns of crest cells (peripheral nerve primordia) in vertebrate embryos. in all the vertebrate embryos examined, the cephalic crest cell streams adhere to the even - numbered rhombomeres (r2, r4, r6, and posterior) of the hindbrain, whereas in the trunk, crest cell streams are subdivided into a somitomeric pattern by the presence of somites (s). abbreviations : mb, midbrain ; ot, otic vesicle ; sc, spinal cord ; sp, spinal nerve primordia ; v, vii + viii, ix, and x, cranial nerve primordia. modified from kuratani (2004). if the head mesoderm in vertebrates differs from somites in capability for developmental patterning, is there evidence of the remnants of head mesodermal segments in, for example, the head cavities ? balfour (1878) first described three pairs of epithelial cavities in the head of early pharyngula shark embryos. they were termed, from anterior to posterior, the premandibular, mandibular, and hyoid cavities (reviewed by g. northcutt in this issue) but were not clearly demarcated from the mesoderm in the pharynegal arches. the pharynegal arches are tube - like epithelial cavities ventrally confluent with the primordial pericardium. van wijhe (1882) redefined the head cavities as the dorsal paraxial moiety of the epithelial cavity, which he compared with the paraxial mesoderm, and the more ventrally located pharyngeal mesoderm was compared with the lateral mesodermal derivatives that are visceral. such head cavities are evident in mid - pharyngula as epithelial cysts floating in a loose mesenchyme in the paraxial domain of the head (van wijhe 1882 ; kuratani and horigome 2000 ; kuratani. 2000). of the head cavities, goodrich (1930) explained that the platt 's vesicle represented the ventral counterpart of the premandibular cavity (fig. historically, there has often been confusion about the definition of head cavities and the concept of head mesodermal domains. the mandibular cavity is thought to be the paraxial portion of the mandibular mesoderm and to be ventrally attached to the mandibular arch mesoderm. thus, in many vertebrate embryos, it corresponds to the ventral, mandibular arch mesoderm that engrailed cognates are expressed (hatta. 1990 ; holland. this expression persists in some of the muscles of the mandibular arch in gnathostomes and lampreys. by definition, this en expression can not be regarded as a mandibular cavity homologue (paraxial part) in the amphioxus as was once held (holland., the mandibular mesoderm mostly represents the pharyngeal arch of the mesoderm, and if this animal possesses a mandibular cavity - equivalent portion, it will be found only in a small dorsal portion of this mesoderm, which has not yet been identified. en expression in the mandibular mesoderm of the lamprey is, thus, most likely to represent a signal for specification of the mandibular arch muscle, as in gnathostomes, not for mesodermal segmentation. expression of a t - box gene, amphitbx15/18/22, in amphioxus somites (beaster - jones. 2006) appears to induce segmentation in the paraxial mesoderm, similar to amphien expression. in vertebrates, however, no pseudosegmental expression patterns of t - box genes have been reported for head mesoderm in vertebrates, although these genes are often reported to be expressed in crest - derived ectomesenchyme (haenig. expression of hox gene cognates by vertebrates and amphioxus may indicate that their mesodermal domains or neuraxial levels are similar (holland 1988, 1996a, 1996b, 2000). at that taxonomic level, however, morphological homology can not be definitively established because the morphological identities of structures may not be tightly linked to hox gene expression. even among vertebrates, hox gene expressions along the neuraxis are not always identical, although morphological homologies can be established among rhombomeres and cranial nerves (murakami. it is difficult to rule out the possibility of a vertebrate ancestor whose rostral paraxial mesoderm may have been segmented in a similar fashion to that of somites. however, animals with a common hox code does not necessarily mean that an identical set of morphological patterns will be established downstream of the code, as shown by fritzsch and northcutt (1993). thus, it does not seem possible at present to support a homology between rostral somites in amphioxus and head cavities in gnathostomes, as was first implied by van wijhe (1906) (cited by franz 1927) (for incomplete homology, see owen 1866 ; gegenbaur 1898 and tautz 1998 ; see also yasui. head cavities in the form of the epithelial cysts in elasmobranch embryos are also present in holocephalans, some actinopterygeans (premandibular and mandibular cavities in amia and sturgeon ; de beer 1924 ; kuratani. 2000) and in amniotes, including humans (usually only the premandibular cavity ; reviewed by kuratani 2003). no such epithelial cysts appear in the head mesoderm of the lamprey after disappearance of the original enterocoel. the presence of an enterocoel does not dictate a head cavity as the premandibular cavity of the chicken is not preceded by any enterocoelic precursor. it seems reasonable to assume that on the phylogenetic tree, head cavities represent a gnathostome synapomorphy and disappeared in a caudal to rostral direction in the more crown groups of vertebrates (reviewed by kuratani 2003, 2004). in this regard, although eye muscle primordia in the lamprey embryo were illustrated by koltzoff, they appear to be mesenchymal and no clear epithelial cysts were described to be associated with these muscle anlagen (koltzoff 1901). i have been unable to detect primordia of this particular eye muscle in l. japonicum embryos or larvae so far. the developmental fate of head cavities is not well understood except that they appear to differentiate into extrinsic eye muscles at histological levels, which is consistent with the one - to - one relationships between the eye - moving cranial nerves and the head cavities (van wijhe 1882). in avian embryos, the prechordal plate, the putative precursor of the premandibular cavity, has been shown by labeling of cells to differentiate into extrinsic eye muscle (jacob. it was suggested that their function is to regulate the rapid growth of the eye in some gnathostomes, but this does not explain why they are poorly developed in some amniotes that have larger eye primordia than do the chondrichthys (reviewed by kuratani 2003). it is intriguing that the head cavities of elasmobranch embryos occupy positions similar to those of the head mesodermal domains of the lamprey. the premandibular cavities and premandibular mesoderm are both located rostral to the notochord, the right and left moieties are united in the midline, the dorsal part of the mandibular mesoderm in the lamprey, and the head cavity are dorsal to the mandibular arch between the ophthalmic and maxillomandibular branches of the trigeminal nerve, and the dorsal hyoid mesoderm and hyoid cavity are dorsal to the hyoid arch (kuratani. 1999, 2000 ; kuratani and horigome 2000). this implies that the development of head cavities is under the same constraint that causes regionalization of the head mesoderm in the lamprey. because of the absence of a proper out - group, it is not easy to speculate about the ancestral state of the mesoderm of the vertebrate head. from the perspective of generative constraint, the head mesoderm of all the vertebrate species (even if it develops into head cavities) appears incapable of metamerical patterning of peripheral nerves as serial homologues of spinal nerves. thus, the primary pattern of the vertebrate head, which was explained by an archetypal segmental concept, is not determined by vestigial somites in the head. rather, a neurepithelial segmental mechanism (rhombomeres) and pharyngeal pouches determine the iterating pattern of the branchiomeric cranial nerves. all the vertebrate species, including cyclostomes and elasmobranchs, share the basic morphology of branchial nerves at embryonic stages (kuratani 1997). in the search for an ancestral somitomeric pattern in the mesoderm, the vertebrate head should be conceptualized as a modified plesiomorphic pattern similar to that of amphioxus, whose peripheral nerves are mostly patterned under somitomeric generative constraint (yasui. this does not imply, however, that the rostral nerves of amphioxus are homologous to spinal nerves. without rhombomeres and epibranchial placodes, it is, therefore, logically impossible to include vertebrate - like branchiomeric nerves and amphioxus - like rostral somites in segmentalist schemes, all of which are rather similar to goodrich 's scheme (jefferies 1986) (fig. 1a). to formulate the developmental patterning of vertebrates as a crown group of chordates, the absence of mesodermally derived, segmental generative constraint should be emphasized, not vice versa. developmentally, the expression patterns and functions of vertebrate hox genes, conspicuous in pharyngeal arch ectomesenchyme, and rhombomeres in the head and in somites in the trunk (reviewed by hunt and krumlauf 1991 ; hunt. 1993 ; graham 2001), as well as the contrasting homeotic responses to all - trans retinoic acid (pharyngeal arch - derivatives in the head, vertebral elements in the trunk) strongly imply that the vertebrate body plan is clearly and characteristically dissociated into head and trunk. this simultaneously emphasizes the lack of overt somitomerism (absence of the source of generative constraint) in the head. the head cavities are paraxial mesodermal structures that become evident secondarily in mid - pharyngula in concert with the regionalization pattern of the head mesoderm. however, histologically overt head cavities are only observed in gnathostomes and are likely to be a synapomorphy that defines this group. mesodermal segmentation in the head, which has not even been shown to exist, can not be regarded as a trait for defining vertebrates as a crown group of chordates. | because of its basal position on the phylogenetic tree of vertebrates, the lamprey embryo would be expected to exhibit segmental head mesoderm. recent observations, however, show that the lamprey does not have any somite - like segments in the head. coelomic head cavities that are most conspicuous in elasmobranch embryos, do not appear to represent universal vertebrate traits. from the perspective of generative constraint, segmental structures in the vertebrate body can be classified into primary segments, which arise as segmental embryonic primordia such as somites and pharyngeal pouches, and secondary segments whose patterns are determined by the presence of primary segments. secondary segments include neural crest derivatives and epibranchial placodes that are not initially segmented. the head mesoderm of vertebrates is secondarily regionalized into several domains that do not impose any secondary segmental patterns on other structures. thus, the vertebrate head is characterized by a lack of segmental generative constraint in its mesoderm. classical segmental theories are now refuted because they attempted to equate the vertebrate head with that of the amphioxus, whose rostral somites are considered primary segments, which are absent from vertebrates. |
in 2002, a global study reported that the us had among the highest reported age standardized incidence rate of bladder cancer (24.1/100,000). the national cancer institute (nci) estimated that in 2009, there would be approximately 70,980 new cases and 14,330 deaths from bladder cancer in the usa. despite reduced exposure to established risk factors such as smoking, aromatic arylamines (occupation), and schistosomal infection, the incidence of bladder cancer in the usa remains high. this is consistent with other studies suggesting that these risk factors only partly explain bladder cancer etiology [4, 5 ]. one etiologic study investigating regional variation in bladder cancer rates across the usa also considered diet as a possible risk factor. several studies [714 ] have reported that dietary factors such as fruit and vegetables may be protective against bladder cancer. however, there is a lack of consistent evidence between the intake of related micronutrient components of fruits and vegetables and reduced risk of the disease. the second expert report on nutrition and cancer by the world cancer research fund / american institute of cancer research states that the evidence was too limited to conclude that any food or nutrient directly influences the risk of bladder cancer. conversely, a recent systematic review as part of a who consultancy report found that nutrient components of specific fruits and vegetables have a possible inverse association with bladder cancer. it is these dietary micronutrients or their metabolites that actually come into direct contact with the bladder epithelium and thus in theory could be protective. vitamin a is obtained from the diet as preformed vitamin a from animal products and as carotenoids including both provitamin a and non - provitamin a carotenoids from plant foods. an inverse association between vitamin a intake and bladder cancer has been reported in some studies [17, 18 ], but not others [3, 1921 ]. some recent studies suggest that it is the carotenoids [8, 2224 ] (-carotene, -carotene [8, 25 ], -cryptoxanthin, lycopene, lutein / zeaxanthin, and not retinol [10, 23, 26 ] that influences the risk of bladder cancer. although, other studies have reported the opposite effects, with some finding that retinol may actually be protective [18, 2729 ] and others that the carotenoids have no effect [3, 10, 19, 30, 31 ]. the relationship between bladder cancer and vitamin c and e also appears to be unclear with some studies reporting potential inverse associations (vitamin c : [29, 32, 33 ] ; vitamin e : [20, 27 ]) and others no associations at all (vitamin c : [3, 19, 21, 23, 25 ] ; vitamin e : [3, 19, 21, 23, 25, 31 ]). deficiencies in folic acid, vitamins b3 (niacin), b6, b12, have been reported to mimic radiation damage to dna and possibly lead to the initiation of cancer. however, there is only limited literature available on the association between water - soluble b - group vitamins and risk of bladder cancer, and the findings have been inconsistent [20, 21, 23, 25, 28, 35 ]. he concluded that the potential for certain dietary minerals to influence urine composition and hence bladder carcinogenesis observed in animal studies needed further evaluation before extrapolation could be made to human populations. to date, few epidemiological studies have investigated the effect of dietary minerals such as sodium [20, 23 ], potassium, calcium [20, 23, 26 ], magnesium, and iron [20, 26 ] on the risk of developing bladder cancer. micronutrient deficiencies could provide a possible explanation for why an estimated 25% of the us population who consume the least fruit and vegetables have double the cancer rate. the aim of our study was to investigate the association between major dietary minerals and vitamins and the risk of bladder cancer in a us population from a region with a high incidence rate. a population - based case control study was conducted in new hampshire, usa. briefly, bladder cancer cases were identified from the new hampshire state department of health and human services cancer registry as histologically confirmed, primary bladder cancer diagnosed between 1 july 1998 and 31 december 2001. to be eligible for inclusion in the study, all cases had to be new hampshire residents aged between 25 and 74, have a listed phone number and speak english. physician consent was obtained before contacting potential participants. of the 472 potentially eligible cases we contacted,, controls were shared with another study on non - melanoma cancer covering a reference period from 1 july 1997 to 30 march 2000. controls less than 65 years of age were selected from lists obtained from the new hampshire department of transportation. controls 65 years of age and older were chosen from data files provided by the centers for medicare & medicaid services (cms) of new hampshire. a total of 526 controls (76%) were interviewed from a potential 694 confirmed eligible participants. extensive in - person interviews were conducted with consenting participants, usually in the homes of the participants. data were collected on participants socio - demographic information such as education level, residence, occupation (history), medical history, lifestyle factors (including tobacco smoking), household water supply, and family history of cancer. interviewers were blind to case / control status and interviews were tape recorded with subjects consent (less than 5% refused to have the interview taped) to ensure consistent quality of the interview and clarification of details. cases were also asked if they held a driver s license or a medicare enrollment card for comparability with controls. interviews which included dietary assessment took place between 2000 and 2003 for both cases and controls (for 95% of subjects). as data were unavailable for 150 cases and 455 controls ; a total of 322 cases and 239 controls were included in the dietary analyses of this study. subjects from whom dietary data were collected were comparable to those who did not provide dietary data with respect to age, sex, and smoking history (data not shown). dietary information was collected from a 121-item semi - quantitative food frequency questionnaire (ffq) that was developed by willett. for the nurses health study. these included dairy, fruit, vegetables, eggs and meat, breads, beverages, and baked goods. there was a list of nine common mineral and vitamin supplements to choose from which included the following : multiple vitamins (participants were asked to report the individual vitamins within this category), vitamins a, c, b6 and e, selenium, iron, zinc, and calcium. additional questions covered folic acid, vitamin d, vitamin b complex, cod liver oil, omega 3 fatty acids, iodine, copper, brewer s yeast, -carotene, magnesium, and an open ended option, other. participants were provided with options for duration of supplement intake ranging from 0 to 10 + years. nutrient intake was calculated by multiplying frequency of each food item on the ffq by the nutrient content for the specific portion size. food composition data were based primarily on data from the united states department of agriculture (usda). we investigated vitamins that had a biologically plausible association with bladder cancer and macro - minerals, which are required in quantities greater than 100 mg per day e.g., sodium, potassium, calcium, phosphorus, and magnesium. an exception was iron, which was included due to its physiological function and potential to influence the risk of bladder cancer. we investigated the association between total mineral and vitamin intake, which included micronutrients from both dietary and supplemental sources and bladder cancer risk. the intake of carotenoids, folate, and vitamin b12 were measured in micrograms (g) per day. all other vitamins including, b1 (thiamin), b2 (riboflavin), b3 (niacin), vitamin c and vitamin e, and minerals (sodium, potassium, calcium, phosphorus, magnesium, and iron) were calculated in milligrams (mg) per day except for vitamin d intake, which was calculated in international units (iu). adjustment was made for a number of possible confounding variables including sex, age, cigarette smoking status (current / non - current smoker), pack years of cigarette smoking (categories : 0 < 10, 10 < 20, 20 < 30, 30 < 40, 40 < 50, 50 ; calculated by dividing the number of cigarettes smoked per day by 20 and multiplying by the number of years smoked) and total energy intake (quartiles for kcal / day). additional adjustment for other potential confounders, total fat intake (fat - soluble vitamins), alcohol and coffee consumption (water - soluble vitamins) did not alter the associations between micronutrients and bladder cancer. odds ratios and 95% cis were calculated to investigate potential differences between invasive and non - invasive bladder cancer. we also performed additional analyses to assess the effects of minerals and vitamins from both dietary and supplemental sources separately. intake of nutrients from dietary sources were analyzed in quartiles using the same statistical model for total intake of minerals and vitamins. we estimated the intake of minerals and vitamins from supplements by subtracting dietary intake from total nutrient intake. due to the relatively small contribution to mineral and vitamin intake by supplements in this study population likelihood ratio tests were conducted to test for trend by assigning an integer to each quartile of the nutrient e.g., 14 then fit the term as a continuous variable in the model. the joint effects of specific nutrients and smoking status / history and age were also investigated. nutrients and age were dichotomized into high and low categories based on median values of the controls. smoking status was dichotomized into never / ever smoker, cigarettes per day into high and low categories (< 20/20 cigarettes per day) and duration of smoking (< 27 years/27 years). we used the likelihood ratio test to evaluate interaction, i.e., comparing the log likelihood of regression models with and without the interaction term included. the false - positive report probability (fprp) was employed to assess the probability of no true association between nutrients and the risk of bladder cancer given a statistically significant finding. this test takes into account three factors that determine the probability that a statistically significant result may actually be a false - positive finding. these are (1) prior probability of an association ; (2) the alpha - level, and (3) statistical power to detect an odds ratio for the alternative hypothesis at a given alpha - level or p value. we used previously reported arbitrary values for these factors : 0.05 (alpha - level), 0.25 (prior probability), and 0.5 (false - positive report probability) [46, 47 ]. all reported p values were two - sided and values less than or equal to 0.05 were considered to be statistically significant. the statistical software package stata / se 10.0 (stata corporation, college station, tx, usa) was used for all statistical analyses. a population - based case control study was conducted in new hampshire, usa. briefly, bladder cancer cases were identified from the new hampshire state department of health and human services cancer registry as histologically confirmed, primary bladder cancer diagnosed between 1 july 1998 and 31 december 2001. to be eligible for inclusion in the study, all cases had to be new hampshire residents aged between 25 and 74, have a listed phone number and speak english. physician consent was obtained before contacting potential participants. of the 472 potentially eligible cases we contacted,, controls were shared with another study on non - melanoma cancer covering a reference period from 1 july 1997 to 30 march 2000. controls less than 65 years of age were selected from lists obtained from the new hampshire department of transportation. controls 65 years of age and older were chosen from data files provided by the centers for medicare & medicaid services (cms) of new hampshire. a total of 526 controls (76%) were interviewed from a potential 694 confirmed eligible participants. extensive in - person interviews were conducted with consenting participants, usually in the homes of the participants. data were collected on participants socio - demographic information such as education level, residence, occupation (history), medical history, lifestyle factors (including tobacco smoking), household water supply, and family history of cancer. interviewers were blind to case / control status and interviews were tape recorded with subjects consent (less than 5% refused to have the interview taped) to ensure consistent quality of the interview and clarification of details. cases were also asked if they held a driver s license or a medicare enrollment card for comparability with controls. interviews which included dietary assessment took place between 2000 and 2003 for both cases and controls (for 95% of subjects). as data were unavailable for 150 cases and 455 controls ; a total of 322 cases and 239 controls were included in the dietary analyses of this study. subjects from whom dietary data were collected were comparable to those who did not provide dietary data with respect to age, sex, and smoking history (data not shown). dietary information was collected from a 121-item semi - quantitative food frequency questionnaire (ffq) that was developed by willett. for the nurses health study. these included dairy, fruit, vegetables, eggs and meat, breads, beverages, and baked goods. there was a list of nine common mineral and vitamin supplements to choose from which included the following : multiple vitamins (participants were asked to report the individual vitamins within this category), vitamins a, c, b6 and e, selenium, iron, zinc, and calcium. additional questions covered folic acid, vitamin d, vitamin b complex, cod liver oil, omega 3 fatty acids, iodine, copper, brewer s yeast, -carotene, magnesium, and an open ended option, other. participants were provided with options for duration of supplement intake ranging from 0 to 10 + years. nutrient intake was calculated by multiplying frequency of each food item on the ffq by the nutrient content for the specific portion size. food composition data were based primarily on data from the united states department of agriculture (usda). we investigated vitamins that had a biologically plausible association with bladder cancer and macro - minerals, which are required in quantities greater than 100 mg per day e.g., sodium, potassium, calcium, phosphorus, and magnesium. an exception was iron, which was included due to its physiological function and potential to influence the risk of bladder cancer. we investigated the association between total mineral and vitamin intake, which included micronutrients from both dietary and supplemental sources and bladder cancer risk. the intake of carotenoids, folate, and vitamin b12 were measured in micrograms (g) per day. all other vitamins including, b1 (thiamin), b2 (riboflavin), b3 (niacin), vitamin c and vitamin e, and minerals (sodium, potassium, calcium, phosphorus, magnesium, and iron) were calculated in milligrams (mg) per day except for vitamin d intake, which was calculated in international units (iu). adjustment was made for a number of possible confounding variables including sex, age, cigarette smoking status (current / non - current smoker), pack years of cigarette smoking (categories : 0 < 10, 10 < 20, 20 < 30, 30 < 40, 40 < 50, 50 ; calculated by dividing the number of cigarettes smoked per day by 20 and multiplying by the number of years smoked) and total energy intake (quartiles for kcal / day). additional adjustment for other potential confounders, total fat intake (fat - soluble vitamins), alcohol and coffee consumption (water - soluble vitamins) did not alter the associations between micronutrients and bladder cancer. odds ratios and 95% cis were calculated to investigate potential differences between invasive and non - invasive bladder cancer. we also performed additional analyses to assess the effects of minerals and vitamins from both dietary and supplemental sources separately. intake of nutrients from dietary sources were analyzed in quartiles using the same statistical model for total intake of minerals and vitamins. we estimated the intake of minerals and vitamins from supplements by subtracting dietary intake from total nutrient intake. due to the relatively small contribution to mineral and vitamin intake by supplements in this study population, these nutrients were dichotomized according to average intake of controls. likelihood ratio tests were conducted to test for trend by assigning an integer to each quartile of the nutrient e.g., 14 then fit the term as a continuous variable in the model. the joint effects of specific nutrients and smoking status / history and age were also investigated. nutrients and age were dichotomized into high and low categories based on median values of the controls. smoking status was dichotomized into never / ever smoker, cigarettes per day into high and low categories (< 20/20 cigarettes per day) and duration of smoking (< 27 years/27 years). we used the likelihood ratio test to evaluate interaction, i.e., comparing the log likelihood of regression models with and without the interaction term included. the false - positive report probability (fprp) was employed to assess the probability of no true association between nutrients and the risk of bladder cancer given a statistically significant finding. this test takes into account three factors that determine the probability that a statistically significant result may actually be a false - positive finding. these are (1) prior probability of an association ; (2) the alpha - level, and (3) statistical power to detect an odds ratio for the alternative hypothesis at a given alpha - level or p value. we used previously reported arbitrary values for these factors : 0.05 (alpha - level), 0.25 (prior probability), and 0.5 (false - positive report probability) [46, 47 ]. all reported p values were two - sided and values less than or equal to 0.05 were considered to be statistically significant. the statistical software package stata / se 10.0 (stata corporation, college station, tx, usa) was used for all statistical analyses. table 1 presents the frequency distributions of selected study characteristics for the 322 cases and 239 controls for whom dietary data were available for analyses in this study. bladder cancer cases consisted of approximately three times more men (74%) than women. a majority of both cases and controls were in the older age group (60 years and over). there was a higher prevalence of current smokers and greater number of both cigarettes smoked per day and years smoked among cases compared with the controls. we detected no statistically significant differences between cases and controls for calorie, total fat, alcohol, or coffee intake.table 1distribution of bladder cancer cases and controls by demographic characteristics and bladder cancer risk factorscases number (%) controls number (%) total322239sex men237 (74)138 (58) women85 (26)101 (42)age (years) [mean (sd)]62 (9.2)60.7 (10.6)smoking status never smokers56 (17.4)95 (39.7) ex - smokers162 (50.3)103 (43.1) current smokers104 (32.3)41 (17.2) years smoking [mean (sd)]32.2 (14)27.4 (15) cigarettes / day [mean (sd)]26.7 (14.8)22.3 (15.6)total calories kcal / day [mean (sd)]1962.56 (790.72)1887.74 (706.64)total fat g / day [mean (sd)]71.74 (32.96)69.77 (31.76)total alcohol g / day [mean (sd)]7.91 (15.84)8.29 (14.45)total coffee g / day [mean (sd)]227.41 (210.13)216.57 (192.88)tumor stage at diagnosis carcinoma in situ (%) 16 (5) non - invasive / low grade (%) 190 (59) non - invasive / high grade (%) 23 (7) invasive (%) 93 (29)sd standard deviation statistically significant differences between cases and controls ; p < 0.05 distribution of bladder cancer cases and controls by demographic characteristics and bladder cancer risk factors sd standard deviation statistically significant differences between cases and controls ; p < 0.05 table 2 shows the ors and 95% cis for bladder cancer and total intake (including dietary and supplemental sources) of fat - soluble and water - soluble vitamins. we observed a borderline statistically significant inverse association between total intake of vitamin e and bladder cancer (highest vs. lowest quartile, or : 0.66 ; 95% ci : 0.361.20 ; p trend = 0.09). no statistically significant ors were observed when comparing the highest quartile of intake with the lowest quartile for carotenoids as a group or for any of the individual carotenoids, -carotene, -carotene, -cryptoxanthin, lycopene, or lutein / zeaxanthin. although the point estimate for vitamin d was well below one, this also did not reach statistical significance (or : 0.58 ; 95% ci : 0.311.06 ; p trend = 0.22).table 2calculated odds ratios (95%) for total intake of vitamins and bladder cancerdaily intakeq1q2q3q4p trendalpha - carotene (g)3.4404404.1647.13647.141104.701104.710.26 cases / controls76/5989/6077/6177/59 ors (95% ci)1.001.33 (0.772.30)1.19 (0.682.11)1.48 (0.832.64)beta - carotene (g)292.292294.212294.223773.803773.815517.205517.210.90 cases / controls94/5978/6165/6079/59 ors (95% ci)1.000.87 (0.501.50)0.80 (0.461.39)0.99 (0.561.72)beta - cryptoxanthin (g)052.6052.61125.77125.78215.71215.720.65 cases / controls109/5976/6159/5878/61 ors (95% ci)1.000.69 (0.401.20)0.66 (0.371.16)0.87 (0.501.53)lycopene (g)03092.683092.69482748287746.437746.440.74 cases / controls89/5973/6072/6088/60 ors (95% ci)1.000.96 (0.551.66)0.70 (0.391.24)0.98 (0.551.73)lutein (g)0.931261.481261.491942.441942.453134.853134.860.66 cases / controls116/5861/6174/6070/59 ors (95% ci)1.000.52 (0.300.90)0.76 (0.451.31)0.80 (0.461.40)total carotenoids (g)398.268879.228879.2312900.6012900.6117932.7217932.730.47 cases / controls107/5973/6052/6087/60 ors (95% ci)1.000.70 (0.411.21)0.49 (0.270.86)0.90 (0.511.59)vitamin d (iu)15.53171.74171.75388.90388.91641.12641.130.22 cases / controls89/5972/60100/6061/60 ors (95% ci)1.000.78 (0.441.38)1.03 (0.601.76)0.58 (0.311.06)vitamin e (mg)1.137.267.2720.9420.95193.35193.360.09 cases / controls77/5795/5881/5754/57 ors (95% ci)1.001.19 (0.682.08)0.88 (0.491.56)0.66 (0.361.20)thiamin (mg)0.211.321.332.112.123.343.350.50 cases / controls91/5863/5999/6367/58 ors (95% ci)1.000.53 (0.290.96)0.97 (0.561.68)0.63 (0.351.14)riboflavin (mg)0.251.631.642.752.764.044.050.61 cases / controls85/6074/5991/6171/59 ors (95% ci)1.000.73 (0.411.29)1.02 (0.581.77)0.75 (0.421.34)niacin (b3) (mg)5.7721.9121.9233.0733.0846.5046.510.23 cases / controls90/5868/6093/6067/60 ors (95% ci)1.000.59 (0.331.06)0.94 (0.541.64)0.56 (0.311.02)vitamin b6 (mg)0.321.941.953.283.294.884.890.41 cases / controls93/5975/5977/6175/59 ors (95% ci)1.000.67 (0.381.18)0.82 (0.471.44)0.72 (0.411.27)vitamin b12 (g)0.755.515.5210.8210.8318.018.10.75 cases / controls79/6089/6065/6087/59 ors (95% ci)1.001.30 (0.752.26)0.84 (0.471.51)1.05 (0.601.84)folate (g)48.41339.03339.04579.10579.11819.50819.510.43 cases / controls82/5982/6185/5971/60 ors (95% ci)1.000.83 (0.471.46)0.91 (0.531.56)0.74 (0.401.35)vitamin c (mg)14.61113.03113.04190.25190.26366.10366.110.42 cases / controls98/5878/6080/6162/59 ors (95% ci)1.000.83 (0.481.43)0.84 (0.481.45)0.78 (0.451.38)or odds ratios, ci confidence intervals, adjusted for age, sex, smoking status : current versus non - current smoker, pack years smoked (categories : 010, 1020, 2030, 3040, 4050, 50 +), and total energy intake (quartiles kcal / day) calculated odds ratios (95%) for total intake of vitamins and bladder cancer or odds ratios, ci confidence intervals, adjusted for age, sex, smoking status : current versus non - current smoker, pack years smoked (categories : 010, 1020, 2030, 3040, 4050, 50 +), and total energy intake (quartiles kcal / day) among the water - soluble vitamins, the or for niacin and bladder cancer was of borderline statistical significance (highest vs. lowest quartile or : 0.56 ; 95% ci : 0.311.02). no other associations were observed between bladder cancer and any other b - group or water - soluble vitamins, which included thiamin, riboflavin, b6, b12, and folate or vitamin c. similarly, no associations were observed between bladder cancer and total intake for any of the minerals reported in table 3, including sodium, potassium, magnesium, calcium, phosphorus, and iron. nor were there any detectable differences in the effects of any of the minerals and vitamins between invasive and non - invasive bladder cancer.table 3calculated odds ratios (95%) for total intake of minerals and bladder cancerdaily intakeq1q2q3q4p trendsodium (mg)344.611484.831484.841898.641898.652372.652372.660.32 cases / controls78/5864/6059/60119/60 ors (95% ci)1.001.17 (0.612.23)1.00 (0.482.12)1.66 (0.703.92)potassium (mg)535.632382.52382.62874.242874.253781.333781.340.58 cases / controls101/5945/6095/6180/59 ors (95% ci)1.000.50 (0.270.91)1.03 (0.532.0)0.65 (0.291.45)calcium (mg)86.63600.36600.37918.23918.241346.41346.50.27 cases / controls99/5973/6090/5960/60 ors (95% ci)1.000.60 (0.351.05)0.88 (0.501.53)0.60 (0.321.13)magnesium (mg)58.76259.03259.04328.08328.09418.58418.590.20 cases / controls95/5962/6183/6080/59 ors (95% ci)1.000.65 (0.361.15)0.79 (0.431.44)0.56 (0.271.16)phosphorus (mg)177.85965.2965.31195.671195.681556.51556.60.46 cases / controls93/6065/5981/6183/59 ors (95% ci)1.001.02 (0.551.92)0.93 (0.441.98)0.70 (0.281.75)iron (mg)2.1211.2511.2615.5615.5723.2023.210.93 cases / controls82/5975/6077/6086/60 ors (95% ci)1.001.04 (0.571.90)0.92 (0.491.73)1.06 (0.571.96)or odds ratios, ci confidence intervals, adjusted for age, sex, smoking status : current versus non - current smoker, pack years smoked (categories : 010, 1020, 2030, 3040, 4050, 50 +), and total energy intake (quartiles kcal / day) calculated odds ratios (95%) for total intake of minerals and bladder cancer or odds ratios, ci confidence intervals, adjusted for age, sex, smoking status : current versus non - current smoker, pack years smoked (categories : 010, 1020, 2030, 3040, 4050, 50 +), and total energy intake (quartiles kcal / day) analyses were repeated for all minerals and vitamins using measures taken from both dietary sources (excluding supplements) and supplements alone (data not shown). the only nutrient of interest to emerge from dietary sources alone when comparing highest quartiles of intake with lowest quartiles of intake, although not statistically significant was phosphorus (or : 0.49 ; 95% ci : 0.211.17 ; p trend = 0.13). no other associations or trends could be observed for any of the other dietary or supplemental sources of minerals and vitamins. we continued analyses for minerals and vitamins where an association had been indicated either from borderline associations and biological plausibility or from previous studies. minerals and vitamins that met the criteria were investigated for interactions with smoking characteristics and age, which were both adjusted for each other. the joint effects of nutrient intake and established risk factors e.g., smoking status (ever / never), cigarettes per day (< 20/20/day), duration of smoking (< 27 years/27 years), and age (< 63 years/63 years) are presented in table 4. the or for heavy cigarette smokers (20 cigarettes per day) in the highest group for total intake of vitamin e, was 0.58 (95% : 0.340.99 ; p interaction 0.03). inverse associations of borderline statistical significance were also observed among heavy smokers for the highest intake of total carotenoids (or : 0.62 ; 95% ci : 0.361.09 ; p interaction 0.08) and niacin (or : 0.66 ; 95% ci : 0.391.14 ; p interaction 0.08). interaction between thiamin intake and the number of cigarettes smoked per day was also of borderline statistical significance. possible reductions in risk of bladder cancer for older participants were also associated with higher intakes of total carotenoids (or : 0.59 ; 95% ci : 0.350.99), vitamin d, thiamin, and niacin (p interaction : 0.04, 0.02, and 0.03, respectively). no other statistically significant associations were observed, although there were reduced odds of bladder cancer among the older age group for the highest intake of vitamin e (or : 0.61 ; 95% ci : 0.371.02) and interactions between vitamin c and ever - smoking (p = 0.08) and vitamin c and age (p = 0.08) were borderline (data not shown).table 4odds ratios (95% confidence intervals) for the joint effects of total intake of selected nutrients (highest vs. lowest) and smoking characteristics and agecarotenoidsvitamin dvitamin ethiaminniacin<12900.6 g12900.6 gors (95% ci)<388.91 iu388.91 iuors (95% ci)<20.7 (mg)20.7 (mg)ors (95% ci)<2.12 (mg)2.12 (mg)ors (95% ci)<33.08 (mg)33.08 (mg)ors (95% ci)cases / controlscases / controlscases / controlscases / controlscases / controlscases / controlscases / controlscases / controlscases / controlscases / controlssmoking status never28/4328/520.66 (0.321.35)29/5427/411.04 (0.502.15)33/5120/420.64 (0.311.33)23/5033/441.35 (0.662.77)27/5329/411.10 (0.542.26) ever155/76111/680.75 (0.481.17)132/65134/790.82 (0.541.24)140/64117/730.73 (0.481.13)134/70132/740.91 (0.591.39)135/66131/770.80 (0.521.23) p interaction0.980.410.870.270.26cigarettes / day < 2025/3026/231.65 (0.683.99)19/2632/271.58 (0.663.79)20/2731/231.57 (0.633.88)19/2832/251.85 (0.784.41)20/2731/251.64 (0.683.95) 20127/4484/440.62 (0.361.09)111/38100/500.73 (0.431.23)118/3685/480.58 (0.340.99)114/4197/470.78 (0.461.34)114/3897/500.66 (0.391.14) p interaction0.080.140.030.100.08years smoking < 2751/3845/341.19 (0.582.47)49/3247/400.78 (0.401.52)49/3645/321.08 (0.542.16)46/3050/420.71 (0.351.43)47/3049/410.70 (0.351.41) 27101/3764/330.62 (0.331.16)81/3284/381.00 (0.551.81)89/2770/400.61 (0.331.12)87/3978/311.41 (0.762.59)87/3578/351.02 (0.561.85) p interaction0.200.580.240.190.51age < 6373/5969/540.91 (0.491.68)76/6966/441.55 (0.882.74)79/6156/471.12 (0.622.01)69/6573/472.02 (1.113.68)69/6373/481.59 (0.882.87) 63110/6070/660.59 (0.350.99)85/5095/760.65 (0.401.08)94/5481/680.61 (0.371.02)88/5592/710.75 (0.451.24)93/5687/700.64 (0.381.07) p interaction0.180.040.180.020.03iu international units, mg milligrams, low intakes were categorized below median and high intakes above mediansadjusted for age, sex, smoking status (current / non - current), number of cigarettes per day, and years of smoking adjusted for age, sex, and kcal only odds ratios (95% confidence intervals) for the joint effects of total intake of selected nutrients (highest vs. lowest) and smoking characteristics and age iu international units, mg milligrams, low intakes were categorized below median and high intakes above medians adjusted for age, sex, smoking status (current / non - current), number of cigarettes per day, and years of smoking adjusted for age, sex, and kcal only the statistically significant inverse associations observed between total intake of carotenoids, vitamin d, thiamin, and niacin and the risk of bladder cancer for older individuals remained robust following the fprp test. each inverse association had less than a 50% likelihood of being false positive for an odds ratio of 1.5 and based on a prior probability of 25%, table 1 presents the frequency distributions of selected study characteristics for the 322 cases and 239 controls for whom dietary data were available for analyses in this study. bladder cancer cases consisted of approximately three times more men (74%) than women. a majority of both cases and controls were in the older age group (60 years and over). there was a higher prevalence of current smokers and greater number of both cigarettes smoked per day and years smoked among cases compared with the controls. we detected no statistically significant differences between cases and controls for calorie, total fat, alcohol, or coffee intake.table 1distribution of bladder cancer cases and controls by demographic characteristics and bladder cancer risk factorscases number (%) controls number (%) total322239sex men237 (74)138 (58) women85 (26)101 (42)age (years) [mean (sd)]62 (9.2)60.7 (10.6)smoking status never smokers56 (17.4)95 (39.7) ex - smokers162 (50.3)103 (43.1) current smokers104 (32.3)41 (17.2) years smoking [mean (sd)]32.2 (14)27.4 (15) cigarettes / day [mean (sd)]26.7 (14.8)22.3 (15.6)total calories kcal / day [mean (sd)]1962.56 (790.72)1887.74 (706.64)total fat g / day [mean (sd)]71.74 (32.96)69.77 (31.76)total alcohol g / day [mean (sd)]7.91 (15.84)8.29 (14.45)total coffee g / day [mean (sd)]227.41 (210.13)216.57 (192.88)tumor stage at diagnosis carcinoma in situ (%) 16 (5) non - invasive / low grade (%) 190 (59) non - invasive / high grade (%) 23 (7) invasive (%) 93 (29)sd standard deviation statistically significant differences between cases and controls ; p < 0.05 distribution of bladder cancer cases and controls by demographic characteristics and bladder cancer risk factors sd standard deviation statistically significant differences between cases and controls ; p < 0.05 table 2 shows the ors and 95% cis for bladder cancer and total intake (including dietary and supplemental sources) of fat - soluble and water - soluble vitamins. we observed a borderline statistically significant inverse association between total intake of vitamin e and bladder cancer (highest vs. lowest quartile, or : 0.66 ; 95% ci : 0.361.20 ; p trend = 0.09). no statistically significant ors were observed when comparing the highest quartile of intake with the lowest quartile for carotenoids as a group or for any of the individual carotenoids, -carotene, -carotene, -cryptoxanthin, lycopene, or lutein / zeaxanthin. although the point estimate for vitamin d was well below one, this also did not reach statistical significance (or : 0.58 ; 95% ci : 0.311.06 ; p trend = 0.22).table 2calculated odds ratios (95%) for total intake of vitamins and bladder cancerdaily intakeq1q2q3q4p trendalpha - carotene (g)3.4404404.1647.13647.141104.701104.710.26 cases / controls76/5989/6077/6177/59 ors (95% ci)1.001.33 (0.772.30)1.19 (0.682.11)1.48 (0.832.64)beta - carotene (g)292.292294.212294.223773.803773.815517.205517.210.90 cases / controls94/5978/6165/6079/59 ors (95% ci)1.000.87 (0.501.50)0.80 (0.461.39)0.99 (0.561.72)beta - cryptoxanthin (g)052.6052.61125.77125.78215.71215.720.65 cases / controls109/5976/6159/5878/61 ors (95% ci)1.000.69 (0.401.20)0.66 (0.371.16)0.87 (0.501.53)lycopene (g)03092.683092.69482748287746.437746.440.74 cases / controls89/5973/6072/6088/60 ors (95% ci)1.000.96 (0.551.66)0.70 (0.391.24)0.98 (0.551.73)lutein (g)0.931261.481261.491942.441942.453134.853134.860.66 cases / controls116/5861/6174/6070/59 ors (95% ci)1.000.52 (0.300.90)0.76 (0.451.31)0.80 (0.461.40)total carotenoids (g)398.268879.228879.2312900.6012900.6117932.7217932.730.47 cases / controls107/5973/6052/6087/60 ors (95% ci)1.000.70 (0.411.21)0.49 (0.270.86)0.90 (0.511.59)vitamin d (iu)15.53171.74171.75388.90388.91641.12641.130.22 cases / controls89/5972/60100/6061/60 ors (95% ci)1.000.78 (0.441.38)1.03 (0.601.76)0.58 (0.311.06)vitamin e (mg)1.137.267.2720.9420.95193.35193.360.09 cases / controls77/5795/5881/5754/57 ors (95% ci)1.001.19 (0.682.08)0.88 (0.491.56)0.66 (0.361.20)thiamin (mg)0.211.321.332.112.123.343.350.50 cases / controls91/5863/5999/6367/58 ors (95% ci)1.000.53 (0.290.96)0.97 (0.561.68)0.63 (0.351.14)riboflavin (mg)0.251.631.642.752.764.044.050.61 cases / controls85/6074/5991/6171/59 ors (95% ci)1.000.73 (0.411.29)1.02 (0.581.77)0.75 (0.421.34)niacin (b3) (mg)5.7721.9121.9233.0733.0846.5046.510.23 cases / controls90/5868/6093/6067/60 ors (95% ci)1.000.59 (0.331.06)0.94 (0.541.64)0.56 (0.311.02)vitamin b6 (mg)0.321.941.953.283.294.884.890.41 cases / controls93/5975/5977/6175/59 ors (95% ci)1.000.67 (0.381.18)0.82 (0.471.44)0.72 (0.411.27)vitamin b12 (g)0.755.515.5210.8210.8318.018.10.75 cases / controls79/6089/6065/6087/59 ors (95% ci)1.001.30 (0.752.26)0.84 (0.471.51)1.05 (0.601.84)folate (g)48.41339.03339.04579.10579.11819.50819.510.43 cases / controls82/5982/6185/5971/60 ors (95% ci)1.000.83 (0.471.46)0.91 (0.531.56)0.74 (0.401.35)vitamin c (mg)14.61113.03113.04190.25190.26366.10366.110.42 cases / controls98/5878/6080/6162/59 ors (95% ci)1.000.83 (0.481.43)0.84 (0.481.45)0.78 (0.451.38)or odds ratios, ci confidence intervals, adjusted for age, sex, smoking status : current versus non - current smoker, pack years smoked (categories : 010, 1020, 2030, 3040, 4050, 50 +), and total energy intake (quartiles kcal / day) calculated odds ratios (95%) for total intake of vitamins and bladder cancer or odds ratios, ci confidence intervals, adjusted for age, sex, smoking status : current versus non - current smoker, pack years smoked (categories : 010, 1020, 2030, 3040, 4050, 50 +), and total energy intake (quartiles kcal / day) among the water - soluble vitamins, the or for niacin and bladder cancer was of borderline statistical significance (highest vs. lowest quartile or : 0.56 ; 95% ci : 0.311.02). no other associations were observed between bladder cancer and any other b - group or water - soluble vitamins, which included thiamin, riboflavin, b6, b12, and folate or vitamin c. similarly, no associations were observed between bladder cancer and total intake for any of the minerals reported in table 3, including sodium, potassium, magnesium, calcium, phosphorus, and iron. nor were there any detectable differences in the effects of any of the minerals and vitamins between invasive and non - invasive bladder cancer.table 3calculated odds ratios (95%) for total intake of minerals and bladder cancerdaily intakeq1q2q3q4p trendsodium (mg)344.611484.831484.841898.641898.652372.652372.660.32 cases / controls78/5864/6059/60119/60 ors (95% ci)1.001.17 (0.612.23)1.00 (0.482.12)1.66 (0.703.92)potassium (mg)535.632382.52382.62874.242874.253781.333781.340.58 cases / controls101/5945/6095/6180/59 ors (95% ci)1.000.50 (0.270.91)1.03 (0.532.0)0.65 (0.291.45)calcium (mg)86.63600.36600.37918.23918.241346.41346.50.27 cases / controls99/5973/6090/5960/60 ors (95% ci)1.000.60 (0.351.05)0.88 (0.501.53)0.60 (0.321.13)magnesium (mg)58.76259.03259.04328.08328.09418.58418.590.20 cases / controls95/5962/6183/6080/59 ors (95% ci)1.000.65 (0.361.15)0.79 (0.431.44)0.56 (0.271.16)phosphorus (mg)177.85965.2965.31195.671195.681556.51556.60.46 cases / controls93/6065/5981/6183/59 ors (95% ci)1.001.02 (0.551.92)0.93 (0.441.98)0.70 (0.281.75)iron (mg)2.1211.2511.2615.5615.5723.2023.210.93 cases / controls82/5975/6077/6086/60 ors (95% ci)1.001.04 (0.571.90)0.92 (0.491.73)1.06 (0.571.96)or odds ratios, ci confidence intervals, adjusted for age, sex, smoking status : current versus non - current smoker, pack years smoked (categories : 010, 1020, 2030, 3040, 4050, 50 +), and total energy intake (quartiles kcal / day) calculated odds ratios (95%) for total intake of minerals and bladder cancer or odds ratios, ci confidence intervals, adjusted for age, sex, smoking status : current versus non - current smoker, pack years smoked (categories : 010, 1020, 2030, 3040, 4050, 50 +), and total energy intake (quartiles kcal / day) analyses were repeated for all minerals and vitamins using measures taken from both dietary sources (excluding supplements) and supplements alone (data not shown). the only nutrient of interest to emerge from dietary sources alone when comparing highest quartiles of intake with lowest quartiles of intake, although not statistically significant was phosphorus (or : 0.49 ; 95% ci : 0.211.17 ; p trend = 0.13). no other associations or trends could be observed for any of the other dietary or supplemental sources of minerals and vitamins. we continued analyses for minerals and vitamins where an association had been indicated either from borderline associations and biological plausibility or from previous studies. minerals and vitamins that met the criteria were investigated for interactions with smoking characteristics and age, which were both adjusted for each other. the joint effects of nutrient intake and established risk factors e.g., smoking status (ever / never), cigarettes per day (< 20/20/day), duration of smoking (< 27 years/27 years), and age (< 63 years/63 years) are presented in table 4. the or for heavy cigarette smokers (20 cigarettes per day) in the highest group for total intake of vitamin e, was 0.58 (95% : 0.340.99 ; p interaction 0.03). inverse associations of borderline statistical significance were also observed among heavy smokers for the highest intake of total carotenoids (or : 0.62 ; 95% ci : 0.361.09 ; p interaction 0.08) and niacin (or : 0.66 ; 95% ci : 0.391.14 ; p interaction 0.08). interaction between thiamin intake and the number of cigarettes smoked per day was also of borderline statistical significance. possible reductions in risk of bladder cancer for older participants were also associated with higher intakes of total carotenoids (or : 0.59 ; 95% ci : 0.350.99), vitamin d, thiamin, and niacin (p interaction : 0.04, 0.02, and 0.03, respectively). no other statistically significant associations were observed, although there were reduced odds of bladder cancer among the older age group for the highest intake of vitamin e (or : 0.61 ; 95% ci : 0.371.02) and interactions between vitamin c and ever - smoking (p = 0.08) and vitamin c and age (p = 0.08) were borderline (data not shown).table 4odds ratios (95% confidence intervals) for the joint effects of total intake of selected nutrients (highest vs. lowest) and smoking characteristics and agecarotenoidsvitamin dvitamin ethiaminniacin<12900.6 g12900.6 gors (95% ci)<388.91 iu388.91 iuors (95% ci)<20.7 (mg)20.7 (mg)ors (95% ci)<2.12 (mg)2.12 (mg)ors (95% ci)<33.08 (mg)33.08 (mg)ors (95% ci)cases / controlscases / controlscases / controlscases / controlscases / controlscases / controlscases / controlscases / controlscases / controlscases / controlssmoking status never28/4328/520.66 (0.321.35)29/5427/411.04 (0.502.15)33/5120/420.64 (0.311.33)23/5033/441.35 (0.662.77)27/5329/411.10 (0.542.26) ever155/76111/680.75 (0.481.17)132/65134/790.82 (0.541.24)140/64117/730.73 (0.481.13)134/70132/740.91 (0.591.39)135/66131/770.80 (0.521.23) p interaction0.980.410.870.270.26cigarettes / day < 2025/3026/231.65 (0.683.99)19/2632/271.58 (0.663.79)20/2731/231.57 (0.633.88)19/2832/251.85 (0.784.41)20/2731/251.64 (0.683.95) 20127/4484/440.62 (0.361.09)111/38100/500.73 (0.431.23)118/3685/480.58 (0.340.99)114/4197/470.78 (0.461.34)114/3897/500.66 (0.391.14) p interaction0.080.140.030.100.08years smoking < 2751/3845/341.19 (0.582.47)49/3247/400.78 (0.401.52)49/3645/321.08 (0.542.16)46/3050/420.71 (0.351.43)47/3049/410.70 (0.351.41) 27101/3764/330.62 (0.331.16)81/3284/381.00 (0.551.81)89/2770/400.61 (0.331.12)87/3978/311.41 (0.762.59)87/3578/351.02 (0.561.85) p interaction0.200.580.240.190.51age < 6373/5969/540.91 (0.491.68)76/6966/441.55 (0.882.74)79/6156/471.12 (0.622.01)69/6573/472.02 (1.113.68)69/6373/481.59 (0.882.87) 63110/6070/660.59 (0.350.99)85/5095/760.65 (0.401.08)94/5481/680.61 (0.371.02)88/5592/710.75 (0.451.24)93/5687/700.64 (0.381.07) p interaction0.180.040.180.020.03iu international units, mg milligrams, low intakes were categorized below median and high intakes above mediansadjusted for age, sex, smoking status (current / non - current), number of cigarettes per day, and years of smoking adjusted for age, sex, and kcal only odds ratios (95% confidence intervals) for the joint effects of total intake of selected nutrients (highest vs. lowest) and smoking characteristics and age iu international units, mg milligrams, low intakes were categorized below median and high intakes above medians adjusted for age, sex, smoking status (current / non - current), number of cigarettes per day, and years of smoking adjusted for age, sex, and kcal only the statistically significant inverse associations observed between total intake of carotenoids, vitamin d, thiamin, and niacin and the risk of bladder cancer for older individuals remained robust following the fprp test. each inverse association had less than a 50% likelihood of being false positive for an odds ratio of 1.5 and based on a prior probability of 25%, we investigated an extensive range of micronutrients in relation to the risk of bladder cancer. possible inverse associations of borderline statistical significance were observed between bladder cancer and total intake of vitamin e and dietary phosphorus. we found some evidence that total intake of vitamin e, total carotenoids, niacin and possibly thiamin might be modified by cigarette smoking in that inverse associations were observed largely among heaviest smokers. higher intakes of carotenoids, vitamin d, thiamin, niacin, and vitamin e were also associated with reduced odds of bladder cancer in the older age group. the potentially protective effect of vitamin e that we observed against developing bladder cancer was consistent with findings from two other us studies [20, 27 ]. however, we detected no association between risk of bladder cancer overall and the intake of carotenoids either as individual carotenoids (-carotene, -carotene, -cryptoxanthin, lycopene, and lutein / zeaxanthin) or collectively as total carotenoid intake. our findings were in agreement with an intervention study, a meta - analysis, two prospective studies [3, 20 ], and one case control study. conversely, four case control studies [8, 22, 23, 48 ] observed inverse associations with carotenoid intake and two other prospective studies [21, 25 ] found specific carotenoids, dietary beta - carotene and beta - cryptoxanthin to be possibly protective. like previous studies, we investigated the most common carotenoids from an estimated 40 carotenoids present in the human diet. while some carotenoids have potential to form vitamin a (provitamin a carotenoids include, -carotene, -carotene, and -cryptoxanthin) others do not have this capability (non - provitamin a carotenoids are lycopene, lutein, and zeaxanthin). contrary to an earlier study, we did not find either of these two categories of carotenoids to be associated with the risk of bladder cancer (data not shown). any chemo - preventive effect may be difficult to detect for individual carotenoids either due to the relatively small sample size, small size of the effect or because carotenoids possibly only work in synergy with each other or other dietary factors. where a relatively large prospective study of us men and a case control study both reported no association between vitamin d and bladder cancer, we observed reduced odds of bladder cancer with higher intake of vitamin d ; although this was not statistically significant. the anti - proliferative effect of vitamin d has been shown in various in vitro and in vivo investigations. however, as vitamin d is available from both dietary sources and uv - b radiation, it is difficult to estimate the full effect of vitamin d without taking into account sunlight exposure. although b - group vitamins are thought to possibly offer protection via their role in genomic stability, dna repair, and regulation of cell division, we did not detect any associations between any b - group vitamin and bladder cancer overall. this was consistent with three previous prospective studies [20, 21, 25 ] and one retrospective study but disagreed with two other recent case control studies [28, 35 ]. we also failed to detect any association between bladder cancer and another water - soluble vitamin with antioxidant potential, vitamin c. once again, there were conflicting reports relating to the effect of vitamin c on bladder cancer risk among the various studies [3, 8, 1921, 23, 29, 32, 33 ]. when reviewing the existing literature on vitamin c and many of the other vitamins, any evidence for an association generally appeared to be weaker in prospective studies compared with case control studies. lack of association between some micronutrients and bladder cancer might also be due to collinearity among nutrients as a result of shared food sources. for instance, many fruits and vegetables rich in carotenoids are also good sources of vitamin c, potassium, and folate. to our knowledge, phosphorus has been investigated in relation to bladder cancer in only one other epidemiological study that reported no association following multivariate analyses. although we observed an approximate 50% reduction in the odds of bladder cancer associated with higher dietary intake of phosphorus, it was not statistically significant. given this ubiquitous micronutrient is an important physiological component of dna, rna, atp, and cell membranes, it may be worthy of further consideration. in our study, a higher intake of vitamin e related to a reduced risk of bladder cancer among heaviest smokers, a finding consistent with those reported in another american case control study. the reduced odds of bladder cancer we observed for the highest quartile of total carotenoids among the heaviest smokers also agrees with results presented in another case control studies [22, 48 ] also reported increased risk of bladder cancer among smokers with the lower intakes of total and specific carotenoids, lutein, and zeaxanthin.. suggested that carotenoids provide protection against bladder cancer for smokers by detoxifying arylamines. borderline statistically significant interactions between the number of cigarettes smoked per day and intakes of thiamin and niacin may warrant further investigation. our findings indicate that higher intakes of vitamin d, thiamin, niacin, carotenoids, and possibly vitamin e may be beneficial to participants in the older age group. older individuals may have limited access to regular sunshine (e.g., living in institutions) and therefore have to rely strongly on dietary sources of vitamin d and supplementation. bladder cancer is a disease that typically affects older people, and bioavailability of b - group vitamins may be compromised in this demographic by certain drugs (e.g., acid lowering agents). additionally, vitamin e, like carotenoids acts as an antioxidant and, as suggested by our results, could be more beneficial under conditions of the greatest oxidative stress such as smoking and aging. like most case control studies, potential for recall bias is a consideration in dietary estimation. however, as dietary factors are not commonly associated with bladder cancer etiology recall of dietary intake is likely to be non - differential between cases and controls. other potential limitations relating to measurement of dietary exposures include only one dietary collection period and the possibility of preclinical disease altering dietary intake. although there was only one collection period, dietary data were collected using a detailed and validated ffq. as for dietary change, a majority of the cases in our study were diagnosed with superficial bladder cancer, and it is considered unlikely that preclinical disease would alter dietary behavior. although we can not totally exclude the possibility of recall bias, dietary intake for major dietary components ; calories, total fat, alcohol, and coffee were comparable between cases and controls suggesting that it should be minimal. finally, controls were drawn from two sources department of transportation (for those under 65 years) and medicare (for those 65 years and older). these are considered nearly complete sources of records in our population. among cases, over 95% report having a driver s license (for those under 65 years) as do over 95% (of those 65 years and older) report being enrolled in medicare (data not shown). we examined the characteristics of controls younger than 65 years and over 65 years and found them to be comparable with respect to sex, smoking, and medical access variables (i.e., visit to a doctor in the past 2 years) ; however, older subjects had less than a college education (40% of those under 65 years and 50% of those 65 years and older). however, level of education is not a strong risk factor for bladder cancer (including in our own data) and thus was not considered a potential confounder in our analysis. while multiple comparisons were made in this study, we attempted to address this issue by utilizing the fprp. statistically significant associations between higher intakes of carotenoids, vitamin d, thiamin, and niacin and risk of bladder cancer among the older individuals remained robust following this test. although it is worth noting that statistical power was low and determining appropriate prior probabilities is difficult. biologically plausible mechanisms of action and similar results reported from other studies provide additional support for some of our findings [8, 20, 27 ]. we investigated an extensive range of micronutrients in relation to the risk of bladder cancer. possible inverse associations of borderline statistical significance were observed between bladder cancer and total intake of vitamin e and dietary phosphorus. we found some evidence that total intake of vitamin e, total carotenoids, niacin and possibly thiamin might be modified by cigarette smoking in that inverse associations were observed largely among heaviest smokers. higher intakes of carotenoids, vitamin d, thiamin, niacin, and vitamin e were also associated with reduced odds of bladder cancer in the older age group. the potentially protective effect of vitamin e that we observed against developing bladder cancer was consistent with findings from two other us studies [20, 27 ]. however, we detected no association between risk of bladder cancer overall and the intake of carotenoids either as individual carotenoids (-carotene, -carotene, -cryptoxanthin, lycopene, and lutein / zeaxanthin) or collectively as total carotenoid intake. our findings were in agreement with an intervention study, a meta - analysis, two prospective studies [3, 20 ], and one case control study. conversely, four case control studies [8, 22, 23, 48 ] observed inverse associations with carotenoid intake and two other prospective studies [21, 25 ] found specific carotenoids, dietary beta - carotene and beta - cryptoxanthin to be possibly protective. like previous studies, we investigated the most common carotenoids from an estimated 40 carotenoids present in the human diet. while some carotenoids have potential to form vitamin a (provitamin a carotenoids include, -carotene, -carotene, and -cryptoxanthin) others do not have this capability (non - provitamin a carotenoids are lycopene, lutein, and zeaxanthin). contrary to an earlier study, we did not find either of these two categories of carotenoids to be associated with the risk of bladder cancer (data not shown). any chemo - preventive effect may be difficult to detect for individual carotenoids either due to the relatively small sample size, small size of the effect or because carotenoids possibly only work in synergy with each other or other dietary factors. where a relatively large prospective study of us men and a case control study both reported no association between vitamin d and bladder cancer, we observed reduced odds of bladder cancer with higher intake of vitamin d ; although this was not statistically significant. the anti - proliferative effect of vitamin d has been shown in various in vitro and in vivo investigations. however, as vitamin d is available from both dietary sources and uv - b radiation, it is difficult to estimate the full effect of vitamin d without taking into account sunlight exposure. although b - group vitamins are thought to possibly offer protection via their role in genomic stability, dna repair, and regulation of cell division, we did not detect any associations between any b - group vitamin and bladder cancer overall. this was consistent with three previous prospective studies [20, 21, 25 ] and one retrospective study but disagreed with two other recent case control studies [28, 35 ]. we also failed to detect any association between bladder cancer and another water - soluble vitamin with antioxidant potential, vitamin c. once again, there were conflicting reports relating to the effect of vitamin c on bladder cancer risk among the various studies [3, 8, 1921, 23, 29, 32, 33 ]. when reviewing the existing literature on vitamin c and many of the other vitamins, any evidence for an association generally appeared to be weaker in prospective studies compared with case control studies. lack of association between some micronutrients and bladder cancer might also be due to collinearity among nutrients as a result of shared food sources. for instance, many fruits and vegetables rich in carotenoids are also good sources of vitamin c, potassium, and folate. to our knowledge, phosphorus has been investigated in relation to bladder cancer in only one other epidemiological study that reported no association following multivariate analyses. although we observed an approximate 50% reduction in the odds of bladder cancer associated with higher dietary intake of phosphorus, it was not statistically significant. given this ubiquitous micronutrient is an important physiological component of dna, rna, atp, and cell membranes, it may be worthy of further consideration. in our study, a higher intake of vitamin e related to a reduced risk of bladder cancer among heaviest smokers, a finding consistent with those reported in another american case control study. the reduced odds of bladder cancer we observed for the highest quartile of total carotenoids among the heaviest smokers also agrees with results presented in another case control studies [22, 48 ] also reported increased risk of bladder cancer among smokers with the lower intakes of total and specific carotenoids, lutein, and zeaxanthin. borderline statistically significant interactions between the number of cigarettes smoked per day and intakes of thiamin and niacin may warrant further investigation. our findings indicate that higher intakes of vitamin d, thiamin, niacin, carotenoids, and possibly vitamin e may be beneficial to participants in the older age group. older individuals may have limited access to regular sunshine (e.g., living in institutions) and therefore have to rely strongly on dietary sources of vitamin d and supplementation. bladder cancer is a disease that typically affects older people, and bioavailability of b - group vitamins may be compromised in this demographic by certain drugs (e.g., acid lowering agents). additionally, vitamin e, like carotenoids acts as an antioxidant and, as suggested by our results, could be more beneficial under conditions of the greatest oxidative stress such as smoking and aging. like most case control studies, potential for recall bias is a consideration in dietary estimation. however, as dietary factors are not commonly associated with bladder cancer etiology recall of dietary intake is likely to be non - differential between cases and controls. other potential limitations relating to measurement of dietary exposures include only one dietary collection period and the possibility of preclinical disease altering dietary intake. although there was only one collection period, dietary data were collected using a detailed and validated ffq. as for dietary change, a majority of the cases in our study were diagnosed with superficial bladder cancer, and it is considered unlikely that preclinical disease would alter dietary behavior. although we can not totally exclude the possibility of recall bias, dietary intake for major dietary components ; calories, total fat, alcohol, and coffee were comparable between cases and controls suggesting that it should be minimal. finally, controls were drawn from two sources department of transportation (for those under 65 years) and medicare (for those 65 years and older). these are considered nearly complete sources of records in our population. among cases, over 95% report having a driver s license (for those under 65 years) as do over 95% (of those 65 years and older) report being enrolled in medicare (data not shown). we examined the characteristics of controls younger than 65 years and over 65 years and found them to be comparable with respect to sex, smoking, and medical access variables (i.e., visit to a doctor in the past 2 years) ; however, older subjects had less than a college education (40% of those under 65 years and 50% of those 65 years and older). however, level of education is not a strong risk factor for bladder cancer (including in our own data) and thus was not considered a potential confounder in our analysis. while multiple comparisons were made in this study, we attempted to address this issue by utilizing the fprp. statistically significant associations between higher intakes of carotenoids, vitamin d, thiamin, and niacin and risk of bladder cancer among the older individuals remained robust following this test. although it is worth noting that statistical power was low and determining appropriate prior probabilities is difficult. biologically plausible mechanisms of action and similar results reported from other studies provide additional support for some of our findings [8, 20, 27 ]. in conclusion, the effects of vitamin e, carotenoids, vitamin d, thiamin, and niacin in relation to the risk of developing bladder cancer may warrant further investigation. future studies should focus on optimal doses and combinations of these micronutrients particularly for high risk groups such as heavy smokers and older individuals. | objectivealthough the effect of fruit and vegetables on the risk of bladder cancer has been widely studied, little is known about their micronutrient components. our aim was to investigate associations between minerals and vitamins and bladder cancer.methodsa case control study was conducted in new hampshire, usa. dietary data were collected from 322 cases and 239 controls using a 121-item food frequency questionnaire. odds ratios (ors) and 95% confidence intervals (cis) were calculated using logistic regression adjusting for sex, age, smoking characteristics, and energy intake.resultsthe ors (95% ci) for highest quartile versus lowest quartile for total intake of vitamin e was 0.66 (0.361.20 ; p trend = 0.09) and 0.49 (0.211.17 ; p trend = 0.13) for dietary phosphorus. the odds of bladder cancer for heavy smokers with the highest total intake of vitamin e, carotenoids, and niacin were 0.58 (0.340.99), 0.62 (0.361.09), and 0.66 (0.391.14), respectively. higher total intakes of carotenoids, vitamin d, thiamin, niacin, and vitamin e were inversely related to bladder cancer risk among older individuals.conclusionour findings suggest further investigation of the effect of vitamin e, carotenoids, vitamin d, thiamin, and niacin on bladder cancer risk may be warranted. future studies should focus on high risk groups such as heavy smokers and older individuals. |
malignant melanoma is the sixth most common uk cancer whose incidence continues to increase year on year. whilst new therapies are being developed, advanced metastatic melanoma is invariably fatal. the number of transplant procedures performed annually continues to increase, partly owing to our ageing population. moreover, improved surgical techniques and refined posttransplantation medical care have prolonged the longevity of organ transplant recipients (otrs) and have permitted patients to receive transplants at older ages. organ transplant recipients (otrs) are predisposed to many types of malignancies, most prominently skin cancers, owing to the considerable doses of immunosuppressive medications required [2, 3 ]. nonmelanoma skin cancers (nmscs) are most notably increased, with an increased nmsc risk of up to 250 times than that of the normal population and reversal of the ratio of basal cell carcinoma to squamous cell carcinoma. as such, the national institute of clinical excellence advocates annual skin surveillance should be undertaken in dedicated secondary care transplant clinics. as melanoma is purported to be an immunologically mediated tumour which can be treated using immunotherapy, immunosuppressive medications used by the otr cohort could be expected to alter its natural history. melanoma in otrs can arise in three principal contexts : melanoma may preexist prior to transplantation, develop following transplantation or may be derived from the organ donor. the largest study of otrs with a history of melanoma prior to transplantation suggests no increased risk of recurrence of local or metastatic melanoma (with mean followup of 10.5 years after original melanoma diagnosis) compared to the control population. this study interrogated database records of the mayo clinic and identified melanoma in 59 patients (61 cases) prior to transplantation. these findings supported a smaller retrospective analysis of 9 melanomas by the skin care in organ transplant patients in europe (scope) group with no recurrences reported with a mean of 60 month followup. a previous interrogation of the cincinnati transplant tumour registry (cttr) identified 31 otrs as having a diagnosis of melanoma prior to transplantation, of whom six developed recurrent disease and died 6 to 30 months posttransplantation (32 month followup), leading the author to recommend an interval of five years following treatment prior to considering solid organ transplantation. apparent differences in reported studies may be due to paucity of histological data available. whilst breslow thicknesses were reported in 25% of the mayo clinic records and 67% of the scope cohort, they were not available for any of the cttr group which may have been of greater depth. none of these studies provide conclusive evidence for recommending an optimal interval postmelanoma prior to transplantation. amongst the otr population, incidence of primary melanoma has been reported as between the same as up to eight times to that of the general population [1013 ]. such large variation is in part attributable to low absolute numbers of cases within each study. the largest cohort study used data from the us scientific registry of transplant recipients (19872008) and included 175,732 patients with renal, cardiac, liver, and lung allografts. 381 cases of melanoma were observed, suggesting an increased risk of 2.6 times than that of the general population. similarly, a large combined australasian registry - based prospective cohort study captured 28,855 patients with up to 42 years of followup and suggested an increased standardised incidence ratio of melanoma of 2.53 amongst the renal transplant recipient population. interrogation of mayo clinic records revealed 638 patients with posttransplantation melanoma (724 cases) with an average time of 5.5 years between first transplant and melanoma diagnosis. interestingly, skin cancers are the second most common form of malignancy after lymphoproliferative disorders in the paediatric posttransplant population. a greater proportion of these skin cancers (12%) are malignant melanoma in the paediatric versus the adult posttransplant population. where melanoma occurs posttransplantation, overall and melanoma cause - specific 3-year survival is significantly worse in otrs compared to the nontransplanted control population, with the difference most marked in cases of greater breslow thickness. brewer. reported three - year survival of 51.2% amongst patients with breslow thickness 1.513.00 mm compared to expected survival of 87.4% from patients with melanoma in nontransplanted control population. subgroup analysis of cardiac transplant patients, who typically take higher doses of immunosuppressants and are at greater risk of squamous cell carcinomas, suggest statistically significant melanoma cause - specific mortality with breslow thickness greater than 1.51 mm. these findings suggest that immunosuppressants are more likely to exacerbate tumours of greater breslow thickness, affording further credence to the hypothesis that melanoma is an immunologically mediated tumour. these findings were mirrored in an earlier retrospective, multicentre analysis of 91 melanomas occurring posttransplantation. compared to demographic- (age and sex) and phenotypic- (tumour thickness and ulceration status) matched controls, otrs developing posttransplantation melanomas had similar survival outcomes for t1 and t2 tumours (less than 2 mm thickness), but significantly worse for t3 and t4 tumours (thickness greater than 2 mm). there appears to be no site predilection for posttransplant melanoma, unlike that for backs in males and lower limbs in females in the nontransplant population [8, 9 ]. initial treatment for posttransplant primary melanoma does not differ significantly from the nontransplant population, where early diagnosed primary melanoma can be completely surgically excised, with depth of invasion governing the excision margin and need for sentinel lymph node biopsy [18, 19 ]. an expert consensus survey issued by the international transplant skin cancer collaborative (itscc) and the skin care in organ transplant patient europe (scope) advocated reduction of immunosuppression in patients with numerous or life - threatening skin cancers. the guidance attempts to balance the risk of tumour burden and risk of metastasis versus the increasing likelihood of organ rejection observed with more aggressive reduction of immunosuppression. reduction of immunosuppression may represent dose reduction or withdrawal of one immunosuppressive agent of several being used. the types of transplanted organ (heart, liver, and kidney) were each considered separately as each imparts differing immunogenicity potential, and failure of each type of graft has different implications with respect to organ replacement therapy and clinical outcomes. recommendations for reduction of immunosuppression for various stages of melanoma in various organ recipients are shown in table 1, graded mild, moderate, and severe. in the case of renal allografts, many experts had a lower threshold for reduction of immunosuppression as dialysis was felt to offer a reasonable alternative to organ failure. for liver recipients, doses of immunosuppressive medication can be slowly reduced to lower levels, as the liver has regenerative potential following insults such as acute rejection. owing to poor renewal potential of cardiac muscle and inevitable fatality should, the graft will be rejected and subsequently fail. in all cases, as morbidity and mortality of skin cancer rises, transplant physicians are more likely to accept the risk of graft compromise associated with reduction of immunosuppression. in nmsc, switching from calcineurin - based immunosuppression (e.g., tacrolimus and ciclosporin) to a mammalian target of rapamycin (mtor) inhibitor (e.g., sirolimus and everolimus) in renal transplant recipients with a previous squamous cell carcinoma is associated with prolonged tumour - free survival and significantly fewer cutaneous squamous cell carcinomas at two years. initial observations in murine models suggest that mtor inhibitors may have an antitumoral effect against melanoma, whilst offering protection against transplanted hearts. owing to the relative rarity of melanoma, larger cohort studies will be needed to see whether such an approach would be clinically advantageous in cases of melanoma. modifications of immunosuppressive regimes must be made by joint consultation of the patient with oncology, dermatology, and transplant medicine teams, assessing the risk of altering the medication upon the timecourse of the tumour and implications of organ rejection in that particular individual. malignancy inadvertently derived from the donor organ is a rare event relative to the number of transplants occurring annually. donors are not believed to have a significantly different incidence of skin cancer to the nondonor population. metastatic melanoma following homologous transplant of a metastatic nodule was first reported in 1965. as the number of organ transplants performed annually continues to increase and otrs are surviving for longer periods, there is an amassing body of case reports of such cases of donor - derived melanoma [2839 ]. affected recipients developed melanoma from three months to three years after transplantation [30, 38 ]. many organ donors, later believed to have occult melanoma at the time of organ harvesting, have been initially diagnosed with a cerebrovascular accident (cva) or primary brain tumour as a cause of death ; in retrospect, this is likely attributable to metastatic melanoma [21, 25 ]. a previous diagnosis of melanoma had not been made in all cases. in a recent review combining case series and case reports, 17 donors have provided organs to 44 recipients, of whom 35 developed melanoma, and 24 died from metastatic disease. where possible, resection of the donor organ (containing melanoma) and associated metastases should be performed together with cessation or reduction of immunosuppression. this may only be a viable option in renal transplant recipients, owing to lack of long - term organ replacement therapy in the case of heart, liver and lungs. following removal of the donated organ, retransplantation may then be possible [33, 40 ]. when considering organ donors, in cases of young death purported to be from cva or primary cerebral neoplasm, the index of suspicion for melanoma should be raised, particularly in individuals with a history of multiple previously excised moles. it has been suggested that where there is a prior history of melanoma, even if fully excised and recurrencefree for several years, this should be an absolute contraindication to organ donation, owing to the possibility of latent melanoma or ultralate recurrence [41, 42 ] which may be as much as 32 years after initial diagnosis and excision. additionally, comprehensive examination of the skin and evaluation for lymphadenopathy and organomegaly should be performed to exclude melanoma prior to organ donation. patients who have thin melanomas removed prior to organ transplantation appear not be at increased risk of recurrence following transplantation. immunosuppressive medications taken by otrs appear to predispose to de novo melanoma and are associated with worse outcomes in tumours of greater breslow thickness. cases of donor - derived melanoma underline the need for careful donor selection and exclusion of melanoma prior to transplantation. as increasing numbers of organ transplants are performed, future studies may better characterise outcomes of melanoma occurring in otrs to help inform treatment strategies in the immunocompromised population, particularly with respect to reduction and alteration of immunosuppressive medications. | the incidence of melanoma continues to increase year on year. with better surgical techniques and medical management, greater numbers of organ transplants are being performed annually with much longer graft survival. the authors review our current understanding of the incidence of melanoma amongst organ transplant recipients, outcomes compared to the immunocompetent population, and management strategies in this burgeoning group. |
wurtzite gallium phosphide (wz gap) has been predicted to exhibit a direct bandgap in the green spectral range. optical transitions, however, are only weakly allowed by the symmetry of the bands. while efficient luminescence has been experimentally shown, the nature of the transitions is not yet clear. here we apply tensile strain up to 6% and investigate the evolution of the photoluminescence (pl) spectrum of wz gap nanowires (nws). the pressure and polarization dependence of the emission together with a theoretical analysis of strain effects is employed to establish the nature and symmetry of the transitions. we identify the emission lines to be related to localized states with significant admixture of 7c symmetry and not exclusively related to the 8c conduction band minimum (cbm). the results emphasize the importance of strongly bound state - related emission in the pseudodirect semiconductor wz gap and contribute significantly to the understanding of the optoelectronic properties of this novel material. |
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it is vital to fully define the normal oral microbial flora before we begin to understand the role of bacteria in oral cancer as there is a distinctive predominant healthy bacterial flora of the oral cavity that is site - specific and highly diverse before delving into their apparent role in tumorigenesis. more than 700 bacterial species or phylotypes, of which over 50% have not been cultivated, have been detected in the oral cavity. it is important to note that micro - organisms in the oral cavity are responsible for various oral diseases, and an existence of an inter - relationship between the two is strongly hinted at. takahashi attempted to study the microbial ecosystem of the oral cavity and its relationship with various oral diseases. gendron.,(2000) opined that oral cavity is a reservoir of bacterial pathogens that can provoke focal infections. hooper. (2006) studied viable bacteria present within oral squamous cell carcinoma tissue. chambers., (2005) conducted a pilot study to examine elevated levels of mutans streptococci in xerostomic cancer patients after pilocarpine therapy. hooper. (2007) studied viable bacteria present within oral squamous cell carcinoma tissue. hsu., (2004) studied the induction of apoptosis in oral cancer cells. lax., (2002) attempted to explain how bacteria could cause cancer. (2010) explored the relevance of human papillomavirus (hpv) infection to carcinogenesis of oral tongue cancer. mager.,(2005) tried to determine if the salivary counts of 40 common oral bacteria in subjects with an oral squamous cell carcinoma (oscc) lesion would differ from those found in cancer - free (oscc - free) controls. mager.,(2006) attempted to study the alleged relationship between bacteria and human cancers. nagy., contended that since alteration in the oral microflora demographics consequently led to local and systemic infections in patients suffering from oral neoplasms, an investigative study on the inhibition of biofilm present on the surfaces of oral squamous cell carcinomas (oscc 's) was warranted. thus, anticancer therapy, irradiation, chemotherapy or surgery compromises the defense mechanism of the oral mucosa and is accompanied by a proliferation of the mucosal biofilm with an overgrowth of yeast and bacteria. this study was designed to probe the inhibition of the biofilm present on the surface of osccs. concerning the organisms found at the tumor site before and after rinsing (with gaba deutschland manufacture meridol (basel, switzerland), s. mitis, staphylococcus aureus and enterococcus faecalis were isolated from at least twice as many tumor surfaces before the rinsing. of the aerobic gram - negative species isolated, haemophilus influenzae, neisseria spp., and serratia spp. furthermore, campylobacter, actinobacillus actinomycetemcomitans and capnocytophaga were found more frequently and porphyromonas at the same frequency before meridol rinsing. of the gram - positive anaerobes, candida albicans were isolated in every sample before rinsing, but only in two patients after rinsing. this study has shown that the cancer lesion itself may greatly increase the local and systemic infection risk to oral cancer patients, even before specific tumor treatment. sharma conducted a comparative study of saliva from patients with oscc and healthy individuals to try and establish the conjecture that the saliva of patients with oral cancers have raised concentrations of certain bacteria and these bacteria can be used as possible diagnostic tools in oral cancer detection. a comparative study of saliva from 45 patients with oscc and 229 healthy controls showed that levels of six common bacteria species p. melaninogenica, leptotrichia buccalis, capnocytophaga ochracea, c. gingivalis, eubacterium saburreum, and s. mitis were significantly higher in patients than in controls. when three of these species (c. gingivalis, p. melaninogenica, and s mitis) were used as diagnostic indicators, they correctly predicted 80% of individuals with oral cancer and 83% of controls. the authors state that alterations in tumor - cell receptors could change the adhesion of some species of bacteria. donna mager (the forsyth institute, boston, ma, usa) is unsure whether these bacteria are simple markers for oscc or have so co - carcinogenic effects. this encouraging study gives credence to the belief that a reliable noninvasive tool for oral cancer detection may not be a pipe dream anymore. sixou., ventured out to review literature devoted to quantitative and qualitative variations in the flora of the oral cavity during immunosuppressive treatment of cancer patients. cloke., assessed patients who contracted wound infection following tissue transfer in head and neck oncological treatments. sakamoto., attempted to investigate the association between postoperative infection, colonization of certain bacterial species and its translocation to the cervical lymph nodes in oral cancer patients. mccarthy., (1965) analyzed the indigenous oral flora of man. meurman., (2010) assessed the infectious and dietary risk factors of oral cancer. rawlinson., (1993) discovered new data on the microbial flora associated with adult periodontitis. rosenquist., (2005) described risk factors for oral and oropharyngeal squamous cell carcinoma. sakamoto., (1999) isolated and studied bacteria from cervical lymph nodes in patients with oral cancer. muthu., (2004) studied oropharyngeal flora changes in patients with head and neck malignancy post radiotherapy. fbin., have opined that the genomics, transcriptomics and proteomics of saliva and the oral cavity became increasingly popular subjects of research in the current era as they represent a host of advantages by being a safe, painless and inexpensive source of complex genetic information. wijers., (2001) tested the hypothesis that aerobic gram - negative bacteria (agnb) play a crucial role in the pathogenesis of radiation - induced mucositis ; consequently, selective elimination of these bacteria from the oral flora should result in a reduction of the mucositis. meurman opined that in the oral cavity, knowledge of the role of microbiota in carcinogenesis is rudimentary at best. inflammation caused by infections may be the most important preventable cause of cancer in general. however, in the oral cavity the role of microbiota in carcinogenesis is not known. as shown in figure 1, several bacteria and candida strains in the mouth convert ethanol to carcinogenic acetaldehyde thus explaining the epidemiological evidence between heavy drinking, smoking, and development of cancer. both the commonly encountered oral streptococci and yeasts possess metabolic pathways for this conversion. proposed paradigm for bacterial involvement in carcinogenesis hooper. regard, tumorous and nontumorous mucosal tissue specimens (approximately 1 cm) were harvested from 10 oscc patients at the time of surgery. differences between the composition of the microbiotas within the tumorous and nontumorous mucosae were obvious which pointed to the selective growth of bacteria within carcinoma tissue. the group concluded that whether the presence of these bacteria within the mucosa has any bearing on the carcinogenic process is a concept worthy of further investigation. chocolatewala., attempted to collate all the scientific data pertinent to the apparent association between micro - organisms and oral cancer. they contended that despite the widening interest in the possible association between bacteria and different stages of the evolution of cancer, much still needs to be done in this regard. in recent times, bacteria - laden with smart polystyrene nanoparticles, which can transport genes, drugs, nanosensors or other cargo into the interior of host cells, are being used to precisely position cargo inside the cells for the early diagnosis and treatment of cancer and other diseases. to summarize, recent research has provided us with a host of information regarding the bacterial mechanisms purported to cause or cure cancer. however, many doubts linger. do microbial infections initiate cancer, or is it the preexisting cancer that compromises the host 's immunity facilitating secondary microbial colonization ? will the site - specific colonization of certain bacteria be of any estimable value in the diagnosis or treatment of oral cancer ? could attenuated bacteria be employed in vaccines to modulate host 's immunity against cancer ? this renders further exploration on this subject mandatory as it would enable us to clearly fathom the role of the micro - organisms, not only in prevention or early diagnosis of oral cancers, but also in providing an effective treatment and improving the survival of the afflicted individual. some viruses reported to cause human cancers include some genotypes of : hpv, hepatitis b virus (hbv), hepatitis c virus, ebv, kaposi 's sarcoma - associated herpes virus and human t - lymphotropic virus. the recently discovered human cancer virus is actually, a polyomavirus (merkel cell polyomavirus) purported to be responsible for a rare form of skin cancer dubbed merkel cell carcinoma. saito., assessed the current issues and perspectives of the rapidly evolving anticancer therapy, dwelling on the potential applications of a distinctive and naturally occurring oncolytic virus. grinde and olsen contended that the focus has traditionally been on bacteria and fungi when dissecting the microbiological aspects of oral disease in detail. wu., have established that sensitive and reliable early diagnostic markers for oscc remain unavailable. herrero., attempted an extensive exploration on the alleged link between hpv and oral cancer. hpv, the causal agent of cervical cancer, apparently has a role to play in the etiology of cancer of the oral cavity and oropharynx. hpv is consistently and more frequently detected in cancers of the oropharynx and tonsil than at other head and neck sites, and hpv-16 tends to be the predominant type detected. results of a few case control studies and a cohort study point to a likely role of hpv in some cancers at certain anatomical sites. however, many aspects of the association remain to be investigated to better define the precise contribution of hpv to the etiology of these tumors. in addition to mounting epidemiologic evidence, extensive laboratory evidence supports the association between hpv and a subset of cancers of the oropharynx. the prospect of hpv vaccine development offers hope for prevention of cervical and anogenital cancers and possibly also for a substantial number of cancers of the oropharynx and oral cavity. recent studies from the md anderson center support an etiologic role for hpvs in at least a sub - set of head and neck squamous cell carcinoma, especially in tonsillar carcinoma. dna technology has shown that a substantial portion of oscc and premalignant lesions contain hpv sequences, often of the high risk genotypes (mainly hpv-16 and hpv-18). hpv detection is higher when analyzed by in situ hybridization and polymerase chain reaction (pcr), and studies with these techniques have disclosed hpv 11, 16 or 18 dna sequences in up to 60% of oscc. analysis of hpv e7 mrna in oscc and cell lines by reverse transcriptase - pcr showed that hpv e7 mrna was present in 90% of patients with oscc. they concluded that though hpv may be implicated in some tumors but clearly not in all oscc where tobacco and alcohol are more important etiological factors. thus, hpv is clearly neither necessary nor sufficient for all tumor production, and it must be remembered that much oscc is induced by the known risk habits involving tobacco and alcohol. they maintained that certain strains of hpv have been shown to be etiologically related to the development of uterine cervical and other genital cancers, but their plausible involvement in the development of malignancies at other sites is still a question mark. the team concluded that in the head and neck region, hpv - associated scc was site specific with the viral dna commonly found in tumors of the waldeyer 's tonsillar ring. patients with hpv - positive tumors were reported to have been afflicted with a higher stage of disease than patients with hpv - negative tumors, but there was no discernable difference in the 3-year survival rates between these two groups of patients., argued that there is a possibility that viral agents other than hpv could contribute to the development of oscc as squamous cell carcinoma of the oral cavity has not been associated with hpv dna, which suggested alternate etiologic factors. hermann., reported and reviewed a case highlighting the presence of hpv-18 and ebv in a squamous cell carcinoma of the tongue in a 20-year - old patient. they acknowledged that this was the first case of co - infection in carcinoma of the tongue to be reported, and they attempted to review the present data and theories concerning viral oncogenesis of oral carcinomas. to find evidence for the presence of hpv or ebv, the team performed hpv - pcr - elisa and ebv - pcr - elisa. an explanation for the failure to detect any ebv dna in some studies may be the hit and run theory. it suggests that only the viral dna is needed to induce malignant transformation. after triggering the carcinoma, the viral dna is lost during the uncontrolled cell cycles of the host cell. a co - infection of high - risk hpv and ebv is mainly found in nasopharyngeal carcinomas. assuming that ebv plays a role in oral carcinogenesis, an interesting hypothesis of viral interaction in triggering malignant transformation has been propounded. a protein homolog, encoded by ebv, might result in a local immunosuppression and lead to or facilitate an infection of epithelial cells by high - risk hpvs. butel, (1999) attempted to establish an etiologic role for virus in causing cancers. the rna and dna tumor viruses have made fundamental contributions to two major areas of cancer research. viral systems support the concept that cancer development occurs by the accumulation of multiple cooperating events. viruses are now scientifically accepted as a credible etiologic factor in the development of human cancer. the infectious nature of viruses distinguishes them from all other cancer - causing factors ; tumor viruses establish long - term persistent infections in human beings, with cancer occurring as a potentially lethal side effect of viral replication strategies. more information needs to be learned about environmental co - factors that synergize with viral infections to stimulate tumor production. both the identification of carcinogenic co - factors in specific virus systems and studies of mechanisms of action of such factors in target tissues warrant investigation. guiding examples are the probable role of aflatoxin in cooperation with hbv chronic infections on induction of hepatocellular carcinoma in certain geographic areas and the involvement of dietary nitrosamines in ebv - induced neoplasia. cruz., (1997) carried out a study using pcr technique to assess the prevalence of epstein - barr virus in oral squamous carcinomas, premalignant lesions and normal mucosa. higa., (2003) analyzed ebv - related oral squamous carcinoma patients simultaneously infected with hpv in okinawa (southern japan). maeda., (1998) investigated the presence of ebv dna in forty - five cases of oral squamous cell carcinoma. shamaa., (2008) gave a detailed exposition of the significance of epstein barr virus (ebv) and dna topoisomerase ii alpha (dna - topo ii alpha) immunoreactivity in normal oral mucosa, oral epithelial dysplasia (oed) and oral squamous cell carcinoma (oscc). sanjaya., scrutinized the data that allegedly links candida and oral cancer. the team hypothesized a causal role for candidiasis in oral precancer and cancer albeit an indirect one while also implying that candida along with other co - factors has a say in initiation and promotion of carcinogenesis. the authors suggest that nitrosation potential of the c. albicans results in the production of carcinogenic nitrosamine thus predisposing the oral epithelium to dysplastic changes leading o carcinoma. further contributing factors include the integrity of the oral mucosa and tobacco smoking habits, which might enhance the virulence of the organism. the literature review has suggested a role of candida in causing preneoplastic changes of the oral mucosa. cawson (1973) demonstrated the ability of c. albicans to elicit epithelial hyperplasia in chick chorioallantoic membrane. several other studies have confirmed the hyperplastic response of the epithelium when invaded by candida. (1998) demonstrated a significant positive correlation between fungal infection and moderate and severe epithelial dysplasia. krogh (1990) commented on the role of yeasts in oral cancer by means of endogenous nitrosation. (1989) postulated that nitrosamine compounds produced by candida species may directly or in combination with other chemical carcinogenesis, activate specific proto - oncogenes and thus initiate the development of a malignant lesion. they further postulated that the progression of the activated cell into a tumorigenic cell might be linked to the amplification and over - expression of oncogenes. the authors propose a pathogenesis for the role of candida in oral precancer and cancer, wherein certain factors such as immunocompromised state may lead to the activation of various biotypes of c. albicans that have nitrosation ability to form nitrosamines from their precursors. these nitrosamines then act on the normal epithelium leading to dysplasia and further development of oral carcinoma thereby suggesting a causal role of candida species. thus, it can be postulated that c. albicans in association with tobacco will enhance the process of carcinogenesis. major environmental risk factors for upper digestive tract cancers are excessive smoking of tobacco, increased alcohol consumption and unsatisfactory maintenance of oral hygiene. consequently, this led to raised ach levels in saliva, which has been shown to be carcinogenic. they postulated that colonization of oral mucosa with candida glabrata (a non - c. albicans species) which is widely deemed to have the potential to produce carcinogenic amounts of ach from both ethanol and glucose may play a considerably vital role in the development of oral cancer. nagy., (1998) and (2000) investigated the inhibition of the biofilm present on the surface of osccs. bakri., (2010) revisited the association between candidal infection and carcinoma, particularly oral squamous cell carcinoma. napenas., (2007) studied the relationship between mucositis and changes in oral microflora during cancer chemotherapy. soysa.,(2004) discussed the clinical and laboratory findings on the relationship between cytotoxics, radiotherapy and oral candidiasis, possible mechanisms of pathogenicity following such therapy, as well as precautions that could be taken to minimize such recalcitrant yeast infections. siikala., (2011) explored the ability of the oral microbiome to produce acetaldehyde in ethanol incubation., (1981) studied the yeast flora of the mouth and skin during and after irradiation for oral and laryngeal cancer. yamamoto., reported some interesting findings on a patient suffering from oral carcinoma. a patient with oral carcinoma underwent chemo radioimmunotherapy for the same and, approximately 4 weeks right from the start of the therapy, the patient had to grapple with severe oral mucositis evidently caused by the chemoradiotherapy, consequently leading to candidal pneumonia. randomly amplified polymorphic dna analysis and dna sequence examination of strains isolated from the oral cavity 1-week before the onset of pneumonia and autopsied lung revealed the identity of both strains as c. albicans, and the dna analysis confirmed aspiration of oral candida. prnnen., set out to study oral mucosal e - cadherin (e - cad) degradation by clinical and reference strains of c. albicans and c. glabrata. this study was carried out to test the ability of two candida strains to degrade human e - cad from the perspective of the candida virulence factor. thus, degradation of e - cad may result in a significant lack of cellular adhesion, which is often associated with cancer cells. we have seen in detail, the various data and the existing literature that strongly suggest a definitive link between micro - organisms and oral cancer. all the three major types of bacteria, fungi and viruses provide certain species, which seem to agree with the aforementioned hypothesis. notwithstanding the burgeoning interest in the possible association between bacteria and different stages of cancer development, our definitive knowledge in its relation to oral cancers remains inadequate although various theories have been put forth by various scientists. similarly, the role of viruses and fungi in causing oral cancer has also been examined in painstaking detail and unanimously, it can be concluded that further study is warranted. new and improved scientific developments coupled with numerous precise methods of intra - cellular study hints at a future that is optimistic and full of possibilities. hopefully, the next few years will provide definitive answers to the most pertinent questions examined here. | oral cancer which is a subtype of head and neck, cancer is any neoplastic tissue growth in the oral cavity. it comprises an abnormal mass of cells that foists genetic mutation and impedes the normal cell cycle, resulting in its unrestrained growth. various studies on the plausible link between oral microbial flora and cancer notwithstanding, our understanding of their link remains obscure and inadequate. the multitude of mechanisms by which the microflora initiate or spur carcinogenesis are still under study and scrutiny. as is widely known, the oral cavity is an abode to a wide assortment of microbes, each present in contrasting amounts. it is observed that increased growth of the microflora is concomitant with known clinical risk factors for oral cancer. manifold bacterial species have been found to interfere directly with eukaryotic cellular signaling, adopting a style typical of tumor promoters. bacteria are also known to impede apoptosis thereby potentially promoting carcinogenesis. the viral role in carcinogenesis (by annulling of p53 tumor suppressor gene and other cellular proteins with subsequent alteration in host genome function) is well documented. furthermore, the changes occurring in the commensal microflora in accompaniment with cancer development could possibly be used as a diagnostic indicator for early cancer detection. the intention of this review is to obtain a better understanding of the role that micro - organisms play in oral cancer etiology. |
native horses of the iberian peninsula have played an important role in the development of current american horse breeds. approximately 10,000 years ago, north and south american equus species became extinct for several reasons (grooves and ryder, 2000). horses returned to the americas during the second trip of christopher columbus to the island of hispaniola in 1493 (present - day dominican republic and haiti) (laguna - sanz, 1991 ; rodero. consequently, the first horses brought to america by columbus were not carefully selected, as revealed by complaints from christopher columbus to the catholic kings about the quality of these animals (cabrera, 1945 ; sponenberg, 1992). it is likely that the original horses were replaced before their departure to america by peasant stock breeds called jacas located in sevilla (rodero., 1992 ; sponenberg, 1992). in the years following the expeditions by columbus, conquering the new world became more important and the quality of the horses brought to america improved. as a result, horse breeds brought to america during this period varied in quality and origin, although most came from the southern iberian peninsula (cabrera, 1945 ; rodero. traditionally, iberian horses have been classified into two groups : celtic ponies, in reference to breeds distributed throughout the north atlantic iberian region, and iberian horses located throughout the southern iberian peninsula (aparicio, 1944 ; jordana., 1995 ; several studies have postulated that iberian celtic ponies share a common ancestry with british ponies, whereas iberian horses share a common ancestry with the barb horse (garcia - dory, 1980 ; sotillo and serrano, 1985 ; lvarez llana, 1995 ; martinez. the high degree of genetic diversity among iberian horse breeds suggests that the iberian peninsula served as a refugium for wild horses and has contributed considerably to local domestic stock, as well as to the domestic horse breeds of central europe and the united kingdom (warmuth., 2011). one route was established by the expeditions of alonso de ojeda and diego de nicuesia in 1509 (diaz, 1988) and went through panama via the gulf of uraba to nueva granada (= new granada ; present - day colombia). the second route was established by spanish expeditions through coro (present - day venezuela) to the eastern plains region known as llanos orientales in nueva granada, in the current departments of arauca, casanare and meta (montoya b, 1988, undergraduate final work, universidad de los llanos, villavicencio, colombia). the horses that arrived via the first route are known today as the colombian paso fino horse breed and are found throughout most of colombia. the marshy areas from which the paso fino breed came favored the development of a typical gait very suitable for agriculture or riding. through years of selection, the colombian paso fino breed has been aptly divided into four lineages based on their natural gait : the colombian paso fino, colombian trocha, colombian trocha - gallop and colombian trot - gallop (fedequinas, 2006). horses that arrived in colombia via the second route gave rise to the colombian creole cattle horse breed (known as criollo de vaquera). this breed occurs in three geographic regions, including the departments of arauca, casanare and meta, which make up part of the llanos orientales near the colombian - venezuelan border. traditionally, these horses have been used in agriculture and have been kept isolated in a state of semi - freedom in herds formed by mares, colts and stallions, without much selective pressure (sandoval rfa and torres mti, 1996, undergraduate final work, universidad de los llanos, villavicencio, colombia). the mitochondrial dna control region has been widely used to understand the origin and diversification of domestic horses (vil., 2001 ; mirol., 2002 ; luis., 2006 ; kavar and dov, 2008 ; warmuth., 2011). previous studies have discovered high levels of mitochondrial dna variability within and among horse breeds, without a clear geographical pattern of distribution (hill., 2002). recent studies have confirmed the iberian peninsula as a geographic maternal and genetic refugium, a conclusion supported by the finding that several pre - domestic iberian maternal lineages survive in modern horses of iberian descent (warmuth., 2011). (2002), is well represented in both southern iberian and new world breeds, thus highlighting the importance of iberian breeds during re - population of the new world by horses (royo., 2005 ; luis., 2006). despite the clear connection between horses found in the iberian peninsula and horses found in the americas today, the influence of the iberian horse gene pool on colombian creole horses has not yet been analyzed. the aim of this study was to analyze the two principal horse populations in colombia, the colombian paso fino and the colombian creole cattle horse, in order to clarify their maternal genetic origin and the current matrilineages in both breeds. in total, 116 blood samples were collected from unrelated animals representing the colombian paso fino (cpf), the colombian creole cattle horse (from the casanare (cvc) and meta (cvm) populations) and the following five iberian horse breeds : asturcon (ast), caballo gallego (cga), jaca navarra (jna), losino (los) and pura raza espaola (pre) (table 1). dna was extracted using a qiagen dneasy blood & tissue kit (qiagen, germany) according to the manufacturer s instructions. a 364-bp fragment of the mtdna d - loop that included positions 1546915832 (xu and arnason, 1994) was sequenced. the primer sequences were designed using the primer 0.5 software (gcg software package, university of wisconsin, usa). the primers used were d - loopf (5-ccaccatcaacacccaaagc-3) and d - loopr (5-gccctgaagaaagaaccagat-3). the amplified fragments were purified using a purelink pcr purification kit (invitrogen), according to the manufacturer s instructions. sequencing was done using an abi prism 310 genetic analyzer (applied biosystems) and the sequences were aligned using clustalx (thompson., 1997). the mean number of pairwise differences (mnpd), nucleotide diversity and fst distances were obtained using mega 4.1 (tamura., 2007) and arlequin 3.11 (excoffier., 2005) softwares. partitioning of the total genetic variance into components based on intergroup (colombian and spanish), interbreed and inter - individual differences was done using analysis of molecular variance (amova) implemented in arlequin 3.11 (excoffier., the sequencing of mtdna from the 116 blood samples of colombian and spanish horse breeds revealed 51 haplotypes defined by 69 polymorphisms (52 transitions and 17 transversions) distributed among 63 distinct sites. five sites contained transitions and transversions and one site had two unique transversions (figure s1). for each breed, we identified between six and 15 haplotypes. the colombian creole cattle horse from casanare (cvc) (15) and asturcon (ast) (10) showed the highest number of haplotypes among the colombian creole horse populations and spanish breeds, respectively (table 1). the cvc and ast populations also had a higher frequency of haplotypes exclusive of the other breeds studied (60% and 70%, respectively). seven haplotypes were common to spanish and colombian horse breeds (haplotypes 2, 3, 8, 13, 15, 16 and 21). only 10 haplotypes were found in more than one breed and 41 haplotypes were unique to a single breed (table 1). the cvc (9) and jaca navarra (jna) (7) populations had a higher number of exclusive haplotypes among colombian and spanish breeds, respectively. haplotypes 2 (24%) and 3 (10%) had a total frequency of 34%, whereas the frequencies of all other individual haplotypes varied from 0.9% to 5% of the total haplotypes (table 1). thirty - eight haplotypes coincided with jansen s haplogroup classification (jansen., 2002) and were assigned to nine previously defined haplogroups (a1, a3, a5, a6, c1, c2, d1, d3 and f1) (table 1) ; the remaining 13 haplotypes were classified as new haplotypes (table 1). the most frequently identified haplotypes in the colombian and spanish breeds were type d1 (47%), a3 (10%) and c2 (9%). haplotype d1 was considered to be representative of iberian and north african breeds and was found in every breed analyzed except jaca navarra (table 2). the skeleton network revealed four main clusters that corresponded to the four haplogroups (a, c, d, and f) described by the classification system of jansen. the haplotypes belonging to haplogroup d showed a star - like skeleton in which the center of the star corresponded to the haplotype with the highest frequency (2) surrounded by a number of peripheral haplotypes of lower frequency. most of the different horse breeds were represented in all four haplogroups, the exceptions being pura raza espaola, which was absent in haplogroup c, and losino and jaca navarra, which were absent in haplogroup f. the haplotype diversity, nucleotide diversity and mean number of pairwise differences were similar for the colombian and iberian breeds (table 3). however, the colombian paso fino breed had lower nucleotide diversity than the other colombian and spanish breeds. haplotype diversity ranged from 0.97 in pura raza espaola to 1 in the gallego breed. among colombian breeds, the colombian creole cattle horse from meta had the highest value (0.92) whereas the colombian paso fino breed had the lowest nucleotide diversity (0.0114) and the lowest mean number of pairwise differences (4.2) the average fst distance between any two breeds ranged from just under 3% to 12% (table 3). the colombian creole horse populations had values at the lower end of this range (2.74.9%) whereas the spanish breeds had higher values ranging from 6.2% to 11.5%. amova of the various haplotypes revealed no significant genetic differences among the spanish and colombian breeds (fct = 1%) (table 4). however, this lack of genetic differentiation between groups was not reflected in the differences among breeds within groups or among breeds between different groups (fst = 5% and fsc = 5%). several studies have confirmed the importance of iberian horses in the repopulation of the new world with equus species (mirol., 2002 ; luis., 2006). however, this is the first genetic analysis of the two principal colombian equine breeds (colombian creole cattle horse or criollo de vaquera and colombian paso fino). haplotypes of haplogroup d were found more often than any other haplotype in the colombian and iberian horse breeds analyzed. haplogroup d is representative of the ancestral horse populations of the iberian peninsula, thus supporting historical records of iberian ancestry in the development of colombian horse breeds (miretti., 2004 ; primo, 2004 ; luis., 2006). haplogroup a has previously been found in the marismeo breed, which is an ancient semi - feral population that occurs in doana natural park in spain (royo., 2005). we have identified haplotype a in some of the colombian horse breeds analyzed, including the sevillan jacas breed, which is closely related to the extant marismeo horse breed, thus explaining the origin of haplogroup a in colombian horse breeds (rodero., 1992 ; sponenberg, 1992). this finding confirms historical documents that report the presence of the sevillan jacas breed among the horses brought to america. the identification of haplogroup c in the colombian horse breeds studied here most likely reflects the authorization to import celtic horses to nueva granada, which included present - day panama and colombia (luis., 2006). haplogroup f was not identified in the colombian horse breeds, but the closely - related haplotypes 11 and 19 were found in the colombian creole cattle horse (criollo de vaquera) breed. haplogroup f, which is found in middle eastern breeds, could be related to the berber phenotype found in colombian breeds. further research in this area should shed light on the corresponding haplotype - phenotype relationships. the proportion of the total variance explained by the genetic differences among iberian and colombian horse breeds was low (1% and data not shown) as a consequence of the iberian origin of the colombian breeds. nevertheless, the variance explained by the differences between breeds was higher (6%) because of reproductive isolation after the colonization event and because of genetic drift. the reduced diversity of new world horse breeds when compared with iberian breeds is consistent with an iberian origin. previous studies that included south american horse breeds support this theory (luis., 2006). as expected, the colombian paso fino breed had a lower degree of genetic diversity than iberian breeds. in contrast, the colombian creole cattle horse breed showed a similar degree of genetic diversity to that of the iberian breeds analyzed. the colombian paso fino breed is considered to have undergone selective breeding because of its characteristic gait, which renders it very suitable for riding. conversely, the colombian creole cattle horse breed has been kept in relative isolation with very little selective pressure. the different selection processes applied to colombian breeds could explain the high genetic diversity found in the colombian creole cattle horse relative to the colombian paso fino horse. our network analysis of the mtdna d - loop sequences discovered 51 haplotypes that could be classified into four main clusters that formed the four principal haplogroups (a, c, d and f). the heterogeneity of the marismeo breed, in which haplogroup a has previously been identified, could explain the high number of unidentified haplotypes (six of 13) that aligned within this haplogroup. in conclusion, four principal matrilineages have been found in the two principal colombian horse breeds. haplogroup d, which is representative of the ancestral iberian horse population, was discovered most often in colombian breeds, thus supporting the ancestral iberian origin of colombian horses. the higher degree of genetic diversity found in the colombian creole cattle horse when compared with either the colombian paso fino or other south american horse breeds is a consequence of significantly different selective pressures in both populations. the following online material is available for this article : figure s1 - variable sites in a 364-bp fragment of the mitochondrial dna d - loop of colombian and spanish horse breeds. | in order to understand the genetic ancestry and mitochondrial dna (mtdna) diversity of current colombian horse breeds we sequenced a 364-bp fragment of the mitocondrial dna d - loop in 116 animals belonging to five spanish horse breeds and the colombian paso fino and colombian creole cattle horse breeds. among colombian horse breeds, haplogroup d had the highest frequency (53%), followed by haplogroups a (19%), c (8%) and f (6%). the higher frequency of haplogroup d in colombian horse breeds supports the theory of an ancestral iberian origin for these breeds. these results also indicate that different selective pressures among the colombian breeds could explain the relatively higher genetic diversity found in the colombian creole cattle horse when compared with the colombian paso fino. |
pseudoexfoliation syndrome (pes) is characterized by the production and accumulation of fibrillar extracellular material within the ocular tissue. the presence of pes is of paramount clinical importance in patients undergoing cataract surgery, because it is associated with a high rate of complications such as zonular dialysis, rupture of the posterior lens capsule, vitreous loss and dislocation of the intraocular lens (iol). pes has also been identified as a main factor predisposing to secondary open - angle glaucoma. spontaneous dislocation of a posterior chamber iol (pc / iol) represents an unusual, severe complication, which mostly occurs in the late postoperative period and requires exchange surgery of the pc / iol or pc / iol repositioning with scleral fixation [2, 3, 4 ]. in all cases reported in the literature, an in - the - bag pc / iol was subluxed posterior into the vitreous cavity. here, we report on a patient with pes who developed partial acute angle - closure glaucoma (acg) with a marked myopic shift due to anterior dislocation of the pc / iol almost 16 months after an uneventful phacoemulsification. to the best of our knowledge, this is the first case of late - onset anterior pc / iol dislocation with partial acg and a marked myopic shift, which resulted in a permanent alteration of the postoperative target refraction. a 70-year - old male underwent an uneventful clear - cornea phacoemulsification with pc / iol implantation in the right eye (od) in january 2006. preoperative routine examination disclosed a bilateral corticonuclear cataract as well as features of pes od (fig. there was no other medical history, and the patient did not receive any medication., we implanted a capsular tension ring (ctr) in order to stabilize the posterior capsule and prevent zonular - capsular complications. a pmma ctr (type 14, 10-mm closed diameter ; morcher gmbh, stuttgart, germany) was implanted, which, according to our personal experience, represents a simple and reliable solution for cases with moderate risk. finally, a foldable pc / iol (hydrophilic acryl) was inserted in the bag, without any complication occurring during the surgical procedure. after one week, the patient 's best corrected visual acuity (bcva) was 1.0 (refraction 0.25/0.5/110). slit - lamp examination demonstrated a marked shallowing of the anterior chamber, without any other visible pathological findings (fig. intraocular pressure (iop) was 29 mm hg od and 11 mm hg os. evaluation of refraction unravelled a significant myopic shift od ; bcva was 1.0 with 3.75/0.5/110. the refractive status os was 0.25/0.75/115 and bcva was 1.0. scheimpflug examination documented a prominent axial anterior dislocation of the pc / iol od, without fluid accumulation between the posterior capsule and the pc / iol (fig. the pc / iol was sitting in the capsular bag os, and the anterior chamber depth was 5.21 mm (fig. iop elevation resisted to topical therapy with -2 receptors agonists (brimonidine eye drops) and, therefore, one week later nd : yag iridotomy was performed. iop was successfully regulated od after nd : yag iridotomy (direct postoperative iop : 14 mm hg). nevertheless, the shallowing of the anterior chamber did not significantly improve, and the anterior dislocation of the pc / iol remained unaltered (refraction 2.75/0.75/110, bcva 1.0). although the myopic shift od was not eliminated, the patient was satisfied with the monovision, which was achieved unintentionally and, therefore, we did not proceed to an exchange surgery of the pc / iol. pes is associated with a high incidence of pc / iol dislocation in cataract surgery, whether it is an in - the - bag pc / iol or an out - of - the - bag pc / iol. this unusual complication may occur with any type of pc / iol on average 8.5 years after its implantation. it is important to note that all cases reported up to date in the literature manifested a posterior pc / iol dislocation in the vitreous cavity. our patient presented with a myopic shift and acg due to delayed anterior pc / iol dislocation od. slit - lamp examination did not reveal an anterior capsular opacification of elements of phimosis which could explain the anterior dislocation of the pc / iol - ctr complex. during a thorough preoperative explanatory discussion, the patient was informed about the complication risk due to the underlying pes, and he strongly insisted that we should implement any necessary precautionary measures during the operation to minimize this risk. therefore, we decided to implant a ctr during the operation in order to stabilize the posterior capsule and prevent zonular - capsular complications, although we did not observe phacodonesis or difficulties in pupil dilation preoperatively. von der lippe. first postulated that zonular weakness in some patients with pes may lead to anterior subluxation of the lens with consecutive ciliary block acg. use of a ctr during the cataract surgery did not prevent this complication, since ctr implantation in pes - associated zonular weakening does not guarantee long - term zonular stability. in our patient, nd : yag iridotomy resulted in a satisfactory iop regulation, but the emmetropic refractive status was not restored. our patient was satisfied with the monovision (od refraction 2.75/0.50/110, od bcva 1.0 ; os refraction 0.25/0.75/115, os bcva 1.0) and, therefore, we did not proceed to pc / iol exchange or pc / iol repositioning. to the best of our knowledge, this is the first report of late - onset anterior pc / iol dislocation in pes, generating a permanent myopic shift. ophthalmologists should be aware of this potential complication, which may occur in patients with pes within a considerable period of time after cataract surgery, and these patients should be warned accordingly. | here, we report on a patient with pseudoexfoliation syndrome who developed acute angle - closure glaucoma with a marked myopic shift due to anterior dislocation of the posterior chamber intraocular lens almost 16 months after an uneventful phacoemulsification. examination with a scheimpflug camera was extremely useful in confirming the diagnosis. this is the fist case of late - onset angle - closure glaucoma with a significant myopic shift due to anterior dislocation of the posterior chamber intraocular lens, which resulted in a permanent alteration of the postoperative target refraction. |
internet access has grown rapidly, to over 75%, and 80% of those with internet access seek health information online.13 fewer than 10% of patients, however, communicate with their provider by e - mail, although many would like to.410 for example, sittig and colleagues observed that only 6% of patients reported e - mailing a doctor, although 50% would e - mail their doctor if given the chance.6 physicians have concerns over the effect of doctor patient e - mail on workload and payment for services and the security of doctor patient e - mail.7 one clinical trial showed no effect of e - mail on decreasing call volume for providers.8 patients also have concerns about the security of doctor patient e - mail,11 and how long it would take doctors to respond online.4 other researchers have observed that physicians often do not respond to e - mail from patients12 and often use doctor patient e - mail inappropriately, conveying urgent or sensitive matters that are better addressed face - to - face.13 doctor patient online communications can be of many types, from simple communications (e.g., for prescription renewals, lab test results, and billing questions) to more detailed interactions requiring back - and - forth communication and clinical decision making (e.g., for a new symptom).8 despite the barriers and limitations of the medium, many insurers are moving toward reimbursing online consultations.1416 we carried out the present study to describe the frequency that patients in the united states saw physicians who did internet or e - mail consults and to describe patient and provider characteristics associated with this activity. no previous study has included nationally representative data at both the patient- and doctor - level, to address this question. we analyzed complete case data from the 2001, 2002, and 2003 national ambulatory medical care survey (namcs), an annual survey of outpatient practices in the united states. all physicians in namcs complete a one - time physician induction interview (pii) telephone interview. for each physician, 1 week was randomly selected, during which 20 to 100% of patient visits were sampled. after each visit, physicians completed an encounter form, detailing patient demographics, diagnoses made and clinical services provided. we excluded physicians who did not submit patient encounter forms (approximately 65% per year) and who did not answer the question on e - mail consults (less than 5% per year). each complete survey included approximately 900 physicians and 18,500 patient visits, for a total of 2,725 physicians and 55,658 visits.17,18 the dependent variable was an indicator of whether the physician engaged in internet or e - mail consults with patients from the pii. in 2001, physicians were asked on average, about how many encounters of the following type do you make with patients each week, and one category was internet / e - mail consults. physicians responded with a number, although the data were collapsed for the data file released to the public and reported as yes if the response was greater than 0, no if the response was equal to 0, and unknown if the response was blank or unknown. in 2002 and 2003, the question wording was changed slightly from on average to during your last complete week of practice. this main outcome measure, therefore, characterizes whether the provider engaged in internet or e - mail consults, not whether the specific patient encounter was an internet or e - mail consult. the following items from the pii were also included in the analysis : specialty was categorized into primary care (e.g., family practice) versus specialty care (e.g., surgery). office setting was categorized into private solo or group practice versus other (e.g., hmo). patient diagnoses were grouped to identify the following major chronic conditions : diabetes, depression, obesity, hypertension, hypercholesterolemia, and asthma, using standard icd-9 codes.1719 patient age, gender, race, smoking status, and reason for the visit were also recorded for each visit. the univariate, bivariate, and multivariate analyses, including physician, patient, and visit characteristics, were done at the visit level. overall, 6.9% of visits were with a provider who conducted internet or e - mail consults (9.2% in 2001, 5.8% in 2002, and 5.5% in 2003), but this decline was not statistically significant. the likelihood of seeing a provider who conducted internet or e - mail consults was greater among visits to primary care providers, for patients seen in the west, for patients aged 4564, for male patients, for nonminority patients, for patients seen for pre-/postsurgical care and for patients who saw only a physician, rather than a nurse in addition to a physician. no significant differences existed, however, between the 4 different types of primary care providers (general practitioners, medicine / pediatrics, general internal medicine, family practice). no significant association was observed between internet or e - mail consults and patient chronic conditions or other provider (e.g., office setting) or visit (e.g., number of medications prescribed) characteristics (table 1). table 1association of physician, patient, and visit characteristics with likelihood of seeing a physician who conducted email consultationscharacteristicsnumber of physicians reporting information about themselves or about patients they sawdid physician do e - mail consults ? (% yes)odds ratio (95% ci)year of survey20018259.21.0020029705.80.58 (0.301.10)20039305.50.56 (0.291.08)physician characteristicsphysician specialty primary care6178.31.66 (1.012.70) specialty care2,1085.71.00type of office setting private solo or group practice2,4836.51.00 all others25310.61.56 (0.733.34)ownership of practice physician or physician group2,4066.81.00 all others3387.30.96 (0.332.82)employment status of physician owner2,0986.91.00 employee or contractor6577.00.85 (0.391.84)geographic region northeast5657.11.74 (0.833.66) midwest6548.82.28 (0.965.41) south9043.81.00 west6029.52.36 (1.095.11)metropolitan area yes2,3727.31.91 (0.784.68) no3534.01.00patient characteristicsgender male2,4708.11.25 (1.091.42) female2,6596.21.00age 18241,5025.30.99 (0.751.29) 25442,4447.11.23 (0.971.57) 45642,5647.51.21 (1.011.44) 65 + 2,3556.31.00race / ethnicityminoritynon - white race or hispanic ethnicity2,0135.31.00white race, non hispanic ethnicity2,4467.11.41 (1.031.94)white race, ethnicity not reported7068.21.28 (0.692.38)tobacco useno2,4196.91.00yes1,8417.51.01 (0.851.21)has 1 or more chronic conditionsyes1,6277.30.99 (0.761.29)no2,6636.81.00visit characteristicsmajor reason for visitacute problem2,3467.61.38 (0.902.12)chronic problem, routine2,3696.71.28 (0.831.98) chronic problem, flare - up1,6757.21.43 (0.922.20)pre-/postsurgery1,1928.52.01 (1.223.31)preventive care1,0615.11.00saw rn / lpnyes9844.61.00no2,3697.81.74 (1.062.87)visit dispositionfollow - up planned2,6476.70.96 (0.681.34)follow - up not planned2,2837.41.00medications prescribed022,6746.50.74 (0.531.03)3 + 1,7568.21.00paymentinsurance2,6526.81.00self - pay / no charge1,0226.20.89 (0.551.45)unknown / other / not stated1,0099.31.36 (0.672.75)physicians may appear in multiple rows that describe patient characteristics. for example, 2,470 physicians saw at least 1 male patient, and 2,659 physicians saw at least 1 female patient.odds ratios presented are multivariable, adjusted for all other variables presented in this table.p <.05p <.01race was noted to be white but the question on hispanic ethnicity was not answered.overall, 23.8% of patients saw a nurse (rn / lpn), and 96% of these also saw a physician.#p <.001 association of physician, patient, and visit characteristics with likelihood of seeing a physician who conducted email consultations physicians may appear in multiple rows that describe patient characteristics. for example, 2,470 physicians saw at least 1 male patient, and 2,659 physicians saw at least 1 female patient. odds ratios presented are multivariable, adjusted for all other variables presented in this table. race was noted to be white but the question on hispanic ethnicity was not answered. overall, 23.8% of patients saw a nurse (rn / lpn), and 96% of these also saw a physician. only 9.2% of outpatient visits in the united states in 2001, 5.8% in 2002, and 5.5% in 2003 were to physicians who engaged in internet or e - mail consults. this represents one of the largest published studies of the availability of physicians who do internet or e - mail consults to date. similarly, large studies have been done by private research firms,5,9,10 but no previous published study included patient, provider and visit - level characteristics. the proportion of visits in which patients saw providers who reported doing internet or e - mail consults is similar to other studies, although results are difficult to compare as the question wording differs.48,10,21 gaster and colleagues observed that 72% of academic physicians averaged 7.7 e - mails to patients each month.22 hobbs and colleagues observed that 75% of academic physicians exchanged e - mail, but most with only 15% of their patients.7 brooks and colleagues observed that 16.6% of physicians in florida exchanged e - mail with patients, but less than 3% did this on at least half of all business days.21 surveys by deloitte and manhattan research observed that 25% of physicians reported e - mailing patients, though frequency was not noted.9,10 surveys of patients have observed lower rates. moyer, sittig, and fox and jupiter research, respectively, observed that 10, 6, 7, and 3% of individuals had ever e - mailed a doctor.2,4,6 the current study, however, examined internet or e - mail consults, rather than simply exchanging e - mail with patients. a study by jupiter research, which similarly described the activity as a consult, observed that only 3% of adults with internet access reported having online clinical consultations with their doctor in 2003.5 the main observation was the low overall rate in the proportion of visits to providers who reported doing internet or e - mail consults and lack of an increase in the rate. the lack of an increase between 2002 and 2003, years in which the question wording was consistent, is somewhat surprising given the simultaneous growth in internet access and online health information seeking.2,3,23,24 this may be because of the looming implementation of the health insurance portability and accountability act25,26 in 2003 and the lack of secure online communication tools and health plan reimbursement at the time.15,27,28 access to providers who conducted e - mail consults was higher among male patients, though the reasons for this are not clear. adjusting for whether or not the provider was an obstetrician gynecologist did not remove the gender difference. although namcs now collects provider demographic information, the namcs in 20012003 did not, so we are unable to test whether these differences are due to demographic differences in the providers seen by men and women. also, patients who saw primary care providers and patients seen for pre-/postoperative care were more likely to see a provider who conducted internet or e - mail consults. primary care providers, whose practice includes a significant amount of chronic disease management, may find e - mail helpful for streamlining communication with patients.13,29 similarly, physicians whose practice includes a significant amount of pre-/postsurgical care may find e - mail useful for patients ; they may not be seeing the patient on an ongoing basis to send laboratory results.8,30 it is somewhat surprising that the number of medications was not associated with likelihood that the patient saw a provider who did e - mail consults, as one important use of e - mail is medication refills.8 this indicates that patients using more medications were not more likely to seek out such providers, perhaps because other factors are more important influences on choice of provider. although it was reassuring that access to physicians who did internet or e - mail consults did not differ among patients of differing insurance status, access to these physicians was less among minority patients. the namcs did not include data regarding patient education level or household income, so residual confounding can not be excluded. our analysis had the following strengths : (1) the sample was nationally representative and multiyear and (2) patient-, visit- and provider - level data were included. first, there is no clear definition of a consult via internet or e - mail, given that these technologies are still evolving. as reimbursement is likely to increase the use of these more formal consults, future studies will be needed to assess the impact and comparability to face - to - face services, as has been done to compare primary care physicians and primary care nurse practitioners.31 second, although certain patients were more likely to see a provider who engaged in internet or e - mail consults (e.g., men), we do not know whether these patients were more likely to engage in an internet or e - mail consult with their provider. this is a limitation of the data, as physicians were only asked once, not after every visit, whether they engaged in internet or e - mail consults. third, we could not examine some patient (e.g., household income) and doctor (e.g., age) characteristics that may be related to whether doctors conduct internet or e - mail consults. we do not believe that these limitations are likely to impact the main findings of the study that internet or e - mail consult rates were generally low and did not appear to be increasing, despite strong growth in other internet - related health activities. | backgrounde - mail communication has the potential to improve communication between patients and doctors.objectivethe objective of the study is to describe the access of patients to physicians who conduct e - mail consults.methodswe analyzed data from the national ambulatory medical care survey (namcs), a nationally representative cross - sectional survey of office - based physician visits, in 2001, 2002, and 2003. the main outcome measure was the percentage of visits to a provider who reported doing internet or e - mail consults.resultsthere was fewer than 1 in 10 outpatient visits in 2001 (9.2%) to physicians who reported doing internet or e - mail consults, and this did not increase in 2002 (5.8%) or 2003 (5.5%). access to these physicians was greater among patients who were male, nonminority, lived in the western united states, seen for pre-/postoperative care, seen by a primary care provider, and not seen by a nurse during their visit. access to physicians who conducted internet or e - mail consults was independent of other patient (e.g., chronic conditions), provider (e.g., office setting), and visit (e.g., medications prescribed) characteristics.conclusionsaccess to physicians who do internet or e - mail consults is generally low and did not increase between 2001 and 2003, despite growth in internet access and in other internet - related activities. |
water, the most abundant molecule in living cells, is required for various vital life functions including the regulation of cell and organelle volume. changes in cell volume are known to occur during cell secretion, in erythrocyte maturation, during cell growth and differentiation, and in cell migration and metabolism. similarly, intracellular swelling of membrane - bound secretory vesicles [4, 5 ] has been demonstrated in the regulated release of vesicular contents during cell secretion. all these studies demonstrate that rapid swelling of cells or secretory vesicles occurs as a result of rapid water entry through membrane - associated water channels or aqps. platelets are 23 m in diameter anuclear cells that contain lysosomes, granules and dense granules, and secrete a variety of essential molecules including growth factors. impaired platelet function is causal to bleeding or clotting, resulting in adverse outcomes such as myocardial infarction, stroke or pulmonary embolism. because regulation of platelet volume significantly affects its function, this study was undertaken to determine the molecular mechanism of platelet volume regulation. volume regulation in cells and secretory vesicles has previously been examined [4, 5 ]. live pancreatic acinar cells in near physiological buffer, when imaged using the atomic force microscope (afm), revealed at nanometre resolution the presence of membrane - bound secretory vesicles called zymogen granules (zgs) lying immediately below the surface of the apical plasma membrane. within 2.5 min. of exposure to a secretory stimulus, the majority of zgs within cells swell, followed by a decrease in size, and a concomitant discharge of intra - vesicular contents. these studies directly demonstrate intracellular swelling of secretory vesicles following stimulation of cell secretion, and vesicle deflation following partial discharge of vesicular contents. a similar mechanism of synaptic vesicle (sv) swelling for neurotransmitter release has also been reported. this direct estimation of vesicle size dynamics at nanometre resolution under various experimental conditions has enabled determination of some of the molecular components participating in secretory vesicle swelling. studies using isolated zgs demonstrate the presence of cl and atp - sensitive k selective ion channels at the zg membrane, whose activities are implicated in zg swelling. these studies further demonstrate that secretion of zg contents from pancreatic acinar cells require the presence of both k and cl. heterotrimeric gi3 protein has been implicated in the regulation of both k and cl ion channels in a number of tissues. analogous to the regulation of k and cl ion channels at the plasma membrane in cells, the regulation of these channels at the zg membrane by a gi3 protein has been demonstrated. isolated zgs from exocrine pancreas swell rapidly in response to gtp and naf, suggesting the involvement of rapid water entry into zgs following gtp exposure. as opposed to osmotic swelling, membrane - associated aqp channels are involved in rapid water entry into cells. the presence of aqp1 and gi3 protein at the zg membrane [4, 5 ], and aqp6 and go protein at the sv membrane [4, 5 ], and their involvement in gtp - mediated vesicle water entry and swelling have previously been reported [4, 5 ]. mastoparan, an amphiphilic tetradecapeptide from wasp venom, potentiates the gtpase activity of gi / go proteins [13 - 15 ]. stimulation of g proteins is believed to occur by the peptide inserting into the phospholipid membrane and forming a highly structured -helix resembling the intracellular loops of g - protein coupled receptors. analogous to receptor activation, mastoparan is thought to interact with the cooh - terminal domain of the g protein subunit. active mastoparan (mas7) in the presence of [-p]gtp demonstrate a significant increase in swelling and gtpase activity in zg, establishing the presence of gi proteins with zgs. similarly, mastoparan has also been demonstrated to stimulate sv swelling. studies further report that vh - atpase present at the sv membrane is responsible for the generation of electrochemical h gradient (ph 5.25.5) within the sv required for transport of neurotransmitters into the sv lumen. in addition to the established role of vh - atpase in neurotransmitter transport into sv, vh - atpase has been suggested to participate in the secretion of stored neurotransmitters [20, 21 ]. because sv swelling is go - mediated, and is required for cell secretion, the involvement of vh - atpase at the sv membrane in go - mediated water gating through the aqp6 channel was hypothesized and tested in a recent study. in agreement, results from the study demonstrated that sv - associated vh - atpase is required for gtp - go mediated swelling of sv. in view of these earlier studies, the involvement of aquaporin and heterotrimeric g - proteins in the regulation of platelet volume was hypothesized and tested in this study. mercuric chloride is known to inhibit most aqps except aqp6, which is stimulated by the compound. exposure of platelets to hgcl2-induced cell swelling in a dose - dependent manner, suggesting the presence of aqp6 in platelets. immunoblot analysis of platelet protein confirmed the presence of aqp6. to further determine if g proteins were involved in aqp6 function in platelets, g - protein specific immunoblot analysis and mastoparan - stimulated swelling was examined. exposure of platelets to mastoparan results in a dose - dependent swelling of platelets, demonstrating for the first time the involvement of g - proteins in platelet volume regulation. immmunoblot analysis further confirm the presence of aqp6 in platelets, and the presence of go, gi1 and gi3 proteins in platelets as previously demonstrated [24, 25 ]. results from this study demonstrate for the first time that aqp6, go, gi1 and gi3 proteins are involved in platelet volume regulation. all animal studies were approved by the iacuc at wayne state university. to isolate red blood cells (rbc), the blood sample was diluted in 10 volume of pbs ph 7.4, and spun at 120 g for 10 min. at 4c. the resulting pellet was resuspended in 2 volume of pbs, and washed three times, followed by their final resuspension in 10 volume of pbs prior to use. the purity of rbc preparation was examined using light and scanning electron microscopy (fig. 1a). to isolate platelets, blood was dispersed in acid citrate - dextrose solution (acd ; 0.1 m citric acid, 0.2 m sodium citrate, 0.4 m dextrose, ph 6.8), which functions as an anti - coagulant and anti - stimulant for platelets. 1a, i). whole blood with an equal amount of acd was centrifuged at 100 g for 15 min. and platelet - rich supernatant was mixed with an equal amount of buffer a (20 mm hepes - naoh ph 7.4, 3.3 mm nah2po4, 2.9 mm kcl, 1 mm mgcl2, 128 mm nacl, 5.5 mm d - glucose, ph 7.4). the supernatant was centrifuged at 1000 g for 10 min. to obtain a pure platelet pellet. isolated platelet and rbc preparations for electron microscopy were fixed using 2% paraformaldehyde and 1% glutaraldehyde for 1 hr at room temperature followed by washing in pbs. isolated pure platelet preparations were used in dynamic light scattering experiments, and both platelet and rbc preparations were solubilized using 1% triton / lubrol for immunoblot analysis (fig. light and scanning electron micrographs of isolated platelets from rat blood, and presence of aqp6, go, gi1 and gi3 proteins in the preparation. (a, ii) light and (a, iii) scanning electron micrographs of purified platelet preparation. (a, iv) light and (a, v) scanning electron micrographs of purified rbc preparation. (b, i) immmunoblot analysis of platelet homogenate, demonstrates the presence of aqp6, and (b, ii) go, gi1 and gi3 proteins. total rbc homogenate demonstrates the presence of aqp1 and the absence of aqp6. to perform light microscopy on isolated platelets, cells were allowed to settle and stick to the slide surface at room temperature, and the unattached cells rinsed gently three times using 200 l of pbs. similarly, to perform scanning electron microscopy on isolated platelet preparations, 25 l of aldehyde fixed isolated platelets was spread on 13 mm thermonox cover slips coated with 0.2% poly - l - lysine. cells were allowed to settle and stick to the surface of the cover slip overnight at 4c, and the excess sample rinsed gently by dipping into four changes of milli q dh2o., each in increasing concentrations of ethanol (35%, 50%, 70%, 85% and 95%) followed by four times 5 min.. the sample was dried and mounted onto one large aluminium specimen stub in puddles of liquid graphite, sputter coated with gold and palladium and imaged with a joel jsm 6060 sem operating at 25 kv, spot size 30, wd810 mm at various magnifications up to 28,000 (fig. platelet size dynamics were determined using real - time, right angle light scattering in a hitachi f-2000 spectrofluorimeter. real - time scattered light intensities at excitation and emission wavelengths of 530 nm were used to measure platelet size dynamics. in these experiments, isolated platelets were suspended in water, and rapid changes in their size was monitored before and after addition of hgcl2 (an aqp1 inhibitor and an aqp6 stimulator), followed by mastoparan exposure. student 's t - test was performed for comparison between groups, with significance established at p < 0.01. sample aliquots solubilized in laemmli sample preparation buffer were resolved using 10% sds - page. the nitrocellulose membranes were incubated in blocking buffer (5% non - fat milk in pbs with 0.1% tween) for 30 min. at room temperature, followed by immunoblotting for 1 hr at room temperature with specific antibodies. primary polyclonal antibodies (santa cruz biotechnology, santa cruz, ca, usa) were used at a dilution of 1:200 (aqp-1, aqp-6, go, gi1 and gi3) in blocking buffer. immunoblotted nitrocellulose membranes were washed in pbs and incubated in horseradish peroxidase conjugated secondary antibody (santa cruz biotechnology) at a dilution of 1:1000 for 1 hr at room temperature. after washing in pbs, the nitrocellulose membranes were processed for enhanced chemiluminescence (amersham bioscience, piscataway, nj, usa) and developed using a kodak 440 image station. analogous to a number of other mammalian cell types, platelet volume significantly affects its function, such as platelet activation and the secretion of a variety of essential molecules. impaired volume regulation in platelets negatively influences blood clotting, resulting in adverse outcomes such as myocardial infarction, stroke or pulmonary embolism. because water channels or aquaporins have previously been implicated in cellular and organelle volume regulation, their presence in platelets and their potential role in platelet volume regulation was hypothesized and investigated. platelets were isolated from rat blood, and their purity determined by both light and scanning electron microscopy (fig. exposure of platelets to hgcl2 induced swelling in a dose - dependent manner, suggesting the presence of aqp6 in platelets (fig. hgcl2 induced a dose- and time - dependent increase in platelet size, as demonstrated using dynamic light scattering. dynamic light scattering further demonstrated that 300 m hgcl2 was the optimal dose in stimulating platelet size increase. in the first 15 sec. after addition of 300 m hgcl2, increase of platelet swelling is a linear function of time (fig. 2a, iii), suggesting that influx of water and ions is unrestricted and rapid at this initial period. after this point, platelet swelling slows down, and changes in platelet volume assume a logarithmic form that can be expressed by a first - order equation, with a rate constant k = 2.3 10/sec. similarly, analogous to hgcl2, dynamic light scattering further demonstrated that mastoparan induces a dose- and time - dependent increase in platelet size (fig. dynamic light scattering demonstrated that 40 m mastoparan was the optimal dose in stimulating platelet size increase (fig. after addition of 40 m mastoparan, increase of platelet swelling is a linear function of time (fig. 2b, iii), again suggesting that influx of water and ions is unrestricted and rapid at this initial period. after this point, platelet swelling slows down, and changes in platelet volume assume a logarithmic form that can be expressed by a first - order equation, with a rate constant k = 24 10/sec. these studies suggested that both aqp6 and gi / go proteins are present in platelets and participate in platelet volume regulation. this is further confirmed, because immunoblot analysis performed on total platelet proteins using specific antibodies to aqp6, and to gi and go proteins, demonstrate their presence in platelets (fig. immunoblot analysis further demonstrates that aqp6 is present in platelets ; however, the rbc water channel aqp1 is absent. similarly, aqp6 is absent in rbc. to further determine if g proteins are present in platelets, g - protein these immunoblot studies confirm the presence of go, gi1 and gi3 proteins in platelets (fig. these results demonstrate for the first time that functional aqp6, go, gi1 and gi3 proteins are present in platelets and are involved in cell volume regulation. it is possible that one or all of the above g - proteins are involved in platelet volume regulation. analogous to secretory vesicle volume dynamics, we have now demonstrated that the regulation of platelet volume involves both water channels and heterotrimeric g - proteins, driving the active and rapid transport of water through the aqp6 channels at the platelet plasma membrane. hgcl2- and mastoparan - induced dose- and time - dependent increase in platelet size, demonstrated using dynamic light scattering. (a, i) dynamic light scattering, demonstrating hgcl2 dose - dependent increase in platelet size over control. (a, ii) exposure of 300 m hgcl2 to isolated platelets, demonstrates a time - dependent increase in platelet size. (a, iv) graph depicts the first - order kinetics of increase in platelet size following exposure to 300 m hgcl2. (b, i) dynamic light scattering demonstrating the mastoparan dose - dependent increase in platelet size over control. (b, ii) exposure of 40 m mastoparan to isolated platelets, demonstrates a time - dependent increase in platelet size. (b, iv) graph depicts the first order kinetics of increase in platelet size following exposure to 40 m mastoparan., performed majority of the experiments, both contributing equally to the study. m.t. and d.j.t. | abstractthe regulation of platelet volume significantly affects its function. because water is the major molecule in cells and its active transport via water channels called aquaporins (aqps) have been implicated in cellular and organelle volume regulation, the presence of water channels in platelets and their potential role in platelet volume regulation was investigated. g - protein mediated aqp regulation in secretory vesicle swelling has previously been reported in neurons and in pancreatic acinar cells. mercuric chloride has been demonstrated to inhibit most aqps except aqp6, which is stimulated by the compound. exposure of platelets to hgcl2-induced swelling in a dose - dependent manner, suggesting the presence of aqp6 in platelets. immunoblot analysis of platelet protein confirmed the presence of aqp6, and also of go, gi1 and gi3 proteins. results from this study demonstrate for the first time that in platelets aqp6 is involved in cell volume regulation via a g - protein mediated pathway. |
the term acute renal failure (arf) is currently substituted by acute kidney injury (aki). aki is a reversible condition in which there is a sudden decline in renal function, manifested by hourly / daily / weekly elevation in serum creatinine and blood urea nitrogen (bun). different organizations such as acute dialysis quality initiative (adqi), acute kidney injury network (akin) and kidney disease international global outcome (kdigo) have provided different definitions for acute kidney injury. among these definitions of aki, the most acceptable one is that of kdigo : aki is any of the followingincrease in serum creatinine (scr) by 0.3 mg / dl (26.5 orincrease in scr to 1.5 times baseline, which is known or presumed to have occurred within the prior 7 days orurine volume < 0.5 ml / kg / h for 6 h. increase in serum creatinine (scr) by 0.3 mg / dl (26.5 mol / l) within 48 h or increase in scr to 1.5 times baseline, which is known or presumed to have occurred within the prior 7 days or urine volume < 0.5 ml / kg / h for 6 h. the incidence of aki in the community is 2147 and 4085 per million populations per year (pmp) in developing and developed nations.4, 5 recent reports in the developed world indicate that aki is seen in 3.29.6% of hospital admissions with overall mortality of 20%50% in icu patients.6, 7 aki demanding renal replacement therapy is 56% with a high in - hospital mortality rate of 60%. it is estimated that nearly 2 million people die of aki every year globally.8, 9 those who survive aki are at a greater risk for later development of chronic kidney disease (ckd). the causes of aki could be pre - renal, renal and post - renal ones. pre - renal failure, the commonest form of acute renal failure, is due to decrease in renal blood flow primarily as a result of hypovolemia. it is reversible if the cause of the decreased renal blood flow can be identified and rectified before kidney damage occurs. intrinsic renal causes of aki include diseases that affect the renal parenchyma which can be divided based on the compartment of the kidney that is affected like tubular injury, tubulointerstitial diseases, diseases of the renal microcirculation and glomeruli and diseases of larger renal vessels. post - renal causes of aki are diseases associated with urinary tract obstruction which account for 5% of renal failures. the management of arf is mainly supportive and in refractory patients renal replacement therapy (rrt) may be the best option. rrt refers to life supporting measures which include hemodialysis, peritoneal dialysis, hemofiltration and renal transplantation. in spite of rrt, mortality in patients with aki remains high and this may be due to the severity of the disease or the adverse effects of rrt. as there is no effective pharmacotherapy for aki, more than 221 plants have been screened for nephroprotective activity both in acute and chronic renal failure models. some of the medicinal plants such as ocimum sanctum, aerva lanata, aegle marmelos, pongamia pinnata, salviae radix, ginkgo biloba, allium sativum, cassia auriculata, nigella sativa, drynaria fortune, tribulus terrestris and others are reported to have nephroprotective activity. azima tetracantha (at) has been traditionally used for many diseases including renal disease. it belongs to the family, salvadoraceae and known as mulchangu in siddha and kundali in ayurvedha. its common names are needle brush (english), kantagurkamai (hindi) and cankakiranam (tamil). at has been reported to have antimicrobial, antifungal, anti - inflammatory, analgesic, antioxidant, antipyretic, antiulcer, anticancer, anti - snake venom, diuretic and hepato - protective activities.19, 20, 21, 22, 23 though at is traditionally used for nephroprotective effect, it has not been evaluated by either preclinical or clinical studies. hence this study was undertaken to evaluate its action in animal models of renal failure. root and fruit : glycosides n - methoxy-3-indolylmethyl - glucosinolate, iodole glucosinolate and n - methoxy indole 3- corbinol. root and stem : flavonoids myricetin, quercetin, rutin, isorhamnetin, rhamnetin and rhamnazin. the objectives were to evaluate the nephroprotective activity of ethanolic extract of a. tetracantha root in glycerol - induced acute renal failure in wistar albino rats and to find out the probable mechanism. chemicals for in vitro and in vivo antioxidants assays were purchased from sigma aldrich, new delhi, india. the plant was collected from tindivanam, villupuram district, tamil nadu, india and it was identified and authenticated by dr. narasimhan, associate professor of botany, madras christian college, chennai, tamil nadu. one kg of roots was thoroughly washed with water and air dried under shade at room temperature for 30 days and milled into a coarse powder. the dried powder obtained was subjected to continuous extraction with 95% v / v ethanol in soxhlet apparatus for 15 cycles. thirty healthy male wistar albino rats weighing between 150 and 250 g were obtained from the central animal house of chettinad hospital and research institute. the antioxidant activity of atr ethanolic extract was evaluated by 2,2-diphenyl-1-picrylhydrazyl (dpph) assay, nitric oxide (no) assay, hydrogen peroxide (h2o2) and ferric reducing antioxidant power (frap) assay. all assays were performed using spectrophotometer and the values were calculated by the following formula.percentageinhibition=absorbanceofcontrolabsorbanceoftest100absorbanceofcontrol the atr extract was prepared in increasing concentrations from 25 to 1600 g / ml for all the antioxidant assays. one ml of atr extract solution was mixed with 1 ml of ethanolic solution of dpph (200 m). another 1 ml dpph solution was mixed with 1 ml of ethanolic solution of vitamin- c (200 g / ml). a mixture of 1 ml of ethanolic solution of dpph (200 m) and 1 ml of ethanol served as control. after mixing, all the solutions were incubated in dark for 2 min and absorbance was measured at 517 nm. the experiments were carried out in triplicate and the average percentage of inhibition was calculated by the above formula. three ml of 10 mm sodium nitroprusside in phosphate buffer is added to 2 ml of atr extract and the reference compound.. 5 ml of the incubated sample and 5 ml of griess reagent (1% sulphanilamide, 0.1% naphthyl ethylenediamine dihydrochloride in 2% phosphorous acid) were added and absorbance of the chromophore formed was measured at 540 nm. two ml atr extract was mixed with 0.6 ml of 4 mm hydrogen peroxide solution prepared in phosphate buffer (0.1 m ph 7.4) and incubated for 10 min. the assay mixture contained 2.5 ml of 300 mm acetate buffer at ph 3.6, 0.25 ml of 10 mm 2, 4,6-tripyridyl - s - triazine solution in 40 mm hcl, 0.25 ml of 20 mm ferric chloride and atr extract at various concentrations in 0.1 ml of methanol. frap values were measured by comparing the absorption change in the test mixture with that obtained from increasing concentrations of fe. female rats (150200 g), nulliparous and non - pregnant were randomly distributed to four groups (14) of 3 each. the rats were fasted overnight and given orally 5, 50, 300 and 2000 mg / kg of atr extract. they were observed continuously for the first 2 h and hourly for the next 6 h and at 24, 48, and 72 h for general behavior, convulsions and mortality. no mortality or toxicity was observed up to the dose of 2000 mg / kg. based on the acute toxicity studies 250 and 500 mg / kg of the ethanolic extract were used for the current study. induction of arf : arf was induced by intramuscular injection of single dose of 50% hypertonic glycerol (8 ml / kg) into both the hind limbs in equal volume as per the standard methods prescribed.30, 31 rats were divided into 5 groups of 6 animals in each. group 1 was treated with normal saline (10 ml / kg) per oral, group 2 with single dose of hypertonic glycerol (8 ml / kg) by intramuscular injection into both the hind limbs, group 3 with glycerol and atr extract 250 mg / kg, group 4 with glycerol and atr extract 500 mg / kg and group 5 was treated with atr extract 500 mg / kg alone. the atr extract was given by oral route 60 min prior to glycerol injection. urine output was measured for all the animals for 24 h. after 24 h, the blood samples were collected by retro orbital sinus puncture and tested for serum creatinine, blood urea nitrogen, total proteins and albumin. the antioxidants, superoxide dismutase (sod), reduced glutathione (gsh), glutathione reductase (gr), glutathione peroxidase (gpx), and catalase (cat) were determined using standard methods prescribed. kidneys were rinsed with normal saline and then fixed in 10% neutral buffered formalin solution, embedded in paraffin and used for histopathological examination. the sections were 2 m thick, deparaffinized, hydrated and stained with haematoxylin and eosin. the renal sections were examined for the extent of damage to glomeruli, tubules and interstitium as well as for capillary congestion and hemorrhage. one way analysis of variance (anova) traced by tukey 's multiple range test was applied to calculate the statistical significance among different groups. the results from hptlc finger print scanned at wavelength 254 nm for ethanolic extract of atr showed 15 polyvalent phytoconstituents (graph) and corresponding retarding fraction (rf), height, area and lambda max (table 1). ethanolic extract of a. tetracantha root in graded concentrations was tested for antioxidant activity in four different in vitro methods and compared with the standard antioxidant, vitamin c. percentage of inhibition of dpph radicals by different concentrations of atr extract was 46% at 25 g / ml and 89% at 1600 g / ml. standard drug vitamin- c showed 50 % inhibition at 25 g / ml and 95% at 1600 g / ml (table 2). the percentage inhibition of no by atr extract was 24% at 25 g / ml and 91% at 1600 g / ml. vitamin - c showed 28 % and 95% inhibition at 25 and 1600 g / ml respectively (table 3). atr extract showed 23% and 88% at 25 and 1600 g / ml and vitamin c at the same concentration showed 28 and 99% of inhibition (table 4). atr extract had shown 22% inhibition at 25 g / ml and 87% inhibition at 1600 g / ml. vitamin - c showed 28 % and 98% inhibition at 25 and 1600 g / ml respectively (table 5). nephroprotective activity was assessed by the levels of serum creatinine, blood urea nitrogen, total proteins, albumin and urine output as well as histopathological changes (table 6). increased levels of serum creatinine, bun and decreased total proteins, albumin and urine output in glycerol treated animals were seen (group 2) compared with normal saline treated animals (group 1) confirming renal failure. in atr extract treated groups there was a significant decrease (p < 0.05) in serum creatinine, blood urea nitrogen and increase in total proteins, albumin and urine output at both the doses of 250 and 500 mg / kg. the group treated with atr extract alone (group 5) did not show any change in the above parameters. sod, gsh, gr, gpx and cat levels were significantly (p < 0.05) increased in group 3 and 4 compared with group 2. atr extract at 500 mg / kg had shown higher antioxidant levels than 250 mg / kg (fig. 2, in vivo antioxidant activity). rat kidney in the control group had shown normal glomeruli with intact bowman 's capsule, normal proximal convoluted and distal convoluted tubules and there was no capillary congestion or hemorrhage (group 1). severe distortion of glomeruli, capillary congestion, hemorrhages and apical blebbing were seen in glycerol treated group indicating renal damage. in group 3 and 4, treated with 250 and 500 mg / kg of atr extract showed markedly reduced capillary congestion, tubular damage and glomerular distortion compared to glycerol treated group. the protective effect was found to be better with 500 mg / kg than 250 mg / kg. group 5 animals were not sacrificed as they did not show abnormalities in biochemical parameters. the present study has evaluated the effect of a. tetracantha root extract in glycerol - induced acute renal failure and its antioxidant potential both in vitro and in vivo. glycerol - induced acute renal failure is one of the commonest animal models used for evaluation of nephroprotective activity.30, 31 the mechanisms involved in glycerol - induced renal failure include oxidative stress, inflammation and apoptosis38, 39 which lead to rhabdomyolysis resulting in myoglobinemia, myoglobinuria and cast formation. rhabdomyolysis causes cytokine activation and release of inflammatory mediators such as il-1, icam-1 and tnf. these pro - inflammatory cytokines mediate the inflammatory reaction causing afferent and efferent renal arteriolar vasoconstriction eventually leading to renal failure. in general, about 1040% of patients with rhabdomyolysis develop arf. as oxidative stress and inflammatory damage have been mainly attributed to the development of acute kidney injury, any compound or drug having antioxidant & anti - inflammatory activity will have a protective action against renal damage. in our study atr extract has shown a protective effect evidenced by its inhibition of alteration of renal biochemical parameters and reversal of histopathological changes. prior administration of atr extract has caused reduction in serum creatinine, blood urea nitrogen and increase in total proteins and albumin. the histopathological examination has shown significant reduction in cellular infiltration, capillary congestion, glomerular damage, tubular necrosis and cast formation induced by glycerol. the possible mechanisms for the nephroprotective effect of atr extract could be due to the antioxidant and anti - inflammatory actions. the root extract has shown antioxidant activity both in vitro and in vivo assays. the in vivo antioxidant effect of ethanolic extract atr on the levels of sod, gsh, gpx, gr and cat is shown in fig. 2. glycerol treatment has reduced the levels of these antioxidants, which get utilized in scavenging the free radicals generated during glycerol - induced oxidative damage of renal parenchyma. atr extract has reduced the oxidative damage as evidenced by the dose dependent increase in the level of the antioxidants. the importance of protecting the antioxidants pool in preventing renal damage has been emphasized by singh. the in vitro antioxidant effect exhibited in different assays has further supported the antioxidant property of atr extract. the phytochemicals found to be present in the root extract are the flavonoids, terpinoids, alkaloids, tannins, saponins & glucosinolates.45, 46 among them tannins, triterpinoids, flavonoids and saponins could be responsible for antioxidant property as these phytoconstituents are already reported to have antioxidant activity. the anti - inflammatory activity could be due to the presence of flavonoids which are well known to have anti - inflammatory action. this could be a reason for using atr extract as a diuretic agent in traditional systems. hence terpinoids, tannins and flavonoids present in the root extract may have contributed to the protection from renal damage induced by glycerol by their anti - inflammatory, antioxidant and vasodilatory as well as diuretic actions. freidelin has been established to have antioxidant, free radical scavenging, hepato - protective ant - inflammatory, analgesic and antipyretic activities. beta sitosterol, also a terpinoid has been reported to have antipyretic and anti - inflammatory activity. the glucosinolate and n - hydroxy-3-indolylmethyl - glucosinolate were detected in atr and not in the other parts of the plant. glucosinolates have vasodilatory effect like tannins and flavonoids.51, 52 renal vasodilatation by these components can improve renal blood flow, reduce the ischemic changes and also increase urine output. thus the in vitro and in vivo findings suggest that protection from oxidative damage, anti - inflammatory and vasodilatory actions of the various phytochemicals present in atr extract could have contributed to the nephroprotective actions of a. tetracantha root extract in acute renal failure. between the 2 doses administered, 250 and 500 mg / kg, 500 mg / kg is found to have significant nephroprotective action. the ethanolic extract of atr is found to have nephroprotective effect in glycerol - induced acute renal failure in wistar albino rats. atr extract is found to have significant action at 500 mg / kg compared to 250 mg / kg. the possible mechanisms could be the antioxidant, anti - inflammatory, vasodilatory and diuretic activity of its phytochemical constituents such as flavonoids, terpinoids, tannins, glucosinolate and saponins. | the gravity of the impact of renal failure on human health is well known and as there is no specific pharmacotherapy for renal failure, the current study was undertaken to evaluate the effect of root extract of azima tetracantha, an ancient medicinal plant used in siddha and ayurvedhic systems of medicine.the experiment was done in glycerol - induced acute renal failure in wistar albino rats. thirty rats were divided into five groups. group 1 was given normal saline (10 ml / kg) per oral, group 2 with single dose of hypertonic glycerol (8 ml / kg) by intramuscular injection into the hind limbs, group 3 with glycerol and ethanolic extract of a. tetracantha root (atr) 250 mg / kg, group 4, glycerol and atr 500 mg / kg and group 5, 500 mg / kg atr. extract was given orally 60 min prior to glycerol injection. 24 h urine output, serum creatinine, blood urea nitrogen, total proteins and albumin were measured for all the groups. kidneys were examined for histopathological changes.the antioxidant activity of the extract was tested in vitro and in vivo. rats treated with atr showed significant improvement in biochemical parameters and histopathological changes compared to glycerol treated group. the protective effect was highly significant at 500 mg / kg. both in vitro and in vivo assays showed significant antioxidant activity. the in vitro activity was comparable to vitamin-c.the ethanolic extract of atr has nephroprotective effect in glycerol - induced acute renal failure and the mechanism of action could be the antioxidant effect. |
tuberculosis (tb) is a serious public health problem and bcg vaccination remains an essential part of tb prevention strategy especially in children. bcg scar is a surrogate marker of vaccination and an important index in the vaccination program. however scar failure is a well - known phenomenon with prevalence varying from 1% to 20% in term infants in different studies worldwide. apart from protection against severe forms of tb, bcg also has a non - tb - related beneficial effect on child survival. studies suggesting association of bcg scar with decreased childhood mortality in developing countries have rekindled the interest on bcg scar. the true magnitude of scar failure and whether the scar negative infants need to be monitored remains unclear. this study was undertaken to determine the scar failure rate and tuberculin conversion in infants vaccinated with bcg within the first month of life. this prospective cohort study was carried out at the immunization clinic of a medical college hospital after obtaining permission from the institutional ethics committee. our cohort was the infants recruited for study from july 2010 to december 2010 and the study period was from july 2010 to march 2011. preterms, babies weighing less than 2 kg, hiv - exposed infants and those with family history of tb or suffering from any acute illness were excluded from the study. all study subjects were administered 0.1 ml of bcg by a single trained staff nurse on the left arm just above the insertion of deltoid muscle intradermally with a 26-gauge needle and tuberculin syringe. bcg vaccine containing moscow bcg - i (russian) strain manufactured at the serum institute of india, pune was used. this vaccine was licensed in india in 2001 and subsequently prequalified by who in 2003 for use in developing countries. the freeze - dried vaccine was reconstituted with normal saline and was used within 3 hours. all vaccinated babies were inspected for the presence of a wheal by one of the investigators. simultaneously, oral polio vaccine and hepatitis b vaccines were also administered as per the national immunization schedule. the parents were given a follow up date 3 months (+ 1 week) from the date of vaccination. on follow up, infants were examined for the presence of scar or local reaction at the vaccination site. bcg scar size was measured both across and along the arm in millimeter using a plastic ruler and the average was calculated. tuberculin skin test was done with purified protein derivative (ppd) solution (5tu ppd/0.1 ml, span diagnostic limited, surat) 0.1 ml injected intradermally with a 26-gauge needle on the flexor aspect of left forearm 2 to 3 inches below the elbow. tuberculin skin test was read after 48 to 72 hours by a ball point pen technique. infants showing a transverse diameter of > 5 mm were considered tuberculin converted following bcg vaccination. continuous variables were summarized using means and standard deviations, while categorical variables were summarized using percentages. the chi - square test was used to determine the association between bcg scar and tuberculin skin test as well as the association of bcg scar with age at vaccination and sex. an unpaired t - test was used to determine the significant difference between mean bcg scar size and tst reaction size of subjects vaccinated within 7 days and after 7 days as well as males and females. continuous variables were summarized using means and standard deviations, while categorical variables were summarized using percentages. the chi - square test was used to determine the association between bcg scar and tuberculin skin test as well as the association of bcg scar with age at vaccination and sex. an unpaired t - test was used to determine the significant difference between mean bcg scar size and tst reaction size of subjects vaccinated within 7 days and after 7 days as well as males and females. of the 85 term neonates recruited, 2 died at home before completion of the study due to unrelated causes and 13 could not complete the follow up as per protocol. only 18 (25.7%) babies received vaccination within 48 hours of birth, which was significantly low (t = 5.510, p = 0.000). majority (72.9%) of infants were vaccinated within 7 days of life, while 15.7% were vaccinated between 8 to 14 days and 11.4% after 14 days of age. sixty - four (91.4%) out of 70 infants exhibited a visible scar after 12 weeks of vaccination representing a scar failure rate of 8.6%. the mean s.d. of scar size was 4.93 2.01 (range : 010 mm ; 95% c.i : 0.91, 8.95). the presence of bcg scar was not significantly affected by age at vaccination (7 days or > 7 days) or gender. similarly, tst positivity was also not affected by age or sex [table 1 ]. tuberculin test was positive (> 5 mm) in 50 (71.4%) infants. mean s.d. of tst reaction size at 12 weeks was 6.01 3.224 (95% c.i : 0.43, 12.46). the association between scar formation and tuberculin positivity was highly significant (p < 0.001). one infant had a positive tuberculin test without a bcg scar [table 2 ]. there was no significant difference in the scar or tst reaction size in the different age groups or sexes [table 3 ]. association of bcg scar and tst with sex and age at vaccination association of bcg scar and tst comparison of scar and tst reaction size according to sex and age at vaccination there was moderate positive but highly significant correlation between scar size and tst reaction size (r = karl pearson correlation coefficient = 0.401, p = 0.001). no adverse reactions to bcg were found in the study subjects. to check dependency of tuberculin positivity, parameters like age at vaccination, sex and presence of scar were considered in multivariate analysis. binary logistic regression showed that scar is the best and significant predictor of tst positivity (p = 0.014) [table 4 ]. the burden of tb is high in developing countries and bcg vaccination continues to remain an important armamentarium in the prevention of serious childhood tb. it is one of the oldest and most commonly used vaccines despite the controversies surrounding it. bcg is generally considered to protect against tuberculous meningitis and miliary tb among infants and young children. as per who recommendation, bcg vaccine should be administered as soon as possible after birth and before 1 month of age for maximum protection. in this study, although majority (72.9%) of infants were vaccinated within 7 days only 25.7% were vaccinated within 48 hours which was significantly low (p < 0.05). this may be because bcg is given only 2 days a week in our clinic as a measure to prevent wastage. nevertheless, it is important that due care is taken to vaccinate infants at the earliest and create better awareness regarding the timing of vaccination especially countries where tb is endemic. in the present study, scar failure rate was 8.6%. this was comparable to other indian studies on term infants by rani (10%) and lakhar (6.1%). higher scar failure rate (55%) has been reported in low birth weight babies from india. a study from pakistan reported scar failure rate of 19.6%, whereas the studies from lima peru, nigeria, and brazil showed much lower scar failure rate of 1.4%, 3.7% and 3.1%, respectively. this difference could be accounted for by variability in the study design and differences in the demographic characteristics of subjects. development of bcg scar depends on the strain, injected dose and technique of administration. other factors like quality of vaccine, proper transport, storage and undiagnosed underlying immune disorder in infants are also responsible for the absence of scar formation. we did not find any association between age and scar positivity unlike surekha 's study where scar failure was more common in infants vaccinated within 48 hours. the presence of bcg scar is the only simple and successful way of determining previous vaccination in clinical settings as well as in health surveys to assess vaccine uptake in spite of studies indicating that scar development is not a reliable indicator of the immunological response to bcg. the mean scar size in this study was 4.93 mm which was quite similar to that observed by aggarwal. the scar size is usually related to the dose and strain of bcg vaccine and has no relation to induced immunity. in aggarwal 's study on timing and dose of vaccination in infants, subjects were divided into three groups : group a : newborns vaccinated with 0.05 ml of bcg, group b : newborns vaccinated with 0.1 ml bcg, group c : infants vaccinated at 46 weeks with 0.1 ml bcg. they observed scar positivity in 93.8%, 100% and 97.1% in groups a, b and c, respectively. they found a significant difference in scar size in group a as compared to groups b and c. the mean tst reaction also was significantly higher in infants who received 0.1 ml of bcg as compared to those who received 0.05 ml of bcg. the authors therefore concluded that dose of bcg in neonates should be 0.1 ml. a mean scar size of 3.4 mm and 3.3 mm at 6 months following 0.1 ml bcg was observed by the study form peru in average weight and low birth weight babies, respectively. thayyil sudhin reported tuberculin conversion to be 80% in term infants and 80.7% in preterms. on the contrary, a low tst positivity (4468%) our finding is similar to aggarwal., who reported tuberculin conversion in 74.7% term newborns vaccinated with 0.1 ml bcg. the wide variations in tuberculin conversion following bcg between different studies is attributable to factors like difference in vaccine strain, potency, strength of tu used, age groups examined, time of tst after bcg, environmental mycobacteria etc. it has been shown that tuberculin positivity increased significantly when repeated at 1 year in infants who were tuberculin negative at 3 months post vaccination. faridi m (2009) in their study concluded that tst is not a reliable indicator to assess bcg response either at 12 weeks or 6 months although they demonstrated a higher positivity at 6 month as compared to 12 weeks. in variance another recent study from taiwan found good correlation between abscess formation at bcg site and tuberculin positivity but failed to show any correlation between scarring and tuberculin positivity. despite its drawbacks, tst following bcg vaccination has been used as a measure of post vaccine allergy in a number of studies. although in vitro assays like leukocyte migration inhibition test (lmit) is more sensitive than tst in assessing the cmi response to bcg, it is not easily available or feasible and in its absence tst remains the best surrogate of efficacy of bcg. the absence of scar should not remain a mere observation of parents but should be part of health surveys. infants who fail to develop a scar is a matter of concern and whether these infants need to be followed and evaluated remain unresolved. recent studies have suggested that bcg scar and positive tuberculin tests are associated with better survival in early childhood in countries with high child hood mortality. the beneficial effect of bcg is attributed to the non - specific enhancement of both antibody and cellular immune responses. considering these studies it seems prudent to revaccinate children without a bcg scar in developing countries with high under 5 mortality. moreover, we have followed up the infants up to 12 weeks only and done a single tuberculin test. it is possible that positivity of scar as well as tst might have increased if we had longer follow up at least for 6 months. there was moderate positive but highly significant correlation between scar size and tst reaction size. larger studies to reveal the true magnitude of the problem and regular evaluation of bcg vaccination programs are recommended. the finding of bcg scar associated with reduced childhood mortality need to be evaluated in further research in developing countries. | background : there is considerable variation in bcg scar failure rate on available data and correlation between bcg scar and tuberculin conversion remains controversial. through this study we aimed to determine the scar failure rate and tuberculin conversion in term infants vaccinated with bcg within the first month.materials and methods : a prospective cohort study was conducted among 85 consecutive infants weighing > 2 kg attending the immunization clinic of a medical college hospital. fifteen subjects who could not complete the follow up were excluded. total of 70 cases were analyzed. all babies were administered 0.1 ml of bcg and examined at 3 months (+ 1 week) for scar. tuberculin test was done with 5tu ppd. an induration of > 5 mm was considered positive. statistical analysis was done using microsoft excel and spss-22.results:out of the 70 infants, 41 (58.6%) were males. although majority (72.9%) of infants were vaccinated within 7 days, only 18 (25.7%) received bcg within 48 hours of birth. sixty - four (91.4%) had a visible scar at 12 weeks post vaccination representing a scar failure rate of 8.6%. tuberculin test was positive in 50 (71.4%). the mean s.d. for scar and tuberculin skin test (tst) reaction size was 4.93 2.01 mm and 6.01 3.22 mm, respectively. the association between scar formation and tuberculin positivity was highly significant (p < 0.001). there was significant correlation between scar size and tst size (r = 0.401, p = 0.001)conclusions : less than 10% of infants fail to develop a scar following bcg vaccination. there is good correlation between scar positivity and tuberculin conversion. |
hypertension is the most frequently treated disease, and can lead to cardiovascularand renal complications in patients who could not achieve their blood pressure goals. the activation of renin - angiotensin - aldosterine system (raas) is one of the established pathogenesis of hypertension, and blocking agents of renin, angiotensin converting enzyme (ace), angiotensin ii (ang ii) type 1 (at1) receptor, and aldosterone of raas are useful medications in the clinical settings. however, there are some discrepancies between the expected results and the real clinical results and treatment responses in raas. despite the diabetic nephropathy is a prototype disease of raas activation, that showed low serum renin levels compared to that of normal controls1). and, it has been hypothesized that complete inhibition of raas with direct renin inhibition and acei or arb leads to the prevention of cardiovascular events. however, in the recently published aliskirentrial in type 2 diabetes mellitus using cardiorenal end points (altitude), aliskiren was associated with a trend toward higher composite cardiovascular events compared to placebo2). the recent discovery of ace - related carboxypeptidase (ace2), angiotensin 1 - 7 ang-(1 - 7) and its receptor of mas has changed the way in which raas is viewed. since they have been known to play an opposite function of ace - ang ii - at1 receptor axis, they are reevaluated to be an important pathogenesis and a treatment tool for hypertension. here we review the difference between intrarenal raas and systemic raas, and the recent update of hypertension related ace2-ang-(1 - 7)-mas receptor axis. the main source of systemic raas is liver angiotensinogen and is converted to ang i by juxtaglomerular (jg) cell secreting renin. the ang i is cleaved by ace from lung and kidney to eight peptide of ang ii. it is known that the concentration of serum ang ii is 50 - 100 pmol / l. ang ii increases sodium and water retention by tubular reabsorption, and is accelerated when aldosterone is secreted by the adrenal gland. ang ii stimulates anti - diuretic hormone secretion in the pituitary gland leading to increased water reabsorption in the collecting duct. those systemic effects of ang ii increase water and sodium retention and the effective circulating volume result in increased perfusion to jg apparatus leading to decreased renin secretion by negative feedback. the kidney contains all the elements of raas, and intrarenal formation of ang ii is independent of the circulating raas. the concentration of intrarenal ang ii is 3 - 5 nm and it has 50 - 100 times higher concentration compared to systemic ang ii3). in addition, renin is secreted from jg cells, connecting duct, and collecting duct, and ace is generated from the proximal tubule to the collecting duct, it is also expressed well in the endothelial cell. the at1 receptor is widely distributed among vascular, glomerular, and tubular elements of the kidney, consistent with this receptor 's role in regulating renal hemodynamics, glomerular filtration, sodium reabsorption, and renin release. urinary angiotensinogen is used as the marker of intrarenal raas activation because the molecular weight of angiotensinogen is 44kda and can not be filtrated by normal glomeruli4). however, recent reports demonstrated that liver angiotensinogen is highly expressed in the kidney when there is an abnormal increase in the permeability of the glomerular capillary wall to angiotensinogen, which characterizes proteinuric kidney diseases. liver angiotensinogen is also an important source of the synthesis of renal angii in the kidney angiotensinogen knock out animal model5). it is highly regulated and is easily stimulated by slight changes such as, a decrease in renal perfusion pressure, a decrease in the delivery of cl to the macula densa, or by sympathetic nerve stimulation. in addition to jg cells, renin mrna and protein have been found in the connecting tubule and collecting duct. unlike jg cells where ang ii inhibits renin released via the at1 receptor, in the collecting duct ang ii stimulates renin expression via the at1 receptor. it is a well - demonstrated phenomenon in the two kidney one clip animal model as a classic ang ii increase hypertension model. the jg cellular renin is increased clipped kidney in an early phase of the two kidney one clip model. after the acute stage, however, renin secretion is also increased in the collecting duct in non - clipped kidney. during periods of jg renin suppression, upregulated renin is produced in the distal nephron and it may be able to support continued intrarenal ang ii formation leading to amplification or maintenance of the hypertensive state6). therefore, unlike acei or arb treatment increases the renin expression in jg cell, decreases renin mrna and protein levels in distal nephron. second, proximal tubular production of angiotensinogen is activated by ang ii via at1 receptor. increased renal angiotensinogen is converted to ang ii by intrarenal renin and ace, and intrarenal ang ii increases renal vasculature tone and tubular sodium reabsorption. elevated reactive oxygen species (ros) in hypertension or diabetes mellitus is known to produce oxidized form of angioten - sonogen. it was demonstrated recently that the oxidized angiotensinogen has a more potent capability to bind renin leading to readily undergo a transition to angiotensin release and increase blood pressure in pre - eclampsia, which is a serious hypertensive complication of pregnancy7). the infusion of ang ii increases the proximal tubular secretion of ace and ace binding activity. the absence of kidney ace reduces renal ang ii accumulation, sodium and water retention in response to ang ii infusion. metabolizes ang ii and produces ang-(1 - 7), thereby contributing to the regulation of blood pressure and progression of renal disease. ace2 is a type 1 integral membrane glycoprotein that is found in most tissues, with its highest expression observed in the kidneys, endothelium, and heart8,9). ace2 is an exopeptidase that catalyzes the conversion of ang i to the nonapeptideang-(1 - 9) and the conversion of ang ii to the heptapeptideang-(1 - 7). the primary role of ace2 is to convert ang ii into ang-(1 - 7) with an efficacy > 400-fold greater than that of the hydrolytic action of ace2 in forming ang-(1 - 9)10). ace2 is associated with a reduction in ang ii and an increase in ang-(1 - 7) levels. ace2 protein levels are significantly decreased in the kidneys of hypertensive patients, and patients with late diabetic nephropathy11,12). the heptapeptide ang-(1 - 7), generated from either ang i or ang ii, acts by opposing the vasoconstrictor, proliferative, and profibrotic actions of ang ii in the circulation and in cardiac, vascular, and renal tissues13,14). ang-(1 - 7) also binds to the mas receptor, a seven transmembrane protein with domains containing sequences characteristic of g - protein coupled receptors. the mas receptor is expressed in renal proximal tubular cells, afferent arterioles, cardiac myocytes, and neuronal cells. therefore, this is a review of the recent update of ace2-ang-(1 - 7)-mas receptor axis related hypertension. the ace polymorphism is one of the most frequently studied subjects to identify the association between pathogenesis of disease and genetic polymorphism. the dd allele of the ace gene is highly associated with diabetic nephropathy, however, the effect of the d allele on hypertension was not noticeable because hypertension itself has multi - factorial pathogenesis15). however, ace among raas component has the strongest association with hypertension after an adjustment for age, sex, and weight in genome wide association study (gwas) of korea and other countries16). a recent gwas study of ace2 polymorphism among chinese and caucacians reported significant associations in women with htn or diastolic blood pressure17). in korea, gwas in seongnam and anseong cohort did not show significant associations between ace2 single nucleotide polymorphism and hypertension. to evaluate the role of ace2 in hypertension, various genetically manipulated animal models were developed for the ace2 deletion or overexpression. in ace2 knockout mice, circadian dipping is still preserved, suggesting that at least in the healthy state, dipping is not regulated by ace2 but showed a mild increase in blood pressure. the increase of blood pressure is dependent on the background of the mice model. in the c57bl/6 background, ace2 deficiency was associated with a modest increase in blood pressure, whereas the absence of ace2 had no effect on baseline blood pressures in 129/svev mice17). interestingly, ace2 knockout mice have highlighted hypertensive responses to ang ii infusion associated with exaggerated accumulation of ang ii in the kidney. after acute ang ii infusion, an increase of plasma and kidney concentrations of ang ii was confirmed by maldi - tof mass spectrometry in ace2-deficient mice than in controls. although the absence of functional ace2 causes enhanced susceptibility to ang ii - induced hypertension, there are few evidences that ace2 plays a role in the regulation of cardiac structure or function. the transgenic ace2 overexpression in spontaneous hypertensive rat (shr) showed a reduction of mean arterial pressure irrespective of heart rate. this ace2 overexpression model showed an increase of ang-(1 - 7) in plasma and kidney, attenuated blood pressure elevation response to ang ii infusion. ang-(1 - 7) has hypotensive action by vasodilatory effects through the release of bradykinin, prostaglandin, endothelium - dependent nitric oxide via mas receptor18). the opposing actions of ang-(1 - 7) on ang ii are not limited to the prevention of vasoconstriction. there are various actions in ang-(1 - 7) such as anti - growth actions, natriuresis via inhibition of the renal tubular na - k atpase pump, attenuate oxidative stress via inhibition of the nadph oxidase. acei increases the production of ang i, which is converted to ang-(1 - 7) by ace2, and endopeptidase. the antihypertensive actions of acei are obtained by increased excretion of ang-(1 - 7), which was observed in urine samples of patients with essential hypertension whose blood pressure was controlled by 6 months of treatment with captopril19). it is well known that acei can reduce the urinary protein excretion in diabetes mellitus, however, in ace2 knock - out mice disappear the antiproteinuric effect of acei. arb may be particularly important because the increase in the concentration of ang ii will stimulate greater production of ang-(1 - 7). in addition, the low affinity binding of ang-(1 - 7) to the at1 receptor may allow the peptide to act as an antagonist in the presence of ang ii. the blockades of ace - ang ii - at1 receptor axis are well known pathways as the anti - hypertensive and anti - proteinuric medication in clinic. recently, some patients have been evaluated as the activators of ace2-ang-(1 - 7)-mas receptor axis, which can be divided into two main classes : (1) aimed to increase the activity of ace2, which will impact the system by increasing the inactivation of ang ii and (2) increasing the production of ang-(1 - 7) and directed to stimulate the ang-(1 - 7) receptor mas (fig. a leading compound ace2 activator, xnt, reduces the blood pressure and improves cardiac function in shr. recombinant human ace2 was also developed as an alternative approach to the therapeutic potential of ace2. treatment with rhace2 attenuates diabetic nephropathy though a mechanism involving a reduction in ang ii and an increase in ang-(1 - 7) signaling21). ave 0991 was the first nonpeptide synthetic compound developed with the objective to stimulate the ang-(1 - 7) receptor. this compound was orally active mas agonist and mimics the effects of ang-(1 - 7) in vessels, kidney, and heart. ave0991 prevents end organ damage in shr, induced by l - name treatment by preserving left ventricular contractility, preventing blood pressure elevation, and decreasing urinary protein excretion22). cgen-856 and cgen-857 are two novel peptides for the other activator of g protein coupled receptor, and were designed for having high specificity for mas receptor23). the ace polymorphism is one of the most frequently studied subjects to identify the association between pathogenesis of disease and genetic polymorphism. the dd allele of the ace gene is highly associated with diabetic nephropathy, however, the effect of the d allele on hypertension was not noticeable because hypertension itself has multi - factorial pathogenesis15). however, ace among raas component has the strongest association with hypertension after an adjustment for age, sex, and weight in genome wide association study (gwas) of korea and other countries16). a recent gwas study of ace2 polymorphism among chinese and caucacians reported significant associations in women with htn or diastolic blood pressure17). in korea, gwas in seongnam and anseong cohort did not show significant associations between ace2 single nucleotide polymorphism and hypertension. to evaluate the role of ace2 in hypertension, various genetically manipulated animal models were developed for the ace2 deletion or overexpression. in ace2 knockout mice, circadian dipping is still preserved, suggesting that at least in the healthy state, dipping is not regulated by ace2 but showed a mild increase in blood pressure. the increase of blood pressure is dependent on the background of the mice model. in the c57bl/6 background, ace2 deficiency was associated with a modest increase in blood pressure, whereas the absence of ace2 had no effect on baseline blood pressures in 129/svev mice17). interestingly, ace2 knockout mice have highlighted hypertensive responses to ang ii infusion associated with exaggerated accumulation of ang ii in the kidney. after acute ang ii infusion, an increase of plasma and kidney concentrations of ang ii was confirmed by maldi - tof mass spectrometry in ace2-deficient mice than in controls. although the absence of functional ace2 causes enhanced susceptibility to ang ii - induced hypertension, there are few evidences that ace2 plays a role in the regulation of cardiac structure or function. the transgenic ace2 overexpression in spontaneous hypertensive rat (shr) showed a reduction of mean arterial pressure irrespective of heart rate. this ace2 overexpression model showed an increase of ang-(1 - 7) in plasma and kidney, attenuated blood pressure elevation response to ang ii infusion. ang-(1 - 7) has hypotensive action by vasodilatory effects through the release of bradykinin, prostaglandin, endothelium - dependent nitric oxide via mas receptor18). the opposing actions of ang-(1 - 7) on ang ii are not limited to the prevention of vasoconstriction. there are various actions in ang-(1 - 7) such as anti - growth actions, natriuresis via inhibition of the renal tubular na - k atpase pump, attenuate oxidative stress via inhibition of the nadph oxidase. acei increases the production of ang i, which is converted to ang-(1 - 7) by ace2, and endopeptidase. the antihypertensive actions of acei are obtained by increased excretion of ang-(1 - 7), which was observed in urine samples of patients with essential hypertension whose blood pressure was controlled by 6 months of treatment with captopril19). it is well known that acei can reduce the urinary protein excretion in diabetes mellitus, however, in ace2 knock - out mice disappear the antiproteinuric effect of acei. arb may be particularly important because the increase in the concentration of ang ii will stimulate greater production of ang-(1 - 7). in addition, the low affinity binding of ang-(1 - 7) to the at1 receptor may allow the peptide to act as an antagonist in the presence of ang ii. the blockades of ace - ang ii - at1 receptor axis are well known pathways as the anti - hypertensive and anti - proteinuric medication in clinic. recently, some patients have been evaluated as the activators of ace2-ang-(1 - 7)-mas receptor axis, which can be divided into two main classes : (1) aimed to increase the activity of ace2, which will impact the system by increasing the inactivation of ang ii and (2) increasing the production of ang-(1 - 7) and directed to stimulate the ang-(1 - 7) receptor mas (fig. a leading compound ace2 activator, xnt, reduces the blood pressure and improves cardiac function in shr. recombinant human ace2 was also developed as an alternative approach to the therapeutic potential of ace2. treatment with rhace2 attenuates diabetic nephropathy though a mechanism involving a reduction in ang ii and an increase in ang-(1 - 7) signaling21). ave 0991 was the first nonpeptide synthetic compound developed with the objective to stimulate the ang-(1 - 7) receptor. this compound was orally active mas agonist and mimics the effects of ang-(1 - 7) in vessels, kidney, and heart. ave0991 prevents end organ damage in shr, induced by l - name treatment by preserving left ventricular contractility, preventing blood pressure elevation, and decreasing urinary protein excretion22). cgen-856 and cgen-857 are two novel peptides for the other activator of g protein coupled receptor, and were designed for having high specificity for mas receptor23). since the discovery of renin from renal extracts in 1898, the raas is still an interesting research field in hypertension, cardiovascular and kidney disease. the new findings of intrarenal raas activation without negative feedback via at1 receptor may be able to support continued intrarenal ang ii formation leading to amplification or maintenance of the hypertensive state. the recent discovery of ace2 and ang-(1 - 7) has modified the way in which the raas is viewed from a purely deleterious system in cardiovascular disease to an useful system to protect target organs. targeting the ace2-ang-(1 - 7)-mas receptor axis may be a novel therapeutic strategy for hypertension and kidney disease. | the activation of renin - angiotensin - aldosterine system(raas) is one of the main pathogenesis of hypertension. all the components of raas are present in the kidneys at higher concentrations compared to plasma levels, and intrarenal formation of angiotensin ii (ang ii) is independent of the systemic raas. there are some unique features in intrarenal raas compared to systemic raas. unlike jg cells where ang ii inhibits renin release via the angii type 1 (at1) receptor by negative feedback, in the collecting duct ang ii stimulates renin expression via the at1 receptor. upregulated renin produced in the distal nephron may be able to support continued intrarenal ang ii formation leading to amplification or maintenance of the hypertensive state.the recently discovered angiotensin - converting enzyme - related carboxypeptidase 2 (ace2)-angiotensin-(1 - 7) ang-(1 - 7)-mas receptor axis has an opposing function to that of the ace - ang ii - at1 receptor axis.the ace2 deficiency was associated with an increase in blood pressure, and ace2 knockout mice have highlighted hypertensive response to ang ii infusion associated with exaggerated accumulation of ang ii in the kidney. recently, several numbers of patients have been evaluated as the activators of ace2-ang-(1 - 7)-mas receptor axis, which can be divided into two main classes : aimed to increase the activity of ace2, and directed to stimulate the ang-(1 - 7) receptor mas. in order to investigate new targets for hypertension and kidney disease, further research on the function of the ace - ang-(1 - 7)-mas receptor axis is required. |
dipeptidyl peptidase (dpp)4 inhibitors enhance the activity of endogenous incretin hormones1, which are important prandial stimulators of insulin secretion, and exhibit other physiological actions, such as reducing bowel motility1. in addition to their glucoselowering effects, dpp4 inhibitors have potential adverse effects due to their diverse and pleiotropic actions3. herein we present three cases of ileus in diabetes patients that may be associated with the use of a dpp4 inhibitor. the first patient was a 70yearold japanese man with a > 10year history of diabetes without diabetic nephropathy or retinopathy. he had undergone surgery for appendicitis. in addition, the patient had been treated for parkinson 's disease with levodopa carbidopa tablets for 2 years, and his condition was stable with mild rigidity. the patient presented at the hospital complaining of persistent nausea, vomiting, and diarrhea for 2 days. the patient had been taking mitiglinide for approximately 20 months to treat his diabetes, but had been prescribed alogliptin (25 mg / day) instead of mitiglinide 11 days prior to his presentation at hospital. the patient passed multiple diarrheal stools and his air fluid levels subsequently resolved without intervention. the second patient was a 61yearold japanese woman with myeloperoxidase antineutrophil cytoplasmic antibody (anca)positive rapidly progressive glomerulonephritis being treated with prednisolone. the patient was in a stable condition (estimated glomerular filtration rate [egfr ] 29 ml / min per 1.73 m). the patient had undergone surgery for early gastric cancer (iic) 25 years previously. she had a > 10year history of type 2 diabetes and her diabetes had been treated with mitiglinide and sitagliptin ; miglitol (150 mg / day) was added to her antidiabetes regimen to control postprandial hyperglycemia, and the sitagliptin was discontinued. the patient refused insulin and had been treated instead with a halfdose of vildagliptin (50 mg / day) in addition to mitiglinide (30 mg / day) and miglitol (225 mg / day) for 4 months. the patient 's prednisolone dose was decreased to 10 mg / day (from 15 mg / day) and she was eventually prescribed a full dose of alogliptin (25 mg / day) instead of vildagliptin (50 mg / day). she was identified with air fluid levels in her colon and was admitted to the surgical unit for further assessment. xray and ct imaging indicated that her ileus was becoming worse (figure 1). gastrointestinal decompression was performed via a nasoenteric tube ; however, this was not effective. so, 3.5 days later, surgical decompression and reconstructive surgery were performed for a collapsed small intestine, which revealed an internal hernia. an upright abdominal xray (a) and ct scan (b) demonstrate small bowel obstruction. the third patient was a 78yearold japanese man with a > 10year history of type 2 diabetes. this patient 's right leg had been amputated 11 years ago because of atherosclerosis obliterans. the patient had chronic kidney disease (egfr 46 ml / min per 1.73 m). the patient had undergone total gastrectomy with partial pancreatosplenectomy and left adrenalectomy 12 years ago for advanced gastric cancer. he had had an ileus that was resolved with conservative treatment 5 years ago. one day prior to admission, the patient experienced intermittent lower left abdominal pain with nausea and vomiting. xray and gastrointestinal decompression via a nasoenteric tube was successful and the patient was discharged 3 weeks after admission. his diabetes had been treated with glargine, metformin (500 mg / day), miglitol (225 mg / day), repaglinide (0.75 mg / day), and sitagliptin (50 mg / day). the patient was also taking amlodipine, aliskiren, calcium polystyrene sulfonate, camostat mesilate, liver hydrolysate, and mosapride citrate hydrate. his diabetes was relatively stable, with an hba1c of 8.0% (ngsp), but random sampling revealed blood sugar levels > 300 mg / dl. a diabetologist had asked the patient to use rapid insulin instead of an oral hypoglycemic agent, but the patient did not like the idea of multiple insulin injections. thirtythree days prior to his admission for the ileus, the patient was prescribed a full dose of vildagliptin (100 mg / day) instead of sitagliptin (50 mg). the first patient was a 70yearold japanese man with a > 10year history of diabetes without diabetic nephropathy or retinopathy. he had undergone surgery for appendicitis. in addition, the patient had been treated for parkinson 's disease with levodopa carbidopa tablets for 2 years, and his condition was stable with mild rigidity. the patient presented at the hospital complaining of persistent nausea, vomiting, and diarrhea for 2 days. the patient had been taking mitiglinide for approximately 20 months to treat his diabetes, but had been prescribed alogliptin (25 mg / day) instead of mitiglinide 11 days prior to his presentation at hospital. the patient passed multiple diarrheal stools and his air fluid levels subsequently resolved without intervention. the second patient was a 61yearold japanese woman with myeloperoxidase antineutrophil cytoplasmic antibody (anca)positive rapidly progressive glomerulonephritis being treated with prednisolone. the patient was in a stable condition (estimated glomerular filtration rate [egfr ] 29 ml / min per 1.73 m). the patient had undergone surgery for early gastric cancer (iic) 25 years previously. she had a > 10year history of type 2 diabetes and her diabetes had been treated with mitiglinide and sitagliptin ; miglitol (150 mg / day) was added to her antidiabetes regimen to control postprandial hyperglycemia, and the sitagliptin was discontinued. the patient refused insulin and had been treated instead with a halfdose of vildagliptin (50 mg / day) in addition to mitiglinide (30 mg / day) and miglitol (225 mg / day) for 4 months. the patient 's prednisolone dose was decreased to 10 mg / day (from 15 mg / day) and she was eventually prescribed a full dose of alogliptin (25 mg / day) instead of vildagliptin (50 mg / day). she was identified with air fluid levels in her colon and was admitted to the surgical unit for further assessment. xray and ct imaging indicated that her ileus was becoming worse (figure 1). gastrointestinal decompression was performed via a nasoenteric tube ; however, this was not effective. so, 3.5 days later, surgical decompression and reconstructive surgery were performed for a collapsed small intestine, which revealed an internal hernia. an upright abdominal xray (a) and ct scan (b) demonstrate small bowel obstruction. note that multiple air the third patient was a 78yearold japanese man with a > 10year history of type 2 diabetes. this patient 's right leg had been amputated 11 years ago because of atherosclerosis obliterans. the patient had chronic kidney disease (egfr 46 ml / min per 1.73 m). the patient had undergone total gastrectomy with partial pancreatosplenectomy and left adrenalectomy 12 years ago for advanced gastric cancer. he had had an ileus that was resolved with conservative treatment 5 years ago. one day prior to admission, the patient experienced intermittent lower left abdominal pain with nausea and vomiting. xray and ct scans revealed air gastrointestinal decompression via a nasoenteric tube was successful and the patient was discharged 3 weeks after admission. his diabetes had been treated with glargine, metformin (500 mg / day), miglitol (225 mg / day), repaglinide (0.75 mg / day), and sitagliptin (50 mg / day). the patient was also taking amlodipine, aliskiren, calcium polystyrene sulfonate, camostat mesilate, liver hydrolysate, and mosapride citrate hydrate. his diabetes was relatively stable, with an hba1c of 8.0% (ngsp), but random sampling revealed blood sugar levels > 300 mg / dl. a diabetologist had asked the patient to use rapid insulin instead of an oral hypoglycemic agent, but the patient did not like the idea of multiple insulin injections. thirtythree days prior to his admission for the ileus, the patient was prescribed a full dose of vildagliptin (100 mg / day) instead of sitagliptin (50 mg). case 1 presented as a firsttime user of a dpp4 inhibitor. in cases 2 and 3, a dpp4 inhibitor had been used chronically and had subsequently been changed for strengthened dpp4 inhibition5. these two patients had also been treated with miglitol, possibly inducing defects in gastrointestinal movement. there was no clear information regarding either diabetic gastroenteropathy or neuropathy in any of the cases. two cases had a history of major abdominal surgery ; one of these patients also had a history of ileus. two of the three patients had other chronic diseases, namely ancaassociated vasculitis7 and parkinson 's disease8, which could potentially affect the motility of the gastrointestinal system, although quantitative evaluations for bowel motility had not been performed. despite the fact that a causal relationship between ileus and the use of a dpp4 inhibitor was unclear, in all patients the onset of ileus occurred within 40 days after strengthened dpp4 inhibition (see figure s1 available as supplementary material to this paper). in all three cases, the dpp4 inhibitors were discontinued : case 1 was treated with mitiglinide only, case 2 was prescribed insulin detemir with repaglinide, and case 3 was given insulin glulisine three times per day and basal glargine. the glycemiclowering action of incretinassociated drugs results from their insulinotropic effects, as well as reduced bowel motility1. however, the incretinassociated drug effects on reduced bowel motility could be harmful if produced in excess through interactions with underlying conditions, such as a history of abdominal surgery, microangiopathy in the gastrointestinal system, or autonomic defects. all three patients in the present series were given a stronger dpp4 inhibitor before the onset of ileus, indicating pathological interactions between defects in basal bowel movement and dpp4 inhibitorreduced bowel motility. there is no case report in the literature describing an association between the use of dpp4 inhibitors and ileus. the three cases presented herein demonstrate the potential for adverse effects of dpp4 inhibitors in certain populations. | abstractdipeptidyl peptidase (dpp)4 inhibitors are a new class of antidiabetic drugs that increase incretin hormone levels to enhance blood sugar leveldependent insulinotropic effects, suppress glucagon action, and reduce bowel motility. these incretin effects are ideal for blood sugar control. however, the safety profile of dpp4 inhibitors is not yet established. herein, we present three cases of ileus, considered to be closely related to the use of dpp4 inhibitors, in diabetic patients. each of the three patients exhibited some risk of a deficiency in bowel movement ; the onset of ileus was within 40 days after strengthened inhibition of dpp4. the use of a dpp4 inhibitor could be safe, although the cases presented herein enable us to inform the scientific community to some of the potential adverse effects of the use of dpp4 inhibitors in select populations. |
the progress currently observed in the field of organic electronics is a result of a combined effort of several communities. synthetic chemists have identified classes of promising compounds, ranging from small conjugated molecules to self - assembling oligomers and conjugated polymers, and developed new synthetic routes, improving both stability and processability of the materials. at the same time, material processing, such as doping, annealing, use of a secondary solvent, and composition tuning, has been adjusted to the demands of the field. in parallel, increased device efficiencies could be achieved, e.g., by optimizing light in- and out - coupling and introducing tandem concepts. compound design requires an in - depth understanding of elementary processes occurring in organic semiconductors.(16) in particular, linking the chemical structure to charge dynamics is a nontrivial task, since several factors can influence charge carrier mobility : the molecular electronic structure, the relative positions and orientations of neighboring molecules, and spatial inhomogeneities in the morphology, which determine charge carrier pathways on a macroscopic scale.(17) furthermore, the choice of the model hamiltonian depends on the specific situation.(18) for perfectly ordered defect - free crystals at low temperatures, the drude model based on band theory(19) or its extensions, which account for local electron phonon coupling, are often used. at ambient conditions, however, the thermal fluctuations of the transfer integral, i.e., the nonlocal electron phonon coupling, are on the same order of magnitude as its average value, and charge transport should be treated as diffusion limited by thermal disorder. this can be achieved using semiclassical dynamics based on a hamiltonian with interacting electronic and nuclear degrees of freedom. if nuclear dynamics are much slower than the dynamics of charge carriers and electronic coupling is weak, charge transport can be described by a hamiltonian with static disorder, based on simple assumptions on the electronic density of states and on the hopping rates between localized states. the latter approach is by now routinely used to study charge transport in amorphous and partially disordered small - molecule - based organic semiconductors. its key ingredients are material morphology and intermolecular charge transfer (hopping) rates.(41) the rates not only depend on the molecular electronic structure but are also sensitive to the mutual positions and orientations of molecules. hence, in order to evaluate the rates, the material morphology must be known at an atomistic resolution. this can be achieved by performing molecular dynamics simulations and thus relies on force - field development for new compounds. if the required time and length scales exceed the range available to atomistic molecular dynamics, coarse - graining techniques can be used.(42) these techniques need to be capable of back - mapping the coarse - grained representation to an atomistic resolution. charge transfer rates can be postulated on the basis of intuitive physical considerations, as is done in the gaussian disorder models. alternatively, charge transfer theories can be used to evaluate rates from quantum chemical calculations. in spite of being significantly more computationally demanding, the latter approach allows one to link the chemical and electronic structure, as well as the morphology, to charge dynamics. the high temperature limit of classical charge transfer theory is often used as a trade - off between theoretical rigor and computational complexity. it captures key parameters which influence charge transport while at the same time providing an analytical expression for the rate. within this limit, the transfer rate for a charge to hop from a site i to a site j reads : where t is the temperature, ij = ijint + ijout is the reorganization energy, which is a sum of intra- and intermolecular (outer - sphere) contributions, eij is the site - energy difference, or driving force, and jij is the electronic coupling element, or transfer integral.(54) a more general, quantum - classical expression for a bimolecular multichannel rate is derived in the supporting information. all of the ingredients entering eq 1 can be calculated using electronic structure techniques, classical simulation methods, or their combination. with the rates at hand, one can study charge transport by solving the differential (master) equation, e.g., by using the kinetic monte carlo method, which is capable of simulating charge dynamics of non - steady - state systems. altogether, the task of charge transport characterization is rather tedious and time - consuming to perform, even for a single compound. the main aim of this work is to introduce a software package which implements a set of techniques for charge transport simulations as well as provides a flexible modular platform for their further development. the paper is organized as follows. in section, we describe the workflow and the basic ideas behind each method. as an illustration, we study charge transport in the bulk of amorphous tris-(8-hydroxyquinoline)aluminum (alq3). the first step is the simulation of an atomistic morphology (section), which is then partitioned into hopping sites (section). the coordinates of the hopping sites are used to construct a list of pairs of molecules (neighbor list). for each pair, an electronic coupling element (section), a reorganization energy (section), a driving force (section), and eventually the hopping rate are evaluated. the corresponding master equation is solved using the kinetic monte carlo method (section), which allows one to explicitly monitor the charge dynamics in the system as well as calculate time or ensemble averages of occupation probabilities, charge fluxes, correlation functions, and field - dependent mobilities (section). ground state geometries, partial charges, and a refined force field are used to simulate atomistically resolved morphologies (section). after partitioning into conjugated segments and rigid fragments (section), a list of pairs of molecules (neighbor list) is constructed. transfer integrals (section), reorganization (section) and site energies (section), and eventually hopping rates are calculated for all pairs from this list. a directed graph is then generated, and the corresponding master equation is solved using the kinetic monte carlo method (section). there is no generic recipe on how to predict a large - scale atomistically resolved morphology of an organic semiconductor. the required methods are system - specific : for ultrapure crystals, for example, density - functional methods can be used provided the crystal structure is known from experimental results. for partially disordered organic semiconductors, however, system sizes much larger than a unit cell are required. classical molecular dynamics or monte carlo techniques are then the methods of choice. in molecular dynamics, atoms are represented by point masses which interact via empirical potentials prescribed by a force field. force fields are parametrized for a limited set of compounds, and their refinement is often required for new molecules. in particular, special attention shall be paid to torsion potentials between successive repeat units of conjugated polymers or between functional groups and the -conjugated system. first - principles methods can be used to characterize the missing terms of the potential energy function. the parametrization must take into account existing force - field contributions, e.g., due to nonbonded interactions. if q is the degree of freedom of interest, constrained geometry optimizations must be performed using both first principles and the force - field levels, yielding the total energies ufp(q) and uff(q), respectively. then, the missing force - field terms are fitted to their difference, ufp(q) uff(q), using a prescribed functional form. for several identical or coupled degrees of freedom, a multidimensional fit can be used. note that force - field validation is as important as its refinement. for instance, x - ray scattering and solid - state nmr provide information about averaged molecular arrangements to which simulation results can be compared. as an example, the refinement of the opls force field for alq3 is described in the supporting information. in total the glass transition temperature and bulk density of amorphous alq3 were compared to the experimental values. an amorphous morphology of alq3 was then obtained by quenching the system after equilibrating it above the glass transition temperature. self - assembling materials, such as soluble oligomers, discotic liquid crystals, block copolymers, partially crystalline polymers, etc., are the most complicated to study. the morphology of such systems often has several characteristic length scales and can be kinetically arrested in a thermodynamically nonequilibrium state. for such systems, the time and length scales of atomistic simulations might be insufficient to equilibrate or sample desired morphologies. in this case, systematic coarse - graining can be used to enhance sampling.(42) note that the coarse - grained representation must reflect the structure of the atomistic system and allow for back - mapping to the atomistic resolution. with the morphology at hand, the next step is the construction of the effective electronic hamiltonian of the system. in a static disorder approximation, this is equivalent to partitioning the system into hopping sites, or conjugated segments, and calculating charge transfer rates between them. physically intuitive arguments can be used for the partitioning, which reflects the localization of the wave function of a charge. for most organic semiconductors, the molecular architecture includes relatively rigid, planar -conjugated systems, which we will refer to as rigid fragments. a conjugated segment can contain one or more such rigid fragments, which are linked by bonded degrees of freedom. the dynamics of these degrees of freedom evolves on time scales much slower than the frequency of the internal promoting mode. in some cases, e.g., glasses, it can be frozen due to nonbonded interactions with the surrounding molecules. to illustrate the concept of conjugated segments and rigid fragments, it consists of a conjugated core to which side chains are attached to aid self - assembly and solution processing. in this case, the orbitals localized on side chains do not participate in charge transport, and the -conjugated system is both a rigid fragment and a conjugated segment. the concept of conjugated segments and rigid fragments. dashed lines indicate conjugated segments, while colors denote rigid fragments. (a) hexabenzocoronene : the -conjugated system is both a rigid fragment and a conjugated segment. (b) alq3 : the al atom and each ligand are rigid fragments, while the whole molecule is a conjugated segment. in alq3, a metal - coordinated compound, a charge carrier is delocalized over all three ligands. individual ligands are relatively rigid, while energies on the order of kbt are sufficient to reorient them with respect to each other. thus, the al atom and the three ligands are rigid fragments. in the case of a conjugated polymer, one molecule can consist of several conjugated segments, while each backbone repeat unit is a rigid fragment. since the conjugation along the backbone can be broken due to large out - of - plane twists between two repeat units, an empirical criterion, based on the dihedral angle, can be used to partition the backbone on conjugated segments.(36) however, such intuitive partitioning is, to some extent, arbitrary and shall be validated by other methods. after partitioning, an additional step is often required to remove bond length fluctuations introduced by molecular dynamics simulations, since they are already integrated out in the derivation of the rate expression. this is achieved by substituting respective molecular fragments with rigid, planar systems optimized using first - principles methods. centers of mass and gyration tensors are used to align rigid fragments, though a custom definition of local axes is also possible. such a procedure also minimizes discrepancies between the force - field and first - principles - based ground state geometries of conjugated segments, which might be important for calculations of electronic couplings, reorganization energies, and intramolecular driving forces. two segments are added to this list if the distance between centers of mass of any of their rigid fragments is below a certain cutoff. this allows neighbors to be selected on a criterion of minimum distance of approach rather than center of mass distance, which is useful for molecules with anisotropic shapes. the electronic transfer integral jij entering the marcus rates in eq 1 is defined aswhere and are diabatic wave functions, localized on molecules i and j, respectively, participating in the charge transfer, and is the hamiltonian of the formed dimer. within the frozen - core approximation, the usual choice for the diabatic wave functions is the highest occupied molecular orbital (homo) in the case of hole transfer and the lowest unoccupied molecular orbital (lumo) in the case of electron transfer, while is an effective single particle hamiltonian, e.g., a fock or kohn sham operator of the dimer. as such, jij is a measure of the strength of the electronic coupling of the frontier orbitals of monomers mediated by the dimer interactions. intrinsically, the transfer integral is very sensitive to the molecular arrangement, i.e., the distance and the mutual orientation of the molecules participating in charge transport. since this arrangement can also be significantly influenced by static and/or dynamic disorder, it is essential to calculate jij explicitly for each hopping pair within a realistic morphology. considering that the number of dimers for which eq 2 has to be evaluated is proportional to the number of molecules times their coordination number, computationally efficient and at the same time quantitatively reliable schemes are required. in general, information about three objects is needed : the two monomer wave functions and and the dimer interaction hamiltonian. an approximate method based on zerner s independent neglect of differential overlap (zindo) has been described in ref (51). this semiempirical method is substantially faster than first - principles approaches, since it avoids the self - consistent calculations on each individual monomer and dimer. this allows one to construct the matrix elements of the zindo hamiltonian of the dimer from the weighted overlap of molecular orbitals of the two monomers. together with the introduction of rigid segments, only a single self - consistent calculation on one isolated conjugated segment is required. this molecular orbital overlap (moo) method has been applied successfully to study charge transport, for instance, in discotic liquid crystals, polymers,(36) or partially disordered organic crystals. while the use of the semiempirical zindo method provides an efficient on - the - fly technique to determine electronic coupling elements, it is not generally applicable to all systems. for instance, its predictive capacity with regards to atomic composition and localization behavior of orbitals within more complex structures is reduced. moreover, transition or semimetals are often not even parametrized. in this case, ab initio based approaches, e.g., density - functional theory, can remedy the situation. within the dimer projection method described in detail in ref (50), explicit quantum - chemical calculations are required for every molecule and every hopping pair in the morphology. as a consequence, the code currently contains scripts which support an evaluation of transfer integrals from quantum - chemical calculations performed with the gaussian and turbomole packages. as an example, distributions of transfer integrals calculated using zindo and dft (with the gradient - corrected b p functional and a tzvp basis set(65)) methods are shown in figure 3. while both distributions are similar, zindo integrals are, on average, smaller than dft ones. the reorganization energy ij takes into account the change in nuclear (and dielectric) degrees of freedom as the charge moves from donor i to acceptor j. it has two contributions : intramolecular, ijint, which is due to a reorganization of nuclear coordinates of the two molecules forming the charge transfer complex, and intermolecular (outersphere), ijout, which is due to the relaxation of the environment. in what follows, we discuss how these contributions can be calculated. if intramolecular vibrational modes of the two molecules are treated classically, the rearrangement of their nuclear coordinates after charge transfer results in the dissipation of the internal reorganization energy, ijint. it can be computed from four points on the potential energy surfaces (pes) of both molecules in neutral and charged states, as indicated in figure 4. adding the contributions due to discharging of molecule i and charging of molecule j yields(49)here, uinc is the internal energy of the neutral molecule i in the geometry of its charged state (small n denotes the state and capital c the geometry). similarly, ujcn is the energy of the charged molecule j in the geometry of its neutral state.(66) note that the pess of the donor and acceptor are not identical for chemically different compounds or for conformers of the same molecule. in this case, thus, ijint is a property of the charge transfer complex and not of a single molecule. potential energy surfaces of (a) donor and (b) acceptor in charged and neutral states. after the change of the charge state if all vibrational modes are treated classically, the total internal reorganization energy and the internal energy difference of the electron transfer reaction are ijint = icn + jnc and eijint = ui uj, respectively. in alq3 molecular conformations are then frozen due to nonbonded interactions in an amorphous glass. the internal energies entering eq 3 were calculated after optimizing molecular geometries of all 512 molecules in charged and neutral states with the soft degrees of freedom constrained to their average values (see the supporting information for details). the distribution of ijint for holes, which is shown in the supporting information, is sharply peaked with a maximum at 0.21 ev and variance of 0.03 ev. computing ijint from the pes of two unconstrained molecules leads to a similar value of 0.23 ev. since alq3 has high energetic disorder arising from its large dipole moment, this small variance in reorganization energy does not affect charge carrier mobility or poole frenkel behavior. during the charge transfer reaction, also the molecules outside the charge transfer complex reorient and polarize in order to adjust for changes in electric potential, resulting in the outersphere contribution to the reorganization energy. assuming that charge transfer is much slower than electronic polarization but much faster than nuclear rearrangement of the environment, ijout can be calculated from the electric displacement fields created by the charge transfer complex:(67)where 0 is the permittivity of free space, di, f(r) are the electric displacement fields created by the charge transfer complex in the initial (charge on molecule i) and final (charge transferred to molecule j) states, v is the volume outside the complex, and cp = 1/opt 1/s is the pekar factor, which is determined by the low (s) and high (opt) frequency dielectric permittivities. equation 4 can be simplified by assuming spherically symmetric charge distributions on molecules i and j with total charge e. integration over the volume v outside of the two spheres of radii ri and rj centered on molecules i and j leads to the classical marcus expression for the outersphere reorganization energy : where rij is the molecular separation. while eq 5 captures the main physics, e.g., predicts smaller outersphere reorganization energies (higher rates) for molecules at smaller separations, it often can not provide quantitative estimates, since charge distributions are rarely spherically symmetric. the difference of the displacement fields at the position of an atom bk outside the charge transfer complex (molecule k i, j) can be expressed aswhere qain (qaic) is the partial charge of atom a of the neutral (charged) molecule i in a vacuum. the partial charges of neutral and charged molecules are obtained by fitting their values to reproduce the electrostatic potential of a single molecule (charged or neutral) in a vacuum. assuming a uniform density of atoms, the integration in eq 4 can be rewritten as a density - weighted sum over all atoms excluding those of the charge transfer complex. using eq 6, ijout / cp was calculated for all pairs from the neighbor list of a system of 512 alq3 molecules. the neighbor list was constructed using a cutoff of 0.8 nm for the centers of mass of the three alq3 ligands, which results in an average of 12 neighbors in the first coordination shell. the electrostatic potential of a single molecule in a vacuum was calculated using the b3lyp functional and a 6 - 311g(d, p) basis set. the resulting distribution of ijout / cp is shown in figure 5, together with a fit to eq 5. the fit yields ralq3 = 0.57 nm and predicts negative ijout for separations smaller than this radius, which is unphysical. outersphere reorganization energy divided by the pekar factor as a function of the distance between two molecules and its fit to eq 5. the remaining unknown needed to calculate ijout is the pekar factor, cp. in polar solvents s opt 1, and cp is on the order of 1. in most organic semiconductors, however, molecular orientations are fixed, and therefore the low frequency dielectric permittivity is on the same order of magnitude as opt. hence, cp is small, and its value is very sensitive to differences in the permittivities. for alq3, s = 3.0 0.3 is the experimentally measured dielectric constant at low frequencies,(69) while at optical frequencies below electronic absorption, opt = 2.9 0.1.(70) thus, cp = 0.01 0.04, yielding outersphere reorganization energies of ijout < 0.08 ev, which are small compared to ijint. similar results have been reported for other organic semiconductors and different methods for computing ijout. a charge transfer reaction between molecules i and j is driven by the site energy difference, eij = ei ej. since the transfer rate, ij, depends exponentially on eij (see eq 1), it is important to compute its distribution as accurately as possible. the total site energy difference has contributions due to an externally applied electric field, electrostatic interactions, polarization effects, and internal energy differences. in what follows, we discuss how to estimate these contributions by making use of first - principles calculations and polarizable force fields. the contribution to the total site energy difference due to an external electric field f is given by eijext = q(frij), where q = e is the charge and rij = ri rj is a vector connecting molecules i and j. for typical distances between small molecules, which are on the order of 1 nm, and moderate fields of f < 10 v / m, this term is always smaller than 0.1 ev. variations of the local electric field can result in large electrostatic contributions to the energetic disorder.(74) when the atomic partial charges of charged and neutral molecules are used, as introduced in section, eijel can be computed from the site energies(31)where raibk = |rai rbk| is the distance between atoms ai and bk and s is the static relative dielectric constant. the first sum extends over all atoms of molecule i, for which the site energy is calculated. the second sum reflects interactions with all atoms of neutral molecules k i. by using eq 7, one assumes that the influence of conformational changes on partial charges and changes of the molecular geometry upon charging are small. in order to minimize finite size effects, we do not use a spherical cutoff but apply the nearest image convention, that is, sum over all neutral molecules in the box after centering the box around the charged molecule. for alq3, with long - range interactions due to its large dipole moment, this procedure converges already for a few hundred molecules. the resulting distribution of the site energy differences without screening (s = 1), shown in figure 6, is gaussian, with variance of = 0.30 ev. note that eijel is constructed on the basis of the neighbor list as described in section. distributions of site energy differences without (blue) and with (red) polarization effects for pairs from the neighbor list. the dashed line corresponds to a parametrized distance - dependent (r) according to eq 8. the influence of polarization effects on the coulomb interactions can be taken into account by using a relative dielectric constant in eq 7. bulk values of s = 25 for typical organic semiconductors uniformly scale all site energies but are not capable of describing polarization effects on a microscopic level. the contribution to eiel from the first coordination shell is then underestimated due to overscreening, and as a result, the site - energy differences become artificially small. alternatively, one can introduce a phenomenological distance - dependent screening function (raibk) in eq 7(30)where the parameter s is the inverse screening length. for a monovalent ion in water, for example, s = 80 and s = 3 nm.(75) this screening function ensures that neighboring atoms interact via an unscreened coulomb potential (1), while the electrostatic interaction between atoms at large separations is screened as in the bulk. while phenomenological distance - dependent screening is computationally efficient, it can not be used for inhomogeneous systems or systems with anisotropic molecular polarizabilities. moreover, s and s are not known for newly synthesized compounds. a more general approach relies on self - consistent methods to obtain polarization fields.(76) here, we use a polarizable force field based on the thole model(77) as implemented in the tinker package.(78) the polarization contribution is refined iteratively. after evaluating the electric field at atom a in molecule i, fai(0), created by all atomic partial charges (s = 1, nearest image convention), the induced dipole moments, ai(0), are computed. during this first step,. induced dipole moments are then iteratively refined as ai(k+1) = fai(k)ai + (1)ai(k), where ai is the isotropic atomic polarizability and = 0.5 is a damping constant for successive over - relaxation. the new electric fields are computed using the induced dipole moments, which now interact with each other also within molecules, allowing for anisotropic molecular polarizabilities. such self - consistent calculations can, however, become computationally prohibitive for large systems. for homogeneous systems and isotropic molecular polarizabilties, one can perform self - consistent calculations for small systems, parametrize eq 8 accordingly, and use (r) to study larger systems. to this end, mosotti relation(79)where is the molecular polarizability volume and n / v is the number density. using this value of s, the parameter s is then fitted to reproduce the distribution of site - energy differences for molecules from the neighbor list. for a neutral alq3 molecule, the thole model (using atomic polarizabilities h, c, n, o, al = 0.696, 1.75, 1.073, 0.837, 5.5, respectively, and a damping factor of a = 0.39 for interactions with induced moments(78)) gives a practically isotropic polarizability volume tensor with = 54.9. this agrees with dft calculations (b3lyp functional and 6 - 311g(d, p) basis set), yielding = 55.2.(80) using n = 512 molecules in a cubic box of length l = 67.8, we obtain s = 2.84, which reproduces the experimental value of 3.0 0.3.(69) the corresponding distributions of site energy differences, shown in figure 6, are practically gaussian. for the thole model, the variance of 0.21 ev is obviously larger than the 0.10 ev obtained using bulk screening with s = 3.0 (not shown) and is smaller than 0.30 ev for s = 1. a fit to eq 8 gives s = 1.3 nm, which is significantly smaller than the inverse screening length of water, 3 nm. the contribution to the site energy difference due to different internal energies (see figure 4) can be written aswhere uicc(nn) is the total energy of molecule i in the charged (neutral) state and geometry. ui corresponds to the adiabatic ionization potential (or electron affinity) of molecule i, as shown in figure 4. for one - component systems and negligible conformational changes eijint = 0, while it is significant for donor acceptor systems. in alq3, significant conformational changes (see section) lead to a gaussian distribution of eijint with a small variance of = 0.01 ev. the internal energy disorder is small compared to the electrostatic (including polarization) energetic disorder and hence does not affect the charge carrier mobility. long - range, e.g., electrostatic and polarization, interactions often result in spatially correlated disorder,(81) which affects the onset of the mobility - field (poole frenkel) dependence. to quantify the degree of correlation, one can calculate the spatial correlation function of ei and ej at a distance rijwhere e is the average site energy. c(rij) is 0 if ei and ej are uncorrelated and 1 if they are fully correlated. for a system of randomly oriented point dipoles, the correlation function decays as 1/r at large distances.(84) for systems with spatial correlations, variations in site energy differences, eij, of pairs of molecules from the neighbor list are smaller than variations in site energies, ei, of all individual molecules. since only neighbor list pairs affect transport, the distribution of eij rather than that of individual site energies, ei, should be used to characterize energetic disorder. for alq3, the spatial correlation function of the electrostatic contribution to site energies, which is calculated for 512 molecules, is shown in figure 7. it qualitatively reveals strong correlations due to the large dipole moment of the meridional isomer of alq3 of approximately 4 debye. quantitatively, this result is not converged with respect to the system size, and bigger systems will exhibit even longer - ranged correlations. the inset of figure 7 shows that distributions of eij for all and neighbor - list - only pairs are clearly different. note that respective distributions of internal site energies are identical, indicating that this type of disorder is spatially uncorrelated. electrostatic site energy correlation function (eq 11) calculated for pairs from the neighbor list without (blue) and with polarization effects from the thole model (red) as well as using a parametrized distance - dependent (r) according to eq 8 (dashed line). inset : gaussian fits to electrostatic site energy distributions for all pairs (= 0.30 ev) and for pairs from the neighbor list (= 0.21 ev). having determined the list of conjugated segments (hopping sites) and charge transfer rates between them, the next task is to solve the master equation, which describes the time evolution of the systemwhere p is the probability of the system to be in a state at time t and is the transition rate from state to state. a state is specified by a set of site occupations, { i }, where i = 1 (0) for an occupied (unoccupied) site i, and the matrix can be constructed from rates ij. in particular, for a system with only one charge carrier, each state is uniquely characterized by the index i of the site the carrier occupies. in other words, only states of type i { 0,..., 0, i = 1, 0,..., 0 } are possible, and the corresponding probabilities pi and the transition rates ij are identical to site occupation probabilities pi and the transfer rates ij, respectively. while being efficient for stationary, low charge carrier density cases (one charge carrier per simulation box), this approach can become unstable for systems with high energetic disorder, where rates vary by several orders of magnitude. in more general cases, such as multiple charge carriers, expressing the state picture (eq 12) in terms of site occupations is required because of an extremely large total number of states. for multiple charge carriers, the master equation can still be rewritten in terms of occupation probabilities (see the supporting information) by assuming only site - blocking charge charge interactions and by using a mean - field approximation.(85) the analogue of eq 13 becomes, however, nonlinear and requires special solvers. if, in addition, several different types of carriers, such as holes, electrons, and excitons, are present in the system and their creation / annihilation processes take place, it is practically impossible to link state and site occupation probabilities and the corresponding rates. instead, the solution of eq 12 can be obtained by using kinetic monte carlo (kmc) methods. kmc explicitly simulates the dynamics of charge carriers by constructing a markov chain in state space and can find both stationary and transient solutions of the master equation. the main advantage of kmc is that only states with a direct link to the current state need to be considered at each step. since these can be constructed solely from current site occupations, extensions to multiple charge carriers (without the mean - field approximation), site - occupation dependent rates (needed for the explicit treatment of coulomb interactions), and different types of interacting particles and processes are straightforward. to optimize memory usage and efficiency, a combination of the variable step size method(86) and the first reaction method is implemented as described in the supporting information. predictions of charge - carrier mobilities in partially disordered semiconductors rely on charge transport simulations in systems which are only several nanometers thick. as a result, simulated charge transport might be dispersive for materials with large energetic disorder, and simulated mobilities are system - size - dependent. in time - of - flight (tof) experiments, however, a typical sample thickness is in the micrometer range, and transport is often nondispersive. in order to link the simulation and experiment, one needs to extract the nondispersive mobility from simulations of small systems, where charge transport is dispersive at room temperature. such extrapolation is possible if the temperature dependence of the nondispersive mobility is known in a wide temperature range. the mobility - temperature dependence can then be parametrized by simulating charge transport at elevated temperatures, for which transport is nondispersive even for small system sizes. this dependence can then be used to extrapolate to the nondispersive mobility at room temperature.(32) for alq3, the charge carrier mobility of a periodic system of 512 molecules was shown to be more than 3 orders of magnitude higher than the nondispersive mobility of an infinitely large system.(32) furthermore, it was shown that the transition between the dispersive and nondispersive transport has a logarithmic dependence on the number of hopping sites n. hence, a brute - force increase of the system size can not resolve the problem for compounds with large energetic disorder, since n increases exponentially with. spatial distributions of charge and current densities can provide better insight into the microscopic mechanisms of charge transport. if o is an observable which has the value o in a state, its ensemble average at time t is a sum over all states weighted by the probability p to be in a state at time tif o does not explicitly depend on time, the time evolution of o can be calculated asif averages are obtained from kmc trajectories, p = s/s, where s is the number of markov chains ending in the state after time t, and s is the total number of chains. if the total occupation time of the state is, thenwhere = is the total time used for time averaging. for ergodic systems and sufficient sampling times, for example, an ensemble average over several kmc trajectories with different starting conditions corresponds to averaging over injected charge carriers in a time - of - flight experiment. in what follows, we focus on the single charge carrier (low concentration of charges) case. for a specific type of particles, the microscopic charge density of a site i is proportional to the occupation probability of the site, piwhere, for an irregular lattice, the effective volume vi can be obtained from a voronoi tessellation of space. for reasonably uniform lattices (uniform site densities), this volume is almost independent of the site, and a constant volume per cite, vi = v / n, can be assumed. in the macroscopic limit, the charge density can be calculated using a smoothing kernel function, i.e., a distance - weighted average over multiple sites. site occupations pi can be obtained from eq 14 or eq 16 by using the occupation of site i in state as an observable. if the system is in thermodynamic equilibrium, that is without sources or sinks and without circular currents (and therefore no net flux), a condition known as detailed balance holdsit can be used to test whether the system is ergodic or not by correlating log pi and the site energy ei. indeed, if ij = ji, the ratios of forward and backward rates are determined solely by the energetic disorder, ji/ij = exp(eij / kbt) (see eq 1). if the position of the charge, r, is an observable, the time evolution of its average r is the total current in the systemsymmetrizing this expression, we obtainwhere rij = ri rj. symmetrization ensures equal flux splitting between neighboring sites and the absence of local average fluxes in equilibrium. it allows one to define a local current through site i asa large value of the local current indicates that the site contributes considerably to the total current. the distribution of currents is very inhomogeneous, and some pathways are sampled more frequently than the others, which is a direct consequence of a rough and correlated energy landscape.(91) isosurface of the current density for amorphous alq3 (512 molecules, external field fz = 10v / m, distance - dependent dielectric constant, dft - based transfer integrals). currents have filamentary structure due to large correlated energetic disorder.(91) the red streamtraces depict interpolated charge pathways. for a single particle, e.g., a charge or an exciton, a zero - field mobility can be determined by studying particle diffusion in the absence of external fields. using the particle displacement squared, ri, as an observable, we obtainhere, ri is the coordinate of the site i ; d is the diffusion tensor,, = x, y, z ; and d = 3 is the system dimension. using the einstein relationone can, in principle, obtain the zero - field mobility tensor. equation 22, however, does not take into account the use of periodic boundary conditions when simulating charge dynamics. in this case, the simulated occupation probabilities can be compared to the solution of the smoluchowski equation with periodic boundary conditions (see the supporting information for details). alternatively, one can directly analyze time - evolution of the kmc trajectory and obtain the diffusion tensor from a linear fit to the mean square displacement, ri,ri, = 2ddt. the charge carrier mobility tensor,, for any value of the external field can be determined either from the average charge velocity defined in eq 19or directly from the kmc trajectory. in the latter case, the velocity is calculated from the unwrapped (if periodic boundary conditions are used) charge displacement vector divided by the total simulation time. projecting this velocity on the direction of the field f yields the charge carrier mobility in this particular direction. in order to improve statistics,, the field dependence of the mobility (poole frenkel plot) is shown in figure 9 for a system of 4096 molecules. to illustrate the role of disorder and correlations, we also show the field dependence for a system without energetic disorder (top panel) and without correlated energetic disorder (randomly shuffled site energies). frenkel behavior for small fields can only be observed if correlated disorder is taken into account. note that, for a system with such large energetic disorder, the absolute values of (nondispersive) mobility are systematically overestimated due to significant finite size effects (see section and ref (32)). the experimentally measured value of the hole mobility at small fields lies between 10 and 10 cm v s.(92) poole mobilities were calculated by averaging over ten, 0.1-s - long (10 s for no disorder), kmc runs and six different field directions. transfer integrals were calculated using dft ; energetic disorder is based on the distance - dependent dielectric constant fitted to the site energy distribution of the thole model. the toolkit is implemented using modular concepts introduced earlier in the versatile object - oriented toolkit for coarse - graining applications (votca).(42) the votca structures are adapted to reading atomistic trajectories, mapping them onto conjugated segments and rigid fragments, and substituting (if needed) rigid fragments with the optimized copies. the molecular orbital overlap module calculates electronic coupling elements between conjugated segments from the corresponding molecular orbitals. it relies on the semiempirical indo hamiltonian and molecular orbitals in the format provided by the gaussian package. an alternative, density - functional - based approach, has interfaces to the gaussian and turbomole packages. an interface to the tinker package is provided for calculations of electrostatic and polarization contributions to energetic disorder. the kinetic monte carlo module reads in the neighbor list, site coordinates, and hopping rates and performs charge dynamics simulations using either periodic boundary conditions or charge sources and sinks. the toolkit is written as a combination of modular c++ code and scripts. the data transfer between programs is implemented via a state file or database, which is also used to restart simulations. analysis functions and most of the calculation routines are encapsulated by using the observer pattern,(93) which allows the implementation of new functions as individual modules. to summarize, we have presented a toolkit for developing and testing methods for charge transport simulations in disordered organic semiconductors. the core of the toolkit is based on a reader and a postprocessor of atomistic trajectories, a fast molecular orbital overlap calculations library, and a kinetic monte carlo code. to illustrate its functionality, we have studied charge transport in amorphous tris-(8-hydroxyquinoline)aluminum, a typical organic semiconductor. the source code of the toolkit is available under the apache license (www.votca.org). | charge carrier dynamics in an organic semiconductor can often be described in terms of charge hopping between localized states. the hopping rates depend on electronic coupling elements, reorganization energies, and driving forces, which vary as a function of position and orientation of the molecules. the exact evaluation of these contributions in a molecular assembly is computationally prohibitive. various, often semiempirical, approximations are employed instead. in this work, we review some of these approaches and introduce a software toolkit which implements them. the purpose of the toolkit is to simplify the workflow for charge transport simulations, provide a uniform error control for the methods and a flexible platform for their development, and eventually allow in silico prescreening of organic semiconductors for specific applications. all implemented methods are illustrated by studying charge transport in amorphous films of tris-(8-hydroxyquinoline)aluminum, a common organic semiconductor. |
our demonstration that the 4,6-o - benzylidene - directed -mannopyranosylation reaction proceeds via the coupling of a highly reactive -mannosyl triflate with an acceptor alcohol without the need for a promoter provided a rare opportunity to study an important class of glycosylation reaction in detail. the rapidity of the reaction at low temperatures and the absence of a suitable chromophore excluded the possibility of simple kinetic measurements for the determination of reaction order and focused our attention on the measurement of kies by nmr methods, preferably at natural abundance so as to avoid the time - consuming and expensive synthesis of labeled compounds. problems of sensitivity and resolution (500 mhz h operating frequency) limited our earlier work to the observation of secondary h kies and even then required the laborious synthesis of samples enriched in deuterium. with an 800 mhz (h operating frequency) nmr spectrometer and cryogenic probe technology at our disposal, we have now succeeded in determining primary c kie s at natural abundance for benzylidene - directed glycosylation reactions in the glucose and mannose series and show that mechanism is a function of donor stereochemistry and in the mannose series differs for the two anomeric products. the 4,6-o - benzylidene protected mannosylsulfoxide 1 was prepared as previously described and converted at 72 c in dichloromethane solution to the -glycosyltriflate 2 by the addition ofan excess of trifluoromethanesulfonic anhydride (tf2o) in the presence of 2,4,6-tri - tert - butyl pyrimidine (ttbp) as a mild, hindered, non - nucleophilic base. the acceptor, 2-propanol, was then added in quantities so as to ensure partial conversion of triflate 2 to the - and -glycosides 3 and 4 before the reaction was quenched at 72 c by the addition of saturated aqueous nahco3(table 1). after work up, the extent of conversion was determined by integration of the h nmr spectrum of the crude reaction mixture (800 mhz) against the internal standard 4,4,5,5-tetramethyl-2-(1-naphthyl)-1,3-dioxolane, and the products isolated chromatographically. the c nmr spectra of the two anomeric products were then recorded and the integrals were determined for the remote benzylidene carbon, employed as an internal standard, and the anomeric carbon, in both the products, and compared with those previously determined in the same manner for the starting sulfoxide. use of the standard equation for kie determination based on the observation of reaction products (eq 1), where f1 is the fractional conversion and r0 and rp the ratio of the anomeric and benzylidene carbon integrals in the substrate and product respectively, afforded the results presented in table 1 for the - and -mannopyranoside products. eq 1.kie=ln(1f1)ln[1(f1rp/r0]=k12ck13c comparison of the results presented in tables 1 reveals that the primary c kie values for the - and -mannoside products are distinctly different, suggesting different mechanisms for the formation of the two anomers. the values for the -anomer 4 fall close to the range (1.031.08) expected for a bimolecular(sn2) reaction, while those for the -anomer 3 are closer to the expected values (1.001.01) of a unimolecular (sn1) reaction. it is also evident from inspection of table 1 that the ratio of the two products depends on the amount of tf2o employed with lower : ratios occurring when more tf2o was employed. we interpret this observation, which was deliberately exploited in order to obtain sufficient amounts of the minor -anomer for the nmr experiments, as being due to the competing triflation of the alcohol, which reduces its concentration and so retards the bimolecular reaction. this is consistent with formation of the -product being zero order in alcohol (sn1), while that of the -product depends on the alcohol concentration (sn2), and therefore with the kie measurements. in contrast to mannopyranosylation, in the glucopyranose series the presence of a 4,6-o - benzylidene acetal results in preferential -glycoside formation most likely reflecting a shift in mechanism. hence, we examined the formation of the - and -glucosides 7 and 8 from the sulfoxides 5, via the triflate 6, by the same c nmr kie technique leading to the data presented in table 2. unlike the case of the mannopyranosides, the observed c kies in the glucose series are at the lower end of the range expected for a bimolecular reaction for both the - and -series. as the formation of mannosyl and glucosyl triflates from sulfoxides is unaffected by either the anomeric configuration or the configuration at sulfur of the sulfoxides employed, for preparative reasons the equatorial sulfoxides 5 were employed for the work in the glucose series (table 2), whereas the axial sulfoxide 3 was used in the mannose series (table 1). to complement the experimental data, and assist in its interpretation, we carried out density functional theory computations of possible transition state structures for glycosylation. we selected the b3lyp/6 - 311++g(3df,3pd)//b3lyp/6 - 31g(d, p) level as a compromise between speed and accuracy as it is sufficient for reproduction of the anomeric effect, but fast enough to survey the dozens of possible trajectories for the reactions of both anomers of each glycosyl donor. we also employed a self - consistent reaction field throughout the structure optimizations to capture any bulk medium effects imposed by the solvent used in the experiments above. the four lowest energy transition state structures identified for the formation of - and -mannosides and the corresponding glucosides are shown in figs 1a d, along with the calculated c (and h) kies (which were indistinguishable whether calculated using the bigeleisen - mayer equation or the ratio of rate constants calculated using transition state theory), pertinent atomic separations corresponding to forming and breaking bonds and the free energies of activation for each process. additional views, as well as other relevant structures are provided in the supplementary information, computational section. the computed transition states in fig 1 represent what may loosely be termed concerted transition states for nucleophilic substitution reactions in which the departing trifluoromethanesulfonate anion retains some degree of association with the putative glycosyl oxocarbenium ion. while intrinsic reaction coordinate (irc) calculations on each of these stationary points confirms that the single imaginary vibrational mode corresponds to nucleophile attachment to, and leaving group departure from, the anomeric carbon (see supplementary information for irc data), the differing degrees of c - otf bond cleavage and c - opr bond formation indicate different relative positions along the reaction coordinate. the transition states for the formation of the -glycosides (figs 1a and 1c) are highly symmetrical, in that the bond to the incoming alcohol is similar in length to that of the departing triflate, whereas those corresponding to the formation of the -anomers (figs 1b and 1d) display nucleophile attachment ahead of leaving group departure. the differences in computed activation energies suggest that formation of the -mannoside 4 should be very strongly favored over the -anomer 3, whereas the same comparison for glucose indicates the -anomer 7 to be considerably favored over the -isomer 8 the same preference observed experimentally in these systems on the preparative scale, and to a lesser extent in the present experiments owing to the necessary use of both excess tf2o and limiting isopropanol ; conditions which erode the selectivity due to suppression of the selective importantly, the computed c kies correspond quite well with the experimental values in these two cases (figs 1b and 1c), i.e. 1.018 versus 1.023 for 4 and 1.025 versus 1.023 for 7. furthermore, the calculated secondary h kie of 1.14 for the formation of the -mannoside 4 is in good agreement with the value of 1.12 some of us measured previously. the formation of the -glucoside 7 through an apparent sn2-like transition state deserves comment as experimentally only the -glucosyltriflate 6 has been observed ; the implication being that a kinetic scheme is in operation in which the more stable -glucosyltriflate is in rapid equilibrium with its less stable but more reactive -isomer, or its functional equivalent a -contact ion pair. this type of kinetic scheme in which the less stable of two isomers, that are in very rapid equilibrium on the reaction timescale, is the more reactive one leads to a situation in which the product distribution bears no relation to the ratio of starting materials is a manifestation of one of the two boundary conditions of the curtin - hammett principle. the glaringly inconsistent result is that obtained for formation of the -mannoside (fig 1a) ; the computed c kie of 1.023 differs significantly from the average experimental value of 1.005 (table 1). the implication is that - mannoside formation does not proceed through the computed sn2-like transition state of fig 1a but rather takes place at the other end of the mechanistic spectrum through a dissociative process that at least approaches the intermediacy of a distinct oxocarbenium ion and a triflate anion. the inability of computational methods to locate such transition states, without resort to artifact such as the inclusion of a further cation to neutralize the charge on the anion, owing to barrier less internal collapse is widely appreciated. this picture is fully consistent with the fact that the anomeric effect is significantly smaller in the glucopyranosides than in the mannopyranosides, from which it follows that the curtin - hammett scenario involving a minor -triflate or a closely related contact ion pair is more likely in the gluco- than in the manno - series. the recent demonstration of the existence of -glycosyltriflates as stable species when the substitution pattern is favorable further supports the possibility of their intervention via a curtin - hammet kinetic scheme. overall, a picture emerges (figure 2) featuring a series of equilibria encompassing at the extremes two covalent glycosy ltriflates, the corresponding contact ion pairs (cip) and the more loosely associated solvent separated ion pairs (ssip). for three of the four cases studied the glycosylation reaction has sn2 character but as the kie is at the lower end of the expected value for such a concerted process the transition states can be considered to be exploded and thus possibly in the grey area defined by reaction on a contact ion pair. for this reason the arrows depicting the formation of the two -products and of the -glucoside in figure 2 originate between the covalent triflates and the corresponding cips. the exception that proves the rule is the formation of the-mannoside 3 that is clearly dissociative in character and involves an intermediate that at least approximates a discrete glycosyl oxocarbenium ion. this picture represents what we consider to be the preferred pathways for the formation of the various isomers ; it does not exclude the possibility of the existence of other minor pathways such as, for example, the formation of a fraction of the -mannoside via an oxocarbenium ion - like species. the transition states located for the formation of the two -glycosides 4 and 8 (figs 1b and 1d) are further characterized by the presence of a hydrogen bond between the acceptor alcohol and o3 of the donor, which prompts the pyranose ring to adopt a b2,5 conformation in these ts structures. similarly, the ts structure for the formation of -glucoside 7 contains a h - bond, but this is now to o2 of the donor in a h3 conformation. whitfield has previously suggested that such h - bonds are important in directing glycosylation reactions. our previous experimental work, however, indicates a commonality of mechanism for the formation of o- and c - glycosides, with no h - bonding possible in the latter, for benzylidene - directed glucosylation and mannosylation. other experimental work from our laboratories confirms the role of c3 substituent in determining stereoselectivity but attributes it to the evolution of the torsional interaction between o2 and o3 as a function of the reaction co - ordinate ; clearly further work is necessary to probe the influence of acceptor - donor hydrogen bonding in these reactions. in conclusion, the agreement between computed and experimentally determined primary c kie values strongly suggests that the 4,6-o - benzylidene directed -mannosylation, and - and -glucosylation reactions proceed through loosely associative transition states in which the incoming nucleophile displaces the leaving group from a loosely bound covalent glycosyl triflate of the opposite configuration. in the case of -glucoside formation the corollary of this observation is the invocation of a covalent -glucosyltriflate, or of a -contact ion pair, and the operation of curtin - hammett - type kinetics. the contrast between the computed kie for the formation of the -mannoside by a concerted pathway and the experimentally observed value strongly suggests a highly dissociative mechanism for this one example, which approaches the intermediacy of a discrete glycosyl oxocarbenium ion. the demonstration of associative transition states for three of the four reactions studied suggests that the concentrations of both glycosyl acceptor and donor may well impact the stereoselectivity of other glycosylation reactions in solution and points the way to the more rational optimization of reaction conditions. by the same token, the stereoselectivities of concentration dependent glycosylations can not be expected to translate directly to reactions conducted on polymeric or other supports. the c nmr spectra for the kie measurements were recorded at 200 mhz using a 90 pulse, an inter scan delay of 30 s, and 512 scans so as to meet the criterion of a minimum 200/1 signal to noise ratio necessary for the precise integrations required. calculations were carried out using the gaussian-09 suite of programs with the results visualized using gaussview 4.0 or 5.0. all preparative, spectroscopic, and computational methods are reported in the supplementary information along with full characterization data for all new compounds. | although arguably the most important reaction in glycoscience, chemical glycosylations are among the least well understood of organic chemical reactions resulting in an unnecessarily high degree of empiricism and a brake on rational development in this critical area. to address this problem primary 13c kinetic isotope effects now have been determined for the formation of - and -manno- and glucopyranosides by a natural abundance nmr method. in contrast to the common current assumption, for three of the four cases studied the experimental values concur with those computed for associative displacement of the intermediate covalent glycosyl trifluoromethanesulfonates. for the formation of the -mannopyranosides the experimentally determined kie differs significantly from that computed for an associative displacement, which is strongly suggestive of a dissociative mechanism that approaches the intermediacy of a glycosyl oxocarbenium ion. the application of comparable experiments to other glycosylation systems should shed further light on their glycosylation mechanisms and thus assist in the design of better reactions conditions with improved stereoselectivity. |
radiotherapy plays an important role in the treatment of thoracic cancers, such as non - small - cell lung cancer (nsclc), small - cell lung cancer (sclc), and esophageal cancer [1, 2 ]. recent advances in accurate diagnosis improve the practice of curative radiotherapy, because patients with unsuspected metastases may avoid unnecessary local therapies and receive necessary systemic treatment. accurate delineation of tumor volume is also important to prevent geographic misses in treatment planning. currently, treatment planning is based on computed tomography (ct) imaging to contour the tumor. tumor delineation is manually performed by each radiation oncologist in clinical practice, which leads to interobserver variability in tumor delineation. accurate delineation of tumor volume requires the identification of anatomic borders of tumors based on accurate diagnosis. f-18 fluorodeoxyglucose positron emission tomography (fdg - pet) and pet / ct have been reported to enable accurate staging and to detect the tumor extension compared with ct alone. fdg - pet imaging has many potential advantages in radiotherapy planning, because it can add biological information combined with anatomical information by ct imaging. radiotherapy planning based on fdg - pet is expected to decrease the interobserver variability amongst thoracic radiation oncologists. in addition to accurate staging, fdg - pet has the potential to improve radiotherapy planning by its precise delineation of primary tumor and lymph nodes. indeed, fdg - pet has been investigated as tracer for radiotherapy planning in many cancer types. in this paper, we focus on the role of fdg - pet in radiotherapy for thoracic cancers, including nsclc, sclc, esophageal cancer, and breast cancer. accurate staging is crucial for lung cancer patients because treatment strategy and prognosis drastically differ according to clinical stage. ct imaging contributes to the initial determination for the staging of nsclc and provides morphologic information of the disease extension. currently, fdg - pet is performed to diagnose the stage and evaluate the effects of the treatment in clinical use for nsclc patients. one of the important roles of fdg - pet is to detect unsuspected lymph nodes or extrathoracic metastases [5, 6 ]. a prospective study has reported that fdg - pet detected unsuspected metastasis in 20% of candidates for radical radiotherapy, changing their treatment strategies. furthermore, the same authors showed that early mortality rate was low in patients staged by fdg - pet compared with those staged by conventional imaging, on the basis of their accurate staging. another prospective study demonstrated that curative radiotherapy in 27% of patients was not qualified by fdg - pet because of distant metastasis or extensive locoregional disease. in this study, 32% of patients also reported that fdg - radiation planning in over 50% of patients staged by ct imaging, due to the detection of unsuspected nodal disease, metastatic disease, or tumor extension from atelectasis. as seen in these findings, the incorporation of fdg - pet improves the accuracy of staging and patient selection, providing better treatment strategy including radiotherapy planning. fdg - pet is increasingly performed to define tumor volume for radiotherapy in patients with nsclc. accurate contouring of organs is important for radiotherapy and pet imaging is currently expected to be useful in the delineation of gross tumor volume (gtv). in this section several methods have been examined for gtv delineation after the introduction of fdg - pet (table 1) [1225 ]. however, this method arbitrarily depends on the window levels set of the pet images. several articles have reported the use of standardized uptake volume (suv) in gtv identification. hong. compared target volume delineation by fdg - pet with that by ct imaging in 19 patients with nsclc, concluding that suv of 2.5 should be used for radiotherapy planning in nsclc patients. erdi. analyzed a phantom with lung lesion by source - to - background ratios. they applied this data to 10 patients with 17 primary or metastatic lung lesions, concluding that the source - to - background ratio was useful in the definition of tumor volumes. compared gtvs obtained from four different methods : visually (gtvvis), applying a 40%-threshold of suv max (gtv40), using an autocontour of suv 2.5 (gtv2.5), and using an algorithm by phantom measurements (gtvbg) in 25 patients with nsclc. in the results, gtvvis, gtv2.5, and gtvbg were relevant to the ct - derived gtv, whereas gtv40 appeared unsuitable for target volume delineation. recently, some studies reported a new gradient - based method relied on the water shed transform and hierarchical cluster analysis. this method provided denoised and deblurred images with an edge - preserving filter and a constrained iterative deconvolution algorithm, leading a better estimation of the gradient intensity. wanet. compared a gradient - based segmentation method, a source - to - background ratio method, and a 40% to 50% of suv max method with surgical specimens of 10 patients with nsclc. all patients underwent image inspections before surgery. among several methods, the gradient - based method achieved the best results, with the lowest average error and smallest standard deviation. werner - wasik. conducted the same experiment using the digital thorax phantom and evaluated the accuracy and consistency of three methods : gradient - based pet segmentation, manual, and constant threshold methods. they concluded that the gradient - based method was the most accurate with the least systematic bias at any phantom size. despite these several techniques for the delineation of tumor volume having been reported in recent years, a gold standard method has not been established to contour gtv automatically. however, fdg - pet has the additional information that aids radiation oncologists to delineate gtv, indicating that fdg - pet should be utilized when tumor volume is delineated in nsclc patients. interobserver variability in regard to gtv definition is a serious problem in the treatment planning for nsclc [26, 27 ]. prospectively evaluated the role of fdg - pet in interobserver variability in 30 nsclc patients. three radiation oncologists contoured gtv using ct alone or fdg - pet registered ct (pet / ct). the mean coefficient of variation for gtv based on pet / ct was significantly smaller than ct alone (p 35% of suv max) of primary tumor. this result may be applied to further radiation therapy of additional doses to the area of higher fdg uptake. elective nodal irradiation to the mediastinal lymph regions in the treatment of stage iii nsclc patients is still a controversy [37, 38 ]. retrospectively evaluated the failure rates in uninvolved nodal regions with involved - field radiotherapy for inoperable 524 patients with nsclc. the 2-year elective nodal control rate was 91% and the authors concluded that involved - field radiotherapy did not cause a significant amount of failure in lymph node regions. rtog0117 also performed involved - field radiotherapy to perform high - dose irradiation without severe side effects. however, some clinicians have raised the concern that omission of elective nodal irradiation requires a further discussion, because recurrence of lymph nodes is usually fatal and microscopic metastasis of lymph nodes occurs substantially in advanced nsclc. fdg - pet can contribute to accurate evaluation of nodal lesions, compared with ct alone [41, 42 ]. conducted a prospective study of involved - field radiotherapy based on fdg - pet for 44 patients. in this study, only 1 patient (2.3%) developed an isolated nodal failure, and the authors concluded that fdg - pet was useful in the involved - field radiotherapy for nsclc patients. furthermore, kolodziejczyk. reported that fdg - pet should be used even in the elective nodal irradiation planning for nsclc, because elective nodal irradiation may not compensate the unsuspected mediastinal lymph nodes. it is controversial whether elective nodal irradiation can be safely omitted by fdg - pet or not. however, this strategy is extremely attractive because it allows radiation - dose escalation without severe side effects. further studies are warranted to establish the utility of fdg - pet in nsclc for radiation planning. sclc often represents an aggressive clinical course and high incidence of distant metastasis because of rapid tumor growth. despite aggressive treatment, although accurate staging is essential for determining the treatment strategy in sclc, it is difficult to evaluate the extension of disease accurately, and especially mediastinum lymph node metastases. prospectively examined the role of pet / ct compared with standard staging (ct and bone scintigraphy) in 29 sclc patients. in their study, pet / ct changed the stage in 5 of the 29 patients (17%), with the authors concluding that pet / ct improves the accuracy of staging in sclc. in other studies, 8.3% to 9.5% of limited - disease sclc staged by the conventional staging procedures was upstaged to extended - disease sclc after fdg - pet information was incorporated [46, 47 ]. arslan. evaluated the accuracy and overall survival staged by fdg - pet or ct imaging. pet scan upstaged 9 (36%) in 25 patients and downstaged 2 (8%) in 25 patients who were staged by ct imaging. furthermore, fdg - pet staging predicted significant survival difference (p = 0.019), while ct imaging did not (p = 0.055). these studies recommended that fdg - pet should be performed for initial staging in limited - disease sclc patients. compared with nsclc, few studies have been performed regarding the delineation based on fdg - pet for sclc. fdg - pet in sclc patients may improve the delineation of tumor volume as well as nsclc. for limited - disease sclc, elective nodal irradiation for mediastinal lymph node regions has been considered necessary to reduce lymph node failure. however, some clinicians have attempted to avoid elective nodal irradiation because extended radiotherapy volume leads to severe side effects. conducted a phase ii study of involved - field radiotherapy for limited - disease slcl staged by ct imaging. also conducted a phase ii trial of involved - field irradiation based on ct imaging for limited - disease sclc. they evaluated overall survival and isolated nodal failure defined as recurrence in regional nodes outside the target volume in the absence of in - field failure. in their study, isolated mediastinal lymph node recurrence was unexpectedly high (11%). given these findings, a report from the international atomic energy agency (iaea) consultants ' meeting indicated that involved - field irradiation in sclc was controversial and should be performed in a prospective clinical trial. currently, fdg - pet is expected to determine whether elective nodal irradiation is necessary or not. two recent studies have shown the usefulness of fdg - pet in omitting elective nodal irradiation in sclc [52, 53 ]. van loon. conducted a prospective study of involved - field irradiation on the basis of fdg - pet for 60 patients with limited - disease sclc. median actuarial overall survival was 19 months and isolated nodal failures was low (3%). the authors concluded that treatment planning based on fdg - pet could decrease isolated nodal failures, compared with their previous results (isolated nodal failure : 11%) treated by ct - based involved - field irradiation. shirvani. reported that involved - field irradiation by intensity - modulated radiotherapy (imrt) for 60 patients with limited - disease slcl was staged by fdg - pet. in this study, the 2-year actuarial overall survival was 58% and isolated elective node was observed in only one patient (3%). these studies concluded that elective nodal irradiation could be safely omitted by fdg - pet staging in sclc. avoidance of mediastinal lymph nodes that are pet - negative can lead to (1) a reduction of toxicity with the same radiation dose or (2) dose escalation with the same toxicity. although involved - field irradiation based on fdg - pet is an attractive treatment in sclc, there is not enough information to recommend the strategy. further prospective studies are required to determine whether elective nodal irradiation can be safely omitted by fdg - pet staging in sclc. another possible role of fdg - pet in sclc is in the evaluation of the therapeutic response. a preliminary study showed that fdg - pet could predict the outcome of treatment by radiotherapy or chemotherapy. in conclusion, although the use of fdg - pet in radiation treatment planning for sclc is still controversial, involved - field irradiation based on fdg - pet is an attractive strategy. fdg - pet - based treatment planning will change the strategy for limited - disease sclc. in the treatment of esophageal cancer, radiotherapy is commonly used in combination with chemotherapy. currently, radiotherapy requires accurate target volume definition based on treatment planning by ct scan. although planning - ct - based target volume definition is considered the gold standard in esophageal cancer, applying fdg - pet to treatment planning may have several advantages, such as accurate staging and delineation of tumor. reported that 70 primary tumors of 74 patients were detected by fdg - pet, with a sensitivity of 95%. the authors showed that fdg - pet had a higher accuracy for stage iv disease compared with conventional modalities (82% versus 64%, p = 0.004). van vliet. performed a meta - analysis to evaluate the diagnostic performance of ct and fdg - pet in staging of esophageal cancer. the sensitivities of ct and fdg - pet for regional lymph node metastases were 0.50 and 0.57, respectively, and their specificities were 0.83 and 0.85, respectively. the authors concluded that each modality plays a distinctive role in the detection of esophageal cancer. the additional information provided by fdg - pet is expected to improve tumor delineation and accurate staging of lymph nodes and distant metastases. several studies have investigated the optimal method for delineating the target volume by fdg - pet. moureau - zabotto. evaluated the effect of the addition of fdg - pet to ct in tumor delineation of 34 esophageal cancer patients. fdg - pet decreased the length of gtv in 12 patients (35%) and increased the length in 12 patients (35%). konski. used suv 2.5 to delineate the tumor extension and evaluated the ct - based tumor length in 25 esophageal cancer patients. the authors concluded that fdg - pet provides additional information for the identification of gtv. compared fdg - pet - based tumor length with surgical specimens and showed that suv 2.5 seemed closest to the pathological length. reported that automated interpretation of fdg - pet using mean activity of the liver plus 2 standard deviations likely affect target definition. compared 11 different methods : suv 2.0, 2.5, 3.0, and 3.5 ; suv max 40%, 45%, and 50% ; mean liver suv plus 1, 2, 3, and 4 standard deviations. the authors concluded that the use of a threshold of approximately 2.5 was the optimal method for the delineation of gtv in esophageal cancer, regardless of suv thresholding method. recent studies indicated that suv 2.5 may be an optimal threshold, but autocontouring using this threshold is not satisfactory. further investigations are required to utilize fdg - pet in the tumor delineation for esophageal cancer in clinical use. another method to utilize fdg - pet in treatment planning is to reduce interobserver variability. evaluated the tumor volumes delineated by fdg - pet in 28 esophageal cancer patients by three radiation oncologists. the authors concluded that fdg - pet might improve target volume definition with less geographic misses, but the effects on interobserver variability were not significant. vesprini. compared fdg - pet / ct with ct alone for the identification of gtv in 10 patients with esophageal cancer by 6 radiation oncologists. muijs. reported that the additional use of fdg - pet led to the modification of ct - based radiotherapy planning in 57% of esophageal cancer patients. furthermore, they showed that fdg - pet significantly changed the radiation dose for organs at risk, such as heart and lung. the additional information provided by fdg - pet has the possibility of improving the local control due to less geographic misses. however, there are no studies that showed whether fdg - pet affects survival or local control rate. further studies are warranted to establish the role of fdg - pet in highly accurate radiotherapy planning for esophageal cancer. one of the limitations in the use of pet imaging in radiotherapy planning is the low spatial resolution. generally, the spatial resolution of current pet scanners (68 mm) is inferior to that of modern ct scanners (1 mm). furthermore, because pet requires several minutes to perform the imaging, tumor motion due to respiration or cardiac action deteriorates pet images and increases diagnostic errors in thoracic cancers. in fact, it is one of the major limitations of fdg - pet in several tumor types and has been largely responsible for the slow acceptance of fdg - pet when major treatment decisions are made on the basis of a negative fdg - pet. sakaguchi. reported that gated imaging acquisition improved the quality of fdg - pet imaging in a moving phantom model. evaluated 48 pulmonary nodules in 43 patients by respiratory - gated pet and ungated method. gated imaging had higher sensitivity and specificity than the ungated method, especially for smaller lesions located in lower lobes. these findings suggested that respiratory - gated pet should be performed for patients with thoracic cancers with respiratory motions. currently, 3-deoxy-3-(18)f - fluorothymidine (flt), a pet imaging marker of proliferation, has been introduced as an alternative to fdg for lung cancer. yang. compared the diagnostic efficacy of flt - pet and fdg - pet in nsclc. the sensitivities of flt- and fdg - pet for primary tumor were 74% and 94%, respectively. in contrast, flt - pet showed better specificity and accuracy than fdg - pet for lymph nodes. although the use of flt - pet is still at an investigational level, further studies are expected to continue to evaluate its efficacy. when pet is used for radiotherapy planning, a range of uncertainties related to technical, physical, biological, and analytical factors must be considered. further investigations are warranted to establish highly accurate radiotherapy based on pet imaging for thoracic cancers. fdg - pet plays a pivotal role in accurate staging and selection of patients to be treated by radiotherapy. furthermore, fdg - pet improves radiotherapy volume and enables dose escalation without severe side effects in lung cancer patients. the main advantage of fdg - pet for esophageal cancer patients is the detection of unrecognized lymph nodes or distal metastases. while fdg - pet was initially expected to result in more accurate target delineation, its efficacy remains controversial and delineation by fdg - pet should not be practiced in clinical use at this stage. further studies are required to confirm the advantage of fdg - pet in radiotherapy planning for thoracic cancers. | radiotherapy plays an important role in the treatment for thoracic cancers. accurate diagnosis is essential to correctly perform curative radiotherapy. tumor delineation is also important to prevent geographic misses in radiotherapy planning. currently, planning is based on computed tomography (ct) imaging when radiation oncologists manually contour the tumor, and this practice often induces interobserver variability. f-18 fluorodeoxyglucose positron emission tomography (fdg - pet) has been reported to enable accurate staging and detect tumor extension in several thoracic cancers, such as lung cancer and esophageal cancer. fdg - pet imaging has many potential advantages in radiotherapy planning for these cancers, because it can add biological information to conventional anatomical images and decrease the inter - observer variability. fdg - pet improves radiotherapy volume and enables dose escalation without causing severe side effects, especially in lung cancer patients. the main advantage of fdg - pet for esophageal cancer patients is the detection of unrecognized lymph node or distal metastases. however, automatic delineation by fdg - pet is still controversial in these tumors, despite the initial expectations. we will review the role of fdg - pet in radiotherapy for thoracic cancers, including lung cancer and esophageal cancer. |
the recurrence of lung cancer after complete resection generally results in a poor prognostic situation with less than one year of life expectancy. when a single lung nodule is detected during follow - up, it is usually one of the manifestations of the systemic metastasis. however, one distinct feature is a second primary metachronous lung cancer appearing after the initial treatment of the primary lung cancer. although there are criteria that can be applied in this situation, it is sometimes very difficult in clinical practice to distinguish between a new lung cancer and a metastatic one. therefore, some researchers have insisted an aggressive surgical approach, whenever possible, as the survival rate after surgery is quite good (1 - 7). on the other hand, many doubts have been expressed concerning the utility of repeated surgery for recurrent lung cancer, as it is associated with a poor overall survival (8, 9). we are convinced of the value of the aggressive surgical approach for all the cases whose the functional condition makes resection possible. the aim of this study is to report the result of re - resection for recurrent lung cancer while focusing on the rationale for this aggressive surgical approach. there were 1,461 patients who underwent operations for primary lung cancer from december 1994 to december 2003. the operations were performed by a group of three thoracic surgeons in a single hospital. every patient was followed - up by the same surgeons on a regular follow - up schedule (every 3 - 4 months for 2 yr, every 6 months for the next 3 yr and then every year). when recurrent disease in the lung was suspected, repeated ct scan were performed at 2 months intervals, and positron emission tomography (pet), bronchoscopy and/or percutaneous needle aspiration (pcna) were also performed. there were 29 consecutive patients who underwent repeated surgical resections for recurrent lung cancer after curative resection for lung cancer. at the time of initial operation, histology and pathological staging are shown in table 1. the diagnostic methods of recurrent lung cancer were chest ct in 11 patients, pet in 9 patients, pcna in 6 patients, and bronchoscopic biopsy in 3 patients. the second primary lung cancers were defined using the criteria outlined by martini and melamed (10). a neoplasm was defined as a metastatic lung cancer if it was a same histology occurring in the same anatomic site within 2 yr after first operation. a second primary cancer was a tumor of different histology, or if histology was the same, the disease free interval of recurrent cancers was at least 2 yr or the second cancer was in different lobe or lung, or origin from carcinoma in situ in the absence of other extrapulmonary or common lymphatic metasitasis. on the repeated lung operations, mediastinoscopy was not performed because most of the patients already had had mediastinoscopy performed at the first lung resection. however, careful intraoperative nodal staging was performed by dissecting the intrapulmonary, hilar and ipsilateral mediastinal nodes. although the extent of resection of recurrent lung cancer in early period was dependent on the surgeon 's preference, thereafter we have made an attempt to avoid limited resection if patients had predictive post - operative fev1 of more than 1.0 l and acceptable cardiac function. postoperatively, the same follow - up plan was applied as for the first lung operation. we analyzed included types of operation, the time between two surgical interventions, operative findings and histology, operative and post - operative complications and the hospital mortality as well as late death. the descriptive statistics are summarized as the median or mean for the continuous variables and as frequencies and percentages for the categorical variables. survival and the disease free rate were analyzed by the kaplan - meier and log - rank methods on spss for window, release 10.0 (spss inc., 29 consecutive patients underwent surgical resections for recurrent lung cancer after curative resection for lung cancer. the median age of patients was 61 yr (range, 45 to 75 yr) and the male / female (22/7) ratio was 3.1:1. the second primary tumor was discovered on the same side as the first tumor in 19 patients. the mean time interval between the first operation and the second operation was 20.315 months (table 1). the mean time interval between the first resection and the second resection was 25.415.1 months for the second primary lung cancers and 8.95.7 months for the metastatic lung cancer. the second operations were completion pneumonectomy in 11 patients, lobectomy in 5 patients, wedge resection in 12 patients, and resection and anastomosis of the trachea in 1 patient (table 2). the causes of death were pulmonary embolism in one patient and respiratory failure in the other patient. major morbidity was observed in 6 (21%) patients after the repeated lung resection ; bronchopleural fistula was noted in 3 patients, myocardial infarction and cerebral vascular accident were noted in 1 patient, pulmonary artery embolism occurred in 1 patient and respiratory failure occurred in 1 patient. tumor recurrence after reoperation was observed in 14 patients (48%). in four of these 14 patients, we performed the third operation ; a completion pneumonectomy in 1 patient, wedge resection in 2 patients and multiple rib resection and chest wall reconstruction in 1 patient. the remaining ten patients were referred for radiation therapy, chemotherapy or supportive care only (table 4). overall follow - up after reoperation ranged from 3 to 103 months with a mean of 41.422.3 months. the overall actuarial 5-yr survival rate was 69% and the 5-yr disease free rate following reoperation was 44% (n=29). no significant difference was detected in survival and the disease free rate between the second primary lung cancer patients and the metastatic lung cancer patients (table 3, fig. 1, 2). the disease free rate following reoperation was significantly different between the wedge resection group and anatomically curative resections such as lobectomy / completion pneumonectomy group (p=0.008), but the survival rate was not significantly different (fig. recent improvements of such diagnostic tools as high - resolution ct scan or pet scan allow physicians to detect recurrent lung cancer much earlier than before. however, there is not yet a consensus for the postoperative follow - up strategy of patients after complete resection for nsclc. (1) reported that intensive follow - up may improve patient survival by detecting recurrences at an asymptomatic stage after surgery for nsclc. it has been reported that for detecting residual or recurrent nsclc, pet had a sensitivity of 100% and a specificity of 61.5 - 92%, but chest ct only had a sensitivity of 67% and a specificity of 85% for the detection of tumor recurrence (12). in our study, when the lesion was centrally located and the percutaneous needle aspiration was difficult to perform, pet was then used as a very effective diagnostic method. in our series, the diagnostic methods of recurrent lung cancer were chest ct in 11 patients, pet in 9 patients, pcna in 6 patients, and bronchoscopic biopsy in 3 patients. when the recurrent lung cancer was difficult to diagnose with using chest ct or pet, we then performed invasive diagnostic methods. the initiation of earlier therapy with the early detection of recurrences may have improved the outcome compared with the therapy given when the patient becomes symptomatic from the tumor (1, 3, 4, 6). major complications occurred in 24% of our patients after the second resections, which is comparable to the previously reported results of pulmonary resection for recurrent lung cancers (3, 4, 17). martini and melamed (10) in 1975 outlined the criteria for differentiation between a second primary lung cancer and a metastatic or recurrent lung cancer. some reports have suggested that patients with a second primary lung cancer have a more favorable prognosis than patients with locally recurrent or metastatic disease (13 - 15). however, other reports did not reveal a significantly different survival between patients operated on for a second primary lung cancer or those patients operated on for locally recurrent or metastatic disease (4, 5, 8, 9). our data showed that the 5-yr survival rates were not different between second primary lung cancer and the possible second primary or metastatic lung cancer. yet the 5-yr disease free rates for lobectomy / completion pneumonectomy group were better than for wedge resection group. it may be explained by the fact that the second operations had been wedge resections for eight patients of the second primary lung cancer subgroup (n=12) who had suffered a second tumor recurrence. the lung cancer study group (16) reported that a resection that is less extensive than lobectomy for primary lung cancer places the patient at an increased risk of local recurrence and this decreases the chances of long term survival. the limited resections used in second primary lung cancer group might have resulted in an increased risk for locoregional recurrence, but the extents of the resection of recurrent lung cancer did not influence the survival in another study (6, 17). (7) reported that pulmonary resection of bronchogenic carcinoma after pneumonectomy was associated with acceptable morbidity and mortality. wedge resection, when feasible, is the treatment of choice. voltolini. (4) has reported on the type of surgical treatment used in case of metachronous second primary lung tumor. the wedge resection was a reasonable and safe alternative to the standard resections in the elderly patients or in those patients with a poor respiratory reserve and after a pneumonectomy. if the tumor was not in the periphery and if it was less than 2 cm in diameter, they consider lobectomy as the treatment of choice even for the second resection. the recurrence rate was lower in the lobectomy / completion pneumonectomy group than in the wedge resection group. the mean disease free time for lobectomy / completion pneumonectomy patients was longer than for the wedge resection patients. eight patients, who had undergone wedge resection of second primary lung cancer, were proven to show recurrence. therefore, lobectomy or completion pneumonectomy is preferred to wedge resection as an operation for a second primary lung cancer. surgical intervention for recurrent lung cancers can be performed with an acceptable mortality and morbidity. if it is tolerable, completion pneumonectomy or lobectomy are recommended procedures of choice for recurrent lung cancer. | we reviewed our experience with resection of recurrent lung cancer to evaluate the benefit and risk of the procedure. from december 1994 to december 2003, 29 consecutive patients underwent pulmonary resections for recurrent lung cancer. the mean duration from the first resection to second surgery was 25.415.1 months for the definite 2nd primary lung cancer (n=20) and 8.95.7 months for metastatic lung cancer (n=9). the procedures at the second operations were completion - pneumonectomy in 11 patients, lobectomy in 5 patients, wedge resection in 12 patients and resection and anastomosis of trachea in 1 patient. morbidity was observed in 6 (21%) of the patients and the in - hospital mortality was two patients (7%) after the repeated lung resection. tumor recurrence after reoperation was observed in 14 patients (48%). the actuarial 5-yr survival rate was 69% and the 5-yr disease free rate following reoperation was 44%. no significant difference was found in overall survival and disease free survival between the 2nd primary lung cancer group and the metastatic lung cancer group. the recurrence rate following reoperation was significantly different between the wedge resection group and lobectomy / completion pneumonectomy group (p=0.008), but the survival rate was not significantly different (p=0.41). surgical intervention for recurrent lung cancers can be performed with acceptable mortality and morbidity. if tolerable, completion pneumonectomy or lobectomy is recommended for resection of recurrent lung cancer. |
the anterior cruciate ligament (acl) is a site injured frequently ; consequently, acl reconstructions are widely performed. despite recent improvements in acl reconstruction, subsequent graft ruptures, residual instability, and limited sports performance after surgery acl remnants may potentially enhance revascularization and cell proliferation and promote the recovery of high - quality proprioception and stability1,2,3,4,5), and to achieve these, a remnant - preserving acl reconstruction technique was recently developed with good outcomes6,7,8). however, poor arthroscopic visualization makes accurate socket placement during remnant - preserving acl reconstruction difficult. since 2007, we have used a three - dimensional (3d) fluoroscopy - based navigation system to position the femoral socket accurately and reproducibly during anatomical acl reconstruction using hamstring tendon grafts or a bone - patellar tendon - bone (btb) graft9,10). this method is particularly appropriate for remnant - preserving acl reconstruction because it enables visualization of the lateral wall and roof of the femoral intercondylar notch, both of which are less easily visualized by arthroscopy. using this system, the surgeon can identify the lateral intercondylar ridge (lir), which is an important topographical landmark used to identify the femoral attachment of the acl with minimal debridement of the remnant11). the present report aimed to describe remnant - preserving acl reconstruction using a 3d fluoroscopy - based navigation system and determine the usefulness of this system in the successful creation of an accurately placed femoral socket. the reference frame (orthopaedic frame hc ; medtronic inc., louisville, co, usa) was attached rigidly to the femur with two half - pins at the beginning of surgery. intraoperative 3d images were acquired with the c - arm of arcadis orbic 3d (siemens ag, erlangen, germany) (fig. the c - arm of the image intensifier was equipped with a wireless tracker (stealth active wireless tracker s / n 130, medtronic inc.) to allow image capture and automatic registration by the navigation computer. the acquired image data were downloaded to the navigation computer (stealthstation tria plus, medtronic inc.) and a 3d image of the distal femur was reconstructed on the computer screen (fig. the medial half of the 3d - reconstructed distal femur was deleted using a specific function of the computer software to provide a better view of the lateral wall and the roof of the femoral intercondylar notch (fig. grafts were selected by a surgeon who took into consideration the activity of patients, the types of sports they may be involved in, and patient preference. btb graft was selected primarily for young male or collision / contact athletes, while hamstring grafts were selected for the others during the study period. btb grafts were 10 mm in width and harvested with bone plugs at both ends from the central portion of the patellar tendon. the femoral bone plug for a rectangular socket was usually 51015 mm as described by shino.12). it was connected to an endobutton (smith & nephew endoscopy, andover, ma, usa) using no. 5 and no. 2 fiberwires (arthrex inc., naples, fl, usa). for the hamstring tendon grafts, the semitendinosus tendon was primarily harvested. when the length or diameter of semitendinosus tendon was insufficient, the gracilis tendon was also harvested. in most cases, the anteromedial (am) bundle consisted of a doubled semitendinosus tendon combined with a doubled gracilis tendon, whereas the posterolateral (pl) bundle consisted only of a doubled semitendinosus tendon. the graft for the am bundle was a minimum of 65-mm in length and 6-mm in diameter, whereas that for the pl bundle was a minimum of 60-mm in length and 5-mm in diameter. each graft was connected to an endobutton cl (smith & nephew endoscopy) in the usual manner. when the remnant tissue of acl was scarred to the posterior cruciate ligament (pcl) or reattached to either the roof of the notch or the lateral wall, the minimal amount of the residual remnant was cleaned from the lower side to place the guide at the anatomical femoral attachment site using a thermal device. during debridement of soft tissues, guide wire placement for femoral socket creation was performed using a femoral guide equipped with a tracker (suretrak2 universal tracker, large passive fighter ; medtronic inc.). we used a far am portal technique13) for femoral tunnel creation. with an arthroscope introduced through a lateral or medial portal, the tip of the femoral guide could be placed arthroscopically through a far am portal at the designated location. when the remnant tissue impeded guide placement during positioning of the femoral guide through the far am portal, the remnant tissue of acl was carefully retracted medially through the medial portal. image - interactive navigation enabled the surgeon to confirm the position of the tip of the femoral guide on the 3d reconstructed image in real time (fig. 3). after the tip of the guide was placed at the target point (center of a socket), and after the position of the guide tip was verified on the computer screen, a guide was inserted slightly, the femoral guide tip was maintained within the femoral footprint, and the knee was fully flexed. accordingly on the navigation computer screen, the surgeon could then identify the entire image of the lateral wall of the femoral notch and lir, even when arthroscopic visualization of the lateral wall of the intercondylar notch was impeded because of the presence of the remnant on deep knee flexion (fig. 3). if the guide tip was displaced and was not arthroscopically recognized, the surgeon could recognize it on the navigation monitor. next, the 3d image was rotated 90 on the navigation screen where the risk of a back wall blowout could be evaluated (fig. finally, the 3d image was rotated 180 to reveal the lateral aspect of the distal femur on the navigation screen (fig. when the btb graft was used, two parallel guide wires were inserted with a 5-mm distance in the center of the femoral insertion along its long axis (fig. conversely, when double - bundle reconstruction with hamstring grafts was performed, two guide wires for the am and pl sockets were inserted separately at the center of each footprint. after insertion of the guide wires during btb grafting, they were overdrilled for an appropriate length using a 5-mm cannulated drill, the two tunnels were interconnected using a dilator (smith & nephew endoscopy), and the lateral femoral cortex was drilled through the center of the two tunnels using an endobutton drill (smith & nephew endoscopy) (fig. each guide wire was overdrilled for an appropriate length by a cannulated drill with a diameter 0.5 mm smaller than that of the graft, each socket was dilated using a dilator with a diameter similar to that of the graft, and the distant cortex was breached with the endobutton drill. the tibial insertion site was arthroscopically determined in reference to the acl remnant, the medial tibial eminence, the anterior horn of the lateral meniscus, the intermeniscal ligament, and the pcl14). tibial tunnel creation and graft passage were performed roughly according to the method described by ochi.15). 11 scalpel blade was used to make a longitudinal slit in an acl remnant tissue. this method allowed not only visualization of the tips of the guide pins or the drill during creation of tibial tunnels but also smooth graft passage. the tibial tunnels were placed in the center of the am and pl footprints for double - bundle reconstruction. meanwhile, two parallel guide wires were positioned within the tibial footprint and overdrilled for the entire length using a cannulated drill, and a dilator (smith & nephew endoscopy) was used to interconnect the two tunnels for rectangular reconstruction using a btb graft. 5 polyester braided suture was passed through the eyelet of a passing pin, which was inserted into the femoral tunnel through the longitudinal slit in the remnant tissue of acl. the graft was passed through the remnant tissue and the endobutton loop was flipped outside the femoral cortex in the usual manner. creation of a longitudinal slit, gradual hooking and pulling up the graft by a probe, and rounding of sharp corners of the bone plug were essential to allow technically challenging graft passage of the btb through the remnant tissue. tibial fixation of the hamstring autografts was accomplished over a suture after fixation using a fully threaded 6.5-mm cancellous screw and washer (meira inc., the am and pl bundle grafts were fixed at full knee extension and the btb graft was also fixed at full knee extension. after graft passage, when residual remnant impingement on the roof or the lateral wall of the femoral notch or the pcl was arthroscopically demonstrated, the impinging part of the remnant was shaved off (fig. the reference frame (orthopaedic frame hc ; medtronic inc., louisville, co, usa) was attached rigidly to the femur with two half - pins at the beginning of surgery. intraoperative 3d images were acquired with the c - arm of arcadis orbic 3d (siemens ag, erlangen, germany) (fig. the c - arm of the image intensifier was equipped with a wireless tracker (stealth active wireless tracker s / n 130, medtronic inc.) to allow image capture and automatic registration by the navigation computer. the acquired image data were downloaded to the navigation computer (stealthstation tria plus, medtronic inc.) and a 3d image of the distal femur was reconstructed on the computer screen (fig. the medial half of the 3d - reconstructed distal femur was deleted using a specific function of the computer software to provide a better view of the lateral wall and the roof of the femoral intercondylar notch (fig. grafts were selected by a surgeon who took into consideration the activity of patients, the types of sports they may be involved in, and patient preference. btb graft was selected primarily for young male or collision / contact athletes, while hamstring grafts were selected for the others during the study period. btb grafts were 10 mm in width and harvested with bone plugs at both ends from the central portion of the patellar tendon. the femoral bone plug for a rectangular socket was usually 51015 mm as described by shino.12). it was connected to an endobutton (smith & nephew endoscopy, andover, ma, usa) using no. 5 and no. 2 fiberwires (arthrex inc., naples, fl, usa). for the hamstring tendon grafts, the semitendinosus tendon was primarily harvested. when the length or diameter of semitendinosus tendon was insufficient, the gracilis tendon was also harvested. in most cases, the anteromedial (am) bundle consisted of a doubled semitendinosus tendon combined with a doubled gracilis tendon, whereas the posterolateral (pl) bundle consisted only of a doubled semitendinosus tendon. the graft for the am bundle was a minimum of 65-mm in length and 6-mm in diameter, whereas that for the pl bundle was a minimum of 60-mm in length and 5-mm in diameter. each graft was connected to an endobutton cl (smith & nephew endoscopy) in the usual manner. when the remnant tissue of acl was scarred to the posterior cruciate ligament (pcl) or reattached to either the roof of the notch or the lateral wall, the minimal amount of the residual remnant was cleaned from the lower side to place the guide at the anatomical femoral attachment site using a thermal device. during debridement of soft tissues, guide wire placement for femoral socket creation was performed using a femoral guide equipped with a tracker (suretrak2 universal tracker, large passive fighter ; medtronic inc.). we used a far am portal technique13) for femoral tunnel creation. with an arthroscope introduced through a lateral or medial portal, the tip of the femoral guide could be placed arthroscopically through a far am portal at the designated location. when the remnant tissue impeded guide placement during positioning of the femoral guide through the far am portal, the remnant tissue of acl was carefully retracted medially through the medial portal. image - interactive navigation enabled the surgeon to confirm the position of the tip of the femoral guide on the 3d reconstructed image in real time (fig. was placed at the target point (center of a socket), and after the position of the guide tip was verified on the computer screen, a guide was inserted slightly, the femoral guide tip was maintained within the femoral footprint, and the knee was fully flexed. accordingly, displacement of the guide tip caused by knee flexion could be mostly prevented. on the navigation computer screen, the surgeon could then identify the entire image of the lateral wall of the femoral notch and lir, even when arthroscopic visualization of the lateral wall of the intercondylar notch was impeded because of the presence of the remnant on deep knee flexion (fig. 3). if the guide tip was displaced and was not arthroscopically recognized, the surgeon could recognize it on the navigation monitor. next, the 3d image was rotated 90 on the navigation screen where the risk of a back wall blowout could be evaluated (fig. finally, the 3d image was rotated 180 to reveal the lateral aspect of the distal femur on the navigation screen (fig. when the btb graft was used, two parallel guide wires were inserted with a 5-mm distance in the center of the femoral insertion along its long axis (fig. conversely, when double - bundle reconstruction with hamstring grafts was performed, two guide wires for the am and pl sockets were inserted separately at the center of each footprint. after insertion of the guide wires during btb grafting, they were overdrilled for an appropriate length using a 5-mm cannulated drill, the two tunnels were interconnected using a dilator (smith & nephew endoscopy), and the lateral femoral cortex was drilled through the center of the two tunnels using an endobutton drill (smith & nephew endoscopy) (fig. 6). when using hamstring tendon(s), each guide wire was overdrilled for an appropriate length by a cannulated drill with a diameter 0.5 mm smaller than that of the graft, each socket was dilated using a dilator with a diameter similar to that of the graft, and the distant cortex was breached with the endobutton drill. the tibial insertion site was arthroscopically determined in reference to the acl remnant, the medial tibial eminence, the anterior horn of the lateral meniscus, the intermeniscal ligament, and the pcl14). tibial tunnel creation and graft passage were performed roughly according to the method described by ochi.15). 11 scalpel blade was used to make a longitudinal slit in an acl remnant tissue. this method allowed not only visualization of the tips of the guide pins or the drill during creation of tibial tunnels but also smooth graft passage. the tibial tunnels were placed in the center of the am and pl footprints for double - bundle reconstruction. meanwhile, two parallel guide wires were positioned within the tibial footprint and overdrilled for the entire length using a cannulated drill, and a dilator (smith & nephew endoscopy) was used to interconnect the two tunnels for rectangular reconstruction using a btb graft. after creation of the tibial tunnel, a no. 3 or no. 5 polyester braided suture was passed through the eyelet of a passing pin, which was inserted into the femoral tunnel through the longitudinal slit in the remnant tissue of acl. the graft was passed through the remnant tissue and the endobutton loop was flipped outside the femoral cortex in the usual manner. creation of a longitudinal slit, gradual hooking and pulling up the graft by a probe, and rounding of sharp corners of the bone plug were essential to allow technically challenging graft passage of the btb through the remnant tissue. tibial fixation of the hamstring autografts was accomplished over a suture after fixation using a fully threaded 6.5-mm cancellous screw and washer (meira inc., the am and pl bundle grafts were fixed at full knee extension and the btb graft was also fixed at full knee extension. after graft passage, when residual remnant impingement on the roof or the lateral wall of the femoral notch or the pcl was arthroscopically demonstrated, between january 2011 and december 2013, we treated 47 patients with remnant - preserving acl reconstruction using the navigation system described above, with the aim of creating a femoral socket with sufficient remnant tissue from the tibia to the femoral notch or the pcl. eighteen patients underwent remnant - preserving acl reconstruction using a rectangular btb graft, while 29 underwent double - bundle reconstruction using hamstring tendon grafts. remnant - preserving acl reconstruction was performed in patients with type 1, 2, or 3 remnant tissue, as described by crain.16). they classified the acl remnants into 4 morphological patterns : type 1, bridging between the pcl and tibia ; type 2, bridging between the roof of the intercondylar notch and tibia ; type 3, bridging between the lateral wall of the intercondylar notch and tibia ; and type 4, no substantial acl remnants. of the 47 patients (22 female and 25 male), 18 had type 1 remnant tissue, 10 had type 2 remnant tissue, and 19 had type 3 remnant tissue. there were no patients with partial acl rupture, including isolated am or pl bundle rupture. the mean age of the patients was 31 years (range, 16 to 55 years) at the time of surgery. the height and weight [meansstandard deviations (sds) ] of patients were 1669 cm and 6212 kg, respectively. the median duration between injury and surgery was 4 months (range, 1 to 348 months). 3d computed tomography (ct) imaging of the operated knee was performed 1 week after surgery and the center of the femoral socket aperture was calculated according to the quadrant technique, as described by bernard.17). the incidence of a back wall blowout and short femoral tunnel (< 25 mm) was evaluated. patients (and their families) were informed that their data would be submitted for publication, and all gave their consent for the same. data in text are given as meansstandard deviations (range, minimum to maximum). in acl reconstruction using a rectangular btb graft, the center of the femoral socket aperture was located at 29.1%4.2% (range, 23.3% to 41.0%) in the horizontal direction and at 51.0%9.8% (range, 34.5% to 71.0%) in the vertical direction. in double - bundle acl reconstruction using hamstring tendon grafts, the center of the am socket aperture was located at 25.5%4.7% (range, 15.2% to 33.4%) in the horizontal direction and 34.3%8.0% (range, 19.6% to 47.2%) in the vertical direction, while that of the pl socket aperture was located at 32.9%5.6% (range, 24.1% to 43.4%) and 57.5%5.8% (range, 48.5% to 72.0%) in the horizontal and vertical direction, respectively. the femoral socket locations were considered to be anatomical in accordance with previous cadaveric studies examining the positions of the acl femoral insertion site18,19) (fig. 8 and table 1). neither back wall blowout nor short femoral tunnel was observed. computer - assisted surgery has recently been introduced to improve the accuracy and reproducibility of socket placement during acl reconstruction20,21,22,23). we have been using a 3d fluoroscopy - based navigation system for accurate and reproducible placement of the femoral socket through a far am portal9,10). using this system, surgeons can identify the lir, which is an important landmark for acl femoral insertion not only on arthroscopy but also on 3d images of the navigation system during primary acl reconstruction. in remnant - preserving acl reconstruction, arthroscopic imaging alone is insufficient to evaluate tunnel placement because the residual remnant impedes visualization of the femoral bone surface. despite poor arthroscopic visualization, this system enables surgeons to visualize the orientation of the lateral wall and the roof of the femoral intercondylar notch. the principle finding of our research was that morphometric analysis of femoral socket placement on postoperative 3d ct images using the quadrant method in this report revealed anatomical femoral socket placements that were similar to cadaveric data previously reported18,19). according to a cadaveric study described by forsythe., the average positions of the center of the am bundle were 21.7% in the horizontal direction and 33.2% in the vertical direction, while those of the pl bundle were 35.1% and 55.3% in the respective directions. calculated from this data, the centers between the am and pl bundles were located at 28.4% in the horizontal direction and 44.3% in the vertical direction. our data were compared with the cadaveric data using 3d ct images in previous studies (table 1). considering these cadaveric data, it was clear that the femoral sockets were anatomically and reproducibly created using 3d imaging - based navigation in the current series. furthermore, no complication such as tunnel back wall blowout or short femoral tunnel was observed in the current series. in our opinion, once the acl has ruptured, the normal tension can not be expected in most cases, even though a part of the acl seems to be intact during arthroscopy. our concept does not refer to the reconstruction of a partially ruptured ligament with the expectation of maintaining tension in the remaining ligament, rather, it is based on the fact that the remnant tissue only has a supplementary effect and that the aim of remnant - preserving reconstruction is coverage by the conserved acl tissue. therefore, the position of the remnant fibers does not affect femoral socket placement using the current technique. furthermore, decreasing the remnant volume by using the double - bundle reconstruction procedure is acceptable, and the current procedure can be applied to patients in whom the remnant is reattached to the pcl or the roof of the notch. the current report did not analyze the clinical results of remnant - preserving acl reconstruction using the navigation system. further follow - up is required to confirm whether or not this technology will result in good clinical outcomes. it will also be necessary to observe whether or not this procedure is associated with complications such as cyclops syndrome or pain and/or limited range of knee motion due to impingement on the femoral intercondylar roof, lateral wall, or pcl. one of the elements that cause the inaccuracy is a registration error. in the navigation system described in this report, registration based on digital fluoroscopic images which are acquired during surgery is automatically performed ; therefore, the registration error can be avoided. according to a laboratory study, 3d fluoroscopic navigation can be performed with an overall application accuracy of 0.470.21 mm24). there are other disadvantages associated with the navigation system described in this report. in this system, a reference frame must be fixed to the lateral femur with two half - pins, which necessitates additional skin incisions and drill holes in the femur. other disadvantages include exposure of the patient and medical staff to radiation at the beginning of the procedure in addition to increased medical costs. 3d fluoroscopy - based navigation can assist surgeons in creating anatomical femoral sockets during technically challenging remnant - preserving acl reconstruction. | introductionrecently, remnant - preserving anterior cruciate ligament (acl) reconstruction has been increasingly performed to achieve revascularization, cell proliferation, and recovery of high - quality proprioception. however, poor arthroscopic visualization makes accurate socket placement during remnant - preserving acl reconstruction difficult. this study describes a surgical technique used to create an anatomical femoral socket with a three - dimensional (3d) fluoroscopy based navigation system during technically demanding remnant - preserving acl reconstruction.surgical techniqueafter a reference frame was attached to the femur, an intraoperative image of the distal femur was obtained, transferred to the navigation system and reconstructed into a 3d image. a navigation computer helped the surgeon visualize the entire lateral wall of the femoral notch and lateral intercondylar ridge, even when the remnant of the ruptured acl impeded arthroscopic visualization of the bone surface. when a guide was placed, the virtual femoral tunnel overlapped the reconstructed 3d image in real time ; therefore, only minimal soft tissue debridement was required.materials and methodswe treated 47 patients with remnant - preserving acl reconstruction using this system. the center of the femoral socket aperture was calculated according to the quadrant technique using 3d computed tomography imaging.resultsthe femoral socket locations were considered to be an anatomical footprint in accordance with previous cadaveric studies.conclusionsthe 3d fluoroscopy - based navigation can assist surgeons in creating anatomical femoral sockets during remnant - preserving acl reconstruction. |
the common causes of chronic liver disease are viral hepatitis, alcohol abuse, and metabolic disorders. these result in hepatocytes damage, the consequence of which may be liver fibrosis, cirrhosis, and/or hepatocellular carcinoma. accurate evaluation of the severity of disease is crucial for treatment planning, that is, commencement of antiviral treatment and prognostication. noninvasive approaches for assessment of liver histology include routine laboratory tests like serum markers, liver functions test, and radiological evaluation of liver. liver histological diagnosis based on needle biopsy determines the inflammatory activity (grading), the extent of fibrosis (staging), and other comorbidities. but the procedure of ultrasound guided liver biopsy is invasive with about 1% risk of significant complications like postinterventional hemorrhage, bile leak, infection, and injury to adjacent organs with less than 0.1% mortality. sampling errors may also be encountered since the liver parenchymal damage in chronic hepatitis is not homogeneous. imaging technologies, particularly ultrasound, are inexpensive, noninvasive, readily available, and acceptable to the patient. it is routinely utilized in evaluation of spectrum of chronic liver disease as it provides useful information on the morphological alterations of the liver and organs affected as a result of portal hypertension ; in addition color doppler flow imaging provides information regarding the liver hemodynamics. the other imaging modalities like computerized tomography (ct) and magnetic resonance imaging (mri) are also helpful, but these are expensive and require contrast administration. a number of ultrasound variables based on liver morphology, hemodynamics, and different techniques of ultrasound like simultaneous use of high and low frequency transducers have been evaluated to predict the liver fibrosis stage with variable accuracy. the purpose of our study was to determine the utility of a simplified scoring system based on routinely evaluated ultrasound features for the evaluation of chronic liver disease and correlate it with the histological findings. the concept is to evolve an effective, simple to understand, applicable, and radiologically relevant scoring system for evaluation of the extent of chronic liver disease. this cross - sectional analytical study was performed in the department of radiology akuh from january 2010 to december 2011. all patients sent to the radiology department of aga khan university hospital for ultrasound guided liver biopsy were included. patients were excluded if the histopathology report of liver biopsy was not available and if the biopsy was performed for focal lesions or autoimmune liver disease. in addition patients unfit for liver biopsy due to jaundice, ascites, and deranged blood profiles were also excluded. prior to the biopsy real time ultrasound using toshiba nemio xg was performed for all patients using 3.55.0 mhz convex transducer by the radiologist on duty in the ultrasound interventional suit having at least 3 years of experience in performing abdominal sonography. ultrasound of the liver was performed, both lobes of liver were evaluated, and a combined impression was derived. in addition size of liver, spleen, and portal vein was also assessed and noted. the ultrasound parameters and scoring system were explained to examining radiologist prior to the procedure and findings recorded on a standard proforma. the ultrasound variables / parameters and their assigned scoring system that was a modified version adopted from the published literature are as depicted in table 1 [7, 8 ]. the sample for the liver biopsy was obtained from the right lobe of liver - anterior segment using bards tru - cut biopsy 18 gauge needle. the histopathology reports were reviewed through the hospital information system and assessed for grading and staging of the biopsy specimen which was analyzed using the batts and ludwig scoring system. grade evaluated the degree and location of inflammation and stage assessing the location and extent of fibrosis in the biopsy specimen. according to the histopathology scoring system stage 0 was described as no fibrosis and with increasing fibrosis a score of 4 was assigned for cirrhosis. for the grading score 0 described portal inflammation only and with increasing lobular inflammation and necrosis, score of 4 denoting severe diffuse hepatocellular damage with bridging necrosis. for the purpose of analysis stage and grade 0 and 1 were taken as mild or no disease and stages 2, 3, and 4 as moderate to severe disease. the ultrasound scoring system was also categorized as a for liver morphological evaluation comprising of liver surface, parenchymal echo texture, and edge and b for the combined score of liver morphology as detailed above and sizes evaluation of liver, spleen, and portal vein. calculated sensitivity of ultrasound for detecting chronic liver disease is 77% with confidence level of 95%, margin of error 10%, and calculated sample size n = 115. this was done by using the following formula n = [z1/(1 p/p) ]. frequencies were calculated, and proportions reported for categorical variables. mean and standard deviations calculated for quantitative variable like age. sensitivity, specificity, positive, and negative predictive values with 95% confidence intervals were reported for ultrasound in detecting chronic liver disease in the patients taking histopathology / biopsy as gold standard. the study population (n = 116) included predominantly males, 74 (64%) with a mean age of 39.54 years sd 12.77, range between 15 and 70 years. data was collected of 116 patients prospectively over a period of two years from a tertiary care center, the aga khan university hospital. out of the 116 patients, 78 (67%) were hepatitis c reactive. sensitivity, specificity, positive and negative predicted values of the liver morphological score denoted as a and combined score of liver morphology and sizes denoted as b was determined using stage and grade as reference standard (table 2). majority of patients 97 (84%) presented with normal liver size, 11 (9%) presented with an enlarged liver, and 8 (7%) presented with a liver smaller in size. the liver surface was smooth in 71 (61%), while 32 (28%) showed a mildly irregular liver surface. liver edge was sharp in 38 (33%), mildly blunted in 66 (57%), and rest 12 (10%) showed a blunted liver edge. portal vein was normal in 112 (97%) and dilated in the remaining of the total sample. sensitivity, specificity, positive and negative predicted values and p - value using chi - square test of the each ultrasound variable were determined using stage and grade as reference standard. current development and improvement in the treatment and management options have stressed a need for prompt diagnosis of cld to identify asymptomatic patients in a population that is high risk, for example, due to high prevalence of viral hepatitis, and hence provide a better patient outcome. accurate estimation of the degree of hepatic damage in fibrosis or cirrhosis before the compensation becomes clinically evident is crucial for treatment, prognosis, and surveillance. the noninvasive methods to assess features of cld include serologic fibrosis markers like fibro test, aspartate aminotransferase - to - platelet ratio index (apri), and radiologic imaging. these tests are regarded to be perfect and ideal only if these are simple, accessible cheap, and exhibit high accuracy. in the present study we attempted to develop a simplified scoring system based on ultrasound parameters routinely evaluated in sonographic studies and likely to be affected during the course of cld like liver morphological appearance and the dimension of liver, portal vein, and spleen. a number of studies have utilized the ultrasound examination for the diagnosis and staging of chronic liver disease making use of different techniques like the conventional gray scale and doppler [12, 13 ] to sophisticate technique of transient elastography and using contrast agent [11, 14 ]. investigated patient with chronic liver disease for the presence of compensated cirrhosis using ultrasound scoring system and achieved the sensitivity and specificity of 78.7% and 80.2%, respectively. a comparison of prior studies using ultrasound scores for the evaluation of chronic liver disease is shown in table 4. our results show a high sensitivity and ppv of liver morphological sonographic evaluation for the staging and grading of cld, respectively, thus supporting it as a screening diagnostic strategy. the two groups of liver fibrosis that is mild / no fibrosis and moderate / severe fibrosis / cirrhosis could be differentiated using this scoring system with high sensitivity and ppv. this is likely to be related to the fact that the simplified scoring system in the present study evaluated the findings on a 3 level scale, that is, 0, 1, and 2 as compared to other studies where findings were evaluated on a 4-point scale ranging from 0 to 4. of the three liver morphology variables, liver surface evaluation depicted specificity of 86.3% for the stage of fibrosis and 91.1% for the grade of inflammation. the result is in keeping with other studies that showed a high specificity of surface nodularity. in this prospective study liver edge was also found to have a high sensitivity and specificity for detection of liver fibrosis and grades of inflammation and differed from other studies in which liver edge was not found to be specific for liver fibrosis evaluation. in this study the cut - off value of the ultrasound score was 2 for liver morphology (category a) and 3 for combination of morphology and sizes (category b). the liver morphology score using 3 variables provided a sensitivity of 90.3%, but a sensitivity of 44.4% was achieved when all 6 variables were assessed and is lower than that reported by using 4 variables. the patients with clinically decompensated cld were excluded in the present study to maximize the efficacy of the ultrasound examination. but in addition to the us signs for assessing liver parenchyma, signs consistent with advanced liver disease like enlarged spleen, shrunken liver, and portal hypertension were also evaluated for their presence in nonsymptomatic patients. the number of patients diagnosed as stage iv fibrosis on histopathology in the present study is 19 (16.4%), while on sonography the frequency of small shrunken liver and splenomegaly is 8 (7%) and 10 (8.6%), respectively. the study results show high sensitivity, but the specificity is low, and hence there is a need to come up with further research to get better diagnostic accuracy. this can be achieved by addressing factors such as intra and inter observer variability, quality assurance of the technique and equipment of ultrasound. since liver histology was taken as gold standard in this study, the possibility of sampling errors and inter- and intraobserver variability in assessment of biopsy specimen can not be ruled out and may have also affected our results. presence of hepatic steatosis significantly affects the liver parenchymal appearance, but this finding was not assessed in the us evaluation of the study group. the simplified ultrasound scoring system evaluated in our study is clinically relevant and reproducible for differentiating patients with cld with mild or no fibrosis to moderate to severe fibrosis. since we also evaluated the sensitivity of these parameters for grading, it is also helpful in determining the prognosis and best possible therapeutic option. | noninvasive approaches for assessment of liver histology include routine laboratory tests and radiological evaluation. the purpose of our study was to determine the utility of a simplified scoring system based on routinely evaluated ultrasound features for the evaluation of chronic liver disease and correlate it with the histological findings. for this cross - sectional analytical study the data was collected prospectively by nonprobability purposive sampling technique. the ultrasound variables / parameters and their assigned scoring system that was a modified version adopted from published literature were evaluated. sensitivity, specificity, positive and negative predictive values of the liver morphological score and combined score of liver morphology and sizes was determined using stage and grade as reference standard. our results show a high sensitivity and ppv of liver morphological sonographic evaluation for the staging and grading of cld respectively thus supporting it as a screening diagnostic strategy. of the three liver morphology variables, specificity of liver surface evaluation was highest for the stage of fibrosis and grade of inflammation. the simplified ultrasound scoring system evaluated in our study is clinically relevant and reproducible for differentiating patients with cld with mild or no fibrosis from moderate to severe fibrosis. |
study area and population - this study was performed in 2009 in the communities of buriti seco and morro grande, which pertain to pedra preta, a little village in a schistosomiasis endemic area of the rural region of montes claros, state of minas gerais, brazil. this area was chosen because the population shows a low migration index (fixed resident population) and the infection rate in 2005 when the last treatment occurred was 12% according to data provided by the control centre of diseases of montes claros. the populations of the two communities were included in this study, which included 201 participants, 93 women and 108 men, aged between one-96 years. diagnostic techniques - an in depth analysis of stool samples, including a combination of parasitological examinations, was performed before treatment to establish the infection rate. four stool samples from all participants were collected and analysed using the kato - katz technique [biomanguinhos - oswaldo cruz foundation (fiocruz), state of rio de janeiro, brazil ] and the commercial test tf - test (immunoassay com ind ltda, brazil) as described by siqueira. the pcr - elisa assay was used on 500 mg of the first stool sample of each participant for comparison with the parasitological tests and evaluation of diagnostic accuracy (fig. 1:flowchart of stool examinations according to different diagnostic techniques for establishing infection rate and the follow - up of positives after treatment. kato - katz - the kk test was applied using 18 slides that were prepared as follows : 12 slides of the first sample and two slides of the second, third and fourth samples were analysed for a total of 750 mg of faeces for each participant (18 x 41.7 mg). tf - test - the tf - test was performed on the first three samples using portions of 500 mg, totalling 1,500 mg of faeces, which was processed with 3 ml of ethyl acetate and one drop of detergent and centrifuged for 2 min at 500 g. pcr - elisa assay - dna from 500-mg stool samples was isolated using the commercial kit qiaamp dna stool mini kit, following the manufacturer s recommendations (qiagen gmbh, germany). a 121-bp repetitive sequence of s. mansoni (hamburger. 1991) was amplified using a forward biotinylated primer (5-gatctgaatccgaccaaccg-3) and reverse primer (5-atattaacgcccacgctctc-3), according to the protocols described by gomes. the amplicons were detected in an elisa plate sensitised with streptavidin using a fluorescein 5-labeled probe (5-tggtttcggagatacaacga-3) with available reagents in the commercial kit pcr plate detection, according to the manufacturer s recommendations (sigma, usa). positive controls extracted from s. mansoni eggs and negative controls (water and negative control of pcr) were added to each pcr - elisa assay. the receiver operating characteristic curve (roc) determined the cut - off value of 0.116. the human beta - actin gene was amplified using the primers aco1 (5-acctcatgaagatcctcacc-3), biotinylated in the 5 region and aco2 (5-ccatctcttgctcgaagtcc-3) as a control for variations in the efficiency of dna extraction and pcr - amplification. products were detected using a fluorescein 5-labeled probe (5-tctccttaatgcacgcacg-3) in the pcr - elisa system as described by gomes. (2010). one stool sample of each treated participant was analysed after treatment to establish the cure rate using the kk technique examining 12 slides per sample and the pcr - elisa assay, which used 500 mg of the same stool sample. comparisons of different diagnostic approaches - diagnostic parameters were calculated using three different approaches for comparison ; (i) two - slide kk examination, which is the current recommendation of the brazilian ministry of health for the national schistosomiasis control program, (ii) 12-slide kk examinations to observe the diagnostic parameters using the same amount of faecal material and (iii) the reference test described above as an approximation of the real infection rate to evaluate test performance. statistical analysis - the software openepi, v.3.03 (dean. co - positivity and co - negativity values were calculated with 95% confidence intervals (ci) for each of the three approaches. the level of diagnostic agreement between different diagnostic techniques was determined using the kappa coefficient. landis and koch (1977) demonstrated poor concordance when this coefficient was lower than 0.20, weak concordance between 0.21 - 0.40, moderate concordance between 0.41 - 0.60, good concordance between 0.61- 0.80 and excellent concordance when higher than 0.81. treatment of positives - all participants who were positive for schistosomiasis as detected using the parasitological techniques were treated with a single dose of 60 mg / kg praziquantel for children and 50 mg / kg for adults. infections with other helminths were treated using a single dose of 400 mg albendazole, as proscribed by the brazilian ministry of health. cure assessment - cure assessments were performed 30, 90 and 180 days post - treatment. sixty - nine of the 72 positive patients were followed up 30 days after treatment, 67 patients were followed up after 90 days and 61 patients were followed up after 180 days. one stool sample from each participant was analysed using the kk technique (12 slides per sample) and the pcr - elisa assay, which was applied to 500 mg of the same stool sample. ethics - the ethical research committee of the research centre ren rachou / fiocruz (cepsh / cpqrr 03/2008) and the national brazilian ethical board (scientific opinion 784/2008, conep 14886) approved this study. the objectives of this study were presented and explained to all participants and written consent was obtained from each individual, who signed a form prior to enrolment in this study. seventy - two of the 201 stool samples examined were positive using the reference test. two - slide kk examination of the same sample, which is the current recommendation of the brazilian ministry of health for the national schistosomiasis control program, detected 16 positives (8%), examination of 12 kk slides detected 29 positives (14.4%), 18 kk slides detected 58 positives (28.9%), 32 positives (15.9%) were detected using the tf - test and 47 positives (23.4%) were detected using pcr - elisa (fig. the comparison of pcr - elisa with two kk slides, which corresponded to 83.4 mg of faeces, revealed that the co - positivity of pcr - elisa was 93.75% (ci : 71.67 - 98.89%) and the co - negativity was 82.7% (ci : 76.6 - 87.47). the comparison with 12 kk slides of the same sample, which corresponded to 500 mg of faeces (41.7 mg x 12 = 500 mg), demonstrated a co - positivity of pcr - elisa of 89.66% (ci : 73.61 - 96.42%) and a co - negativity of 87.8% (ci : 82.06 - 91.87%) (table i) 2:diagnostic performance of parasitological and molecular techniques using stool samples, pedra preta, municipality of montes claros, state of minas gerais, brazil, 2009. table iresults of co - positivity and co - negativity for polymerase chain reaction (pcr)-elisa in relation to two kato - katz (kk) slides and in direct comparison with 12 kk slides for the detection of infection with schistosoma mansoni kk positivity rates % (n / n)pcr - elisa % (n / n) co - positivityco - negativity8 (16/201) (2 slides = 83.4 mg)93.7 (15/16)82.7 (153/185)14.4 (29/201) (12 slides = 500 mg)89.7 (26/29)87.8 (151/172) out of 47 positives detected by pcr - elisa using 500 mg of the first sample, 12 were not identified by the reference test. thirty - seven positives only were detected by the reference test, however the first assay was achieved with a single sample and the parasitological combination with four samples. two kk slides showed moderate agreement with pcr - elisa (kappa index of 0.405) and 12 slides showed a substantial agreement (kappa index of 0.616) (table ii). the pcr - elisa assay showed moderate agreement with the reference test (kappa index of 0.426) (table iii). table iicomparison and agreement between results detected by the polymerase chain reaction (pcr)-elisa in relation to kato - katz (kk) method using two and 12 slides of the same stool sample kk (2 slides = 83.4 mg) kk (12 slides = 500 mg) positivenegativetotal positivenegativetotalpcr - elisa (500 mg of the 1st sample)positive153247positive262147negative1153154negative3151154 total16185201 29172201 kappa index : 0.405kappa index : 0.616 table iiicomparison and agreement between results detected by the reference testa and polymerase chain reaction (pcr)-elisab reference test totalpositivenegativepcr - elisa (500 mg of the 1st sample)positive351247negative37117154total72129201 kappa = 0.426 a : four samples = 18 slides of kato - katz + tf - test ; b : single sample. a : four samples = 18 slides of kato - katz + tf - test ; b : single sample. the cure rate 30 days after treatment using the kk technique was 100% and one positive individual was detected using pcr - elisa (cure rate of 98.5%). three positive individuals were detected using pcr - elisa 90 days after treatment (cure rate of 95.5%), but these patients were not identified using analysis of 12 kk slides. the cure rate 180 days after treatment was 98.4% using kk (1 out of 61) and 96.5% using pcr - elisa (2 out of 61), with a final cure rate of 95.1% (3 out of 61) (table iv). table ivassessment of cure 30, 90 and 180 days after treatment by the kato - katz (kk) and polymerase chain reaction (pcr)-elisa techniquestechniquescure rate % (n / n) 30 days90 days180 dayskk (12 slides = 500 mg)100 (69/69)100 (67/67)98.4 (60/61)pcr - elisa (500 mg)98.5 (68/69)95.5 (64/67)96.5 (59/61) this assay proved a valuable tool for the diagnosis of schistosomiasis despite its relatively high cost. pcr - elisa can be applied to comparisons between diagnostic methods and it is very useful to confirm suspicious cases that were not detected using conventional parasitological methods and evaluate cure control after chemotherapeutic treatment. the pcr technique is widely used for the diagnosis of several human diseases, but its application in neglected diseases, especially schistosomiasis, were explored only recently. 2002) first described the use of pcr in faecal samples for the diagnosis of schistosomiasis. 2009) compared to routine parasitological examinations, such as the kk technique (katz. the positivity rate detected using pcr - elisa was 23.4% in the present study, which was higher than the 14.4% using kk technique (12 slides). however, three participants who exhibited shedding eggs in faeces (diagnosed using the kk technique) were not identified using pcr - elisa, which may be explained by the absence of eggs in the samples examined by pcr assay, the presence of taq dna polymerase enzyme inhibitors or failure in extraction, although the positive internal control (human beta - actin gene) was detected in these samples. further, the dna from these negative pcr - elisa samples were diluted 1:10, 1:20 and 1:40 and assayed again to exclude the possibility of inhibiting the reaction, but the results were identical to the initial assay. pcr - elisa detected 20 positive cases that were not identified using the kk technique (12 slides). (2009) who showed that pcr detected 23% positive cases amongst negative samples for s. mansoni using the kk technique from individuals living in a hypoendemic area. these authors reported that 16 out of 194 participants exhibited positive pcr, but did not present eggs in faeces, whereas two positive participants using the kk method were negative using pcr. (2003) explained that the positive cases that were not detected using pcr could occur during the dna amplification reaction as a result of various factors, such as amplification inhibition by faecal compounds, dna degradation during transportation from the field to the laboratory or the absence of eggs in the sample examined. a study by gomes. (2010) demonstrated that pcr - elisa assay analysis of stool samples from 206 individuals from an endemic area compared with 12 kk slides from a single sample revealed a sensitivity of 97.4% and specificity of 85.1%, which are higher than the values in our study. this difference may be explained by factors that are inherent in the experimental procedures, which may generate errors related to the detection of parasite dna, its degradation during biological sample collection or absence in the sample analysed. the need for well - trained technicians who rigorously apply quality control measures at all stages of the reaction should be stressed to avoid false - negative or false - positive results from contamination. molecular methods may suffer a loss of standardisation (reproducibility) and strength when in house these criteria are required for the possible application of these methods in control programs (gomes. the commercial kit qiaamp dna stool mini kit was used in this study for dna extraction from stool to avoid methodological interferences in the procedure. physical methods (e.g., separation of rooms and materials and the use of laminar flow hood with ultraviolet light) were utilised throughout the experiment to minimise the chances of sample contamination. therefore, it is indicated for diagnosis of schistosoma spp in special situations, when high sensitivity and specificity are required and infrastructure is available (rabello. a greater number of parasitic infections with low viral load are detected using diagnostic methods with increased sensitivity. seventy of the 72 (97.2%) individuals infected by s. mansoni presented low worm burden [1 - 100 eggs per gram (epg) ], one individual showed medium parasite burden (156 epg) and another patient showed a high intensity of infection (555 epg) as evaluated using the reference test. the assessment of cure after treatment using the kk technique and pcr - elisa assay showed a decrease of infected individuals. for example, dna from dead worms or eggs remaining in tissues can be excreted in faeces for a period of time after treatment. another reason is that the sensitivity of the pcr - elisa for the detection of low parasite burdens is greater than the kk technique (wichmann. the lower cure rate six months after treatment may be explained by the temporary interruption of the oviposition by female worms as a result of treatment and a resumption of oviposition at a later time. these individuals also may have been re - infected because of the constant exposure they may suffer in the endemic areas. managers of control programs in areas of low transmission must choose between the kk technique with various samples or pcr - elisa assay to detect the largest number of infected individuals using a single sample. notably, the first technique is less expensive, but the latter technique is more accurate. the cost of pcr reagents is us $ 10 per faecal sample (gomes. 2010), but these costs are greatly reduced when the technique is utilised on a large scale. pcr also provides good precision (reproducibility and repeatability), is genus - specific and may estimate worm burden, which correlates with the values of epg of faeces determined using the kk technique. the results obtained using pcr - elisa support the development of a promising diagnostic method that only uses a faecal sample. this technique represents an important step forward to support studies on epidemiology and disease control, especially when the cost would be reduced by the use of industrial scale production. the assessment of cure in situations of very low parasite load, such as after therapeutic interventions, may require increased numbers of kk slides or a test with higher sensitivity than pcr - elisa. this study also demonstrated a low positivity rate during follow - up after treatment, which indicates that an in - depth diagnostic survey is more effective and it may likely prevent successive treatments. | this study evaluated parasitological and molecular techniques for the diagnosis and assessment of cure of schistosomiasis mansoni. a population - based study was performed in 201 inhabitants from a low transmission locality named pedra preta, municipality of montes claros, state of minas gerais, brazil. four stool samples were analysed using two techniques, the kato - katz (kk) technique (18 slides) and the tf - test, to establish the infection rate. the positivity rate of 18 kk slides of four stool samples was 28.9% (58/201) and the combined parasitological techniques (kk+tf - test) produced a 35.8% positivity rate (72/201). furthermore, a polymerase chain reaction (pcr)-elisa assay produced a positivity rate of 23.4% (47/201) using the first sample. all 72 patients with positive parasitological exams were treated with a single dose of praziquantel and these patients were followed - up 30, 90 and 180 days after treatment to establish the cure rate. cure rates obtained by the analysis of 12 kk slides were 100%, 100% and 98.4% at 30, 90 and 180 days after treatment, respectively. pcr - elisa revealed cure rates of 98.5%, 95.5% and 96.5%, respectively. the diagnostic and assessment of cure for schistosomiasis may require an increased number of kk slides or a test with higher sensitivity, such as pcr - elisa, in situations of very low parasite load, such as after therapeutic interventions. |
mycoplasma pneumoniae is an important cause of respiratory tract infections including community - acquired pneumonia. it has been suggested that the pathogen may also play a role in the development of atherosclerosis. this hypothesis is supported by the ability of m. pneumoniae to cause extrapulmonary manifestations affecting the blood vessels,,. however, others have presented discordant results, so that the role of m. pneumoniae in the pathogenesis of atherosclerosis must still be regarded as unknown,. serum iga antibodies are a more reliable indicator for acute m. pneumoniae infection than igm, because specific igm are not mounted regularly in elderly patients,. importantly, the serum level of specific iga declines to normal values earlier after acute infection than the level of specific igm, with a significant decrease 60 days after the onset of disease. the aim of this study was to investigate the role of m. pneumoniae in the development atherosclerosis by repeated testing of specific iga over a period of 6 month. ninety - one consecutive patients with symptomatic and asymptomatic internal carotid artery (ica) stenosis > 70%, who were admitted for surgery march and july 2004 were enrolled in this study. thirty - nine patients (43%) had asymptomatic ica stenosis, 32 patients (35%) had transient ischaemic episodes and 20 patients (22%) were operated on for non - disabling stroke. all patients were planned for routine eversion endarterectomy (eea) of the ica under regional anaesthesia. as anaesthetic agent 1% lidocaine was used for a combined superficial and deep cervical block. symptomatic patients with stroke had an interval of less than 6 weeks prior to surgery ; symptomatic patients with transient hemispheric and non - hemispheric symptoms were all operated on within 3 weeks after presentation. for all patients, the following data were assessed : age, sex, clinical stage of carotid artery disease (cad) (right / left), degree of stenosis (right / left), body mass index (bmi), alcohol consumption (gram / week), nicotine (cigarettes / day), diabetes mellitus, cholesterol, triglycerides and homocysteine. patients on antihypertensive medication or a blood pressure of 140/75 mmhg on repeat measurements were characterized hypertensive. maximum ica stenosis was assessed by magnetic resonance angiography and duplex and stenosis of 70% were an indication for surgery. serum was obtained by venipuncture of the antecubital vein using standard tubes (vacuette edta tubes, greiner bio - one, kremsmuenster, austria) and centrifugation 10 minutes at 2,000 g. serum was stored at 80c until use. the first serum specimen (s1) was taken before surgery, and the second after an interval of 6 months (s2). specific iga were measured by the seromp iga kit (savyon diagnostics, ashdod, israel) according to the manufacturer s recommendations. as indicated by the manufacturer values > 20 bu (binding units) were considered positive. statistical analysis was performed by chi squared test (yates correction), using epiinfo 2002 (cdc, atlanta, ga, usa) the iga seroprevalence at admission (s1) was 18.7% (17 of 91 patients). in these patients, pneumoniae iga after a period of 6 month (median : 36 bu ; range : 2257 bu). among the 74 patients with negative s1 results, the elisa results and basic clinical data of the 18 patients with 1 result > 20 bu are shown by table 1 (tab. none of the assessed demographic factors or risk factors for atherosclerosis was associated with iga seropositivity, neither were the degree cavk or the degree of stenosis. in this study we found a considerable seroprevalence of iga antibodies to m. pneumoniae in patients with internal carotid artery stenosis. however, the more remarkable finding was that in the majority of our patients iga remained elevated at essentially constant levels through a period of at least 6 month. in contrast, few patients (3) developed specific iga between the taking of the first and the second serum specimen. we have focused on iga determination in the present study, because specific igm formation may be lacking in the elderly. igg antibodies, on the other hand, remain elevated for longtime after infection, which results in a high igg seroprevalence, that can cause interpretation difficulties. determination of specific iga is a suitable tool for the diagnosis of m. pneumoniae infection. it has been shown that after infection specific iga decline to normal values (i.e. levels below the cut - off value of commercially available tests) earlier than specific igm. however, iga - based diagnosis is less established than igm- or igg - based diagnosis, and persistently elevated iga as indicator of chronic infection, although biologically plausible, has to be interpreted with caution. it has to be mentioned that differing figures concerning the seroprevalence of iga antibodies to m. pneumoniae in elderly patients have been published, with seroprevalences up to 79%,. however, the applied assay and the respective cut - off level for positivity is obviously important in this context. by the assay applied in the present study, we have found a seroprevalence of 13% (> 20 bu) and no value > 30 bu among 46 healthy subjects. nevertheless, a significant trend to a decrease in antibody level would be expected if the elevated iga levels in our patients should simply reflect past respiratory tract infection. it is notable that in the 13 patients with persistently high iga levels, 6 showed an increasing and 6 a decreasing iga level, with the level in one patient remaining constant (table 1 (tab. an important study negotiating a potential role of m. pneumoniae in the development of atherosclerosis has been provided by maraha., who failed to detect the pathogen in the great majority of 103 atherectomy specimens and degenerative heart valve specimens. however, it should be considered that some mycoplasma - associated diseases, including severe pneumonia and neurological manifestations, are immunologically mediated and require the presence of the pathogen only in an early stage of the disease,,. therefore, serological methods may be advantageous to explore the association between m. pneumoniae infection and atherosclerosis. in conclusion, we have applied a new serological approach to investigate the role of m. pneumoniae in the development of atherosclerosis. the results can not be explained throughout by the general seroprevalence, or by past respiratory tract infections with the pathogen, so that they suggest a role for m. pneumoniae in the development of atherosclerosis. however, it has to be considered that the association between chronic mycoplasma infection and persistently elevated iga antibodies is not yet proven by large - scale epidemiological investigations. future studies on the potential role of m. pneumoniae in the development of atherosclerosis are warranted. | background : it has been suggested that mycoplasma pneumoniae may play a role in the development of atherosclerosis, but to date this association is still a matter of debate due to conflicting findings.methods : we have investigated the levels of specific iga antibodies to m. pneumoniae in 91 patients with internal carotid artery (ica) stenosis using a commercial kit (seromp iga ; savyon diagnostics, israel ; cut - off value : 20 binding units ; bu). all patients underwent surgery for ica stenosis. from each patient, the first serum sample (s1) was taken before surgery, and the second after an interval of 6 month (s2).results : the s1 seroprevalence was 18.7% (17/91). thirteen of the 17 patients with positive s1 levels also remained positive after six month, whereby no decrease of iga level was seen (median s1 level : 34 bu, range : 2265 bu ; median s2 level : 37 bu, range : 2258 bu). specifically, six of the patients showed an increased level after 6 months, and six a decrease, with the level remaining constant in one patient. in contrast, only 3 of the 74 s1 negative patients became positive for anti - m. pneumoniae iga between the taking of the first and the second serum specimen (p<0.01). none of the assessed demographic factors or risk factors for atherosclerosis was associated with iga seropositivity, neither were the degree cavk or the degree of stenosis.conclusion : these findings can not be explained throughout by the general seroprevalence, or by past respiratory tract infections with the pathogen, and therefore may suggest a role for m. pneumoniae in the development of atherosclerosis, since a chronic infection must be assumed. |
in einstein 's theory of general relativity, linearization of the field equations demonstrates that small perturbations of the metric obey a wave equation. these small disturbances, referred to as gravitational waves, travel at the speed of light. however, some other gravity theories predict a dispersive propagation (see for references). the most commonly considered form of dispersion supposes that the waves obey a klein - gordon - type equation that is given below : (1)1c2(2t22(mgc2)2)=0. physically, the dispersive term is ascribed to the quantum of gravitation having a nonzero rest mass mg, or equivalently a noninfinite compton wavelength g = h / mgc, where is the potential function of the gravitational field. moreover, several of today 's theories like string theory, superstring theory, m - theory and loop quantum gravity, and quantum field theory predict the existence of graviton particles. in quantum field theory graviton is the elementary particle that mediates the gravitational force and is expected to be massless, and that is because the gravitational force itself has an infinite range. furthermore, graviton must be a spin-2 boson, which results from the fact that the source of gravitation is the stress - energy tensor itself. additionally, it can be shown that any massless spin-2 field could give rise to a force that is indistinguishable from gravitation because a massless spin-2 field must couple to the stress - energy tensor in the same way that the gravitational field does. therefore, this result suggests that if a massless spin-2 particle is discovered, it must be the graviton. thus graviton detection remains vital in the validation of the theories and also in the research that strives to unify quantum mechanics with general relativity. although physicists normally speak as if bosons mediating the gravitational force exist, the extremely weak character of the gravitational interaction makes the detection of graviton an extremely hard issue. the detection of a single graviton may in fact be ruled out in the real universe. if researchers answer dyson 's question in an affirmative way, concluding that graviton detection is impossible, this will immediately raise issues in relation to the necessity of gravity quantization. however, attempts to extend the standard model with gravitons have run into serious theoretical difficulties at high energies (processes with energies close to or above the planck scale) because of infinities arising due to quantum effects (in other words, gravitation is nonrenormalizable). since classical general relativity and quantum mechanics are incompatible at such energies, from a theoretical point of view, the present situation is not tenable. some proposed models of quantum gravity attempt to address these issues, but these are speculative theories. there are only a few conceivable sources of graviton production, like black hole decay, spontaneous emission of gravitons from neutral hydrogen, bremsstrahlung from electron - electron collisions in stellar interiors, and conservations of photons to gravitons by interstellar magnetic fields. here we will only briefly touch upon the graviton production by black hole decay, which appears to be the most promising mechanism. black holes of mass m possess a hawking temperature that is equal to (2)tbh = c38kbgm, where kb is the boltzmann constant, g is the gravitational constant, is planck 's constant, and c is speed of light. a primordial black hole with mass m 10 kg can in principle evaporate gravitons of energy e 10 ev or higher and, with no observational constraints on the mass of the primordial black holes applied, can constitute most of the universe 's dark matter. on the other hand, primordial black holes with mass m 10 kg that emit particles with energy higher e 10 ev would have already long been evaporated. recently, finn and sutton have examined the binary pulsar, psr b1913 + 16, of which the observed decay rate coincides with that expected from relativity to approximately 0.3%. a nonzero graviton mass would upset this remarkable agreement altering the predicted orbital decay, implying an upper limit on the graviton mass. the authors have obtained a crude estimate on this bound using dimensional analysis. for a system with characteristic frequency one expects the effects of a graviton mass to appear at second order in m/. for gravitational waves at twice the orbital frequency of psr b1913 + 16, requiring (m/) < 0.003 implies an upper limit of order 10 ev / c. this is comparable to the best limit from solar system observations, m < 0.44 10 ev / c. finn and sutton have examined an extension of linearized general relativity which includes a mass term for the graviton. they have chosen the unique mass term for which the wave equation of the linearized theory takes the standard form with an h - independent source and for which the prepredictions of massless general relativity are recovered by setting m 0 at the end of the calculations. in recent papers by novello and neves as well as neves (2004) and liao the authors express a link between the cosmological constant and the graviton mass mg. similarly, in bousso the author argues that the total observable entropy is bounded by the inverse of the cosmological constant. this holds for all space - times with a positive cosmological constant, including cosmologies dominated by ordinary matter and recollapsing universes. moreover, in mongan, the author examines a vacuum - dominated friedmann universe asymptotic to a de sitter space, with a cosmological event horizon that its area in planck 's units determines the maximum amount of information that will ever be available to any observer. moreover, in a recent paper by mureika and mann, the authors examine the idea of information transfer between a test particle and the holographic screen in entropic gravity. the transfer respects both the uncertainty principle and causality, and a lower limit on the number of information bits in the universe relative to the mass may be derived. their result is found to be in excellent agreement with the current experimental mass bound of photon and graviton, suggesting that entropic gravity might be the result of a recent local symmetry that is softly broken. in particular cosmological holography postulates that all the information content in our universe is encoded on its cosmological horizon. this proposal has been put forward by smoot and in short states that all possible past and future histories of our universe are encoded on its apparent horizon and by that making a connection. moreover, the authors proceed by asking how much the universe as a whole, mass, and information included can tell us about its parts or the lightest possible mass of the elementary particles. the authors further claim that in entropic gravity there is a lower limit to the number of bits of holographic screen which provides the information transfer between the test particle and the screen, which obeys causality as well as the uncertainty principle. similarly, in haranas and gkigkitzis, the authors examine the bekenstein bound of information number n and its relation to cosmological parameters in a universe, where in gkigkitzis. the authors use a recent result for the number of information n derived from landauer 's principle ; they obtain an expression for the cosmological constant. finally, in haranas and gkigkitzis, the authors investigate the number of information n as related to the minimum quantum and gravitational masses in a vacuum - dominated universe. in this contribution we seek to investigate and understand the physics behind any possible relations that might result from the relation of the graviton mass to the surface area of the universe expressed in planck units according to the holographic principle. as a result the number of information n on the horizon of the universe enters our calculation, and therefore the relation of the graviton mass to the number of information n will be established, via the cosmological constant lambda dependence on the number of information. moreover, we use landauer 's principle of information in relating the graviton mass to the mass of the universe and the mass of an elementary particle. finally, we further investigate the relation of the graviton mass to the ricci scalar and basic cosmological parameters of the universe. in a recent paper by das (2014) the author uses the quantum raychaudhuri equation to obtain the quantum corrected friedmann equation for the range of graviton / photon. similarly, with reference to wesson, novello, tajmar, and liao, we write that the proposed relation between the graviton mass and the cosmological constant is (3)1gr2=23=mgr2c22, where is planck 's constant, c is the speed of light, and gr is the graviton wavelength. this equation also follows from einstein 's linearized field equations which include the cosmological constant lambda and also from the equations of motion for a massive spin-2 field that propagates in a de sitter background. using (1) we obtain that the mass of the graviton can be written as (4)mgr = c23. equation (4) is actually the same equation as the one given in wesson, in which mgr = mwes/3. the dependence of the graviton mass on is a property that remains valid not only for de sitter but also for arbitrary background geometries as long as they stay in the setting of einstein 's second description of the equations of motion. we can also construct a mass associated with lambda by using planck 's constant and also the speed of light c (ibid. following novello we write that the total mass of gravitons that exist in the universe is given by (5)mgr = mgrngr = c3gc23=c2g23, where ngr is the total number of gravitons contained inside the observable horizon and it is equal to (6)ngr = c3g=1=max, where p is the planck length and max = c / g is the maximum value of the cosmological constant as it is defined in. therefore we write the total number of gravitons in the universe in the following way : (7)ngr=maxc(23). the holographic principle indicates a possible nonlocality mechanism in any vacuum - dominated friedmann universe. to be more precise, a holographic nonlocal quantum mechanical description can be possible for a finite amount of information in a closed vacuum - dominated universe. today 's theories assume that the universe began by a quantum fluctuation from nothing, underwent inflation, and became so large that it is locally almost flat and that since the inflationary era the vacuum energy density of the universe is constant. this is the case of the existence of a nonzero cosmological constant. more information of such a universe arising in quantum cosmological way is presented in mongan. systems that dynamically evolve in time not only transform but also process information. using the relation given in haranas and gkigkitzis [15, 17 ] we can write the cosmological constant as a function of the number of information to be (8)=3nlp2ln2=3nln2max. substituting (8) into (2) we obtain that (9)mgr = c2nlp2ln2=clp2nln2=0mpln, where 0 = (2/ln2) 3.010 and mpl = (c / g) is the planck mass ; therefore we see that mgr1/n. similarly, the total number of gravitons contained inside the observable horizon can be written as a function of the number of information n in the following way : (10)ngr = c3gnlp2ln23=ln23n, or in other words ngr n. therefore, the total graviton mass in the universe is equal to (11)mgr = mgrngr=0mpln, where 0 = (1/3)(2ln2/) and therefore mgrn. similarly, using (9), we obtain that the number of information n is given by (12)n=2ln2(mplmgr)2. moreover, from (11), we obtain a second expression for the number of information n that reads (13)n=92ln2(mgrmpl)2. as a result we find that the number of information n can be expressed as the square of the ratio of two fundamental masses, namely, the planck mass and the mass of the graviton or the square of the total number of gravitons in the universe over the planck mass. in relation to the laws of physics we say that they determine the amount of information that a given system can register (i.e., number of bits or nats) as well as the number of elementary logic operations that the given system can perform (i.e., number of operations). with reference to landauer [16, 22, 23 ] we can say that information is physical, and that also all information is registered and processed by physical systems. physical systems can be described in terms of information, and information processing is related to the system description by physical laws. landauer 's principle is a physical principle pertaining to the lower theoretical limit of energy consumption of a computation. landauer postulated that any logically irreversible manipulation of information, such as the erasure of a bit or the merging of two computation paths, must be accompanied by a corresponding entropy increase in non - information - bearing degrees of freedom of the information processing apparatus or its environment. landauer 's principle asserts that there is a minimum possible amount of energy required to change one bit of information, known as the landauer limit, and it is equal to (14)emin=kbtln2, where kb = 1.38 10 j / k is boltzmann 's constant and t is the temperature of the circuit. therefore landauer 's energy formula can be written in terms of the information number n in the following way : (15)e = nkbtln2. for example, the equivalence between information and energy can be interpreted using the results obtained in recent experiment by funo.. in their experiment the authors have shown that entanglement can produce an increase or gain of thermodynamic work, where the gain is determined by the change of the information content. similarly, brut. have shown that there is a link between information theory and thermodynamics. landauer 's principle is a simple consequence and its logic emanates directly from the second law of thermodynamics. the law states that the entropy of a closed system can not decrease at the same time with the corresponding temperature. therefore, if one nat of information is lost during a computation, the amount of entropy generated is at least kbln2, and therefore the energy emitted in the environment is e kbtln2. landauer 's principle has been accepted as a physical law, but it has also been challenged by shenker and norton and defended by bennett and ladyman.. next, with reference to alfonso - faus and fullana i alfonso and gkigkitzis., the authors say that all physical systems of mass m and energy mc are equivalent to an amount of information in number of bits of the order of (16)n(mc2h), where m is the mass of the system, c is the speed of light, is planck 's constant, and h is the hubble constant. in alfonso - faus and fullana i alfonso the authors claim that (3) is of universal validity. they further say that the unit of energy that should be taken as the minimum quantum of energy is h. this implies that the relativistic energy of a mass m has n times this minimum quantum of energy nh, where n is the number of information in nats. in other words the product nh corresponds to the energy of all the information number n carried by the system. thus, the expression for the cosmological constant lambda obtained in gkigkitzis. is used below : (17)=3ln2(hcmug), where mu is the total mass of the universe and h is the hubble parameter, and substituting (16) in (2) and simplifying we obtain that (18)mgr=02hgcmu=0(m3/2mu)=0(m3mu)1/2, where m = (h / cg) is the mass of the pion as it is given by weinberg, and therefore we can write the mass of the universe mu in the following way : (19)mu=02(m3mgr2), or in other words as the ratio of two fundamental particle masses in the universe. in order to investigate a possible relation of the graviton mass to time t let us now consider a flat universe, that is, k = 0, where k is the curvature constant and density parameter = 1. we can write that in a flat universe the number of information n evolves as a function of time in the following way : (20)n(t)=4ln2(clp)2t2=(4ln2)(ttp)2. the bound is not fixed but rather grows as time progresses and the horizon expands and encompasses more particles. the expression above is in agreement with the work of lloyd and davies where the authors predict that in a flat universe n t. substituting (20) in (9) we obtain that (21)mgr=(ln28)1/2mpl(tplt), and therefore we find that the mass of the graviton in a flat universe evolves as mgr 1/t. using (21) we find that in a flat universe at the moment where t = t0 where t0 = 4.346 10 s is the age of the universe the mass of the graviton becomes (22)mgr=(ln28)1/2mpl=8.7771062mpl[kg ]. in order to investigate the relation of the graviton mass with fundamental cosmological parameters let us look at haranas and gkigkitzis. in this paper the authors derive the ricci scalar that is defined by [35, 36 ] (23)rsc=(6rc2r+6r2c2r2 + 6kr2).r is the scale factor of the universe. in a flat universe the resulting ricci scalar can be written as a function of the number of information number n in the following way to be (24)rsc=6ln2(q)lp2n, where q and = 1 are the deceleration and density parameters of the flat universe. solving (24) for the deceleration parameter of the universe we obtain (25)q=+ln26lp2rscn. next, in a flat universe filled with nonrelativistic matter, that is, p = 0, corrected for the graviton contribution, we have that the deceleration parameter given by (26)q=rrh2=12 + 14h2(mgrc2)2. therefore, equating (25) and (26), we obtain that the mass of the graviton is given by (27)mgr2=42h2c4[12+ln26lp2rscn ], where the quantity h / c = mhub is the hubble mass defined in. in the present era we have that mhub = h0/c = h(3.8 10) g2.7 10 g when h = 0.71. equation (27) gives the mass of the graviton in terms of the humble parameters h, the density parameter, the planck length p, the ricci scalar rsc, and finally the number of information number n. using (5) in haranas and gkigkitzis and eliminating the number of information n, that is, n = 6(q)/ln2prsc, we obtain the following expression for the mass of the graviton to be (28)mgr=2hc2(q12)1/2=2mhub(q12)1/2. similarly, we find that the mass of the graviton is related to the ricci scalar rsc in the following way : (29)mgr=2c[(q1/2)3(q)rsc]1/2, where rh is the universe horizon, as well as to deceleration parameter q. using the fact that the graviton range is given by gr = / mgrc we obtain an expression for the ricci scalar as a function of graviton range and the cosmological parameter q to be (30)rsc=34gr2(q)(q1/2). we write the expression for the time that the universe expands from a maximal density to a minimal density dominance and is determined by the stage of the nonrelativistic matter dominance to be (31)tmax23mgrc2. therefore, (31) can also be related to the number of information n via the relation of the graviton mass to the number of information, and therefore we find that (32)tmaxln23tpln. solving (32) for the number of information our result is in agreement with that of lloyd and davies where the author predicts that this is equal to the maximum number of bits registered by the universe using matter, energy, and gravity, and it is found with the help of the bekenstein bound and the holographic principle to the universe as a whole. it is given by the square of the ratio of the age of the universe to that of planck time. similarly, using (9) for the total mass of the gravitons, we find that the number of information n can also be written in the following way : (34)n=92ln2(mgrmpl)2. in this paper we have considered an expression for the mass of the graviton as it is given by novello. novello 's expression depends on the cosmological constant lambda. using the result in haranas and gkigkitzis [15, 17 ] we find a relation of graviton mass mgr to the number of information n. as a result we find that the mass of the graviton is inversely proportional to the number of information n ; that is, mgr1/n. furthermore we find that the number of gravitons contained inside the observable horizon is directly proportional to the number of information n ; that is, ngr n. similarly, the total mass of gravitons that exist in the universe is proportional to the number of information n ; that is, mgrn. we find two different expressions for the number of information n, one that is given as the square of the ratio of the planck mass to the mass of the graviton, that is, n (mp / mgr), and another that is equal to the square of the ratio of the total graviton mass in the universe over the planck mass, that is, n (mgr / mp). next, using a recent definition for the number of information n resulting from landauer 's principle, we find that the mass of the graviton can be expressed as the square root of third power of the pion mass m over the mass of the universe mu, which implies that the mass of the universe is further equal to the third power of the pion mass m over the square of the planck mass mpl. moreover we find that in a flat universe the graviton mass varies according to mgr 1/t, and furthermore we find that at time t = t0 (where t0 is age of the universe) the mass of the graviton is mgr = 8.777 10mpl = 1.909 10 kg. this order of magnitude is in agreement with the result given in gershtein. where the authors predict that mgr = 3.2 10 g to 95% confidence level. similarly dgp (dvali - gabadadze - porrati) constraints predict that the mass of the graviton falls in the range mgr10 10 kg. in an effort to establish a relation of the mass of graviton to basic parameters of the universe, wefind that the mass of the graviton is simply twice the hubble mass mh as it is defined by gershtein., times the square root of the quantity q 1/2, where q is the deceleration parameter of the universe. in reference to the geometry of the universe we find that the mass of the graviton varies according to the relation mgrrsc or mgr rsc and therefore mgr obviously controls the geometry of the space time through a deviation of the geodesic spheres from the spheres of euclidean metric. in general relativity, therefore, the graviton mass mgr determines the curvature rsc since rsc mgr and therefore the einstein - hilbert action itself depends on the graviton mass. similarly, we obtain that the ricci scalar scales as rsc 1/gr, and in a similar way the action itself can also depend upon the range of the graviton gr. furthermore, the time interval taken for the universe expansion from a maximum to a minimum density varies as the square root of the number of information, that is, tmaxn, resulting in an expression for the number of information as a function of time t that agrees with that derived by lloyd ; namely, n = (3/ln2)nlloyd. using (2) and (6) we find that number of information n associated with the mass of the graviton is given by (35)n=(32ln2c2lpl2mgr2)=2ln2(mplmgr)24.2010122, where the mass of the graviton is taken to be mgr = 3.2 10 g. this is a rather curious result which suggests that the number of information bits n associated with the graviton mass is of the same order of magnitude as the numbers appearing in cosmology. for example, funkhouser finds a new large number of coincidences as well as a scaling law for the cosmological constant and other quantities which are also investigated. as a result the author claims that the pure numbers originate naturally from basic ratios of fundamental parameters and they do not require arbitrary powers of coefficients. n is associated with the cosmological constant lambda and is equal to 4.661 10, a number in agreement with that given in funkhouser. this is still two orders of magnitude smaller than the number of information that can fit in the universe to which a physical meaning can be attributed. this is a huge number but still small to compare various numbers, one of which is called graham number. finally the entropy of the graviton mass can be estimated using the formula (36)sgr = kblog2(2n)=(2ln2)(mplmgr)2kb4.19110122kb6.01099 j / k. the calculated entropy is of the same order of magnitude as the entropy within the cosmic horizon calculated when matter within is taken into account and has been recently given in a paper by egan and lineweaver to be equal to (2.88 0.16) 10kb4.0 10 j / k. following novello we have that ngr = c / g = 1/p = max/, where ngr is the total number of gravitons in the universe, and therefore the total entropy due to the total number of gravitons is given by (37)sgr(tot)=(2ln2)kbc4g2mgr2. using (6) and (7) along with the fact that p = g / c in (32) we find that the entropy due to the total number of gravitons in the universe as a function of the number of information n is given by the following simplified expression : (38)sgr(tot)=(ln23)kbn2=1.0151024n2. moreover in a flat universe using (18) we obtain that the entropy due to the total number of gravitons in the universe evolves in time in the following way : (39)sgr(tot)=(163ln2)kb(ttpl)4. during a very early era and in particular when t = tpl the total entropy due to the total number of gravitons takes the values (40)sgr(tot)=(163ln2)kb=3.3351022 j / k, where kb = 1.38 10 j / k and tp = g / c5=5.39110 - 44 s. similarly, in the present era when t is the age of the universe, t = tage = 13.798 by = 4.387 1017 s, we obtain that the total entropy due to the total number of gravitons is given by (41)sgr(tot)=3.33610222 j / k. finally, using the relation given in mureika and mann, we write that the graviton mass satisfies the following relation : (42)mgr162(mun), where mu is the mass of the universe and n = nu is the number of events or operations that could have occurred in the age of the universe t = tage. using that, the maximum number of bits using gravitational degrees of freedom as well as conventional matter and energy is equal to the maximum number of elementary operations and using an expression given in gkigkitzis. we write that (43)n(t)nops=4ln2(ttpl)2, and after substituting in (42) we obtain that (44)mgr4ln2(tpl2h2)mu. next substituting for the planck time tpl = (g / c) and for the mass of the universe as it is given in haranas and gkigkitzis and also valev, namely, mu = c / gh, and simplifying we obtain that (45)mgr4ln2(hc2)4ln2mhub, where mhub = h / c is defined in gershtein. following sivaram we define the gravitational self - energy of the graviton to be egse = h (45) which can be further written as (46)mgr4ln2(egsec2)4ln2mgse, where mgse is the corresponding gravitational self - mass of the graviton. in valev the author gives a theoretical estimation of the graviton mass based on the assumption that the compton wavelength of the graviton is close to the hubble distance c / h, which produces a value of the graviton mass that is given by the relation mgr h / c. in this paper we investigate the relation of the graviton mass to the number of information n in the universe, and an n dependence has been found. similarly, we find that the total number of gravitons inside the horizon of the universe is proportional to the number of information n. furthermore, using landauer 's principle, we obtain that the mass of the graviton can also be expressed as the one - third power of the ratio of the mass of the pion over the mass of the universe, from which we obtain that the mass of the universe is related to the cube of the mass of the pion over the square of the graviton mass. moreover, we find that the evolution of the graviton has an inverse time dependence ; that is, mgr 1/t. finally, in relation to the geometry of the universe, we find that the mass of the graviton is related to the ricci scalar in the following way : mgrrsc, where at the same time the ricci scalar depends on the inverse of the graviton range according to the relation rsc 1/gr. | we investigate the relation of the mass of the graviton to the number of information n in a flat universe. as a result we find that the mass of the graviton scales as mgr1/n. furthermore, we find that the number of gravitons contained inside the observable horizon is directly proportional to the number of information n ; that is, ngr n. similarly, the total mass of gravitons that exist in the universe is proportional to the number of information n ; that is, mgrn. in an effort to establish a relation between the graviton mass and the basic parameters of the universe, we find that the mass of the graviton is simply twice the hubble mass mh as it is defined by gerstein. (2003), times the square root of the quantity q 1/2, where q is the deceleration parameter of the universe. in relation to the geometry of the universe we find that the mass of the graviton varies according to the relation mgrrsc, and therefore mgr obviously controls the geometry of the space time through a deviation of the geodesic spheres from the spheres of euclidean metric. |
proper signal transmission by protein kinases requires that they phosphorylate specific substrates at defined sites. multiple mechanisms can act in concert to provide substrate specificity for kinases, including kinase - substrate colocalization, compartmentalization through the use of scaffold proteins, substrate recruitment through kinase adaptor subunits, and direct physical interactions between kinases and their substrates (ubersax and ferrell, 2007). in order for substrate phosphorylation to occur, the residue phosphorylated by the kinase must bind at least transiently to the catalytic cleft of the kinase. accordingly, protein kinases tend to phosphorylate substrates in the context of consensus sequence motifs that have complementarity to the kinase active site (pinna and ruzzene, 1996). such kinase phosphorylation site motifs play an important role in targeting kinases to specific substrates within the cell, as well as directing kinases to phosphorylate specific sites on their substrates. one important aspect of the kinase phosphorylation site motif is the identity of the phosphorylation site residue itself. almost all kinases in eukaryotes phosphorylate protein substrates on ser, thr, or tyr residues. ser - thr kinases, which make up approximately 80% of the human kinome, comprise several groups phylogenetically distinct from tyr - specific kinases (manning., 2002). accordingly, ser - thr kinases possess conserved signature residues within the catalytic domain important for accommodating a small, aliphatic phosphoacceptor residue at the active site (taylor., 1995). likewise, a distinct set of conserved residues characterizes tyr kinases, which must accommodate a large, aromatic residue. interestingly many, though not all, ser - thr kinases are significantly selective for either ser or thr as the phosphoacceptor residue (pinna and ruzzene, 1996). for example, camp - dependent protein kinase (pka) strongly favors ser over thr in peptide substrates, and a large majority (> 90%) of established in vivo phosphorylation sites are at ser residues (aimes., 2000 ; conversely, the kinase lkb1 activates numerous downstream protein kinase substrates by phosphorylation exclusively on thr residues (lizcano., 2004). although substrate specificity studies have predominantly been conducted in vitro, evidence is now emerging indicating that phosphoacceptor residue identity significantly influences substrate phosphorylation efficiency in living cells (kang., 2013). thus, the presence of a preferred phosphoacceptor residue appears to be important for targeting of specific substrates by ser - thr kinases., we show that phosphoacceptor preference of ser - thr kinases is determined largely by the identity of a single residue, which we term this residue is located within the kinase activation segment, a conformationally flexible loop important for kinase regulation. mutagenesis of this residue is sufficient to change the phosphoacceptor preference for kinases from distinct groups in a predictable manner. identification of this residue therefore establishes a simple rule that can be used to predict phosphorylation site preference for a kinase of unknown specificity. we go on to show by x - ray crystallography of kinase - peptide complexes that conformation, rather than binding affinity, drives phosphoacceptor preference. overall, these studies explain how a protein kinase is able to discriminate between ser and thr, two residues that differ only by a single methyl group. we recently analyzed the peptide substrate specificity of a large number of ser - thr kinases from s. cerevisiae (mok., 2010). among these kinases, 32 preferred ser, 10 preferred thr, and 14 lacked substantial phosphoacceptor residue preference (table s1 available online). while about half of the nonselective kinases belonged to a single phylogenetic group (the cdk-, mapk-, gsk3-, and ckii - related [cmgc ] kinase group), possibly attributable to a distinct peptide - binding mode (brinkworth., 2003 ;, 1999 ; mok., 2010), all other kinase groups included both ser- and thr - specific kinases (table s1 and figure s1). thus, in contrast to the clear distinction between tyr kinases and ser - thr kinases, ser - preferring and thr - preferring kinases can not be readily distinguished based on phylogeny. residues within the kinase catalytic domain that determine target specificity covary with corresponding residues in the substrate peptide (li., 2003). computational analysis of our yeast kinase data identified residues that covaried between kinases and their consensus peptide sequences (yip., 2011). one residue correlating strongly with phosphoacceptor preference is found immediately downstream of a conserved asp - phe - gly (dfg) sequence at the n terminus of the kinase activation loop (hereafter referred to as the dfg+1 residue). consistent with a potential role in controlling kinase specificity, this residue is located within the substrate - binding cleft (goldsmith., 2007). among yeast kinases analyzed, ser - selective kinases had larger hydrophobic residues (predominantly leu, phe, and met) at this position, while most thr - specific kinases had the -branched aliphatic residue ile at this position. for example, lkb1 and pdk1, which phosphorylate primarily thr residues in substrates (lizcano., 2004 ; mora., 2004), have the -branched residues val and thr, respectively, at the dfg+1 position. in addition, human protein kinase c isozymes, which prefer ser as the phosphoacceptor (ferrari., 1985 ; house., 1987), have the straight - chain aliphatic residue met at the analogous position. based on its location within the kinase fold and the strong correlation between its identity and kinase specificity, we hypothesized that the dfg+1 residue might be an important determinant of phosphoacceptor specificity for ser - thr kinases. based on this hypothesis, mutation of the dfg+1 residue within ser - thr kinases should alter specificity in a predictable manner. we initially focused on human kinases in the ste7-, ste11-, and ste20-related (ste) group. most kinases in this group, including those in the mammalian ste20-like (mst) kinase family, are thr selective (miller., 2008), while members of the p21-activated kinase (pak) family are ser selective (rennefahrt., 2007). phosphorylation site specificity within these families correlates with dfg+1 residue identity (val for mst kinases and phe for paks). we generated dfg+1 exchange mutants of these kinases (mst4 and pak4) and examined their phosphorylation specificity alongside their wild - type (wt) counterparts using optimized synthetic peptides that differ only at the phosphosite. as anticipated, wt mst4 preferentially phosphorylated the thr peptide, while wt pak4 preferentially phosphorylated the ser peptide (figure 1a and table 1). furthermore, mutation of the dfg+1 residue was sufficient to invert phosphorylation site specificity for both kinases on both consensus peptide substrates and peptide libraries (figures 1a and s2). notably, we found that for a given kinase, the km values for phosphorylation of the ser and thr peptides were similar, but phosphoacceptor residue identity had a large effect on kcat (table 1). these results suggest that phosphoacceptor preference is not mediated by differences in substrate binding affinity. to determine whether the dfg+1 residue controls phosphoacceptor preferences among ser - thr kinases in general, we tested the effect of mutating this residue in two more distantly related kinases : snf1 (the yeast ortholog of amp - activated protein kinase, from the ca / calmodulin - dependent protein kinase - related group) and pka (from the pka-, pkg-, and pkc - related [agc ] group). we generated a series of snf1 mutants in which the dfg+1 residue (leu198) was changed to residues most commonly found in other kinases at this position and evaluated the ability of these mutants to phosphorylate ser- and thr - containing peptides (figure 1b). mutation of leu198 to a small or -branched residue (ser, val, or ile) converted it to a thr kinase, while mutation to phe in snf1 retained a preference for ser. unexpectedly, snf1, predicted to be ser specific, did not discriminate between ser and thr. for pka, mutation of the dfg+1 residue (phe187) to val inverted its selectivity from ser to thr specific (figure 1c). taken together, these data indicate that the dfg+1 residue plays a predominant role in dictating phosphoacceptor specificity across multiple kinase groups. if the dfg+1 residue is the primary determinant of ser - thr kinase phosphoacceptor specificity, the identity of this residue should allow us to predict the preference of an uncharacterized kinase. most kinases within the human agc group appear to prefer ser, and in keeping with this, leu, met, and phe are found most frequently at the dfg+1 position. kinases in the rock family are outliers in that they have a -branched thr dfg+1 residue, suggesting they are likely to prefer thr over ser. using a pair of matched peptides differing only at the phosphoacceptor site, we found that rock1 indeed had a significant (approximately 2-fold) preference for thr as the phosphoacceptor (figure 1d). to assess the contribution of the dfg+1 residue to substrate phosphorylation in cells, we examined the ability of pak4 to phosphorylate an established substrate, the proapoptotic protein bad (gnesutta., 2001), upon mutation of either pak4 or its phosphorylation site in bad (ser112 to thr, resulting in bad). coexpression of wt pak4 catalytic domain with bad in hek293 cells yields robust phosphorylation at ser112 (figure 2). despite having similar catalytic parameters to the wt kinase in vitro on peptide substrates (see table 1), pak4 expression in contrast, pak4 could phosphorylate bad in cells, but wt pak4 phosphorylation of bad was substantially reduced. these results suggest that an optimal combination of phosphorylation site residue and dfg+1 residue is essential for maximal phosphorylation of at least some kinase substrates in living cells. to understand the structural basis for how the dfg+1 residue controls phosphoacceptor specificity, we determined the x - ray cocrystal structures of wt and f461v mutant pak4 catalytic domains in complex with consensus peptide substrates incorporating both their favored and disfavored phosphorylation site residues (paktide - s and paktide - t, figure 3). we also determined an additional x - ray cocrystal structure of wt pak4 with a longer version of the consensus peptide, paktide - s(l) (figure s3). as previously observed for wt pak4 in the absence of bound peptide (eswaran., 2007 ; ha., 2012), in each of the structures pak4 is observed in the active state conformation with the activation loop phosphorylated on residue ser474. the maximal root - mean - square deviation between the five structures is 1.1 over 290 c- atoms (table 2). interestingly, the cocrystallized atp analog was only visible in the electron density for the structure of the wt pak4 complex with paktide - s (figure s3). both structures of wt pak4 in complex with the optimal substrate, paktide - s or paktide - s(l), are highly similar, with small differences observed in the conformation of several side chains within the bound peptide and some crystal packing - induced conformational differences c - terminal to the phosphoacceptor residue (figure s3). the bound peptide has a nearly identical backbone conformation, and, importantly, the residues most strongly selected by the kinase (the p-2 arg and the p0 ser residues [rennefahrt., 2007 ; zhu., 2005 ]) are observed in very similar orientations. as found in other structures of ser - thr kinases with bound peptide substrates (brown., 1999 ;, 2005 ; madhusudan., 1994 ; yang., 2002), in both structures the ser hydroxyl points toward the atp binding site and is engaged in a network of polar contacts with conserved residues in the kinase catalytic loop (figures 3a and s3l). both structures therefore show the pak4-peptide complex in a conformation apparently competent for phosphate transfer to occur. the dfg+1 phe residue in these wt pak4 structures is positioned within 4 of the ser phosphoacceptor -carbon and oriented perpendicular to the substrate peptide backbone (figure 3b). this conformation for the dfg+1 phe would preclude the additional methyl group of a thr phosphoacceptor due to steric hindrance. indeed, in the structure of wt pak4 in complex with paktide - t, the bound peptide is in a nearly identical conformation. however, to accommodate the thr phosphoacceptor residue, the dfg+1 phe undergoes significant conformational movement : it rotates 75 about its - bond to allow the face of the phe aromatic ring to interact with the thr methyl group (figure 3c). this conformation for the dfg+1 residue likely interferes with positioning of the -phosphate of atp (figures s3m s3o). consistent with the kinetic data, the structure does not suggest impaired paktide - t binding to the wt kinase. rather, decreased phosphorylation of thr substrates may occur due to conformational effects within the catalytic center. taken together, these structures provide a likely explanation for why wt pak4, and by extension other kinases with a dfg+1 phe residue, is selective for ser at the phosphorylation site. to understand why kinases with a -branched dfg+1 residue favor thr, we examined the cocrystal structures of pak4 in complex with paktide - s and with paktide - t. in the cocrystal structure of pak4 with paktide - t, the phosphoacceptor thr methyl group stacks snugly against the mutated dfg+1 val residue, positioning the phosphoacceptor hydroxyl in an orientation similar to that observed in the wt pak4-paktide - s complex (figure 3d). the phosphoacceptor thr appears poised for phosphate transfer, and thus the combination of val at the dfg+1 position and thr at the phosphoacceptor site seems to be highly accommodative to positioning an active - like conformation for the phosphoacceptor hydroxyl. in striking contrast, the cocrystal structure of pak4 in complex with paktide - s displays an unexpected conformation of the phosphoacceptor ser residue (figure 3e). although the overall peptide conformation is similar, the phosphoacceptor ser is rotated about its - bond (1) such that the hydroxyl group is pointed away from the active site. the pak4 cocrystal structures therefore suggest that val at the dfg+1 residue favors thr over ser because the additional methyl group allows interplay between the dfg+1 and phosphoacceptor residues, correctly positioning them to promote an optimal conformation for phosphate transfer. our results suggest that discrimination by kinases between ser and thr involves control of proper conformation rather than binding to the active site. our conclusion that ser and thr peptides bind with equal affinity is partly based on our observation that pak4 and mst4 phosphorylate ser and thr substrates with similar km values. however, because the km value for an enzyme reaction is not necessarily equivalent to the substrate dissociation constant, substrate binding, conformation, and product release could all theoretically contribute to phosphoacceptor discrimination. we note, however, that ser and thr peptides appear to bind with equal affinity to the ser - selective kinase pka (aimes., 2000). discrimination between tyr and ser / thr by kinases appears to involve differences in binding affinity as well as conformational control. x - ray crystal structures of tyr kinases in complex with substrates have revealed favorable nonpolar interactions between the phosphoacceptor tyr and conserved tyr kinase - specific residues within the active site cleft (favelyukis., 2001 ; hubbard, 1997). in addition, tyr kinases and ser - thr kinases differ in the conformation of the so - called p+1 loop, which interacts with the peptide backbone of a bound substrate to control its distance from the active site, thus dictating the size of the phosphoacceptor that can be accommodated (hubbard, 1997 ; taylor., 1995). comparative kinetic analyses have been conducted for ser and tyr peptide phosphorylation by casein kinase 2 (ck2), a kinase that belongs to the cmgc ser - thr kinase group but that can also phosphorylate on tyr residues, albeit with greatly reduced catalytic efficiency. these analyses revealed large (100-fold or more) differences in both kcat and km, as well as distinct sequence preferences at residues flanking the phosphoacceptor (marin. these results suggest that ser and tyr peptides bind in distinct modes to the active site of ck2 and imply different binding affinities. in contrast, we found that ser and thr peptides bound to pak4 with an identical backbone conformation and apparently comparable affinities. the preference of a kinase for ser or thr, as reflected in peptide substrate phosphorylation in vitro, is likely to be generally important for substrate targeting in vivo. for example, we observed that most kinases in yeast prefer ser, and based on the identity of their dfg+1 residues, most human kinases are predicted to prefer ser as well. these observations suggest that the preponderance of pser among yeast and human phosphoproteomes (hornbeck., 2012 ; sadowski., 2013) is related to intrinsic biochemical properties of eukaryotic kinases. for some kinases the presence of the preferred phosphoacceptor residue may constitute an essential component of its phosphorylation site motif. however, in many cases the preferred phosphoacceptor, while not absolutely required for phosphorylation by the kinase, may serve to confer robustness on substrate phosphorylation. for example, recent work has shown that exchanging thr for ser at sites of phosphorylation by the mtor kinase could modulate sensitivity to activating stimuli or inhibitors of the kinase (kang., 2013). in addition to influencing recognition by kinases, phosphorylation site identity can also influence downstream signaling as both protein phosphatases and phosphoprotein interaction domains can discriminate between pser and pthr (bremmer. while we have identified the dfg+1 residue as a major determinant for several kinases, other residues within the catalytic domain undoubtedly contribute to phosphoacceptor preference as well. individual kinases vary widely with respect to the stringency with which they discriminate between the two residues, and this is likely to be controlled by additional active site residues. for example, a residue in the glycine - rich loop located proximal to the -phosphate of bound atp was shown to influence the extent to which pka prefers ser (aimes., 2000). in addition, we found that a dfg+1 leu residue correlated with ser preference for most kinases, yet for kinases in the cmgc group it was associated with a lack of phosphoacceptor selectivity. identification of additional modifier residues will facilitate a more complete understanding of structural features controlling kinase specificity. in addition, for some kinases it is possible that phosphorylation site specificity is entirely determined by other residues. nonetheless, our current study suggests that, by inspection, one may determine whether a kinase is selective for ser or thr. to wit, a -branched residue at the dfg+1 position appears to invariably dictate a preference for thr, but phe dictates preference for ser as the phosphoacceptor residue. our study therefore provides a much improved understanding of how protein kinases discriminate between phosphorylation site residues. pak4, mst4, and pka (wt and point mutants) were expressed with n - terminal hexahistidine tags in e. coli and purified by immobilized metal affinity chromatography. pak4 and mst4 were further subjected to anion exchange, followed by size exclusion chromatography or size exclusion chromatography alone, respectively. gst - snf1 (wt and mutant) was expressed in e. coli, purified by one - step glutathione affinity chromatography, and then activated by phosphorylation with recombinant elm1 kinase, as described (lee., 2012). descriptions of expression vectors used and detailed purification procedures are provided in supplemental experimental procedures. kinases were incubated with peptides and [-p]atp at 30c for varying times, and aliquots were spotted on to p81 filter discs. filters were quenched in 75 mm phosphoric acid and then washed three times for 4 min in 75 mm phosphoric acid, washed once briefly in acetone, air - dried, and analyzed by liquid scintillation counting. the amount of product formed was calculated from a standard curve made using [-p]atp at the same specific activity. initial rates were determined by fitting the data to a line with excel, and catalytic constants were calculated by fitting the initial velocity data to the michaelis - menten equation using kaleidagraph software. detailed procedures, including buffer compositions for each kinase reaction, are provided in supplemental experimental procedures. hek293 cells were cultured at 37c and 5% co2 in dulbecco s modified eagle s medium containing 4.5 g / l d - glucose and l - glutamine (invitrogen), 10% fetal bovine serum (fbs), and 1% penicillin / streptomycin. cells in 6-well plates were cotransfected with 0.4 g pegfp - pak4 catalytic domain and 3.6 g pebg - bad using lipofectamine 2000 (invitrogen). after 24 hr, cells were exchanged into reduced serum medium (0.1% fbs) for 16 hr and treated with 250 nm wortmannin for 30 min prior to lysis. cell lysates were prepared by washing briefly with pbs and extracting into lysis buffer (20 mm tris - hcl [ph 7.5 ], 150 mm nacl, 1 mm edta, 1 mm egta, 1% triton x-100, 2.5 mm sodium pyrophosphate, 1 mm -glycerophosphate, 1 mm na3vo4, 1 mm dtt, 1 mm pmsf, 10 g / ml leupeptin, 2 g / ml pepstatin, and 10 g / ml aprotinin) for 10 min at 4c with rotation and centrifuging (10 min at 13,500 g). gst - tagged bad was purified from the supernatant with 20 l glutathione sepharose 4b suspension (ge healthcare). samples were subjected to sds - page followed by immunoblotting with -bad pser112 antibody (cell signaling technology # 9291), -gst antibody (cell signaling technology # 2624) and -gfp antibody (clontech # 632381), and detection by enhanced chemiluminescence. crystals of wt pak4 and pak4 catalytic domain were obtained with both paktide - t (ggrrrrrtwyfgggk) and paktide - s (rrrrswy) using the vapor diffusion hanging drop method. for wt pak4 with peptides, we grew wt pak4 crystals against 0.1 m tris - hcl (ph 7.5) and 1.52.0 m naoac at room temperature and soaked with peptides. for pak4 cocrystals with peptide, we grew against conditions of 0.1 m tris - hcl (ph 7.5) and 1.52.0 m naoac at room temperature. for cocrystals of pak4 in complex with paktide - s(l) (rrrrrswyfdg), we grew against 1.7 m ammonium sulfate, 15% peg400, and 0.1 m tris - hcl (ph 8.0). 24-id - e, national synchrotron light source (nsls) beamline x25, or the swiss light source beamline sls - x10. following structure solution, good electron density was observed for each of the kinase domains and their bound peptides. | summaryeukaryotic protein kinases are generally classified as being either tyrosine or serine - threonine specific. though not evident from inspection of their primary sequences, many serine - threonine kinases display a significant preference for serine or threonine as the phosphoacceptor residue. here we show that a residue located in the kinase activation segment, which we term the dfg+1 residue, acts as a major determinant for serine - threonine phosphorylation site specificity. mutation of this residue was sufficient to switch the phosphorylation site preference for multiple kinases, including the serine - specific kinase pak4 and the threonine - specific kinase mst4. kinetic analysis of peptide substrate phosphorylation and crystal structures of pak4-peptide complexes suggested that phosphoacceptor residue preference is not mediated by stronger binding of the favored substrate. rather, favored kinase - phosphoacceptor combinations likely promote a conformation optimal for catalysis. understanding the rules governing kinase phosphoacceptor preference allows kinases to be classified as serine or threonine specific based on their sequence. |
how signaling molecules enable cells of multicellular organisms to communicate and assemble tissues and organs is a central question in biology. embryo manipulation and molecular genetics established that a surprisingly small number of secreted proteins of only a handful of conserved gene families govern a multitude of cell - fate decisions. much of the diversity in signaling outputs at the level of target gene regulation is attributed to tissue - specific signal integration in elaborate networks. but while the number of known interactions between signaling pathways increases daily, our conventional charts of this crosstalk give little account of how the molecules involved reach their correct localization. in part, this oversimplification is due to the fact that the role of trafficking and its regulation are not well understood. however, advances in cell biology and improved imaging technologies now allow us to follow protein trafficking at high resolution and in real time. imaging, combined with sensitive genetic screens and sophisticated manipulation of protein and membrane trafficking, established that localization is one of the key determinants regulating signal outputs. the present article will review the most recent important findings on how trafficking controls signaling at the level of polarized secretion, protein processing, and endocytosis. as each of these fields are limitless, they are only represented here by illustrative examples to emphasize that they are intertwined, and that they need to be integrated to appreciate how intimately signal regulation is coupled to trafficking. the most basic decision in trafficking is whether a protein is sorted apically or basolaterally in polarized epithelial cells - for example, to position ion channels and proton gradients. the diffusion barrier of tight junctions, which insulate apical from basolateral membranes, can also limit access of ligands to receptors, as shown for transforming growth factor beta (tgf-). protein sorting into distinct exocytic carriers occurs in the trans - golgi network (tgn) or in endosomes. determinants of apical sorting include glycosylphosphatidylinositol (gpi) anchors, which mediate membrane attachment and oligomerization in lipid rafts (reviewed in), as well as crosslinking of carbohydrate side chains by apical sorting receptors such as the mannose - binding lectin vip36 (vesicular integral membrane protein of 36 kda), or the raft - independent transporter, galectin-3 [6 - 8 ]. by contrast, basolateral sorting involves cytosolic tails of transmembrane proteins that bind endosomal ap-1b (adaptor protein complex 1b) or other cytoplasmic adapter proteins interacting with clathrin. depletion of clathrin in mdck (madin darby canine kidney) cells thus disrupts basolateral sorting without diminishing polarized secretion of apical proteins. studies on the morphogens hedgehog (hh) and wingless (wg) in drosophila wing imaginal discs suggest that specific carrier proteins mediate transcytosis from the apical to the lateral plasma membrane. in this system, production of hh in the posterior compartment gives rise to a signaling gradient in anterior cells, whereas wg secreted at the dorso - ventral boundary moves dorsally and ventrally. secretion of wg and hh is regulated by palmitoylation and, in the case of hh, a cholesterol adduct. both wg and hh are released basolaterally in association with lipoprotein particles [13 - 15 ], but how lipoproteins extract their cargo from the plasma membrane is poorly understood. recent work in mammalian cells confirmed that binding of the wg - related protein wnt3a to lipoprotein and basolateral secretion depend on wnt3a palmitoylation. wnt proteins are palmitoylated in the endoplasmatic reticulum by porcupine and exit the tgn upon association with wntless (wls), a transporter that is recycled from endosomes to the golgi by the retromer complex [17 - 20 ]. if endocytosis in drosophila imaginal discs is blocked by dominant negative dynamin, or by deleting the receptors dfz2 (drosophila frizzled 2) and arrow [22 - 24 ], extracellular wg is trapped on the apical plasma membrane, suggesting that wls targets wnt proteins apically. a first hint was that wg fails to move across mutant clones of cells lacking the glypicans dally (dly) and dally - like protein (dlp). glypicans are gpi - anchored heparan sulfate proteoglycans that are internalized by the lipid raft scaffold protein flotillin / reggie via a clathrin- and caveolin - independent route. immunostaining and antibody uptake by a green fluorescent protein - dlp fusion protein showed that dlp initially localizes to the apical membrane, but that it is efficiently recycled basolaterally within less than 90 minutes after endocytosis moreover, in mutant cells lacking dynamin function or dlp, apical - basal transcytosis of dlp or the basolateral targeting of wg, respectively, were blocked, indicating that wg is targeted laterally by dlp via a dynamin - dependent endocytic route. dly and dlp also enhance the activity and spreading of hh, bind lipoprotein particles, and colocalize with hh and its receptor ptc (patched) in vesicles of signal receiving cells. colocalization and stimulation of hh signaling were lost if the gpi anchor of dlp was substituted by a transmembrane domain. does this mean that both wg and hh rely on glypicans for apical - to - basal transcytosis as a gate to board the basolateral lipoprotein shuttle that controls gradient formation ? in support of this model, secretion of wg and hh in the wing imaginal disc also depends on reggie-1/flotillin-2. furthermore, mutant forms of hh that can not be lipid - modified fail to sort basolaterally and instead diffuse apically, giving rise to an abnormally shallow signaling gradient. taken together, these studies suggest that wg and hh are transcytosed from the apical to the lateral plasma membrane and transferred to lipoprotein particles by glypicans (figure 1a). another critical aspect of protein trafficking concerns the mechanisms of signal regulation in endocytic compartments. it is well established that endocytosis is important for ligand - induced receptor degradation during signal attenuation and reduction of the extracellular concentration of morphogens in the extracellular space. however, endocytosis is equally important for signaling molecules to access specific endosomal signaling platforms and sorting stations (for a survey of existing endocytic pathways, see). the best known uptake routes initiate from clathrin - coated pits or caveolae, which are severed from the plasma membrane by dynamin. tgf- receptors enter both compartments, but only coated pits enable access to the smad anchor for receptor activation (sara) on early endosomes, and hence activation and nuclear translocation of cytoplasmic smad transcription factors. by contrast, uptake via caveolae leads to recruitment of the ubiquitin ligase smurf2 and receptor degradation. degradation of the related bone morphogenetic protein (bmp) receptors via a caveolar route increases upon dephosphorylation by dullard, which attenuates the anti - neuralizing function of bmp signaling during xenopus gastrulation. surprisingly, a caveolar uptake has now been found to also mediate ligand - degradation of the epidermal growth factor receptor (egfr), whereas clathrin - mediated uptake allows recycling and sustained egfr signaling. this is unexpected in light of the conclusion from earlier studies that egfr is internalized mainly through clathrin - coated pits (reviewed in). caveolar uptake can also promote signaling, for example, of wnt proteins. to signal via the canonical pathway, wnt ligands bind complexes of frizzled (fz) receptors with the co - receptor lipoprotein - related protein (lrp)5 or lrp6. in response to wnt3a, lrp6 binds caveolin and together with dvl (dishevelled) forms intracellular aggregates that inhibit the kinase gsk3 (glycogen synthase kinase 3 beta) in order to stabilize -catenin and induce target genes. depletion of caveolin in cultured cells inhibits both wnt3a - induced endocytosis and signaling of lrp6, indicating that canonical wnt signaling depends on caveolar uptake. by contrast, in cells depleted of clathrin, canonical wnt signaling was not impaired. however, clathrin and the adapter molecule -arrestin internalize complexes of fz with wnt5a and wnt11, which activate distinct, non - canonical signal transduction pathways to specify planar cell polarity and induce convergence extension movements during gastrulation [39 - 41 ]. interestingly, binding to the secreted wnt antagonist dkk1 (dickkopf-1) redistributes lrp6 from caveolae to a clathrin - dependent uptake route. together, these studies show that receptor endocytosis is essential for wnt signaling, and that uptake by distinct entry pathways is tightly regulated and mediates the activation of different intracellular signaling cascades. during endocytosis, signaling molecules are often processed by proteases to remove an inhibitory propeptide, or to shed a trans - membrane domain or release a cytoplasmic tail. sequential cleavage of notch by furin, adam10 (a disintegrin and metallopeptidase domain 10), and -secretase leads to the release of the notch intracellular domain (nicd), which enters the nucleus to regulate target genes. it is well established that notch is hyperactivated in lethal (2) giant discs (lgd) mutant flies, where transport from early to late endosomes is inhibited [44 - 46 ]. a recent study in drosophila wing and eye imaginal discs now has shown that null mutations in dynamin, rab5 or the endocytic syntaxin avl (avalanche) all diminish the amount of nicd, suggesting that cleavage occurs after endocytosis. consistent with this view, immunostaining of notch and nicd in dividing drosophila sensory organ precursor cells revealed the presence of cleaved notch on sara - positive early endosomes of only one of the asymmetric daughter cells. in addition, nuclear translocation, but not the production of nicd, requires acidification of endosomes by the aquaporin big brain. together, these observations suggest that the production and nuclear translocation of nicd are coupled to endosome maturation, but limited by rapid sequestration of notch in lysosomes (figure 1b). propeptides contain sorting signals, mediate interactions with transporter proteins or sterically hinder precocious binding to receptors. it is important, therefore, that propeptides are removed in the appropriate subcellular compartments. proprotein processing also balances the activities of precursor and mature forms of their substrates, as shown for nodal or the neurotrophic growth factor precursor, respectively. nodal is essential in the implanted blastocyst to inhibit precocious neural differentiation of pluripotent progenitor cells and to pattern the surrounding primitive endoderm, whereas later it induces mesoderm and definitive endoderm formation. endoderm and mesoderm are specified during gastrulation by distinct signaling thresholds of smad2 and smad3 transcription factors, which are phosphorylated by endosomal signaling complexes of activin receptors, processed nodal and a gpi - anchored co - receptor of the egf - like cripto / frl-1/cryptic (cfc) family. but cripto already binds nodal in the endoplasmatic reticulum, in part via the prosegment of the uncleaved precursor. cripto also recruits the subtilisin - like proprotein convertases furin or pace4 (paired amino acid cleaving enzyme 4), which cleave nodal. however, furin and pace4 are not coexpressed with cripto in vivo, but secreted by cells in the microenvrionment. in addition, cripto and nodal access the plasma membrane by an unconventional route bypassing proprotein convertases in the tgn. nodal thus is not processed until arrival at the cell surface. upon maturation, nodal is rapidly endocytosed, whereas in the absence of cleavage it is only slowly internalized and secreted via the tgn. the advantage of maturing in a complex with cripto is that the gpi - anchor of cripto localizes nodal processing to membrane microdomains that access endosomal signaling platforms, whereas cripto - independent nodal processing favors uptake in degradative compartments. (a) the glypicans dally (dly) and dally - like protein associate with lipid rafts and mediate basal transport of the morphogens wingless (wg) and hedgehog (hh) in drosophila wing imaginal disc epithelial cells to access lipoprotein particles in the hemolymph. cleavage of the glycosylphosphatidylinositol (gpi) anchor facilitates endocytosis of glypican and associated cargo in signal - receiving cells. some shedding of dly also occurs apically, but whether any hh protein moves through the apical lumen is controversial (stippled green arrow). left panel : after s1 cleavage by intracellular furin, the notch extracellular domain (orange) is engaged by membrane - bound ligands, which are ubiquitinated in their cytosolic tails by neuralized (neur) and mind bomb (mib). endocytosis of notch ligand by epsin in signal - sending cells (purple arrow) enables activation of the s2 site by adam-10 or -17, followed by -secretase cleavage of the intramembraneous s3 site and nuclear translocation of the notch intracellular domain (nicd). nuclear translocation of nicd in addition requires acidification of the endosome by the aquaporin big brain (bib). loss of bib suppresses notch hyperactivation in lgd mutants, suggesting that nicd matures on endosomes, rather than at the plasma membrane. ubiquitination of transmembrane proteins (green) by e3 ligases during endocytosis is guided by arrestins (arr) and mediates binding to endosomal hrs (hepatocyte growth factor receptor - regulated tyrosine kinase substrate), a subunit of endosomal sorting complex required for transport (escrt)-0. sequential assembly of escrt - i, -ii and -iii complexes culminates in the recycling of ubiquitin (ub) by a deubiquitylating enzyme (dub), followed by invagination of the limiting membrane and associated cargo into the endosome lumen. the endosome membrane is a mosaic of different subdomains, which sort cargo for delivery to lysosomes or other destinations (see text for details). (d) a protein lattice consisting of the escrt - iii components vacuolar protein - sorting (vps)20, vps24 and sucrose non - fermenting 7 (snf7) is sufficient to induce intralumenal budding of the limiting membrane of giant unilamellar vesicles. recruitment of the atpase vps4 by vps2 disassembles escrt - iii components for recycling (after). cell surface receptors are marked for degradation by ubiquitination, a covalent modification that targets the internalized protein to multivesicular bodies (mvbs) for delivery to lysosomes. studies in yeast suggest that substrate specificity of the e3 ubiquitin ligases is conferred by adaptors of the arrestin family (figure 1c). upon arrival in early endosomes, the ubiquitin moieties bind hepatocyte growth factor receptor - regulated tyrosine kinase substrate (hrs), which associates with stam (signal transducing adapter molecule) to form the endosomal sorting complex required for transport (escrt)-0 and thereby initiate the sequential assembly of the multiprotein complexes escrt - i, -ii and -iii. the interactions among escrts culminate in the recruitment of deubiquitylating enzymes and of the aaa+ atpase vacuolar protein - sorting 4 (vps4). deubiquitylation by ubiquitin - specific protease y (ubpy, doa4 in yeast) and ubp2 allows recycling of ubiquitin and advances cargo to intralumenal vesicles of endosomes that mature into mvbs [61 - 63 ] (figure 1c). how intralumenal vesicles are induced to bud from the limiting membrane into the endosome lumen has long remained elusive. a landmark study using a novel in vitro reconstitution assay has now shown that addition of only three yeast escrt - iii subunits [vps20, vps24, and sucrose non - fermenting 7 (snf7) ] to synthetic giant unilamellar vesicles is sufficient to induce budding and scission of intralumenal vesicles from the limiting membrane (figure 1d). the vps2 subunit subsequently recruits vps4 to disassemble and recycle the components of this complex for further rounds of budding. independently demonstrated that the vps4 atpase is required to disassemble the protein lattice of snf7 oligomers in vitro, although in their model vps4-induced disassembly is linked to membrane scission. accordingly, the primary role of escrt-0, -i and -ii probably is to localize escrt - iii to endosomal membrane microdomains loaded with cargo. during development, tgf-s, receptor tyrosine kinases, and notch proteins specify distinct cell fates at different signaling thresholds, which ultimately reflect the concentration and longevity of signaling complexes in endosomes. since most if not all of these signaling molecules are degraded via the mvb pathway (reviewed in), the new mechanistic insights provide a solid foundation to elucidate how the residence time of different receptors or combinations of receptors at the endosome - limiting membrane are regulated. the importance of escrt - mediated signal attenuation during development is further highlighted by the early embryonic lethality of mutant mice lacking hrs or the escrt - iii component chmp5 (charged mvb protein 5), and by the hyperactivation of notch and excess proliferation of epithelial cells in drosophila lacking the escrt - ii subunits vps25, vps22 or vps36 [70 - 73 ]. a future challenge will be to determine how the sorting of signaling molecules to intra - endosomal vesicles controls morphogenesis in specific contexts, and how this process is regulated in vivo. the most basic decision in trafficking is whether a protein is sorted apically or basolaterally in polarized epithelial cells - for example, to position ion channels and proton gradients. the diffusion barrier of tight junctions, which insulate apical from basolateral membranes, can also limit access of ligands to receptors, as shown for transforming growth factor beta (tgf-). protein sorting into distinct exocytic carriers occurs in the trans - golgi network (tgn) or in endosomes. determinants of apical sorting include glycosylphosphatidylinositol (gpi) anchors, which mediate membrane attachment and oligomerization in lipid rafts (reviewed in), as well as crosslinking of carbohydrate side chains by apical sorting receptors such as the mannose - binding lectin vip36 (vesicular integral membrane protein of 36 kda), or the raft - independent transporter, galectin-3 [6 - 8 ]. by contrast, basolateral sorting involves cytosolic tails of transmembrane proteins that bind endosomal ap-1b (adaptor protein complex 1b) or other cytoplasmic adapter proteins interacting with clathrin. depletion of clathrin in mdck (madin darby canine kidney) cells thus disrupts basolateral sorting without diminishing polarized secretion of apical proteins. studies on the morphogens hedgehog (hh) and wingless (wg) in drosophila wing imaginal discs suggest that specific carrier proteins mediate transcytosis from the apical to the lateral plasma membrane. in this system, production of hh in the posterior compartment gives rise to a signaling gradient in anterior cells, whereas wg secreted at the dorso - ventral boundary moves dorsally and ventrally. secretion of wg and hh is regulated by palmitoylation and, in the case of hh, a cholesterol adduct. both wg and hh are released basolaterally in association with lipoprotein particles [13 - 15 ], but how lipoproteins extract their cargo from the plasma membrane is poorly understood. recent work in mammalian cells confirmed that binding of the wg - related protein wnt3a to lipoprotein and basolateral secretion depend on wnt3a palmitoylation. wnt proteins are palmitoylated in the endoplasmatic reticulum by porcupine and exit the tgn upon association with wntless (wls), a transporter that is recycled from endosomes to the golgi by the retromer complex [17 - 20 ]. if endocytosis in drosophila imaginal discs is blocked by dominant negative dynamin, or by deleting the receptors dfz2 (drosophila frizzled 2) and arrow [22 - 24 ], extracellular wg is trapped on the apical plasma membrane, suggesting that wls targets wnt proteins apically. a first hint was that wg fails to move across mutant clones of cells lacking the glypicans dally (dly) and dally - like protein (dlp). glypicans are gpi - anchored heparan sulfate proteoglycans that are internalized by the lipid raft scaffold protein flotillin / reggie via a clathrin- and caveolin - independent route. immunostaining and antibody uptake by a green fluorescent protein - dlp fusion protein showed that dlp initially localizes to the apical membrane, but that it is efficiently recycled basolaterally within less than 90 minutes after endocytosis moreover, in mutant cells lacking dynamin function or dlp, apical - basal transcytosis of dlp or the basolateral targeting of wg, respectively, were blocked, indicating that wg is targeted laterally by dlp via a dynamin - dependent endocytic route. dly and dlp also enhance the activity and spreading of hh, bind lipoprotein particles, and colocalize with hh and its receptor ptc (patched) in vesicles of signal receiving cells. colocalization and stimulation of hh signaling were lost if the gpi anchor of dlp was substituted by a transmembrane domain. does this mean that both wg and hh rely on glypicans for apical - to - basal transcytosis as a gate to board the basolateral lipoprotein shuttle that controls gradient formation ? in support of this model, secretion of wg and hh in the wing imaginal disc also depends on reggie-1/flotillin-2. furthermore, mutant forms of hh that can not be lipid - modified fail to sort basolaterally and instead diffuse apically, giving rise to an abnormally shallow signaling gradient. taken together, these studies suggest that wg and hh are transcytosed from the apical to the lateral plasma membrane and transferred to lipoprotein particles by glypicans (figure 1a). another critical aspect of protein trafficking concerns the mechanisms of signal regulation in endocytic compartments. it is well established that endocytosis is important for ligand - induced receptor degradation during signal attenuation and reduction of the extracellular concentration of morphogens in the extracellular space. however, endocytosis is equally important for signaling molecules to access specific endosomal signaling platforms and sorting stations (for a survey of existing endocytic pathways, see). the best known uptake routes initiate from clathrin - coated pits or caveolae, which are severed from the plasma membrane by dynamin. tgf- receptors enter both compartments, but only coated pits enable access to the smad anchor for receptor activation (sara) on early endosomes, and hence activation and nuclear translocation of cytoplasmic smad transcription factors. by contrast, uptake via caveolae leads to recruitment of the ubiquitin ligase smurf2 and receptor degradation. degradation of the related bone morphogenetic protein (bmp) receptors via a caveolar route increases upon dephosphorylation by dullard, which attenuates the anti - neuralizing function of bmp signaling during xenopus gastrulation. surprisingly, a caveolar uptake has now been found to also mediate ligand - degradation of the epidermal growth factor receptor (egfr), whereas clathrin - mediated uptake allows recycling and sustained egfr signaling. this is unexpected in light of the conclusion from earlier studies that egfr is internalized mainly through clathrin - coated pits (reviewed in). caveolar uptake can also promote signaling, for example, of wnt proteins. to signal via the canonical pathway, wnt ligands bind complexes of frizzled (fz) receptors with the co - receptor lipoprotein - related protein (lrp)5 or lrp6. in response to wnt3a, lrp6 binds caveolin and together with dvl (dishevelled) forms intracellular aggregates that inhibit the kinase gsk3 (glycogen synthase kinase 3 beta) in order to stabilize -catenin and induce target genes. depletion of caveolin in cultured cells inhibits both wnt3a - induced endocytosis and signaling of lrp6, indicating that canonical wnt signaling depends on caveolar uptake. by contrast, in cells depleted of clathrin, canonical wnt signaling was not impaired. however, clathrin and the adapter molecule -arrestin internalize complexes of fz with wnt5a and wnt11, which activate distinct, non - canonical signal transduction pathways to specify planar cell polarity and induce convergence extension movements during gastrulation [39 - 41 ]. interestingly, binding to the secreted wnt antagonist dkk1 (dickkopf-1) redistributes lrp6 from caveolae to a clathrin - dependent uptake route. together, these studies show that receptor endocytosis is essential for wnt signaling, and that uptake by distinct entry pathways is tightly regulated and mediates the activation of different intracellular signaling cascades. during endocytosis, signaling molecules are often processed by proteases to remove an inhibitory propeptide, or to shed a trans - membrane domain or release a cytoplasmic tail. sequential cleavage of notch by furin, adam10 (a disintegrin and metallopeptidase domain 10), and -secretase leads to the release of the notch intracellular domain (nicd), which enters the nucleus to regulate target genes. it is well established that notch is hyperactivated in lethal (2) giant discs (lgd) mutant flies, where transport from early to late endosomes is inhibited [44 - 46 ]. however, whether -secretase cleavage occurs in endosomes has been controversial. a recent study in drosophila wing and eye imaginal discs now has shown that null mutations in dynamin, rab5 or the endocytic syntaxin avl (avalanche) all diminish the amount of nicd, suggesting that cleavage occurs after endocytosis. consistent with this view, immunostaining of notch and nicd in dividing drosophila sensory organ precursor cells revealed the presence of cleaved notch on sara - positive early endosomes of only one of the asymmetric daughter cells. in addition, nuclear translocation, but not the production of nicd, requires acidification of endosomes by the aquaporin big brain. together, these observations suggest that the production and nuclear translocation of nicd are coupled to endosome maturation, but limited by rapid sequestration of notch in lysosomes (figure 1b). propeptides contain sorting signals, mediate interactions with transporter proteins or sterically hinder precocious binding to receptors.. proprotein processing also balances the activities of precursor and mature forms of their substrates, as shown for nodal or the neurotrophic growth factor precursor, respectively. nodal is essential in the implanted blastocyst to inhibit precocious neural differentiation of pluripotent progenitor cells and to pattern the surrounding primitive endoderm, whereas later it induces mesoderm and definitive endoderm formation. endoderm and mesoderm are specified during gastrulation by distinct signaling thresholds of smad2 and smad3 transcription factors, which are phosphorylated by endosomal signaling complexes of activin receptors, processed nodal and a gpi - anchored co - receptor of the egf - like cripto / frl-1/cryptic (cfc) family. but cripto already binds nodal in the endoplasmatic reticulum, in part via the prosegment of the uncleaved precursor. cripto also recruits the subtilisin - like proprotein convertases furin or pace4 (paired amino acid cleaving enzyme 4), which cleave nodal. however, furin and pace4 are not coexpressed with cripto in vivo, but secreted by cells in the microenvrionment. in addition, cripto and nodal access the plasma membrane by an unconventional route bypassing proprotein convertases in the tgn. nodal thus is not processed until arrival at the cell surface. upon maturation, nodal is rapidly endocytosed, whereas in the absence of cleavage it is only slowly internalized and secreted via the tgn. the advantage of maturing in a complex with cripto is that the gpi - anchor of cripto localizes nodal processing to membrane microdomains that access endosomal signaling platforms, whereas cripto - independent nodal processing favors uptake in degradative compartments. (a) the glypicans dally (dly) and dally - like protein associate with lipid rafts and mediate basal transport of the morphogens wingless (wg) and hedgehog (hh) in drosophila wing imaginal disc epithelial cells to access lipoprotein particles in the hemolymph. cleavage of the glycosylphosphatidylinositol (gpi) anchor facilitates endocytosis of glypican and associated cargo in signal - receiving cells. some shedding of dly also occurs apically, but whether any hh protein moves through the apical lumen is controversial (stippled green arrow). left panel : after s1 cleavage by intracellular furin, the notch extracellular domain (orange) is engaged by membrane - bound ligands, which are ubiquitinated in their cytosolic tails by neuralized (neur) and mind bomb (mib). endocytosis of notch ligand by epsin in signal - sending cells (purple arrow) enables activation of the s2 site by adam-10 or -17, followed by -secretase cleavage of the intramembraneous s3 site and nuclear translocation of the notch intracellular domain (nicd).. nuclear translocation of nicd in addition requires acidification of the endosome by the aquaporin big brain (bib). loss of bib suppresses notch hyperactivation in lgd mutants, suggesting that nicd matures on endosomes, rather than at the plasma membrane. ubiquitination of transmembrane proteins (green) by e3 ligases during endocytosis is guided by arrestins (arr) and mediates binding to endosomal hrs (hepatocyte growth factor receptor - regulated tyrosine kinase substrate), a subunit of endosomal sorting complex required for transport (escrt)-0. sequential assembly of escrt - i, -ii and -iii complexes culminates in the recycling of ubiquitin (ub) by a deubiquitylating enzyme (dub), followed by invagination of the limiting membrane and associated cargo into the endosome lumen. the endosome membrane is a mosaic of different subdomains, which sort cargo for delivery to lysosomes or other destinations (see text for details). (d) a protein lattice consisting of the escrt - iii components vacuolar protein - sorting (vps)20, vps24 and sucrose non - fermenting 7 (snf7) is sufficient to induce intralumenal budding of the limiting membrane of giant unilamellar vesicles. recruitment of the atpase vps4 by vps2 disassembles escrt - iii components for recycling (after). cell surface receptors are marked for degradation by ubiquitination, a covalent modification that targets the internalized protein to multivesicular bodies (mvbs) for delivery to lysosomes. studies in yeast suggest that substrate specificity of the e3 ubiquitin ligases is conferred by adaptors of the arrestin family (figure 1c). upon arrival in early endosomes, the ubiquitin moieties bind hepatocyte growth factor receptor - regulated tyrosine kinase substrate (hrs), which associates with stam (signal transducing adapter molecule) to form the endosomal sorting complex required for transport (escrt)-0 and thereby initiate the sequential assembly of the multiprotein complexes escrt - i, -ii and -iii. the interactions among escrts culminate in the recruitment of deubiquitylating enzymes and of the aaa+ atpase vacuolar protein - sorting 4 (vps4). deubiquitylation by ubiquitin - specific protease y (ubpy, doa4 in yeast) and ubp2 allows recycling of ubiquitin and advances cargo to intralumenal vesicles of endosomes that mature into mvbs [61 - 63 ] (figure 1c). how intralumenal vesicles are induced to bud from the limiting membrane into the endosome lumen has now shown that addition of only three yeast escrt - iii subunits [vps20, vps24, and sucrose non - fermenting 7 (snf7) ] to synthetic giant unilamellar vesicles is sufficient to induce budding and scission of intralumenal vesicles from the limiting membrane (figure 1d). the vps2 subunit subsequently recruits vps4 to disassemble and recycle the components of this complex for further rounds of budding. independently demonstrated that the vps4 atpase is required to disassemble the protein lattice of snf7 oligomers in vitro, although in their model vps4-induced disassembly is linked to membrane scission. accordingly, the primary role of escrt-0, -i and -ii probably is to localize escrt - iii to endosomal membrane microdomains loaded with cargo. during development, tgf-s, receptor tyrosine kinases, and notch proteins specify distinct cell fates at different signaling thresholds, which ultimately reflect the concentration and longevity of signaling complexes in endosomes. since most if not all of these signaling molecules are degraded via the mvb pathway (reviewed in), the new mechanistic insights provide a solid foundation to elucidate how the residence time of different receptors or combinations of receptors at the endosome - limiting membrane are regulated. the importance of escrt - mediated signal attenuation during development is further highlighted by the early embryonic lethality of mutant mice lacking hrs or the escrt - iii component chmp5 (charged mvb protein 5), and by the hyperactivation of notch and excess proliferation of epithelial cells in drosophila lacking the escrt - ii subunits vps25, vps22 or vps36 [70 - 73 ]. a future challenge will be to determine how the sorting of signaling molecules to intra - endosomal vesicles controls morphogenesis in specific contexts, and how this process is regulated in vivo. while aberrations in trafficking linked to cell polarization defects occur in a variety of disease syndromes and contribute to tumorigenesis (reviewed in), relatively little is known about the mechanisms regulating the polarized secretion of soluble ligands. the new role of glypicans discussed here during the secretion of wnt and hh proteins raises the question of whether these or similar transporters mediate basolateral delivery of other soluble proteins. it is well established that wnt, tgf-, hh and notch pathways, and other developmental signals are also regulated during endocytosis. that different entry pathways mediate degradation or recycling, or however, irrespective of whether uptake involves clathrin or caveolin, egfr is sorted to lysosomes via early endosomes. so what is the advantage of a triage at the plasma membrane ? why tidy the house if the kids will make a mess again anyway ? a possible answer is that endosomes are orderly kids who do not randomly mix cargo, but instead discriminate between cargo from distinct endocytic origins based on differences in ubiquitination, clustering, or the composition of oligomeric receptor complexes at the plasma membrane. consistent with this view, clathrin - dependent or -independent uptake of wnts involves different co - receptors, and cluster size determines post - endocytic sorting of gpi - anchored proteins. moreover, the limiting membrane of early endosomes is a mosaic of discrete subcompartments (figure 1c). likewise, nodal molecules tethered to cripto are not evenly distributed, indicating that early endosomes are unlikely to haphazardly mix cargo. it is plausible, although not proven, that proteins accumulate in distinct subdomains of the endosome membrane for signaling or for deployment to other destinations. based on their lipid content, endosomes also generate at least two different types of intralumenal vesicles for lysosomal targeting or recycling. it will be exciting to learn how endosomes keep order among their cargo to correctly localize and integrate different signaling inputs. another question is how exocytic and endocytic sorting machineries respond to signaling. in the developing respiratory system of drosophila, wg modulates cell adhesion and intercalation by regulating the recycling of e - cadherin this is achieved through upregulation of the transcription factor spalt (sal), which induces drip11 (drosophila rab11 interacting protein) to stimulate the small gtpase rab11 on recycling endosomes. in vertebrates, fibroblast growth factor signaling induces the transcription factor foxj1, a regulator of intraflagellar transport proteins, which drive the elongation of cilia [80 - 82 ]. known functions of cilia in cell polarity and signaling, and their assembly by membrane trafficking, have been comprehensively reviewed elsewhere. how these sensory organelles control, for example, the balance between canonical and non - canonical wnt signaling, or the shape of hh signaling gradients is still not clear. a better understanding of the relationship between signaling and trafficking will surely help to resolve these questions. | tissue patterning during development relies on cell communication by secreted proteins and receptors that engage in complex signaling crosstalk to induce distinct cell behaviors in a context - dependent fashion. here i summarize recent insights into basic mechanisms that control the distribution and activities of transforming growth factor beta, wnt, hedgehog, and notch proteins, by regulating trafficking decisions during secretion and endocytosis. |
the migration of cells and tissues is fundamental to metazoan life, driving tissue morphogenesis, homoeostasis and defence. cell migration requires dynamic reorganization of the actin cytoskeleton, with filaments providing mechanical support of protrusion of the front and retraction of the rear. the lamellipodium, a thin leaflet of plasma membrane filled with actin filaments, constitutes the key organelle generating the force for directional protrusion of the cell periphery (small, 2002 ; pollard, 2007). importantly, the actin filaments building the lamellipodium are oriented with their fast - growing, barbed ends pointing outwards (small, 1978), allowing the centrifugal growth of the network. work over the years has largely focused on signalling pathways stimulating the formation of these structures (raftopoulou and hall, 2004 ; disanza, 2005), and the molecular players driving actin polymerization to induce and maintain them (pollard and borisy, 2003 ; pollard, 2007). although the increase in knowledge on the biochemical activities of factors implicated in actin polymerization at the cell periphery has been significant, the detailed actin assembly and disassembly kinetics in lamellipodia are unsettled and controversial. breakthroughs in actin biochemistry include the discovery of the arp2/3 complex (machesky, 1994) and formins (woychik, 1990), recognition of their relevance in nucleating actin filaments (reviewed in goley and welch, 2006 ; goode and eck, 2007 ; pollard, 2007), as well as in vitro reconstitution of actin - based motility using purified proteins (loisel, 1999 ; romero, 2004). spire (quinlan, 2005) and cordon - bleu (ahuja, 2007), implicated in vesicle trafficking and neuronal morphogenesis, respectively, add to the list of actin nucleators found in eukaryotes. there is increasing consensus on the list of molecules harbouring key functions in lamellipodium protrusion (small, 2002 ; stradal and scita, 2006), mostly based on manipulations of protein expression or activity. suppression of arp2/3 complex components by rna interference (rnai) or sequestration of the complex in the cytosol inhibits lamellipodia formation (machesky and insall, 1998 ; kunda, 2003 ; steffen, 2006). arp2/3 complex activation in lamellipodia is thought to be mediated by wave complex (stradal, 2004) and cortactin, although the relevance of cortactin in this process is controversial (cosen - binker and kapus, 2006). heterodimeric capping protein is also essential for arp2/3 complex - mediated motility, both when reconstituted in vitro (loisel, 1999) and in vivo, as rnai - mediated knockdown causes excessive filopodia formation at the expense of lamellipodia (mejillano, 2004). another protein family of comparable relevance is adf / cofilin proteins, which were proposed to promote lamellipodial protrusion by driving both actin assembly and disassembly (carlier, 1999 ; ghosh, 2004 ; hotulainen, 2005 ; andrianantoandro and pollard, 2006). all these factors are found to be enriched in lamellipodial protrusions, sometimes with significant spatial variations (small, 2002), but their turnover rates in steady - state protrusion of live lamellipodia are much less well defined. for example, differences of opinion still exist about the site(s) of actin assembly within lamellipodia (wang, 1985 ; theriot and mitchison, 1991 ; small, 1995 ; watanabe and mitchison, 2002). by employing state - of - the - art bleaching and photoactivation approaches combined with a new method of analysis and mathematical modelling our findings, which reveal treadmilling for both actin and arp2/3 complex, but uncoupling of their dynamics from cortactin and cofilin, call for a re - evaluation of the roles of these proteins in lamellipodia assembly. the turnover of lamellipodial actin was initially examined using fluorescence recovery after photobleaching (frap) (wang, 1985) or photoactivation (theriot and mitchison, 1991), leading to the proposal of two distinct models for actin filament turnover in lamellipodia, treadmilling versus nucleation release, respectively. whereas the former mechanism assumes assembly of individual lamellipodial filaments at the front and disassembly at the rear, the latter features short filaments, released from the front, and capable of continuous turnover throughout the lamellipodium (small, 1995). several groups have subsequently studied actin network flow using fluorescent speckle microscopy, which exploits inhomogeneous incorporation of low amounts of fluorescent molecules into polymer (danuser and waterman - storer, 2006). again, analysis of single - molecule speckles in lamellipodia suggested that most of the actin filaments in the lamellipodium are generated away from the tip by a mechanism now termed basal polymerization ' (watanabe and mitchison, 2002), which is inconsistent, at least in part, with more recent studies (hotulainen, 2005 ; nakagawa, 2006 ; iwasa and mullins, 2007). to re - evaluate when and where actin filaments are nucleated within lamellipodia of migrating cells, we bleached or photoactivated actin derivatives expressed in motile b16-f1 melanoma cells at various subcellular locations (figure 1). in initial experiments, a laser scanning device of a confocal microscope was used to bleach rectangular regions in lamellipodia of cells expressing egfp actin, and recovery of fluorescence was recorded with wide - field imaging, enabling high spatial and temporal resolution (figure 1a and supplementary movie 1). at 2 s after bleaching, a sharp rim of actin was observed at the lamellipodium tip, which progressively expanded rearwards into more proximal regions of the lamellipodium with an average speed of 3.78 m / min (figure 1a) (n=13). these data already indicated a strong bias of incorporation of actin at the lamellipodium front, as observed previously (wang, 1985), and are inconsistent with the view deduced from in vitro studies that treadmilling does not contribute much to actin filament turnover in cells (pollard, 2007). to exclude the possibility that energy introduced into the system by bleaching may cause an experimentally induced modification of actin filament dynamics in our cells, we employed two additional methods to study actin network turnover in lamellipodia : photoactivation and a pseudo - fluorescence loss in photobleaching (flip) approach. activation of photoactivatable egfp - tagged actin co - expressed with mrfp actin revealed rapid rearward flow of the activated probe and its dissipation from the rear of the lamellipodium (figure 1b), which largely coincided with exchange of non - activated actin from the front (see supplementary movie 2). consistent with previous observations (wang, 1985 ; hotulainen, 2005 ; nakagawa, 2006), these data supported a treadmilling mode of actin filament turnover in lamellipodia. however, it was recently proposed that frap and photoactivation of lamellipodial actin may lead to erroneous interpretations, due to significant turnover of the network from within the bleached or photoactivated area while flowing rearward (watanabe and mitchison, 2002). to exclude a contribution of intra - lamellipodial assembly / disassembly events to network turnover, we bleached thin lines parallel to the lamellipodial leading edge and established that the bleached lines travelled rearwards with the lamellipodial mesh (not shown) similar to wider bleached regions (see above). in addition, we exploited the rapid translocation of unpolymerized actin to the leading edge discovered recently (zicha, 2003). in this flip type of experiment, we rapidly bleached the lamella in a position several microns behind the lamellipodium and recorded the incorporation of bleached actin monomers into the lamellipodial actin meshwork (figure 1c and supplementary movie 3). using photoactivation, the average speed of actin translocation to the leading edge was determined to be approximately 3.11.5 m / s in b16-f1 cells, with peak rates in individual cells of up to 6 m / s (data not shown). in the flip experiment, this rapid translocation of unpolymerized actin was detected as the incorporation of a narrow line of bleached actin into the lamellipodium tip (figure 1c). if there were an active reorganization of the lamellipodial network by intra - lamellipodial assembly / disassembly (watanabe and mitchison, 2002), we would expect a rapid dissipation of the line of bleached actin, which was not the case. instead, bleached actin from the lamella that incorporated into the lamellipodium tip flowed steadily rearwards in a well - defined line (figure 1c), consistent with treadmilling. a monte carlo model of diffusion was employed to simulate the fluorescence recovery observed in the lamellipodium shown in figure 1a (for details on the model, see supplementary data). the model that best fitted the experimental data is shown in figure 1d (compare figure 1a and d, see supplementary movie 4) and assumed actin assembly probability to be highest at the leading edge (for actin assembly and disassembly probability profiles relative to the distance from the leading edge, see figure 1e). from these data, we conclude that actin predominantly polymerizes at the lamellipodium front, presumably at the interface between plasma membrane and the filament ends in the network abutting the membrane. however, to obtain a more unbiased determination of the dynamic behaviour of actin, applicable also to the regulatory molecules studied here, we developed a quantifiable parameter which we called treadmilling factor ' (tmf) (figure 1f, see supplementary movie 5). to obtain the tmf, we extracted and plotted the recovery of fluorescence in both the front and the rear half of the lamellipodium over time (figure 1 g). upon normalization of fluorescence to compare individual movies (see materials and methods), the tmf was taken as the difference between the two curves averaged over time and constituted a direct measure of biased recovery from the front (figure 1 g). for the movie shown in figure 1a, the tmf was 0.41 (figure 1 g), which compared closely to 0.43 (figure 1h) obtained from the computer simulation depicted in figure 1d. according to definition, the latter value comes close to the highest possible value for a tmf for a component with continuous recovery from the front, at least for the lamellipodium width given (see supplementary data and below). in contrast, proteins with homogenous recovery over the entire lamellipodium will display tmfs close to 0. importantly, the tmf for egfp actin (0.44) obtained from the average curves of six movies recorded with a double scan - headed confocal (see supplementary movie 6) was practically identical to the value observed in the simulation (figure 1i). taken together, these data show that actin assembly in the lamellipodia of these motile cells occurs exclusively at the front, with the actin filament arrays turning over by treadmilling. in addition, rapid severing and re - annealing of actin filaments as proposed recently to drive dynamic network rearrangements in lamellipodia (miyoshi, 2006) can not occur with significant frequency in the lamellipodia of b16-f1 (this study) and mtln3 (see below) cells, as well as fibroblasts (wang, 1985) or neuronal cells (nakagawa, 2006), because such activities would preclude a strongly biased recovery from the front. having developed a system to record and analyse the spatial and temporal features of actin network turnover in b16-f1 lamellipodia, we turned to studying established regulators of lamellipodia protrusion ; initially the actin filament nucleating arp2/3 complex and its lamellipodial activators, the wave complex and cortactin. to ascertain reliable analysis of arp2/3 complex dynamics, both c- and n - terminal fusions of all seven subunits of the complex were screened for proper incorporation into lamellipodia, as described previously (rottner, 2006). of these, the smallest subunits arpc5 (also know as p16) and arpc4 (p20) were selected for further analysis. in contrast to recent observations with fluorescent speckle microscopy, which indicated enrichment only in the distal two - thirds of the lamellipodium (iwasa and mullins, 2007), we observed egfp - tagged arp2/3 complex subunits to accumulate in the entire lamellipodium (supplementary figure 1), consistent with original immunolabelling data (welch, 1997), although lamellipodia were on average narrower than in cells expressing egfp actin (see below). arp2/3 complex is considered to amplify the generation of rapidly growing barbed ends by mediating the branching of daughter filaments off the barbed ends or sides of mother filaments. although arp2/3 complex could in principle be activated by multiple molecules in lamellipodia, the wave complex, which is known to accumulate at the tips of these structures (steffen, 2004 ; stradal, 2004), is currently considered most relevant for their formation. previous analyses of arp2/3 complex speckles indicated dynamics different from actin speckles (watanabe and mitchison, 2002), with a biased incorporation at the lamellipodium front (miyoshi, 2006 ; iwasa and mullins, 2007). irrespective of its activation, arp2/3 complex may also be subject to rapid turnover in deeper lamellipodial regions, for example, upon network debranching and/or dissociation from actin, which may have previously been missed in single - molecule speckle analyses (iwasa and mullins, 2007). to establish lamellipodial arp2/3 complex dynamics, we performed frap experiments with cells expressing arpc4 (not shown) or arpc5 (figure 2a c). as shown in figure 2a and b (see also supplementary movie 7), arp2/3 complex largely recovered from the front, which would be consistent with its activation at the lamellipodium tip where the wave complex is localized (steffen, 2004). analysis of the representative cell in figure 2a showed a significant difference between fluorescence recovery at the front and rear part of the lamellipodium (figure 2b), which was confirmed by the average recovery behaviour of all cells analysed (figure 2c). as expression of arp2/3 complex subunits caused narrowing of the lamellipodium as compared with actin expressers and as the tmf is sensitive to changes in lamellipodium width (see supplementary figure 2a and b), we used the average lamellipodium width of arpc5 expressers for determining the tmfs of both arpc5 and actin (actinnarrow, termed n - actin in figure 2d). interestingly, the tmf obtained for actinnarrow was even less (0.28) than that measured for arpc5 (0.33 ; figure 2d ; see also table i). thus, these data confirm exclusive incorporation of arp2/3 complex at the lamellipodium front and preclude arp2/3 complex - mediated branching along filament sides in deeper regions of the lamellipodium and/or active capping / uncapping dynamics on filament pointed ends (mullins, 1998). arp2/3 complex turns over in the lamellipodium tip region with a half - time of recovery (t1/2) of 7.1 s, which is almost identical to actin (t1/2=7.5 s), as expected for components incorporated into the lamellipodium network by treadmilling. we next asked how this compares to the dynamics of the presumptive activator of arp2/3 complex at the lamellipodium tip, the wave complex. of its five constituents the ubiquitous complex comprises sra-1, nap1, abi-1, hspc300 and wave2 (gautreau, 2004)abl interacting proteins (abi) were the first to be shown to accumulate at the tips of protruding lamellipodia and filopodia (stradal, 2001). bleaching of abi-1 at the lamellipodium tip and analysis of its recovery revealed that abi-1 shows a comparably slow turnover in this location (figure 2e and f, and supplementary movie 8), with a t1/2 almost double that of arp2/3 complex (13.6 s). to examine whether abi-1 dynamics indeed reports back on the turnover of the entire wave complex, we also studied the turnover of wave2. as wave overexpression is known to suppress the protrusion of spontaneous lamellipodia, frap experiments on reasonably bright cells had to be combined with stimulation of lamellipodia formation by aluminium fluoride (alf4) treatment (hahne, 2001). surprisingly, wave2 turnover appeared significantly accelerated (t1/2=8.6) as compared with abi-1 (figure 2 g and h, and supplementary movie 9). the increased turnover was not due to alf4 treatment, as preliminary rates measured in low wave2 expressers without treatment was at least as fast (not shown). comparable turnover rates (t1/2=6.4) on the membrane were recently also reported for the haematopoietic nap1 isoform hem-1 (weiner, 2007). collectively, these data suggest that abi-1 dynamics at the lamellipodium tip does not correlate precisely with the dynamics of the wave complex. however, this does not necessarily indicate dissociations of wave and abi from each other or from other wave complex constituents in the lamellipodium tip. instead, the differential turnover rates may be explained by the fact that abi-1 is able to engage in actin regulatory complexes additional to wave complex (stradal and scita, 2006). thus, individual abi molecules may be retained in the lamellipodium tip by these additional interactions, for instance with eps8 or ena / vasp proteins (tani, 2003). this would be consistent with the similar turnover rates (t1/2=14.4 s) observed for the ena / vasp family member vasp (supplementary figure 3 and movie 10). notwithstanding this, arp2/3 complex turnover at the lamellipodium is worthwhile to be compared with that of its activator wave. although the half - times of fluorescence recovery in the tip region of the lamellipodium are comparable for wave and arp2/3 complex (see also table i), they are not inconsistent with multiple arp2/3 complex activations executed by individual wave complex units, in analogy to what is known for arp2/3 complex activators retained at bacterial or bead surfaces (wiesner, 2003 ; carlier and pantaloni, 2007 ; pollard, 2007). however, prerequisites for this assumption to hold true are as follows : the observed fluorescence intensities of arp2/3 complex and wave reflect comparable amounts of endogenous complexes present, and the actual zone of arp2/3 complex activation at the membrane, corresponding to wave localization, is much more restricted than what we define as the tip region by light microscopy, which is likely. in any case, our data also suggest that wave, abi and vasp are components of perhaps partially associated, larger protein assemblies, which share the feature of being pushed forward by the network of polymerizing actin filaments they are engaged in regulating. the second arp2/3 complex activator enriched in lamellipodia is the type ii nucleation - promoting factor cortactin (cosen - binker and kapus, 2006). in contrast to wave (figure 2 g), cortactin labels the entire lamellipodium (figure 3a and supplementary figure 4a and c), in a manner indistinguishable from arp2/3 complex (figure 2a, see also supplementary figure 1). nevertheless, its dynamics in the lamellipodium significantly differed from that of actin and arp2/3 (supplementary movies 1113). first, cortactin did not recover in a treadmilling manner as observed for actin or the arp2/3 complex (cortactin tmfs=0.15 and 0.16 for two independent constructs, respectively ; figure 3b and supplementary figure 4b). we conclude that the slight deviation from 0 was largely due to the density gradient of cortactin localization from distal to more proximal lamellipodial regions (figure 3a), which tightly follows that of actin (rottner, 1999 ; small, 2002), and not due to partial treadmilling. this view was corroborated by the best linear fit for the rear - half recovery curve, which was exponential for both cortactin constructs (figure 3b and supplementary figure 4b), and not sigmoidal as observed for treadmilling components such as actin and arp2/3 complex (figures 1e and 2c). second, the turnover of cortactin in the lamellipodium was comparably fast, albeit with moderately divergent recovery times measured for the two constructs (t1/2=6.4 and 3.8 s ; compare with t1/2 of actin in the entire lamellipodium=21.3 s ; see also table i). to exclude that the two different egfp cortactin constructs used did not display erroneous behaviour perhaps due to being non - functional, we tested a third, independently generated egfp cortactin, the functionality of which had previously been confirmed by reconstitution of a knockdown phenotype (zhu, 2007). not unexpectedly, this cortactin variant again displayed rapid, and continuous recovery throughout the entire lamellipodium, without convincing bias of recovery from the front (supplementary figure 4c and supplementary movie 13), revealing that cortactin recovery does not follow the treadmilling behaviour in lamellipodia as observed for actin and arp2/3 complex. thus, although the mechanistic subtleties of cortactin function in lamellipodia remain unclear, the dynamics of most cortactin molecules, at least in deeper lamellipodial regions, is inconsistent with their engagement in the promotion of arp2/3 complex activation. through its ability to interact with both f - actin and the arp2/3 complex (cosen - binker and kapus, 2006), cortactin may serve to stabilize arp2/3 complex - induced actin networks rather than drive their (arp2/3-mediated) assembly. two other lamellipodial proteins are considered indispensable for proper lamellipodium protrusion and arp2/3-mediated motility in general (carlier and pantaloni, 2007) : heterodimeric capping protein (mejillano, 2004) and adf / cofilin (kiuchi, 2007). similar to previous observations (mejillano, 2004 ; iwasa and mullins, 2007), we found capping protein enriched in the front region of the lamellipodium (supplementary figure 5), and frap experiments revealed a t1/2 of recovery at the lamellipodium tip of approximately 7.2 s (figure 3c and d, and supplementary movie 14), almost an order of magnitude slower than deduced from speckle analysis (miyoshi, 2006). the reason for this discrepancy is unclear, but could arise from the difficulty to ascertain speckle dynamics for proteins with tight spatial restriction, like capping protein (figure 3c), wave complex components or vasp. vasp not to display any continuous association with the lamellipodium tip (miyoshi, 2006), whereas frap experiments revealed quite slow turnover in this location (applewhite, 2007) (see also supplementary figure 2, supplementary movie 10 and table i). assuming a balance of actin assembly and disassembly in the lamellipodium at steady state, similar turnover rates at the lamellipodium tip for actin (t1/2 of approximately 7.5 s) and capping protein (t1/2=7.2 s) indicate that capping protein does not dissociate from individual filaments until they are disassembled. this is probable at least for filaments short enough to be disassembled within the observed time frame. the dependence of capping protein accumulation in the tip region on actin filaments is corroborated by its loss upon filament depolymerization by latrunculin b (supplementary figure 6 and supplementary movie 15). furthermore, the restriction of capping protein to the front is inconsistent with significant retrograde flow of capped filaments into the lamellipodium mesh. as the absence of actin assembly behind the tip region as observed here precludes the existence of elongating barbed ends throughout the lamellipodium (see above), maintenance of capping behind the tip region (if it exists) may be accomplished by additional factors. potential candidates include eps8 or twinfilin (carlier and pantaloni, 2007), although it should be noted that none of these factors are capable apparently of compensating for an essential aspect of capping protein function in lamellipodium protrusion revealed by rnai (mejillano, 2004). an alternative explanation of the data is that heterodimeric capping protein selectively aborts growth of short, non - productive filaments, perhaps subtending non - productive angles to the protruding lamellipodium front. future work is required to test this possibility. on the basis of in vivo and in vitro observations, adf / cofilin is considered to enhance actin treadmilling by promoting both filament disassembly (loisel, 1999 ; kiuchi, 2007) and assembly (ghosh, 2004 ; andrianantoandro and pollard, 2006). interestingly, kiuchi (2007) concluded that adf / cofilin supports protrusion by maintaining a high actin monomer pool instead of driving nucleation, but they did not examine cofilin dynamics in the lamellipodium. we show here that cofilin, which readily associates with the entire lamellipodium (figure 4a and supplementary movie 16), shows frap dynamics markedly different from actin or arp2/3 complex, with a tmf of 0.061, and a t1/2 for the entire lamellipodium of 6.5 s (figure 4a and b). to exclude that ectopically expressed egfp - tagged cofilin is subject to rapid inactivation by phosphorylation on serine 3 (kiuchi, 2007), with the potential to change its dynamics, we also examined the turnover of a non - phosphorylatable constitutively active mutant (s3a) and an inactive, phosphorylation - mimetic cofilin (s3d). interestingly, both mutants (figure 4e, and supplementary movies 17 and 18) recovered throughout the entire lamellipodium with even faster turnover rates than wild - type cofilin (figure 4e), indicating that phosphorylation / dephosphorylation events may retain the protein in the lamellipodium, but the tmf was virtually identical to the wild - type protein (see also table i). these data show that inactivation (by phosphorylation) can not have caused erratic behaviour of our wild - type cofilin. to prove that egfp tagging had not generally abrogated the functionality of our cofilin, its activity was examined by a number of additional approaches. first, purified, egfp - tagged cofilin quenched the fluorescence of pyrenylated actin filaments in a manner indistinguishable from untagged cofilin (andrianantoandro and pollard, 2006), demonstrating actin filament binding in vitro (figure 4c). in addition, co - immunoprecipitations showed interaction of all expressed egfp - tagged cofilin variants with actin in cell extracts, although, as expected, with less efficiency for the inactive variant (supplementary figure 7). furthermore, filament depolymerization / severing mediated by egfp - tagged wild - type cofilin was demonstrated both by co - sedimentation assay (figure 4d) and by direct observation of fragmentation of individual actin filaments by total internal reflection fluorescence (tirf) microscopy (supplementary figure 8). thus, no indication was obtained to suggest that egfp tagging abrogated the function of the cofilin variants used here, neither in vivo nor in vitro. importantly, and as opposed to previous suggestions (ghosh, 2004), comparison of actin and cofilin dynamics in lamellipodia thus precludes a significant role for cofilin in promoting nucleation, at least in b16-f1 cells. instead, our data are consistent with cofilin supporting protrusion indirectly by promoting f - actin disassembly both in lamellipodia and other cellular locations (kiuchi, 2007). finally, to confirm the general relevance of this observation, we extended our experimental approach to mtln3 mammary carcinoma cells, which are frequently used to study cofilin function in vivo (ghosh, 2004 ; van rheenen, 2007). mtln3 cells expressing egfp actin or cofilin were induced to form lamellipodia with egf and subjected to frap analysis as described above (figure 5 and supplementary movies 19 and 20). interestingly, the actin network in lamellipodia stimulated by egf also turned over by treadmilling, whereas cofilin did not, indistinguishable from the observations made with b16-f1 cells moving on laminin. collectively, from previous work and the data presented here, we provide a comprehensive model of steady - state lamellipodium protrusion (figure 6), in which arp2/3-mediated birth of filaments at the lamellipodium tip activated by wave complex is counterbalanced by capping of unproductive filaments at the tip and actin disassembly from the rear. in this scenario, neither cortactin nor cofilin is engaged in nucleation (ghosh, 2004 ; andrianantoandro and pollard, 2006 ; cosen - binker and kapus, 2006) or severing and re - annealing, as proposed for cofilin recently (miyoshi, 2006). our findings call for a revision of ideas about lamellipodia turnover (watanabe and mitchison, 2002) and the role of cortactin in actin - based motile processes. gst - tagged cofilin constructs were expressed in escherichia coli strain rossetta (promega) and purified from bacterial extracts on glutathione - conjugated agarose (sigma - aldrich, germany) using standard procedures. the gst tags were cleaved by incubating the purified fusion proteins with prescission protease in phosphate - buffered saline (pbs), ph 7.3, supplemented with 1 mm dithiothreitol (dtt) and 1 mm edta overnight at 4c. after cleavage, the gst tags were removed by passing the protein solution over glutathione - conjugated agarose columns. cofilin and egfp cofilin remained in the flow through and were subsequently dialysed against pbs containing 1 mm dtt and 5 mm benzamidine. protein concentrations were calculated from the predicted extinction coefficients (invitrogen ; vector nti software). actin was purified from rabbit skeletal muscle as described (spudich and watt, 1971) and subsequently gel filtered on a superdex 200 column using an kta purifier system (amersham pharmacia biotech). for high - speed actin sedimentation assays, g - actin was first polymerized in actin polymerization buffer containing 10 mm imidazole, 2 mm mgcl2, 0.2 mm cacl2, 1 mm na - atp, 1 mm dtt and 50 mm kcl, ph 7.2 for 2 h at 21c. subsequently, filamentous actin was incubated in actin polymerization buffer either in the presence or absence of untagged or gfp - tagged cofilin for additional 2 h at 21c. the protein mixtures were then sedimented at 100 000 g in a beckman optima table top ultracentrifuge for 30 min, and the pellets brought to the original volume in sds sample buffer. quenching of pyrenyl fluorescence by cofilin was determined by fluorescence spectroscopy on a jasco fp-6500 spectrofluorometer at 343 nm excitation wavelength and 384 nm emission wavelength. briefly, 3 m f - actin (10% pyrenyl labeled) in actin polymerization buffer was measured alone or after the addition of either untagged or gfp - tagged cofilin (1 m each) at 21c. mouse melanoma cells (b16-f1) were from american type culture collection (atcc crl-6323) and maintained in dmem (invitrogen, germany) with 10% fcs (paa laboratories, austria) at 37c in the presence of 5% co2. mtln3 cells, kindly provided by jeff segall and bob van de water, were maintained in dmem, 5% fbs (sigma) and 2 mm glutamine. cells were transfected using superfect (invitrogen) and fugene hd (roche) for b16-f1 and mtln3, respectively. following transfection, b16-f1 cells were plated in ham 's f12 medium (sigma - aldrich) containing 10% fcs onto acid - washed glass coverslips coated with 25 g / ml laminin (sigma - aldrich), and examined on the same day. aluminium fluoride treatment was carried out as described (steffen, 2004). mtln3 cells were seeded on glass overnight and stimulated with egf (5 nm) shortly before movie acquisition. cells were observed in an open, heated chamber (warner instruments, reading, uk) at 37c on inverted microscopes. initial experiments as the one shown in figure 1a were performed on a lsm 510 meta (zeiss, jena, germany), which was equipped with a 100, 1.45na plan - fluar tirf objective (zeiss) and an interline transfer, progressive scan ccd camera (coolsnaphq ; photometrics, tucson, az, usa) driven by metamorph software (molecular devices corp., selected cellular areas covering parts of protruding lamellipodia were bleached (30 iterations at full laser power at 488 nm, 30 mw argon laser) immediately after and before one full - frame scan of respective fields, which was followed by switching to epi - fluorescence imaging using a mercury lamp (100 w) as light source. switching time was approximately 2 s. the majority of experiments was performed using a double - scan - headed confocal microscope (fluoview1000, olympus), allowing simultaneous imaging of egfp- and/or rfp - tagged probes (with 30 mw 488 nm multiline argon and/or 20 mw 561 nm solid - state lasers, respectively) and photobleaching / activation using a 20 mw 405 nm diode laser. circular regions and rectangles were bleached / photoactivated in the tornado and regular line - scanning modes, respectively. egfp and mrfp imaging was carried out at laser powers of approximately 15 and 1020%, respectively. a 100 /1.45na planapo tirf objective (olympus inc.) image analysis was carried out on a pc using fv10-asw 1.6 viewer (olympus inc., olympus, hamburg, germany), imagej (http://rsb.info.nih.gov/ij/), metamorph (molecular devices corp.) and adobe photoshop cs software. frap data were analysed as described in supplementary data, using sigmaplot 10.0 (scientific solutions sa, pully - lausanne, switzerland) and microsoft excel 2000. the diagrams shown in figures 1f and 6 were drawn using microsoft powerpoint 2000 and adobe illustrator cs2, respectively. the simulations of actin recovery in the lamellipodium were carried out as detailed in the supplementary data and using mathematica version 5.2. gst - tagged cofilin constructs were expressed in escherichia coli strain rossetta (promega) and purified from bacterial extracts on glutathione - conjugated agarose (sigma - aldrich, germany) using standard procedures. the gst tags were cleaved by incubating the purified fusion proteins with prescission protease in phosphate - buffered saline (pbs), ph 7.3, supplemented with 1 mm dithiothreitol (dtt) and 1 mm edta overnight at 4c. after cleavage, the gst tags were removed by passing the protein solution over glutathione - conjugated agarose columns. cofilin and egfp cofilin remained in the flow through and were subsequently dialysed against pbs containing 1 mm dtt and 5 mm benzamidine. protein concentrations were calculated from the predicted extinction coefficients (invitrogen ; vector nti software). actin was purified from rabbit skeletal muscle as described (spudich and watt, 1971) and subsequently gel filtered on a superdex 200 column using an kta purifier system (amersham pharmacia biotech). for high - speed actin sedimentation assays, g - actin was first polymerized in actin polymerization buffer containing 10 mm imidazole, 2 mm mgcl2, 0.2 mm cacl2, 1 mm na - atp, 1 mm dtt and 50 mm kcl, ph 7.2 for 2 h at 21c. subsequently, filamentous actin was incubated in actin polymerization buffer either in the presence or absence of untagged or gfp - tagged cofilin for additional 2 h at 21c. g in a beckman optima table top ultracentrifuge for 30 min, and the pellets brought to the original volume in sds sample buffer. quenching of pyrenyl fluorescence by cofilin was determined by fluorescence spectroscopy on a jasco fp-6500 spectrofluorometer at 343 nm excitation wavelength and 384 nm emission wavelength. briefly, 3 m f - actin (10% pyrenyl labeled) in actin polymerization buffer was measured alone or after the addition of either untagged or gfp - tagged cofilin (1 m each) at 21c. mouse melanoma cells (b16-f1) were from american type culture collection (atcc crl-6323) and maintained in dmem (invitrogen, germany) with 10% fcs (paa laboratories, austria) at 37c in the presence of 5% co2. mtln3 cells, kindly provided by jeff segall and bob van de water, were maintained in dmem, 5% fbs (sigma) and 2 mm glutamine. cells were transfected using superfect (invitrogen) and fugene hd (roche) for b16-f1 and mtln3, respectively. following transfection, b16-f1 cells were plated in ham 's f12 medium (sigma - aldrich) containing 10% fcs onto acid - washed glass coverslips coated with 25 g / ml laminin (sigma - aldrich), and examined on the same day. aluminium fluoride treatment was carried out as described (steffen, 2004). mtln3 cells were seeded on glass overnight and stimulated with egf (5 nm) shortly before movie acquisition. cells were observed in an open, heated chamber (warner instruments, reading, uk) at 37c on inverted microscopes. initial experiments as the one shown in figure 1a were performed on a lsm 510 meta (zeiss, jena, germany), which was equipped with a 100, 1.45na plan - fluar tirf objective (zeiss) and an interline transfer, progressive scan ccd camera (coolsnaphq ; photometrics, tucson, az, usa) driven by metamorph software (molecular devices corp., selected cellular areas covering parts of protruding lamellipodia were bleached (30 iterations at full laser power at 488 nm, 30 mw argon laser) immediately after and before one full - frame scan of respective fields, which was followed by switching to epi - fluorescence imaging using a mercury lamp (100 w) as light source. switching time was approximately 2 s. the majority of experiments was performed using a double - scan - headed confocal microscope (fluoview1000, olympus), allowing simultaneous imaging of egfp- and/or rfp - tagged probes (with 30 mw 488 nm multiline argon and/or 20 mw 561 nm solid - state lasers, respectively) and photobleaching / activation using a 20 mw 405 nm diode laser. circular regions and rectangles were bleached / photoactivated in the tornado and regular line - scanning modes, respectively. egfp and mrfp imaging was carried out at laser powers of approximately 15 and 1020%, respectively. a 100 /1.45na planapo tirf objective (olympus inc.) image analysis was carried out on a pc using fv10-asw 1.6 viewer (olympus inc., olympus, hamburg, germany), imagej (http://rsb.info.nih.gov/ij/), metamorph (molecular devices corp.) and adobe photoshop cs software. frap data were analysed as described in supplementary data, using sigmaplot 10.0 (scientific solutions sa, pully - lausanne, switzerland) and microsoft excel 2000. the diagrams shown in figures 1f and 6 were drawn using microsoft powerpoint 2000 and adobe illustrator cs2, respectively. the simulations of actin recovery in the lamellipodium were carried out as detailed in the supplementary data and using mathematica version 5.2. supplementary movie 1 supplementary movie 2 supplementary movie 3 supplementary movie 4 supplementary movie 5 supplementary movie 6 supplementary movie 7 supplementary movie 8 supplementary movie 9 supplementary movie 10 supplementary movie 11 supplementary movie 12 supplementary movie 13 supplementary movie 14 supplementary movie 15 supplementary movie 16 supplementary movie 17 supplementary movie 18 supplementary movie 19 supplementary movie 20 supplementary movie legends | cell migration is initiated by lamellipodia membrane - enclosed sheets of cytoplasm containing densely packed actin filament networks. although the molecular details of network turnover remain obscure, recent work points towards key roles in filament nucleation for arp2/3 complex and its activator wave complex. here, we combine fluorescence recovery after photobleaching (frap) of different lamellipodial components with a new method of data analysis to shed light on the dynamics of actin assembly / disassembly. we show that arp2/3 complex is incorporated into the network exclusively at the lamellipodium tip, like actin, at sites coincident with wave complex accumulation. capping protein likewise showed a turnover similar to actin and arp2/3 complex, but was confined to the tip. in contrast, cortactin another prominent arp2/3 complex regulator and adf / cofilin previously implicated in driving both filament nucleation and disassembly were rapidly exchanged throughout the lamellipodium. these results suggest that arp2/3- and wave complex - driven actin filament nucleation at the lamellipodium tip is uncoupled from the activities of both cortactin and cofilin. network turnover is additionally regulated by the spatially segregated activities of capping protein at the tip and cofilin throughout the mesh. |
each year, streptococcus pneumoniae causes more than 800,000 deaths worldwide in children under 5 years of age. currently marketed s. pneumoniae vaccines, which are based on polysaccharide capsular antigens from the most common strains, have substantially reduced pneumococcal disease rates. however, because serotypes can vary between countries or regions, coverage may be incomplete in some cases. moreover, serotype replacement might eventually render these vaccines less effective. to provide broader, more diverse, and possibly infection stage - specific protection, vaccines based on conserved proteins are being investigated., pneumococcal histidine triad protein d (phtd) is a conserved surface protein that mediates attachment to respiratory epithelial cells and can elicit a protective immune response. in mice, intranasal immunization with phtd generates robust serum antibody and cd4 th1-biased immune memory responses and confers protection against pneumococcal colonization. a second study in mice showed that vaccination with phtd protects against nasopharyngeal and lung colonization. in a primate study, vaccination with phtd and chemically detoxified pneumolysin induced high levels of antibodies and protected against a challenge with s. pneumoniae serotype 19f. a phase i trial in adults 1850 years of age showed that a aluminum phosphate - adjuvanted phtd vaccine candidate was well tolerated, immunogenic, and could be boosted by a second vaccine dose. during development of an enzyme - linked immunosorbent assay (elisa) to measure antibody responses in the phase i trial, we found that individual and pooled serum from unimmunized healthy adults contained substantial phtd - binding antibody (data not shown). to further investigate the immune response elicited by a phtd - based pneumococcal vaccine, we developed a murine passive protection sepsis model for assessing the functional activity of human anti - phtd antibodies. naturally occurring human phtd - binding antibody was purified from a commercial pooled serum (obtained from approximately 200 healthy individuals ; sigma, st. the concentration of anti - phtd antibody was determined by elisa, and its purity and specificity were confirmed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis, western blotting, and competition with recombinant phtd (data not shown). the purified phtd - binding antibodies were passively transferred by intraperitoneal injection (200 l) to 6- to 8-week - old female cba / cahn - btk xid /j (cba / n) mice (jackson laboratories, bar harbor, me). after 1 h, the mice were challenged by intravenous injection with a lethal dose (50 colony forming units [cfu ] in 200 l) of s. pneumoniae strain a66.1 (serotype 3) (obtained from d. briles, university of alabama - birmingham). the proportion of mice surviving at 14 days post - challenge increased with the concentration of anti - phtd antibody (fig. the dose providing 50% survival (ed50) was estimated to be 1679 elisa units (eu)/ml (95% confidence interval [ci ], 14201946) by logistic regression with probit link. 6- to 8-week - old female nave cba / n mice (n = 5/group) received an intraperitoneal injection of 200 l test sample or pbs. after 1 h, mice were challenged intravenously with a lethal dose of s. pneumoniae strain a66.1 (serotype 3), and survival was monitored for 14 days. data were analyzed using logistic regression with probit link under proc glimmix in sas version 8.2 to determine the ed50. in each plot, circles indicate survival data for individual samples, and the best fit regression is shown as a green line, with the upper and lower limits of the 95% confidence interval shown as yellow and red lines, respectively. the horizontal line indicates a median response of 50% survival. in (a), mice were injected with 2.827.5 eu (110 g) of purified anti - phtd antibody or pbs. survival data were from 4 passive protection experiments, which included 13 survival points. in five of the 13 cases (206, 275, 687, 1375, 2063, and 2750 eu / ml), the passive protection experiment was performed twice, and for 2 of these (275 and 2750 eu / ml), the points overlap and appear as a single data point. protection experiments for 3 cases (43, 435, and 2178 eu / ml) were not repeated and were included to provide a more appropriate range of antibody concentrations. in (b), mice were injected with 1:20 to 1:60 post - immune sera. to account for the baseline levels of protection seen in the pre - immune sera, the analysis was corrected for over - dispersion using proc genmod with the scale = option in sas version 9.13 and based on survival data from mice that received pre- and post - immune sera. 6- to 8-week - old female nave cba / n mice (n = 5/group) received an intraperitoneal injection of 200 l test sample or pbs. after 1 h, mice were challenged intravenously with a lethal dose of s. pneumoniae strain a66.1 (serotype 3), and survival was monitored for 14 days. data were analyzed using logistic regression with probit link under proc glimmix in sas version 8.2 to determine the ed50. in each plot, circles indicate survival data for individual samples, and the best fit regression is shown as a green line, with the upper and lower limits of the 95% confidence interval shown as yellow and red lines, respectively. the horizontal line indicates a median response of 50% survival. in (a), mice were injected with 2.827.5 eu (110 g) of purified anti - phtd antibody or pbs. survival data were from 4 passive protection experiments, which included 13 survival points. in five of the 13 cases (206, 275, 687, 1375, 2063, and 2750 eu / ml), the passive protection experiment was performed twice, and for 2 of these (275 and 2750 eu / ml), the points overlap and appear as a single data point. protection experiments for 3 cases (43, 435, and 2178 eu / ml) were not repeated and were included to provide a more appropriate range of antibody concentrations. in (b), mice were injected with 1:20 to 1:60 post - immune sera. to account for the baseline levels of protection seen in the pre - immune sera, the analysis was corrected for over - dispersion using proc genmod with the scale = option in sas version 9.13 and based on survival data from mice that received pre- and post - immune sera. we next examined the activity of vaccine - induced antibodies in the passive protection model. in the phase i clinical trial, adults were vaccinated on weeks 0 and 4 with 6, 25, or 100 g of the candidate aluminum phosphate - adjuvanted phtd vaccine. as described above, we found substantial pre - existing anti - phtd antibodies in pooled sera from vaccine - nave healthy adults. we therefore selected subjects who had the lowest pre - immune passive protection activity and the highest post - immune phtd antibody titers for testing in the passive protection assay. for testing in subsequent experiments, we selected a dilution for each subject where the pre - immune serum was not protective in the passive protection assay. all samples were initially tested at a dilution of 1:20, and survival was compared to control mice injected with pbs. human pre - immune sera that were protective at a 1:20 dilution and for which corresponding post - immune sera (week 8) had an increase in titer of at least 1000 eu / ml were further tested at a 1:40 dilution. pre - immune sera shown to be protective at a 1:40 dilution and for which corresponding post - immune sera had an increase in titer of at least 2000 eu / ml over baseline were further tested at a 1:60 dilution. of the 54 serum pairs tested, we identified appropriate dilutions for 18 (table 1). vaccination increased the anti - phtd antibody titer in all subjects at all vaccine doses (6, 25, and 100 g). 21, 36, 37, 41, 43, 45, 51, 57, 58, and 61) significantly delayed death in the murine challenge model compared to the same dilution of the corresponding pre - immune serum. in addition, 2 of these post - immune sera (from subject nos. 37 and 43) significantly improved survival compared to the corresponding pre - immune serum. as with the naturally acquired antibody, survival increased with the concentration of anti - phtd antibody as determined by elisa (fig. the ed50 in this case was estimated to be 1331 eu / ml (95% ci, 7622038). table 1.anti-phtd titers and survival mediated by the 18 selected paired pre-/post - immune seraanti - phtd titer (eu / ml)mice surviving at day 14 (n / n)significant difference (p<0.05)subjectvaccine dosepre - immune serum (week 0)post - immune serum (week 8)differenceserum dilution testedpre - immune serum (week 0)post - immune serum (week 8)delay to deathprotection46 g163125210881:201/53/566 g853452611:200/50/586 g138225861:200/50/5156 g872141271:200/50/5206 g663632971:201/52/5216 g77157801:200/52/5x236 g2106104001:200/51/52525 g137134912121:401/50/53625 g109136412561:401/54/5x3725 g14510398931:200/55/5xx4125 g204634431:200/50/5x4325 g89247823891:401/55/5xx45100 g69141213431:200/52/5x51100 g256140411481:400/50/5x56100 g302222319211:400/52/557100 g107323931321:600/53/5x58100 g326474344171:406/1010/10x61100 g288323129441:400/104/10xin the phase i clinical trial, healthy adults were vaccinated twice (week 0, 4) with the candidate aluminum phosphate - adjuvanted phtd vaccine. dilutions (1:20) of pre - immune sera (200 l) were passively transferred by intraperitoneal injection to 6- to 8-week - old female nave cba / n mice (n = 5/group). after 1 h, the mice were challenged by intravenous injection of a lethal dose (50 cfu in 200 l) of s. pneumoniae strain a66.1 (serotype 3). pre - immune sera that were protective at a 1:20 dilution and for which corresponding post - immune sera (week 8) had an increase in titer of at least 1000 eu / ml was further tested at a 1:40 dilution. pre - immune sera shown to be protective at a 1:40 dilution and for which corresponding post - immune sera had an increase in titer of at least 2000 eu / ml over baseline were further tested at a 1:60 dilution. using this method, appropriate dilutions for testing paired pre - immune and post - immune sera from these subjects were tested in the passive protection assay, and survival after 14 days is shown. delay to death was assessed by survival distribution functions using the product - limit approach and compared between groups of mice by log - rank test using proc lifetest in sas version 9.13. differences in protection were compared by one - sided fisher 's exact test using proc freq with the exact option in sas version 9.13. anti - phtd titers and survival mediated by the 18 selected paired pre-/post - immune sera in the phase i clinical trial, healthy adults were vaccinated twice (week 0, 4) with the candidate aluminum phosphate - adjuvanted phtd vaccine. dilutions (1:20) of pre - immune sera (200 l) were passively transferred by intraperitoneal injection to 6- to 8-week - old female nave cba / n mice (n = 5/group). after 1 h, the mice were challenged by intravenous injection of a lethal dose (50 cfu in 200 l) of s. pneumoniae strain a66.1 (serotype 3). survival was followed for 14 days. pre - immune sera that were protective at a 1:20 dilution and for which corresponding post - immune sera (week 8) had an increase in titer of at least 1000 eu / ml was further tested at a 1:40 dilution. pre - immune sera shown to be protective at a 1:40 dilution and for which corresponding post - immune sera had an increase in titer of at least 2000 eu / ml over baseline were further tested at a 1:60 dilution. using this method, appropriate dilutions for testing were identified for the 18 subjects shown. paired pre - immune and post - immune sera from these subjects were tested in the passive protection assay, and survival after 14 days is shown. delay to death was assessed by survival distribution functions using the product - limit approach and compared between groups of mice by log - rank test using proc lifetest in sas version 9.13. differences in protection were compared by one - sided fisher 's exact test using proc freq with the exact option in sas version 9.13. finally, we performed ligand competition experiments to investigate the specificity of the vaccine - induced activity. for these experiments, we selected 5 serum pairs (subject nos. 4, 21, 37, 43, and 61) that spanned the different vaccine doses and functional activities. 4 and 61 were selected because the post - immune sera significantly increased survival at day 14 compared to the pre - immune sera, and sera from subject nos. 37 and 43 were selected because the post - immune sera increased survival at day 14 and delayed death compared to the pre - immune serum. 21 because the post - immune serum had weak or no functional activity, as indicated by a non - significant delay to death (p = 0.06) and small increase (79.9 eu / ml) in anti - phtd titer compared to the pre - immune serum. prior to testing in the passive protection model, the selected serum pairs were incubated for 1 h with recombinant phtd to block phtd - binding antibodies. in parallel reactions, sera were incubated with lytb, another s. pneumoniae surface protein, as a control. compared to the pre - immune sera, post - immune sera from all subjects except no. 21 significantly increased mouse survival when incubated with lytb, whereas survival was not significantly increased by any of the post - immune sera incubated with phtd (table 2). therefore, most or all of the protective activity in the post - immune sera was due to anti - phtd antibodies. in summary, our results show that the candidate phtd vaccine induces anti - phtd antibodies that can protect against s. pneumoniae infection. another phtd candidate vaccine, adjuvanted with as02v, was recently reported to be immunogenic and well tolerated in adults and to induce antibodies that passively protect mice against a lethal dose of s. pneumoniae. vaccination with the as02v - adjuvanted candidate vaccine increased the protective activity in sera from older but not younger adults. the authors concluded that the difference between the 2 age groups was due to higher baseline activity in the younger adults. in the current study, we tried to avoid interference from such baseline functional activity by testing serum dilutions at which the corresponding pre - immune sera was not protective. in addition, we performed competition experiments to confirm that the functional activity in the sera was specifically due to anti - phtd antibodies and not to antibodies to other pneumococcal antigens, which could have arisen from a s. pneumoniae infection during the trial. table 2.specificity of vaccine - induced antibodysurvival, n (%) + lytb+ phtdsubjectdilution testedpre - immunepost - immunep - valuepre - immunepost - immunep - value41:204 (26.7)11 (73.3)0.0131 (6.7)3 (20.0)ns211:200 (0)1 (6.7)ns1 (6.7)0 (0.0)ns371:201 (6.7)14 (93.3)<0.0010 (0.0)0 (0.0)ns431:401 (6.7)7 (46.7)0.0180 (0.0)2 (13.3)ns611:400 (0.0)4 (26.7)0.04980 (0.0)0 (0.0)nspaired pre- and post - immune sera from subject nos. 4, 21, 37, 43, and 61 were incubated at the indicated dilutions with 20 g / ml phtd or lytb proteins for 1 h. the sera (200 l) were then administered by intraperitoneal injection to 6- to 8-week - old female nave cba / n mice (n = 15/group). after 1 h, the mice were challenged intravenously with a lethal dose (50 cfu in 200 l) of s. pneumoniae strain a66.1 (serotype 3), and survival was monitored for 14 days. p - values were calculated using a one - sided fisher 's exact test comparing survival in mice injected with matched post - immune and pre - immune sera in sas version 8.2. specificity of vaccine - induced antibody paired pre- and post - immune sera from subject nos. 4, 21, 37, 43, and 61 were incubated at the indicated dilutions with 20 g / ml phtd or lytb proteins for 1 h. the sera (200 l) were then administered by intraperitoneal injection to 6- to 8-week - old female nave cba / n mice (n = 15/group). after 1 h, the mice were challenged intravenously with a lethal dose (50 cfu in 200 l) of s. pneumoniae strain a66.1 (serotype 3), and survival was monitored for 14 days. p - values were calculated using a one - sided fisher 's exact test comparing survival in mice injected with matched post - immune and pre - immune sera in sas version 8.2. ns, not significant (p 0.05). according to logistic analysis, naturally acquired and vaccine - induced antibodies had overlapping activities in the passive protection model (ed50 = 1679 eu / ml [95% ci, 14201946 ] for naturally acquired antibodies vs. 1331 eu / ml [95% ci, 7622038 ] for vaccine - induced antibodies). another study also found that naturally acquired anti - phtd antibody from human serum can protect mice against a lethal s. pneumoniae intranasal challenge ; however, the study did not examine the relationship between functional activity and vaccination, antibody quantity, or antibody quality. accordingly, our current results indicate that the candidate phtd vaccine increases functional activity, at least in part, by increasing the antibody concentration. our preliminary epitope mapping studies and experiments measuring serum binding to bacterial surfaces suggest that vaccination can also expand the epitope repertoire of anti - phtd antibodies (data not shown). collectively, these results showed that human anti - phtd antibodies, whether naturally acquired or induced by the phtd candidate vaccine, are functional. this supports the development of the phtd candidate as part of a broadly protective pneumococcal vaccine. | currently marketed streptococcus pneumoniae vaccines are based on polysaccharide capsular antigens from the most common strains. pneumococcal histidine triad protein d (phtd) is a conserved surface protein that is being evaluated as a candidate for a vaccine with improved serotype coverage. here, we measured the functional activity of human anti - phtd antibodies in a passive protection model wherein mice were challenged with a lethal dose of s. pneumoniae by intravenous injection. this functional activity was compared with anti - phtd antibody concentrations measured by enzyme - linked immunosorbent assay (elisa) to estimate the 50% protective dose (ed50). anti - phtd antibodies affinity purified from pooled normal human sera passively protected mice with an ed50 of 1679 elisa units / ml (95% confidence interval, 14201946). sera from subjects injected with aluminum - adjuvanted phtd in a phase i trial had similar activity per unit of antibody (ed50 = 1331 elisa units / ml [95% confidence interval, 7622038 ]). vaccine - induced activity in the passive protection model was blocked by pre - incubation with recombinant phtd but not by a control s. pneumoniae antigen (lytb). these results show that human anti - phtd antibodies, whether naturally acquired or induced by the phtd candidate vaccine, are functional. this supports the development of the phtd candidate as part of a broadly protective pneumococcal vaccine. |
in 1962, bartter. described a clinical syndrome characterized by hypokalemia, metabolic alkalosis, hyperreninemia, hyperaldosteronism and normal blood pressure. it occurs mostly in childhood or adolescence, and initial presentation in patients over 40 years of age was very rare. neverthless, neverthless, the syndrome has aroused great interest in many clinical investigators because it may provide new insights in to renal electrolyte metabolism and the pathophysiology of hypertension. we recently experienced a case of adult - onset bartter s syndrome who showed hypokalemia, metabolic alkalosis, normotension, hyperreninemia, hyperaldosteronism and juxtaglomerular cell hyperplasia. a 40-year - old woman was admitted to the chonnam university hospital, with a three months history of severe generalized weakness and fatigue. the first episode occurred three years ago and spontaneously remitted from it following bed rest, she denied ingestion of licorice, diuretics, laxative or any other medication, and she had no significant nausea, vomiting or diarrhea. the patient appeared relatively well and had a height of 154 cm and a weight of 52 kg. the pulse rate was 98/min, the blood pressure 100/70 mmhg, and the respiration rate 18/min. meq / l for serum potassium, 32.7 meq / l for serum bicarbonate, 86.1 mmhg for pao2, 48 mmhg for paco2 and 32 mmol / l for total co2 content. serum sodium averaged 139 meq / l, chloride 85 meq / l, total calcium 5.1 meq / l, ionized calcium 2.0 meq / l, inorganic phosphorus 4.7 mg / dl, magnesium 1.6 meq / l, uric acid 13.4 mg / dl and serum osmolality 289 mosl / kg. the blood urea nitrogen was 44.0 mg / dl, serum creatinine 1.7 mg / dl and creatinine clearance 35 ml / min. pertinent studies of the blood and serum revealed a normal hemoglobin level, hematocrit value, white blood cell count, total protein, albumin, alkaline phosphatase and transaminase levels. twenty four - hour urinary excretion of sodium was 183 meq, potassium 67 meq, chloride 247 meq, calcium 120 mg, protein 150 mg, glucose 30 mg and urine amounts 2,300 cc. the plasma renin activity was 48.2 ng / ml / hr and aldosterone level 34.4 ng / dl. fena was 2.6%, fecl, 5.7%, fek 55%, and feuric acid 8.2%. thiazide and furosemide were not detected in her urine by high - performance liquid chromatography at the department of pharmacology, chonnam university medical school, kwangju, korea. juxtaglomerular hyperplasia of varying degrees and multiple vacuolization of proximal tubule due to chronic hypokalemia were noted (fig. 1, 2). the tubules, interstitium and blood vessels were entirely normal with no scarring or atrophy. the constellation of hypokalemia, relative hypotension, increased renin activity, increased aldosterone level and juxtaglomerular hyperplasia substantiated the diagnosis of bartter s syndrome. following the renal biopsy, the patient had been treated with potassium chloride, 40 meq twice a day, spironolactone, 50 mg three times a day, propranolol, 20 mg three times a day, enalapril, 2.5 mg a day and indomethacin, 25 mg three times a day. the administration of medications led to an increase in serum potassium to 3.5 to 4.5 meg / l. in association with this improvement in the serum potassium concentration, the patient s muscle strength was rapidly recovered and the patient did well for the following two months (table 1). bartter s syndrome consists of hypokalemia due to renal potassium wasting, elevated plasma renin activity and aldosterone secretion, normal blood pressure, hyporesponsiveness of blood pressure to infused angiotensin ii and hyperplasia of granular cells of the juxtaglomerular apparatus of the kidney. clinically, bartter s syndrome can be divided into at least two groups : one group with an eary (infancy), and the other with a late onset of symptoms. neonatal bartter s syndrome is characterized by the intrauterine onser of polyuria, leading to polyhydramnios between the 22nd and 24th weeks of gestation. in adults, the primary etiolgy of bartter s syndrome is still unknown. with the findings of abnormalities in plasma and urinary prostaglandins, many investigators considered a defect in prostaglandin homeostasis as the primary defect. the inhibition of chloride transport in the loop of henle by prostaglandins, especially in the face of hyperaldosteronism, would lead to potassium wasting and alkalosis. however, prostaglandin inhibition does not completely cure the defects in bartter s syndrome. also, continued use of prostaglandin inhibitors may not result in continuous benefit to patients with bartter s syndrome. stein reviewed the published material and the data derived in his laboratory with bartter s syndrome, and concluded that bartter s syndrome may have more one underlying etiology. bartter and baehler favored primary chloride wasting as the defect of bartter s syndrome. these hypotheses all consider the resulting potassium deficiency as the cause of the prostaglandin, bradykinin, kallikrein, and vasopressor abnormalities. sodium, chloride, and potassium losses result in volume depletion, increased aldosterone levels, and metabolic alkalosis. magnesium depletion causes kaliuresis, diuretics cause potassium and volume depletion and vomiting causes renal potassium wasting and volume depletion. treatment is generally focused on the repair of hypokalemia by inhibition of the renin - angiotensin - aldosterone or the prostaglandin - kinin system. potassium supplementation, magnesium repletion, propranolol, spironolactone, prostaglandin inhibitors and conventing enzyme inhibitors all have been advocated, but each has met with limited success. in this case, the patient was diagnosed to be adult - onset bartter s syndrome due to hypokalemia, relative hypotension, increased renin activity, increased aldosterone level and juxtaglomerular hyperplasia. in appropriate medical therapy, a positive potassium balance and an increase in serum potassium concentration. in association with this improvement in the serum potassium concentration, the patient s muscle strength was rapidly recovered and the patient did well for the following two months. | bartter s syndrome is characterized by renal potassium wasting with hypokalemia, metabolic alkalosis, increased renin - angiotensin - aldosterone system, normal blood pressure, resistance to the pressor effects of angiotensin ii and juxtaglomerular cell hyperplasia. most of the cases have been noted in the pediatric age group and adult - onset cases are very rare. we report a case of adult - onset bartter s syndrome. |
there were no conflicts of interest, sources of financial support, corporate involvement, patent holdings, etc. involved in the research and preparation of this case report. there were any soure of founding, sponsors for collection, analysis and interpretation of data ; in the writing of the manuscript ; and in the decision to submit the manuscript for publication. for our case report the scientific ethics committee approval was required, the consent of the patient expressing his consent to our medical records. all the case reports was written keeping the anonymity of the patient and not by entering sensitive information, but in our medical records has been given consent for privacy. study concept : militello g., mascolino a., scerrino g., and gulotta g. design : kabhuli k., gulotta e., de marco p., incandela f. data collection : militello g., zabbia g., mascolino a., kabhuli k., gulotta e., de marco p.,scerrino g., and gulotta g. data analysis and interpretation : militello g., zabbia g., mascolino a., kabhuli k., gulotta e., de marco p., incandela f., scerrino g., and gulotta g. writing papers : militello g., de marco p., gulotta g. | the primary mammary chondrosarcoma corresponds to less than 0,5% of the mammary malignancies. for the period ranging from 1967 to 2014, only 18 cases were reported in the literature.a 41 year old woman found a hard nodule on her external right superior quadrant / axillary prolongation through breast self - examination. the vacuum - assisted core biopsy (vacb) revealed high grade extra - skeletal myxoid chondrosarcoma. a skin - nipple - sparing mastectomy with the insertion of a mammary expander was performed. a protocol of adjuvant radiotherapy was also indicated.until 2013, the gold standard was the radical mastectomy. by 2014, there were two cases of conservative approach to quadrantectomy. to our knowledge, this represents the first case in the literature in which a skin - nipple - sparing mastectomy has been performed on a primitive mesenchymal neoplasm of the breast. such an oncoplastic approach was performed considering the young age of the woman, to assure the surgical radicality and a better quality of life to the patient. |
chronic renal failure is accompanied by a deficiency state in both cell - mediated and humoral immunity which is deepened by hemodialysis (hd). a direct contact of the patient s peripheral blood mononuclear cells (pbmc) with an artificial membrane increases the production of pro - inflammatory cytokines : tumor necrosis factor - alpha (tnf-), interleukin (il)-1 and il-6 [2, 3 ]. what s more, hd patients demonstrate a decreased production of il-2 and interferon gamma (ifn-) [4, 5 ]. the deficient production of cytokines is probably related to changes in phenotype of cd4 lymphocytes from hd patients, which exhibit decreased expression of the major co - stimulatory cd28 antigen and main activation markers : cd25 and cd69. recombinant human erythropoietin (rhepo) administered to hd patients to correct the anemia state can influence both the phenotype of t lymphocytes and the production of cytokines. our previous studies have shown that rhepo treatment normalizes the impaired expression of cd28 and cd69 antigens of cd4 lymphocytes. additionally, several months of therapy restores the balance of cytokines by reducing the level of tnf- [8, 9 ] and increasing the level of il-2 and il-10 [4, 8, 9 ] in whole blood cell cultures of hd patients during treatment. the level of tnf- and il-10 changes immediately when the hemoglobin level exceeds the optimal value of 10 g / dl and is stable during treatment, while the level of il-2 increases continuously during treatment. this observation suggests that the normalization of the levels of tnf- and il10 was reached shortly after the start of rhepo therapy. increase of the level of il-2 has been achieved after several months of rhepo administration, so this change was secondary with respect to changes in the level of tnf- and il10. for example, the level of il-2 may depend on increased expression of cd28 on cd4 lymphocytes and/or decreased percentage of cd152 lymphocytes. however, it is still not clear which cytokines are regulated by the presence of erythropoietin in the circulation. in order to better understand these mechanisms, we decided to test how the presence of rhepo in cell culture influences cytokine production in stimulated cd4 lymphocytes from hd patients with a stable hemoglobin level and expression of cd28, cd69 and cd25 antigens of stimulated cd4 lymphocytes similar to those observed in healthy individuals. repetitive hd procedure has also been reported to induce apoptosis of t lymphocytes, which leads to t - cell lymphopenia sometimes observed in these patients. therefore, we also tested if the presence of rhepo in cell culture can protect stimulated cd4 lymphocytes of hd patients from apoptosis. for this study we have chosen 13 hd patients (11 men and 2 women, mean age 59 13.99 years) with a hemoglobin level above 10 g / dl (mean hemoglobin 12.53 1.57 g / dl) and expression of cd28, cd69 and cd25 antigens of stimulated cd4 lymphocytes similar to those observed in healthy controls. 7 hd patients received epoetin alpha or beta and the epoetin doses were adjusted to hemoglobin levels. 6 hd patients who did nt receive rhepo were also included in the study group. these patients did not require rhepo administration, since the level of hemoglobin was maintained at the correct level. they showed similar phenotype of stimulated cd4 lymphocytes and the level of th1 and th2 cytokines in culture supernatants as hd patients treated with rhepo and healthy controls (data not shown). none of the patients suffered from any infection, inflammation, malnutrition, malignancy or blood loss during the study. the study has been approved by the ethical committee of the medical university of gdask. thirty milliliters of venous peripheral blood from each hd patient was collected in tubes containing edta as the anti - coagulant agent before hd session. peripheral blood mononuclear cells (pbmc) were isolated from venous peripheral blood as previously described and stimulated with an immobilized anti - cd3 antibody (125 ng / ml) with or without the addition of rhepo (epoetin alpha, 0.1 u / ml) and incubated for 3 days at 37 c, 5 % co2. stimulated cells were then collected in order to estimate the percentage of apoptotic cd4 lymphocytes. culture supernatants were collected and frozen at 80 c for the assessment of cytokine production. collected cells were stained with the rpe - cy5-conjugated anti - cd4 monoclonal antibody (dako, denmark) and pe - conjugated annexin v (bd pharmingen, usa) and analyzed with flow cytometry on facscan (becton dickinson, usa). cytometric bead array (cba, bd biosciences, usa) was used to estimate the level of th1/th2 cytokines produced by stimulated pbmc. data were analyzed with cyflogic, version 1.2.1 (perttu terho and cyflow ltd). statistical analysis was done using the statistica program, version 8 (statsoft, poland). the significance tests were chosen according to data distribution. the level of significance in all was p 0.05. the expression of erythropoietin receptor (epo - r) has been reported in many non - hematopoietic cells including lymphocytes. we have recently shown that rhepo increases the phosphorylation of signal transducer and activator of transcription 5 (stat5) in stimulated cd4 lymphocytes. therefore, we examined whether the presence of rhepo in cell culture can protect stimulated cd4 lymphocytes of hd patients from apoptosis and we have shown that if present at a concentration similar to the physiological level rhepo promotes the survival of cd4 lymphocytes. hd patients showed a significant decrease in the percentage of apoptotic cd4 cells (cd4annexin v cells) after stimulation with the anti - cd3 antibody combined with rhepo as compared to cells stimulated with the anti - cd3 antibody alone (fig. 1). this fact has been observed in other cell types and our team is being the first to described it in t lymphocytes.fig. 1comparison of the percentage of cd4 cell apoptosis depending on the presence of rhepo in cell culture. midpoints of figures present medians, boxes present the 25 and 75 percentile and whiskers outside visualize the minimum and maximum of all the data, p < 0.05, wilcoxon signed ranks test comparison of the percentage of cd4 cell apoptosis depending on the presence of rhepo in cell culture. midpoints of figures present medians, boxes present the 25 and 75 percentile and whiskers outside visualize the minimum and maximum of all the data, p < 0.05, wilcoxon signed ranks test we also investigated the level of th1 cytokines (il-2, ifn-, tnf-) and th2 cytokines (il-4, il-5, il-10) in culture supernatants of lymphocytes stimulated with the anti - cd3 antibody with or without addition of rhepo. we did nt observe any changes in the level of il-2 in culture supernatants of lymphocytes stimulated with the anti - cd3 antibody in the presence of rhepo. since the phenotype of stimulated cd4 lymphocytes from these patients and the level of il-2 in culture supernatants is similar to those observed in healthy individuals, we suspect that the production of il-2 is dependent on the level of cd28 antigen on cd4 lymphocytes, as confirmed by a positive correlation between these parameters (data not shown). the presence of rhepo in cell culture increased the levels of ifn- and il-10 (fig. 2), whose production can also be enhanced by il-2 [14, 15 ]. since il-2 and epo act through receptors that belong to the same family and have common signaling pathways the level of tnf- was decreased in the presence of rhepo in cell culture from hd patients (fig. 2). it is not clear whether down - regulation of tnf- was caused by rhepo or rather il-10, whose level was increased in the presence of rhepo. not the first time we demonstrate the impact of rhepo on cytokine levels in hd patients. our study shows that rhepo affects the production of cytokines which are found to be regulated through signaling pathways involving stat5, for example. the presence of rhepo appears to regulate levels of tnftnf- and il-10, depending on the initial level of cytokines. healthy controls had a lower percentage of apoptotic cd4 cells after stimulation with the anti - cd3 antibody compared to hd patients so the presence of rhepo in cell culture did nt influence it. healthy controls also did nt show any changes in th1 or th2 cytokine levels when rhepo was added to cell culture but at the same time the level of tnf- was already decreased while the level of il-10 and ifn- was increased compared to hd patients (data not shown).fig. 2comparison of the levels of th1 and th2 cytokines in culture supernatants of stimulated lymphocytes depending on the presence of rhepo. graphs show the level of th1 (il-2, ifn-, tnf-) and th2 (il-4, il-5, il-10) cytokines in hd patients. midpoints of figures present medians, boxes present the 25 and 75 percentile and whiskers outside visualize the minimum and maximum of all the data, p < 0.05, wilcoxon signed ranks test comparison of the levels of th1 and th2 cytokines in culture supernatants of stimulated lymphocytes depending on the presence of rhepo. graphs show the level of th1 (il-2, ifn-, tnf-) and th2 (il-4, il-5, il-10) cytokines in hd patients. midpoints of figures present medians, boxes present the 25 and 75 percentile and whiskers outside visualize the minimum and maximum of all the data, p < 0.05, wilcoxon signed ranks test these results confirm one more time the role of rhepo signaling in t lymphocytes of hd patients. in our opinion, rhepo protects cd4 lymphocytes from apoptosis and restores the balance of cytokines by reducing the level of tnf- and increasing the level of anti - inflammatory il-10, though the mechanism of action of rhepo on t lymphocytes is still unclear. at the same time these results suggest that improved production of il-2 is not directly dependent on rhepo presence but seems to be a consequence of a long - term rhepo treatment. these observations confirm that rhepo administration not only has a beneficial effect on the red blood cells but also regulates the functioning of immune cells. | recombinant human erythropoietin (rhepo) treatment of hemodialyzed (hd) patients normalizes the altered phenotype of cd4 + lymphocytes and restores the balance of th1/th2 cytokines. we decided to test how the presence of rhepo in cell culture modulates cytokine production of cd4 + lymphocytes in hd patients with stable hemoglobin level and expression of activation antigens of stimulated cd4 + lymphocytes similar to those observed in healthy individuals. we also tested whether the presence of rhepo in cell culture protects stimulated cd4 + lymphocytes of hd patients from apoptosis. peripheral blood mononuclear cells (pbmc) of hd patients were stimulated with an immobilized anti - cd3 antibody with or without addition of rhepo. the percentage of apoptotic cd4 + lymphocytes and the level of th1/th2 cytokines in culture supernatants were measured with flow cytometry. hd patients showed a decrease in the percentage of apoptotic cd4 + cells after stimulation with the anti - cd3 antibody combined with rhepo. the level of ifn- and il-10 was increased while the level of tnf- was decreased in the presence of rhepo in cell culture from hd patients. these results confirm the role of rhepo signaling in t lymphocytes of hd patients. |
asymmetric cell division is key to cellular differentiation, and one intriguing aspect of such asymmetry is that a young and immaculate cell can be generated from an aged and deteriorated one (aguilaniu., 2003, higuchi., 2013, hughes and gottschling, 2012, liu., 2010, shcheprova., 2008). in bacteria, yeast, and specific stem cells, the generation of such rejuvenated progeny includes an asymmetrical inheritance of oxidized and aggregated proteins (aguilaniu., 2003, ogrodnik., 2014, the retention of protein aggregates in the yeast progenitor (mother cell) requires the protein remodeling factor hsp104 (erjavec., 2007, liu. 2009), the protein deacetylase sir2 (aguilaniu., 2003, orlandi., 2010), and actin cables together with actin cytoskeletal organizational proteins such as formins, cct, and calmodulin (liu., 2010, the control of damage inheritance is dependent also on spatial deposition of damaged / unfolded proteins into specific protein inclusions (pis), such as ipod (insoluble - protein - deposit), junq (juxta - nuclear - quality - control), and inq (intra - nuclear - quality - control) (burri and lithgow, 2004, kaganovich., 2008, miller., 2015, spokoini., 2012). formation of such pis are factor - dependent processes, requiring calmodulin and functional actin cables alongside hsp104 (liu., 2011, 2014), suggesting that the actin cytoskeleton is imperative for both mother cell - biased segregation of aggregates (liu., 2011, song., 2014) and the formation of specific inclusions (specht., 2011). in addition, two members of the hook family of proteins, btn2 and cur1, control the spatial deposition of misfolded proteins into discrete pis together with chaperones (e.g., sis1) and small heat shock proteins (e.g., hsp42) (malinovska., 2012, park., 2013, specht., 2011). however, the exact mechanisms of how misfolded proteins and aggregates are distinguished and sorted to the different pis and quality - control compartments are not identified (miller., 2015). in addition, the identification of asymmetry - generating genes (aggs) controlling inheritance of aggregates and genes required for pi formation has so far been restricted to candidate approaches, suggesting that many components remain to be discovered. in this work, we report the results of an unbiased, high - throughput screen for aggs and present a catalog of an organism s complement of genes involved in generating age - asymmetry. we discovered a previously unknown role for vesicle trafficking, membrane fusion to the vacuole, and vacuole inheritance proteins in the sequestration and fusion of aggregated proteins into cytoprotective ipod sites that are retained in mother cells. furthermore, we found that the vacuole inheritance adaptor protein vac17 is a limiting member of the aggs and show that this protein regulates asymmetry and replicative lifespan through myo2-dependent effects on endocytosis and spatial protein quality control. in addition, an unbiased proteomics approach identified members of the endosomal vesicle trafficking process, including the dynamin - like protein vps1, as physical interactors of the protein disaggregase hsp104. we further established that vps1 is involved in aggregate retention and formation of protein inclusions and that vac17 fails to affect these activities in the absence of vps1, indicating that these two proteins function along the same pathway. the data highlight that endosomal trafficking to the vacuole is an integral part of a cytoprotective spatial protein quality control affecting the rate of cellular aging. to embark on unbiased, automatized, screens for aggs, we generated a mutant library, using synthetic genetic array (sga) technology (see experimental procedures), in which the hsp104 gene was replaced by the functional hsp104-gfp fusion. the hsp104 gene encodes the heat shock protein hsp104 that binds to protein aggregates (glover and lindquist, 1998), and hsp104-gfp serves as an efficient reporter of such aggregates that can be observed as microscopic intracellular foci and inclusions (erjavec. after robot - assisted transfer of cells of the ordered hsp104-gfp - containing arrays to liquid 96-well plates, aggregate formation was elicited by a transient heat shock (42c for 30 min ; figure s1a). upon returning to 30c, after which no more aggregates were formed, the mother cells were allowed to generate new daughter cells, and the aggregate content in the progenitor and progeny was detected by an online granularity analysis (molecular devices) after obtaining images by high - content microscopy (hcm) (figure s1a). values of the aggregate content in mothers and their daughters were retrieved and mutants (with deletions in aggs) deviating from the wild - type value identified. among the 4,600 mutants analyzed, 111 were scored as aggs when a deviation of 25% from the wild - type was used as the cutoff, meaning a 25% increase in daughters inheriting one or more aggregates (figure 1a ; figure s1a ; table s1). the reduction in aggregate asymmetry in the set of mutants identified was not correlated to a general increase (or decrease) in aggregation (figure s1b). the screen also identified mutants with an enhanced aggregate asymmetry (table s2), and gene ontology annotation analysis suggests that alterations in the processes related to lipid metabolism and dna repair can boost the mother cells ability to retain aggregates during cytokinesis (figure s1c). this set of mutants was not further characterized in this study, which instead focuses on genes (aggs) required to generate proper aggregate asymmetry. aggs were found on all yeast chromosomes but were markedly enriched (25% of the hits) on the right - hand arm of chromosome iii (figure s1d). this is interesting considering that duplications of chromosome iii occur reproducibly in yeast cells grown under heat stress and provide the cell with an enhanced tolerance to proteostatic stress (yona., 2012). cross scoring with gene ontology (go), genetic / physical interaction, and keyword databases established that aggs are enriched for actin cytoskeletal functions / binding and intracellular vesicle trafficking processes (figure 1b ; figures s1e and s1f), specifically, genes required for membrane tethering and fusion to the vacuole. such genes included ccz1 (the guanine nucleotide exchange factor required for membrane tethering and fusion events at the late endosome and vacuole) (nordmann., 2010), the syntaxin - related snares vam3 and syn8 (mediating docking / fusion of late transport intermediates with the vacuole) (burri and lithgow, 2004), and vps8 of the corvet multisubunit tethering complex (involved in endosomal vesicle tethering and fusion of endosomes to the vacuole) (balderhaar and ungermann, 2013) (table s1 ; figures 1b1d). consistently, fab1 mutant cells devoid of phosphatidylinositol-3,5-bisphosphate (pi(3,5)p2), a resident signaling lipid on late endosomes, multivesicular bodies (mvb), and vacuoles, required for vesicle fusion to the vacuole (shaw., 2003) (figures 1c and 1d), were also identified in the screen as an agg. moreover, both vph1, required for vacuolar proton pumping as a subunit of the vacuolar - atpase (v - atpase), and rav1, a member of the rave complex involved in assembly of the v - atpase, were scored as aggs. these aggs may also be linked to vesicle trafficking as vacuole fusion has been shown to require vacuolar acidification by the v - atpase (baars., 2007, coonrod., 2013) (figure 1c ; table s1). that aggs are involved in vesicle and vacuolar fusion is noteworthy as misfolded proteins have been shown to become spatially recruited to ipod inclusion sites at the proximity of the vacuole surface (spokoini., 2012) in addition, we found that the hsp90 chaperone hsp82 acts as an agg and is a central interactor (physical and genetic) of several asymmetry - generating processes, including vesicle trafficking, the tor1/skn7 signaling pathway, cytoskeletal organization, chromatin organization / segregation, and lipid signaling (figure 1c). to test whether the quality - control system of protein aggregates interacts directly with endosomal trafficking systems (or other systems), we performed an unbiased screen for hsp104 interactions during normal growth conditions and heat shock. hsp104-gfp was immunoprecipitated under mild conditions along with an untagged control both at 30c and 37c. immunoprecipitation was confirmed by sds - page (figure s2a), and peptides were identified by liquid chromatography - tandem mass spectrometry (lc - ms / ms). hsp104 was the most abundant protein present by spectral count as expected (table s3). spectral counts in each of the four experiments were compared to the peptideatlas database as described (miao., 2013, several components of the vesicle transport system were found to be enriched among hsp104 interactors, including the guanine exchange factor sec7, the snare disassembly chaperone sec18, and the vacuole anchor vac8 (table s3). analysis of the hsp104 interactions that were seen only during heat shock revealed an even more significant enrichment for proteins involved in vesicle trafficking (figure 1e), suggesting that hsp104 generally associates with the endomembrane system with a more pronounced association during heat shock. these data indicate that the involvement of membrane trafficking genes in the establishment of damage asymmetry could be linked to an hsp104-mediated interaction between aggregated proteins and components of the vesicle trafficking machinery. among the vacuolar trafficking - related genes identified in the screen, we found that vac17 and vac8, required for actin cable - dependent vacuole inheritance (weisman, 2006), act as aggs (figure 1c ; table s1). act1, myo2, vac8, and vac17 encode the four key components of the vacuole inheritance machinery (figure 2a) : vac17 serves as an adaptor protein recruiting vacuole vesicles to the actin cable tracks by its dual interaction with vac8 (on vacuole vesicles) and the myo2 motor protein (on actin cables) (weisman, 2006). manual quantification and complementation of aggregate inheritance defects (liu., 2011, spokoini., 2012) demonstrated that both vac17 (figure 2b) and vac8 (figure 2c), similar to act1 and myo2 (erjavec., 2007, liu., 2010, song., 2014), are required for mother cell - biased segregation of aggregates. by using a protocol previously described to discriminate between effects on aggregate retention and aggregate removal (hill., 2014, 2014), we found that vac17 is predominantly affecting aggregate retention (figure 2d). moreover, while the wild - type allele of vac17 complemented both vacuole and aggregate inheritance defects when reintroduced into vac17 cells, the vac17bd allele, encoding a vac17 protein unable to interact with myo2 (tang., 2003), did not (figure 2b). consistent with a role for vac17-myo2 interaction for aggregate inheritance, cells containing the myo2-n1304s allele of myo2, which lacks the vac17-binding domain (eves., 2012), displayed a reduced ability to retain protein aggregates in mother cells (figure 2e). these data indicate that the role of actin cables in establishing aggregate asymmetry (aguilaniu., 2003, erjavec., 2007, liu., 2010) may be linked to the recruitment of misfolded / aggregated proteins to actin cables by the vac17/8 proteins and myo2. vac17 and hsp104 aggregates co - localized in about one - third of the cells suggesting that vac17 is, at least to some degree, associated with misfolded / aggregated proteins further strengthening a role for this protein in spatial control of heat - induced aggregates (figure 2f). however, the absence of vac17 did not affect inclusion formation or retention of the amyloid, disease - related, huntingtin protein htt103qp (dehay and bertolotti, 2006, wang. previous studies demonstrated that amyloidic proteins and misfolded proteins formed upon heat shock follow different routes for their deposition (specht., 2011), and our results suggest that vac17 is only regulating the latter process (see also data below concerning ubc9). the vac17 vacuole - adaptor protein is only present at around 20 copies / cell and competes with other adaptor proteins for recruitment of cargo to the myo2 motor protein and actin cables (eves., 2012). to approach whether vac17 might be limiting for aggregate retention in mother cells, we constructed a vac17-overproducing strain by exchanging the weak vac17 promoter with the strong pgpd promoter. the levels of vac17 were markedly elevated by this promoter exchange (figure 3a), and the retention of both heat - induced and aging - induced aggregates in mother cells were even more pronounced than in wild - type cells (figures 3b and 3c). this effect was not due to an altered localization of vac17, as the overproduced protein displayed the same localization pattern as endogenously expressed vac17 : during growth at 30c vac17 is predominantly found at the bud neck or within the bud (figures s3a and s3b), consistent with previous observations (eves., 2012, jin., 2009). upon a shift to 38c, however, both endogenously and overexpressed vac17 were relocalized to the mother cell, where 34% and 66% co - localized with hsp104-associated aggregates, respectively (figures 2f, s3a, and s3b). the localization of the low - abundant vac17 was not the result of background fluorescence or signal bleed - through, as evidenced by a non - gfp control (figure s3a). overproduction of vac17 did not affect aggregate inheritance in cells carrying the myo2-n1304s protein unable to bind vac17 suggesting that vac17 acts through its interaction with myo2 also in boosting aggregate asymmetry (figure 2e). interestingly, inheritance of vacuolar content was not boosted above the level of wild - type cells by overproducing vac17 (figures 3d and 3e), and while overproduction of vac17 completely restored vacuole inheritance in a fab1 mutant it failed to re - establish aggregate retention (figure 3f). furthermore, while overproduction of vac17 could not limit aggregate inheritance in the myo2-n1304s mutant, vacuole inheritance was modestly improved (figure 2e). together, these results suggest that vacuolar inheritance defects alone can not explain the defects in aggregate segregation, which therefore may be linked to additional functions of the myo2-vac17 complex (see below). considering the large number of hits in late endosomal trafficking and membrane fusion, we contemplated the possibility that vac17 might affect aggregate segregation by acting in these pathways. consistent with this notion, defects in the hops / corvet (vps16 : vps16 is a subunit of both hops and corvet ; figure 1d) multi - subunit tethering complexes required for fusion to the vacuole caused a drastic defect in aggregate retention, and vac17 overproduction failed to affect aggregate inheritance in the absence of these complexes (figure 3 g). these data, along with the identified requirement for snare proteins (figures 1c and 1d ; table s1), show that fusion to the vacuole might be required for proper vac17-dependent retention of protein aggregates in mother cells. however, we can not rule out the possibility that the mutants analyzed suffer from additional defects in vacuole functions that might impact on aggregate inheritance. the aggregate segregation defect in vac17 cells was accompanied by a reduced ability to deposit misfolded proteins into discrete inclusions (kaganovich., 2008, spokoini., 2012). upon a shift from 30c to 38c, the immediate occurrence of multiple stress foci (sf [spokoini., 2012 ] ; also called q - bodies [escusa - toret., 2013 ], peripheral aggregates [specht., 2011 ], and cytoqs [miller., 2015 ]) and the subsequent deposition of misfolded / aggregated proteins in ipod and the juxta - nuclear - quality control compartment (junq) can be time resolved (figure s3c). using hsp104-gfp together with the sf / ipod / junq - reporter ubc9-rfp, we found that the advancement to the inclusion stage was affected in vac17-deficient cells as these cells displayed multiple hsp104-associated ubc9 sf (figures 4a4d). in contrast, overproduction of vac17 increased inclusion formation (figures 4a, 4c, and 4d). as for aggregate inheritance, the effect of vac17 on inclusion formation required its myo2-binding domain (figure 4b). single - cell analysis demonstrated that vac17 affected the fusion of small sf into inclusions such that the merging of sf was retarded in vac17 cells and accelerated in vac17-overproducing cells (figure 4d ; figure s4 ; movies s1, s2, and s3). co - staining with fm4 - 64 established that the inclusions of wild - type cells and vac17-overproducing cells often localized to the vicinity of the vacuole (ipods [kaganovich., 2008 ]) and that the sf of vac17 cells were accompanied by a fragmented vacuole morphology of this mutant (figures 4e and 4f ; figure s4). analysis of the small heat shock protein hsp42, which is specifically localized to sf and ipods (escusa - toret. 2011), confirmed that vac17 is increasing the efficiency of ipod formation (figure s5a). cells lacking the vps16 subunit of the hops / corvet complexes displayed a drastic defect in inclusion formation, and this defect could not, similar to aggregate retention (figure 3c), be suppressed by vac17 overproduction (figure 5 g ; figure s5c ; movies s4 and s5). these data suggest that defects in vacuole tethering / function inhibit fusion of misfolded proteins / sf into vacuole - proximal ipods in the mother cell, leading to an increased inheritance of damaged proteins by the daughter cell. cells lacking fab1, which display defects in vacuole fusion, fission, and tethering and contain one large vacuole rather than fragmented ones (gary., 1998), also display a defect in inclusion formation that was not affected by vac17 overproduction (figures s5c and s5d ; movies s6 and s7). this indicates that vacuolar fragmentation is not a prerequisite for a failure to form inclusions. indeed, salt - stress - induced vacuole fragmentation (bonangelino., 2002, li and kane, 2009) demonstrated no significant correlation between the number of vacuoles and the number of heat - induced aggregates (figures s6a and s6b). similarly, we found no correlation between vacuole numbers and aggregate numbers in aged mother cells (figure s6c). the yeast dynamin homolog, vps1, a resident protein of endosomes required for proper endocytosis to the vacuole (smaczynska - de rooij., 2010) was identified as an interactor of hsp104 in both untreated and heat - shocked cells (table s3), and the interaction between vps1 and hsp104 was confirmed by immunoprecipitation (figure 5a). similar to the trafficking genes identified in the screen, deletion of vps1 resulted in defects in both damage asymmetry and in the formation of protein inclusions, although the effect on inclusion formation was modest (figures 5b5d). moreover, vac17 failed to affect these processes in cells lacking vps1 (figures 5b5d), suggesting that vps1 and vac17 might act in the same pathway of spatial protein quality control. vac17 molecules are progressively lost from mother cells during the generation of daughter cells as the protein is transported into the protruding bud and degraded to allow for proper vacuole inheritance (weisman, 2006). thus, the vac17 pool needs to be replenished after each cell cycle, and, if not properly compensated for by de novo synthesis, vac17 levels could decline during replicative aging of mother cells. indeed, we found this to be the case (figure 6a) and wondered whether vac17 might become limited in old cells for the management of protein aggregates. in support of this notion, overproduction of vac17 reduced the fraction of mother cells displaying aggregates as well as the number of aggregates per aged cell (figures 6b6d), while the opposite result was observed for cells lacking vac17 (figures 6b6d). elevation of vac17 levels also counteracted age - related vacuolar fragmentation (figure 6e) and the decline in endocytotic capacity (hughes and gottschling, 2012, tang., 2008) (figure 6f), supporting the notion that endocytosis is closely linked to ipod formation and that endocytosis and vesicle fusion to the vacuole is diminished in aged cells. the data indicate that vac17 possesses a previously unknown role in endocytosis, in addition to its role in vacuole inheritance, and we therefore tested whether cells carrying the myo2 protein (myo2-n1304s), specifically unable to bind vac17, display reduced endocytosis and found this to be the case both in young and old cells (figure 6 g). to test whether vac17 also affected vacuole - to - vacuole fusion, we analyzed vacuolar fusion during salt - stress recovery and found no evidence for the fusion of vacuoles being retarded in cells lacking vac17 (figure s5d). interestingly, improvement of aggregate management and endocytosis by vac17 overproduction was accompanied by a robust extension of lifespan, whereas vac17 deficiency modestly accelerated aging (figure 7a). to evaluate whether the effects of vac17 acted through its influence on protein quality control (pqc), we tested whether full activity of cytosolic hsp70 chaperone activity and/or the protein disaggregase hsp104 was required for lifespan extension by vac17. cytosolic hsp70s were needed for lifespan extension upon vac17 overproduction (figures 7b and 7c), while the hsp104 disaggregase was not (figure 7c), suggesting that pqc functions other than protein disaggregation are required for lifespan control by vac17. consistent with the requirement for the hops / corvet complexes for vac17-dependent aggregate fusion (figure 3 g), the vps16 deletion completely blocked lifespan extension by vac17 overproduction (figure 7d). in addition, the myosin - binding domain of vac17 was required for lifespan extension suggesting that interaction with myo2-actin is a prerequisite for vac17-dependent lifespan control, similar to aggregate retention, endocytosis, and ipod formation (figure 6e). the results demonstrating that vac17 overproduction extend lifespan in a hsp70-dependent manner indicates that vac17-dependent sequestration of misfolded proteins and sf into discrete inclusions (figures 4a4d) may be an important process in detoxifying aberrant protein species (cohen., 2006, treusch., 2009), and we therefore tested whether ubc9 became toxic in vac17 cells, as in cells deficient in cytosolic hsp70 chaperone activity (ling., 2014). we found this to be the case ; lack of vac17 reduced the fitness of cells expressing unfolded ubc9 (figure 6f) demonstrating that vac17 is a physiologically relevant aide - de - camp in spatial protein quality control required for mitigating proteotoxicity. in this paper, using an un - biased imaging approach to discover asymmetry - generating genes (aggs), we found that the adaptor protein vac17, which is a limiting factor for vacuole inheritance (weisman, 2006), is a key factor also in (1) limiting inheritance of protein aggregates, (2) generating protein inclusions in both heat - stressed and aged cells, and (3) maintaining endocytosis and vacuole integrity during aging. specifically, defects in late endocytosis and vesicle fusion to the vacuole lead to defects in protein inclusion formation and subsequent inheritance of protein aggregates, indicating that the aberrant management of damaged proteins observed in aged cells might be linked to a prior decline in endocytosis (figures 6f and 7 g). mitigating such a decline in vacuole - directed endocytosis by vac17 overproduction counteracted the accumulation of multiple aggregates in aged cells and extended lifespan. such data are in line with work showing that increased vacuole fusion by osh6 overproduction can retard mother cell aging (gebre., 2012) and that lifespan extension by caloric restriction requires vesicle - vacuole fusion (tang., 2008). we observed a partial colocalization between vac17 and hsp104-associated aggregates and found that vac17 became increasingly localized to the mother cell during heat stress (figures 2f and s3). while such data might indicate that vac17 is affecting inclusion formation and retention by direct interaction with protein aggregates in the mother cell, it is also possible that a fraction of the vac17 pool is aggregating during heat stress. in fact, a role for vac17 in inclusion formation does not require a direct interaction with protein aggregates as vac17 may be acting upstream the final deposition of misfolded proteins at the surface of the vacuole. vac17 may be doing so by affecting the rate of vesicle trafficking as discussed, and our data suggest that vac17 is not required for vacuole - vacuole fusions. in addition, we found that vac17 overproduction can suppress severe defects in vacuole inheritance without affecting aggregate inheritance in some mutants analyzed (figures 2e and 3f). however, although vac17 overexpression can restore vacuole inheritance in some mutants, we can not exclude the possibility that the inherited vacuoles are not fully equivalent, functionally, to wild - type (anand., 2009). therefore, defects in vacuole inheritance may affect aggregate retention not through vacuole inheritance per se, but instead through the generation of daughters and, in turn, mothers with defective vacuoles. the improvement of endocytosis and extension of lifespan by vac17 overproduction suggest that this protein becomes limiting upon aging, and that the decline in both endocytosis and formation of protein inclusions during aging may be due to a failure to maintain a proper titer of vac17 (weisman, 2006). indeed, we found that vac17 levels, normalized to total amount of protein, declined during mother cells aging (figure 6a). it is possible also that the activity of the endocytic apparatus decline during aging due to an age - related degeneration of actin cytoskeletal dynamics (gourlay., 2004) and a collapse in vacuolar acidification (hughes and gottschling, 2012), which is required for vesicle - vacuole fusion (coonrod., 2013). regardless of the nature of system failure, it is clear that elevated levels of vac17 alone results in a robust extension of lifespan, pinpointing vac17 as a yeast gerontogene. we found that vac17 together with the actin - associated protein myo2 is required for the merging of aggregates into ipods, which is interesting since actin, and actin - remodeling proteins such as calmodulin (cmd), is required for heterotypic and homotypic vacuole fusion (eitzen., 2002) (figure 7 g), and actin and cmd have previously been shown to be required for proper formation and asymmetrical inheritance of protein inclusions (liu., 2010, the concerted effect of vac17-myo2 on endocytosis, vacuolar integrity, and aggregate fusion / ipod formation could be a result of misfolded proteins / stress foci interacting with membrane vesicles (meriin., 2003) that could concentrate misfolded proteins at vacuolar - proximal ipod sites with the aid of snare and the hops / corvet tethering complex (figures 1e and 7 g). indeed, we found that the protein disaggregase hsp104 interacted with several protein components involved in endomembrane trafficking, suggesting that misfolded / aggregated proteins interacting with this disaggregase might hitchhike on trafficking routes leading to the vacuole. moreover, hsp104-endomembrane protein interactions became more pronounced upon heat shock, indicating that that disaggregation machinery and vesicle trafficking systems are increasingly engaged with each other upon protein aggregation (figure 1e). the proteins interacting with hsp104 included the dynamin - like protein, vps1, which appears to act on the same pathway of ipod formation as vac17. vps1 is localized on endosomes, and its absence results in a retarded endocytosis to the vacuole and the accumulation of clusters of endosomes surrounding the vacuole (smaczynska - de rooij., although the exact mechanisms remain unclear, the vac17-dependent route for aggregate deposition appears to provide the cell with two important features of protein quality control. first, it ensures that misfolded and potentially cytotoxic proteins are not inherited by the progeny. some misfolded proteins are imported into the nucleus where their deposition serves as a spatial cytoprotective measure and allows for consecutive degradation by the 26s proteasome (miller. it is possible that the same is true for misfolded / aggregated proteins deposited at the vacuolar surface, i.e., that a proportion of the proteins are translocated into the vacuolar lumen for destruction by vacuolar proteases, a possibility that remains to be elucidated. the yeast strains used (s228c background) were grown in ypd or synthetic drop - out media with corresponding antibiotics. sga mating to introduce hsp104-gfp into the deletion collection was performed according to published protocols (costanzo., 2010, cells were subjected to heat treatment to induce protein aggregation, followed by a recovery period and formaldehyde fixation. imaging of fixed cells with hsp104-gfp - bound aggregates was performed using a high - content microscope (imagexpress). acquired images were quantified using metaxpress, and top hits were analyzed for functional enrichment using database for annotation, visualization and integrated discovery (david) (huang. immunoprecipitations using anti - gfp beads (chromotek) were performed on exponentially growing cells expressing hsp104-gfp as well as an untagged control at both 30c and 37c. eluted proteins were digested with trypsin, peptides were identified by lc - ms / ms, and unweighted spectra were compared to the peptideatlas s. cerevisiae database to determine enrichment. protein aggregation was induced at 38c or 42c, followed by live - cell imaging. single - cell analysis of aggregate fusion was accomplished through 4d confocal imaging of cells attached to concanavalin a (sigma)-coated microscope plates. for the aggregation of ubc9-rfp, expression was induced by growing cells in galactose at 30c and repressed by the addition of glucose before transferring cells to 38c (kaganovich., 2008). all figure data are based on the average of at least three individual experiments, error bars representing sd. data were tested using an unpaired two - tailed t test, with p values less than 0.05 considered to be significant. old cells were obtained using the magnetabind biotin - streptavidin approach (sinclair and guarente, 1997, smeal., 1996), consecutively isolating biotin - labeled cells after culturing for 2 days. replicative lifespan was determined through manual dissection using singer msm micromanipulator (egilmez., 1990), and lifespan data were analyzed using a two - tailed mann - whitney u test, with p values less than 0.05 considered as significant. b.l. designed the agg screen, wrote the journal for image analysis, and, together with x.h., s.m.h. compiled and analyzed the screen data and conducted the follow - up experiments, with the aid of j.g., r.j. p.o.w. performed the biochemical analysis of hsp104 interactions and the follow - up experiments on vps1. t.n initiated and coordinated the study, analyzed the data, and, together with b.l. and s.m.h., | summaryage can be reset during mitosis in both yeast and stem cells to generate a young daughter cell from an aged and deteriorated one. this phenomenon requires asymmetry - generating genes (aggs) that govern the asymmetrical inheritance of aggregated proteins. using a genome - wide imaging screen to identify aggs in saccharomyces cerevisiae, we discovered a previously unknown role for endocytosis, vacuole fusion, and the myosin - dependent adaptor protein vac17 in asymmetrical inheritance of misfolded proteins. overproduction of vac17 increases deposition of aggregates into cytoprotective vacuole - associated sites, counteracts age - related breakdown of endocytosis and vacuole integrity, and extends replicative lifespan. the link between damage asymmetry and vesicle trafficking can be explained by a direct interaction between aggregates and vesicles. we also show that the protein disaggregase hsp104 interacts physically with endocytic vesicle - associated proteins, such as the dynamin - like protein, vps1, which was also shown to be required for vac17-dependent sequestration of protein aggregates. these data demonstrate that two physiognomies of aging reduced endocytosis and protein aggregation are interconnected and regulated by vac17. |
approximately 67% of siadh cases are reported to be caused by cancer, the majority of which (70%) are linked to small cell carcinoma of the lung (1). head and neck cancers are responsible for only 1.5% of siadh cases ; however, the majority of these cases have the histology of small cell carcinoma (2). there have been scant reports of squamous cell carcinoma as a cause of siadh and, even more rarely, squamous cell carcinoma of the head and neck. this report describes a patient who was found to have squamous cell carcinoma of the nasopharynx and later developed siadh following neck dissection. a 77-year - old caucasian man presented to the ent clinic with a 3 month history of progressive nonproductive cough. he had a social history of smoking, one pack a day for 50 yr. his regular check - up excludes any other possibility for unknown diseases, including renal failure or heart failure. he had a past surgical history of coronary artery bypass graft (cabg). upon physical examination, a biopsy of the mass was ordered and the tissue sample demonstrated transformation to squamous cell carcinoma. the patient then underwent modified radical neck dissection without sacrificed internal jugular vein, which was carried out by our hospital 's head and neck cancer team. on post - operative day 2, a doppler ultrasound (us) detected deep vein thrombosis (dvt) in the patient 's left arm which prompted us to start him on heparin (lovenox). on post - operative day 6, the patient developed pulmonary edema, and a loop diuretic (furosemide, lasix) was then added to his treatment. the echocardiography showed normal ventricular function. on post - operative day 10, he presented with lethargy, 2 + peripheral edema and a central venous pressure (cvp) of 6 mmhg (fig. mmol / l, a serum k level of 4.7 mmol / l, a serum osmolality of 265 mosm / kg, a ph of 7.46, a pco2 of 53 mmhg and hco3 of 35 mmol / l. the urine analysis showed an osmolality of 390 mosm / kg, a urinary na of 142 mmol / l and fractional excretion of sodium > 1%. this was followed by thyroid function tests and an acth stimulation test, the results of which were found to be within normal limits, eliminating hypothyroidism and adrenal insufficiency as possible causes for the patient 's electrolyte disturbance (fig. a diagnosis of siadh was established, and the patient was treated with 1 g / day of oral salt and 3% hypertonic saline. in addition to this, the diuretic was discontinued to avoid aggravating the hyponatremia. by his post - operative day 15 the oral salt was then placed on hold, and 0.9% normal saline was started. on the morning of post - operative day 20, the patient 's blood pressure temporarily dropped from a normal range to 70/57 mmhg. however, it was quickly discovered that the patient was mistakenly receiving 0.45% hypotonic saline instead of 0.9% normal saline. at this point, the patient had already received approximately 1,320 ml of half - normal saline for 4 hr, and in addition to the drop in his blood pressure, he became confused and then started vomiting. the half - normal saline was instantly stopped and his serum na was checked, and it was found to be 124 mmol / l. unfortunately, his clinical condition deteriorated very quickly, with his blood pressure continuing to drop. cardiopulmonary resuscitation (cpr) siadh was first described in two patients with bronchogenic small cell carcinoma in 1957 by schwartz (3). at the time, schwartz suggested that small cell carcinoma produced some quantity of antidiuretic hormone, which was later found to consist of arginine vasopressin (avp) by bleich hl in 1976. bleich also discovered the avp - regulated water channels in the kidneys that were later termed " aquaporins " (4). schwartz 's hypothesis was proven by detecting ectopic avp in small cell carcinoma (5). this hypothesis was further confirmed by expression analysis of the avp - np ii gene, which controls the production of avp, in small cell carcinoma cells. these findings established the well accepted mechanism of action of siadh caused by small cell carcinoma (6). in 1976, moses was the first to describe siadh in patients with sqamous cell carcinoma, but the mechanism by which it occurs was not discovered and it has not been demonstrated since (7). there has not been a proven or widely accepted theory explaining siadh caused by squamous cell carcinoma. although many researchers have found avp gene not only in small cell carcinoma but also in undifferentiated carcinoma (8) and anaplastic carcinoma (9), they have failed to find avp gene in squamous cell carcinoma. this course is stimulated by neuropeptides such as acetylcholine, histamine, bradykinin, angiotensin ii and neuropeptide y (10, 11). in 2004, lee demonstrated a significant increase of neuropeptide y in the hypothalamus of a mouse that was inoculated with human squamous cell carcinoma (11). these findings led him to suggest that squamous cell carcinoma produced neuropeptide y, which stimulated production of endogenous avp, while small cell carcinoma produced ectopic avp directly. in addition, there has been more compelling evidence to support lee 's theory, such as the finding of increased avp levels in 38% of siadh patients with squamous cell carcinoma, despite no evidence of ectopic avp (12). in the event that neuropeptide y is detected in siadh patients with squamous cell carcinoma, this hypothesis can be proven clinically. in addition to siadh association with a number of malignancies (most notably small - cell cancer of the lung), siadh can also occur following head injury, surgery (following neck dissection and manipulation of the nervous system), drugs (such as morphine, nsaids, and oxytocin) and in a number of pulmonary (pneumonia, abscess, and tuberculosis) and endocrine disease states (hypothyroidism and glucocorticoid deficiency). it should be considered in patients who are euvolemic and hyponatremic with elevated urine sodium (usually greater than 20 meq / l) and urine osmolality. avp is an antidiuretic hormone that maintains homeostasis against hypotension and hypovolemia, which is sensed by the baroreceptors in the neck. when these receptors are stimulated, e.g. via neck dissection, serum avp will rise as a result of the signaling from the baroreceptors. in 1997, mesko observed siadh in 27% of those patients undergoing neck dissection (13). however, when siadh develops in a cancer patient who has undergone neck dissection, it is uncertain whether the siadh is a paraneoplastic syndrome or post - operative siadh. siadh can be a paraneoplastic syndrome caused by squamous cell carcinoma or a post - operative siadh caused by neck dissection. therefore, we will conduct further research on the neuropeptides of squamous cell carcinoma, which may help to prevent siadh post - neck dissection. | the paraneoplastic syndromes include the disorders that accompany benign or malignant tumors but are not directly related to mass effects or invasion by the primary tumor or its metastases. neoplastic cells can produce a variety of peptides that exert biologic actions at local and distant sites and can elicit responses that cause a variety of hormonal, hematologic, dermatologic and neurologic symptoms. almost every type of malignancy has the potential to produce hormones or cytokines or to induce immunologic responses. lung cancers, both non - small cell and small cell, are capable of producing a variety of paraneoplastic syndromes. the majority of such syndromes are caused by small cell carcinomas, including many endocrinopathies. syndrome of inappropriate antidiuretic hormone (siadh) has been commonly associated with small cell carcinoma and is often seen in these patients. however, siadh associated with squamous cell carcinoma has rarely been reported on, and the mechanism for this rare association is still unknown. we present here a case of a 77-yr - old man who developed siadh caused by squamous cell carcinoma of the nasopharynx. |
a case report and review of orbital aspergillosis treatment with voriconazole in the english language literature. amphotericin b with debridement is the current standard of care for orbital aspergillosis ; however, its prognosis is unfavorable. when compared to amphotericin b, voriconazole demonstrates a survival benefit, has less systemic toxicity, and is better tolerated by patients. while a prospective trial comparing amphotericin b to voriconazole in orbital aspergillosis is not feasible, there is evidence to support the use of voriconazole as primary therapy. orbital aspergillosis is a rare and often fatal condition in healthy patients. due to its vague initial symptomatology, it is often misdiagnosed and improperly treated which, in turn, facilitates disease progression. once proper treatment is initiated, the therapy is often poorly tolerated and the mortality rate remains high. in this case report, we will discuss a patient whose symptoms began in july 2010 and follow the historical, clinical, radiographic, and histological course until the diagnosis of invasive orbital aspergillosis was made in november 2010. we will then discuss treatment options presented to this patient and summarize treatment options in the context of the available literature. a 68-year - old white man with a past medical history of glaucoma with remote bilateral trabeculectomies, hypertension, depression, and sinusitis presented with a 1-month history of headaches and pain around his right eye. the patient described the pain as a constant ache behind his right eye which, over the course of the month, had intensified to become a sharp, stabbing pain. during the third week, he began to have blurred vision and ptosis of the right lid. he sought treatment at his local hospital where he was admitted and had a head ct, a head mri, and bilateral temporal artery biopsies. no pathology was seen on imaging, and the temporal artery biopsies were negative. as a result, the patient was presumptively diagnosed with giant cell arteritis (gca) and treated with corticosteroids. the patient presented in our neuro - ophthalmology clinic 4 weeks after the initial onset of his symptoms. at this time, the patient 's medications consisted of amlodipine, valsartan, paroxetine, omeprazole, and oxycodone. his best - corrected visual acuity was 20/60 od and 20/25 os ; his color vision tested with ishihara plates was 1/11 od and 11/11 os. on examination, he had a trace right relative afferent pupillary defect, complete ptosis of the right lid, and a frozen right eye due to paralysis of cranial nerves iii, iv, and vi (fig. the combination of the visual and clinical findings led to the diagnosis of orbital apex syndrome. fundus examination demonstrated pink optic nerves with a cup / disc ratio of 0.8 od and 0.9 os. laboratory testing revealed a c - reactive protein level of 7.9 mg / l, erythrocyte sedimentation rate of 46 mm / h, and white blood cell count of 6,900/l with 93% neutrophils. an mri and ct of the head were ordered which demonstrated an infiltrative process centered in the right pterygopalatine fossa and involving the soft tissues of the nasal posterolateral recess, right orbital apex, right sphenoid sinus, and dura adjacent to the clinoid process (fig. the department of otolaryngology was consulted, and a right trans - sphenoid orbital biopsy was performed. preliminary pathology from the biopsy demonstrated an infiltrate consisting of a mixture of granulocytes, giant cells, and small mature lymphocytes without evidence of infection. the slides were considered suggestive of a nonspecific inflammatory etiology, such as tolosa - hunt syndrome, and were sent for further staining. the patient was discharged on prednisone 80 mg / day (1 mg / kg / day). the following morning, the patient 's best - corrected visual acuity had deteriorated to 20/100 od, and his right eye remained frozen. according to recommendations provided by the division of infectious diseases, he was started on voriconazole 4 mg / kg i.v. the patient 's ophthalmoplegia and visual acuity improved dramatically within 72 h, and after 6 days of therapy the patient 's visual acuity was 20/40 od. after 6 weeks of treatment, the patient returned to our clinic with full extraocular movements, full color vision, and a visual acuity of 20/30 od. the divisions of infectious diseases and dermatology were consulted, and it was found that the patient had developed a drug - induced photosensitivity rash secondary to voriconazole. his therapy was changed to daily intravenous micafungin 150 mg, which he has tolerated well for the last month. the best - established risk factor for orbital aspergillosis is sinusitis of a paraorbital sinus ; however, there is no consensus on which loci confer the greatest risk [1 3 ]. other risk factors include trauma, facial surgery, high - exposure professions (demolition, yard work, or working in endemic areas of aspergillus flavus such as the sudan), and immunocompromised states including aids, absolute neutrophil count 8-mm thickening of the sinus mucosa) with an enhancement of the optic nerve or erosion of the orbital bones should prompt suspicion for orbital aspergillosis. a ct of the orbit without contrast may aid in the diagnosis by screening for bony erosion and examining for intraluminal calcification, which is pathognomonic. to confirm the diagnosis, fungal cultures of the nares have been shown to be unreliable and should not be used for screening. if imaging reveals a paraorbital sinusitis, an anterior rhinoscopy as well as a biopsy may be performed. while anterior rhinoscopy is the least invasive approach, 50% of cases require a second biopsy [1, 8 ]. alternative approaches include orbital fine needle aspiration or trans - sinus orbital biopsy ; these approaches have a higher yield but are more invasive than anterior rhinoscopy. the tissue should be sent for both frozen and permanent sections to look for fungi as well as other pathology. in addition, slides should be sent for periodic acid - schiff stain and gomori methenamine silver stain to look for haemotoxophilic organisms with 45 branching septate hyphae 24 m in width. if pathological testing is initially negative but a high suspicion of aspergillosis is maintained, corticosteroids should be held until cultures are known and empiric treatment for aspergillosis should be initiated. 0.5 mg / kg / day with debridement of focal abscesses [1, 2, 4 ]. in total, 4060% of patients show a response to amphotericin b, including liposomal preparations. despite treatment with amphotericin b, once the infection has manifested as an orbital apex syndrome, the mortality rate remains 7080% [1, 2, 4, 5, 6 ]. due to a high rate of relapse, prolonged treatment for 2 years to life is suggested [1, 2 ]. amphotericin b has numerous side effects, including irreversible nephrotoxicity, and as a result is often poorly tolerated. there is a case report of a patient who died as a direct result of amphotericin b therapy for invasive orbital aspergillosis. itraconazole is better tolerated than amphotericin b, but has similar response, relapse, and mortality rates [1, 4 ]. this drug should not be used if there is pulmonary involvement because a higher mortality has been demonstrated in these cases. concomitant induction of amphotericin b and itraconazole may increase response, and maintenance with itraconazole alone is significantly better tolerated than with amphotericin b. fluconazole 100200 mg / kg / day and rifampin 600 mg / day have also been shown to be effective, but there are too few reports to evaluate their role in therapy [4, 9 ]. ketoconazole on the other hand has been shown to be ineffective in treating orbital aspergillosis. voriconazole is a second - generation triazole antifungal that has become the usual treatment of extraorbital invasive aspergillosis after demonstrating a 22% survival benefit over amphotericin b in a comparative evaluation [10, 11 ]. voriconazole 6 mg / kg for 1 day, then 4 mg / kg / day, has less systemic side effects than amphotericin b, favorable bone penetration, and is better tolerated for maintenance therapy [10, 11, 12 ]. despite its favorable profile, we could only find one case report in the english literature and two abstracts in the japanese literature on voriconazole treatment of invasive orbital aspergillosis [10, 13, 14 ]. in each of these case reports, the patients demonstrated a dramatic improvement within several days of voriconazole therapy. in the english literature, sasindran. presented a case of an 8-year - old boy who developed an orbital apex syndrome after a trauma. the japanese literature presented 2 cases of patients in their 70s being successfully treated for orbital aspergillosis, but the treatment protocols were not discussed in the abstracts [13, 14 ]. despite being the current standard of treatment for invasive orbital aspergillosis, amphotericin b preparations offer an unfavorable prognosis. with optimized amphotericin b therapy, additionally, only 4060% of patients with orbital aspergillosis will respond to treatment with amphotericin b, and patients poorly tolerate prolonged therapy. most patients develop serious complications such as nephrotoxicity, and 1 patient is reported to have died as a direct result of the therapy. while the role of voriconazole as the mainstay of therapy for extraorbital invasive aspergillosis is well defined, its role in orbital aspergillosis has not yet been documented. the use of voriconazole seems intuitive for orbital aspergillosis since it confers a survival benefit, provides better bone penetration, and is better tolerated than amphotericin b preparations in extraorbital invasive aspergillosis. the efficacy of voriconazole has been demonstrated in our patient and the 3 above - mentioned case reports found in the literature. out of the 4 patients, only our patient reported a side effect (photosensitive drug rash) due to voriconazole. our patient 's risk factors for orbital aspergillosis included type 2 diabetes and a history of previous sinusitis. these risk factors are usually associated with noninvasive orbital aspergillosis rather than with the invasive disease as confirmed by mri in our case. our patient may initially have had a noninvasive orbital aspergillosis that was exacerbated by the use of corticosteroids for the treatment of probable gca. the reversible immunosuppression conferred by the steroids may explain the rapid invasive progression and the favorable response to voriconazole. while 4 patients do not demonstrate a statistical benefit, voriconazole can be considered an efficacious alternative to amphotericin b for the treatment of orbital aspergillosis that is better tolerated by patients. | background / aimto describe a case of invasive orbital aspergillosis and evaluate treatments and outcomes.methodsa case report and review of orbital aspergillosis treatment with voriconazole in the english language literature.conclusionamphotericin b with debridement is the current standard of care for orbital aspergillosis ; however, its prognosis is unfavorable. when compared to amphotericin b, voriconazole demonstrates a survival benefit, has less systemic toxicity, and is better tolerated by patients. while a prospective trial comparing amphotericin b to voriconazole in orbital aspergillosis is not feasible, there is evidence to support the use of voriconazole as primary therapy. |
studies have shown that acute stress increases nociceptive thresholds [14 ], leading to the possibility that stress, in general, produces stress - induced analgesia (sia). however, more and more evidence in animal experiments suggests that repeated or prolonged stress can decrease nociceptive thresholds [1, 513 ]. for example, rats that were briefly exposed to short, emotionally arousing nonnoxious stress, such as holding or novel environments, displayed an immediate and transient hyperalgesia in response to thermal and electrical stimuli, which was followed by a longer period of analgesia. in addition, prolonged stress from repeated exposure to a cold environment or restraint induces hyperalgesia [5, 7 ]. exposure to a stressor in a laboratory generates a wide variety of adaptive responses, producing cellular, immune, endocrine, and behavioral effects, including changes in pain threshold. acute stress paradigms, in particular, can induce antinociceptive effects and produce the phenomenon of stress - induced analgesia (sia). unlike sia, the mechanisms involved in stress - induced hyperalgesia (sih) are poorly understood. it was reported that chronic, repeated stress might activate the sympathoadrenal stress axis [14, 1619 ]. the communication box method can produce psychological stress in animals, since they can perceive the responses of other animals exposed to physical stress delivered through an electric foot shock. this kind of intraspecies psychological stress was detected in earlier studies and further confirms that animals subjected to experimental anxiety within the communication box have increased stress hormones (plasma corticosterone level). the present study was designed to induce experimental psychological stress, such as anxiety, in rats using an emotional stress paradigm called intraspecies emotional communication within a communication box. the hypothesis of this study was that psychological stress could induce changes in nociceptive thresholds, and an additional goal of this study was to determine whether nociceptive thresholds could return to normal after the stress is removed. 48 male sprague - dawley rats, weighing from 160 to 180 g (approximately 35 days old) were housed in 80 cm 45 cm 40 cm cages in a temperature - controlled room at 24c under a 12-hour light / dark cycle and were given free access to food and water. the rats were randomly divided into 4 groups : control group (con ; n = 8), foot - shocked groups (further divided into three subgroups as fs-1, fs-2, and fs-3, with 8 rats in each subgroup), psychological stress group (ps ; n = 8), and drug treatment group (dt ; n = 8). it consisted of 16 compartments which were each 16 16 cm and were separated by transparent plastic boards with several small holes. the boards prevented each animal from physical contact but allowed them to receive cues such as visual, auditory, and olfactory sensations from the neighboring animals. each compartment was equipped with a grid floor of stainless steel rods, 5 mm in diameter, placed at intervals of 0.3 cm. an electric generator with 48 voltages (made by biology medical electron department of the fourth military medical university, xi'an, china) was connected to the grid floor to produce an electric current and generate an electric foot shock every 2 seconds. the grid floors of eight compartments were covered by plastic plates to prevent electric foot shock and served as non - foot - shock compartments for the ps rats (figure 1). the whole experiment contains 3 periods : prepsychological stress period, psychological stress period and psychological stress removal period. in the pre - psychological stress period, all rats were individually confined in each compartment of the communication box for one hour without any electric foot shock for one week in order to adapt them to the surroundings. two identical communication boxes were adopted, and the electric foot shocks were introduced to the fs-1 and fs-2 rats (stress senders) from 8:00 am to 9:00 am with foot shock daily. the ps and dt rats (stress responders) confined in the non - foot - shock compartments were then exposed to psychological stress cues from the neighboring fs rats, including shrieks, smells of urine or faeces, and jumping response. consequently, the ps / dt rats were assumed to be in a state of fear or anxiety. at the same time, fs-3 and con rats were in a third communication box without foot shock delivery. this period lasted for 14 days, from 1st day to 14th day. in the psychological stress removal period (from 15th day to 28th day), procedure was similar to that of psychological stress period, but there was no foot shock delivered in all groups. diazepam was chosen to assess the effect of an anxiolytic drug on the masticatory muscle mechanical sensitivity of ps rats. diazepam was dissolved in saline containing 40% propylene glycol. from days 1 to 14, diazepam (1 mg / kg) was injected subcutaneously into dt rats 30 min before the stress stimulation. the rats in ps / dt / con groups were weighed weekly in the experiment. the experimental procedures were reviewed and approved by the ethics committee of the fourth military medical university. the fs (1, 2, and 3) rats were only used to induce psychological stress in the neighboring ps / dt rats and were not included in the following investigations. the elevated plus - maze (epm) apparatus (rd1208, shanghai mobiledatum corporation, shanghai, china) consists of two open arms (50 10 cm) and two enclosed arms (50 10 cm, with 50-cm high walls) extending from a central square platform (10 10 cm). the apparatus was elevated 50 cm above the floor in a quiet room with the temperature controlled at 20c. the light in the room was just bright enough to clearly observe the rats ' movement within a 1.5 meter range. two of the opposing arms (50 cm 10 cm) were enclosed by 40 cm high side and end walls (enclosed arms). the other two arms did not have walls (open arms). at the beginning of the experiment, the animals were placed in the central area (10 cm 10 cm) of the maze facing an enclosed arm. entry into one arm was recorded when an animal placed all four paws past the line dividing the central square from the open arms. the test arena was wiped with a damp cloth after each trial. the number of entries into the open / closed arms and the time spent in open arms / closed arms were measured by an observer who was blinded to the treatment conditions of the animals. then the percent of open arm entries (100 open / open + enclosed entries, oe%) and the time spent in the open arms (100 open / open + enclosed arm time, ot%) of the epm were calculated for each rat. we used oe% and ot% to analyze the anxiety level of each animal [2528 ]. instead of standing on meshed metal or a grid surface habituation required no more than petting the rats, and it was completed within half an hour. the smell that developed on the glove of the handler by handling the animals appeared to facilitate the habituation. this test environment was advantageous in that the rats were not restrained, but their movements were still restricted., two orofacial areas were tested : the temporal muscle region at the central point of the line between the orbit and the tragus and the masseter muscle belly region, at a site 10 mm inferior to the temporal muscle testing point (figure 2). at these locations,, force was applied with a probe - oriented perpendicular to the sagittal plane. head flinching, characterized as sudden quick head withdrawal, or vocalization / crying, was considered to be positive pain responses. the response threshold was defined as the lowest bending force of the filaments that produced at least three positive responses in five trials with 5 s interval. the bending force of the von frey filaments was verified on a balance with a resolution up to 0.001 g before test (ohaus, model gt410d) (table 1). for this study, we defined increased mechanical sensitivity as a statistically significant decreased withdrawal threshold compared to baseline, which was obtained in pre - psychological stress period. from seven days before applying the emotional stressor to the fourteenth day after psychological stress removal, we recorded the mechanical threshold of the masticatory muscle. we used these indexes to compare changes in the degree of mechanical pain before and after psychological stress. for all of the data reported here, the observer was blinded to the treatment conditions for the rats. experimental data were analyzed by a one - way analysis of variance (anova) across the con group, ps group, and the dt group using spss, version 11.0 (spss co., chicago, illinois). the snk - q test was also used to calculate any differences between the two groups. the purpose of this study was to explore the effects of psychological stress upon sensitivity of the masticatory muscles. but the foot - shocked group rats experienced a great part of physical stress, so we did not include this group in the experimental evaluations. initially, the mean body weight of rats did not differ between the ps, dt, and con groups (170.8 1.7 g, 175.0 2.3 g, and 173 3.2 g, resp. there was no significant difference in the body weight gain between the ps, dt and con groups (320.7 7.1, 323.2 5.7, and 325.2 4.8, resp..05) during the whole study, which contained a psychological stress period and a removal of stress period. the anxiety behaviors of rats were measured by oe% and ot% in epm. as shown in figure 4(a), the baseline measurements did not show significant differences among the control (48.13 1.47%), dt (47.56 1.79%) and ps (46.53 1.41%) groups (p >.05). during the psychological stress period, on 1st day, 7th day, and 14th day, ps rats had a smaller percentage (%) of entries into the open arms (open arm entry, oe) (42.15 1.01%, 34.03 1.19%, 38.53 1.27%, resp.) compared to rats in the con group (49.25 1.89%, 49 1.42%, 49.38 1.42%, resp.) (p.05). during the psychological stress period, on 1st day, 7th day and 14th day, the ps (62.06 1.40%, 55.31 1.48%, 58.59 1.24%, resp.) and dt (64.78 1.40%, 65.05 1.58%, 67.6 1.34%, resp.) rats preferred to spend a smaller percentage of time in the open arms (ot%) compared to the con group rats (68.5 1.19%, 67.88 1.43%, 69.49 0.99%, resp.) (p.05). in the prepsychological stress period, the mechanical thresholds of the bilateral masseter and the temporal muscles were assessed by von frey filaments. the head withdrawal threshold gradually increased then stabilized on the 7th day (shown as the pre - stress stage of figures 5(a)5(d)). increased mechanical sensitivity was induced in both the temporal muscles and the masseter muscles on both sides of the head following psychological stress (shown as the on - stress stage of figures 5(a)5(d)). during the psychological stress period, increased mechanical sensitivity of the ps rats was observed in the bilateral masseter and temporal muscles with the peak time on the 7th day. then the mechanical sensitivity was alleviated during the following days till the 14th day at a pain threshold lower than the con rats (p.05). after removing the psychological stress, the mechanical pain threshold of temporal muscles in ps group gradually elevated and returned to normal on the 28th day (p >.05). at the same time, the mechanical sensitivity of masseter muscles in ps group was gradually alleviated and finally returned to the baseline level on the 28th day (p >.05) (shown as the post - stress stage of figures 5(a)5(d)). through the whole experiment, the ps group showed greater mechanical sensitivity than the con and dt groups (p.05). the communication box method can produce anxiety in animals as they perceive the responses of other animals exposed to physical stress from an electric foot shock. in the present study, the communication box was used to simulate a psychologically stressful environment and to determine whether anxiety would induce increased mechanical sensitivity in the masticatory muscles. we also examined whether the nociceptive response could return to normal after the stressor was removed. it is interesting that when the rats were first placed into the narrow chart of communication box which meant new environment to them, they were already under psychological stress actually. as the time went on, the mechanical sensitivity decreased to a stable level considered as this may indicate that the ps group had learned to accommodate themselves to the stressful environment of communication box without foot shock delivered. the stress stimulation was commenced in the psychological stress period. from the very first day with stress, decreased head withdrawal thresholds induced by anxiety were observed in the ps group, but not in the dt and con groups, which may indicate that psychological stress stimuli upregulated the mechanical sensitivity of masticatory muscles. then the mechanical sensitivity was alleviated from 8 to 14 days in the ps group. this may indicate that the ps rats had learned to accommodate to the stressful environment. they found that increased mechanical thresholds (decreased head withdrawal threshold to mechanical pressure) were induced in both the temporal muscles and the masseter muscles on both sides of the head following occlusal interference. the mechanical threshold decreased from the first day and peaked on the seventh day. besides, the present results showed that no increased mechanical sensitivity occurred from 0 to 14 days in the con group, which indicated that this group did not experience psychological stress during the whole period. meanwhile, there was also no evidence of increased mechanical sensitivity in the dt group. it might be attributed to the gradual blocking effect of diazepam, which has the strong potency of suppressing anxiety. in the results of oe% and ot% in epm, we found that the data in dt rats was lower than con rats, but higher than ps rats. it may be due to low doses of diazepam used in our experiment, which could not completely block stress stimulator. we noticed that the results of mechanical test and epm tests were not entirely consistent, which might be caused by subjective error of the experimenter or some unknown factors and still need further study. in the psychological stress removal period, without any foot shock, the increased mechanical sensitivity of ps rats was alleviated from the 15th day and decreased until 28th day without any significant differences to con rats. this trend suggested that removing the stressor would be an effective means for curing diseases induced by anxiety. it suggested that, in this model of chronic stress, the reinstatement of the activity of systems involved in the nociceptive response has different patterns, as shown by the period of time and the types of response that were evaluated (the basal measurement or the nociceptive response to acute stress). prolonged stress could lead to more lasting alterations in the neural systems involved in nociception modulation. changes in the weight of the thymus, spleen and adrenal glands are usually found when stress is induced. ishikawa also observed no changes in the weight of organs or overall body weight in rats exposed to emotional stress. the present study showed that there was no significant difference between the ps, dt, and con groups, which is consistent with the results of rosales 's study. these results may indicate that the effect of the anxiety induced by the communication box was not strong enough to induce a weight gain, but it was sufficient to induce increased mechanical sensitivity in masticatory muscle. unlike stress - induced analgesia, the mechanisms involved in stress - induced mechanical sensitivity increase are not well known. satoh suggested that long - lasting mechanical hyperalgesia (3 days) induced by prolonged cold stress involved peptide - containing primary afferents (substance - p and calcitonin - gene - related peptide). quintero suggested that increased thermal and chemical nociception (8 - 9 days) observed after a subchronic swimming stress might be mediated by changes in the activity of the central serotonergic system. works in human showed that a reduction in the pain threshold after long - term psychoemotional stress was probably due to a reduction in the activity of the brain 's opioid system. preliminary investigations have also found that psychological stress, such as anxiety and tension, could increase the myoelectricity [34, 35 ] and parafunction activities of the masticatory muscle. stress - provoking stimuli are known to activate the dopaminergic system. according to research by ida., the mesoprefrontal dopamine system plays an important role in the control of negative states such as fear and/or anxiety. diazepam has been suggested to induce inhibitory effects on the activation of mesoprefrontal dopamine neurons and the hypothalamic - pituitary - adrenocortical axis the drug also has been used as an antianxiety drug in several stress - related experiments using the communication box [23, 37 ]. throughout the experiment in the current study, the ps group showed greater mechanical sensitivity than the con and dt groups, but they found that the supplementation of serotonergic tone during subchronic stress exposure appears to provide prophylaxis for stress - induced mechanical sensitivity, but this effect does not obviate a role for dopamine. in fact, even though there is a disruption in both the serotonergic and dopaminergic function that occurs within the nucleus accumbens (nac) following chronic stress, the impact on dopamine outlasts the impact on serotonin (5-ht). this difference may be responsible for the persistent expression of stress - induced hyperalgesia after serotonergic function has been normalized. secondly, from the therapeutic implications, benzodiazepines (e.g., diazepam as used in the present experiment) are pharmacologically linked to gabaergic (gamma aminobutyric acid) neurotransmission. on administering diazepam, the benzodiazepine receptors are activated and, since they are allosterically linked to gaba - a receptors, they modulate the chloride channel opening indirectly and induce inhibition of that neuronal function. the experiments in this study indicated that anxiety could induce increased mechanical sensitivity through exposure to repeated stress in rats. we also demonstrated that changes in withdrawal thresholds to mechanical pressure could be blocked by diazepam in repeatedly stressed rats. further studies concerning the mechanisms of stress - induced mechanical sensitivity would be relevant to studies of the etiology of chronic pain disorders. | to explore the relationship between psychological stress and masticatory muscle pain, we created a communication stress animal model to determine whether psychological stress could induce increased mechanical sensitivity in masticatory muscles and to study the changes of mechanical nociceptive thresholds after stress removal. forty - eight male sprague - dawley rats were divided into a control group (con), a foot - shocked group (fs, including 3 subgroups recorded as fs-1, fs-2, and fs-3), a psychological stress group (ps), and a drug treatment group (dt). ps and dt rats were confined in a communication box for one hour a day to observe the psychological responses of neighboring fs rats.measurements of the mechanical nociceptive thresholds of the bilateral temporal and masseter muscles showed a stimulus - response relationship between psychological stress and muscle mechanical sensitivity. the dt rats, who received a diazepam injection, showed almost the same mechanical sensitivity of the masticatory muscles to that of the control in response to psychological stress. fourteen days after the psychological stressor was removed, the mechanical nociceptive thresholds returned to normal. these findings suggest that psychological stress is directly related to masticatory muscle pain. removal of the stressor could be a useful method for relieving mechanical sensitivity increase induced by psychological stress. |
it is very important and significant for doctors to determine the status of vascular - related diseases such as thrombosis and arteriosclerosis and to precisely visualize the three - dimensional (3d) structure of the vasculature before surgery [13 ]. in dental fields, understanding the 3d structure of the vasculature is necessary and important for surgical operations on tumors and cysts. the most precise evaluation technique is conventional angiography (cr) using contrast medium. in cr, if the treatment should be applied for the status of vessels, the technique was continuously followed by the interventional radiology such as the embolization of arteries with arteriosclerosis. therefore, the cr is a firm position for elucidating the status of vascular - related disease such as thrombosis and arteriosclerosis, and precisely visualization of 3d vasculatures at the preoperations. however, the risk of a new focal neurological deficit caused by cr is about 0.14% ~ 0.5%, and this risk rises to 1.3% ~ 2.6% in patients investigated for stroke and transient ischemic attacks. therefore, it is important to identify the 3d structure of the vasculature of the arterial and venous systems as noninvasively as possible. to determine the 3d structure of the vasculature as noninvasively as possible, ultrasonography (us) is easy to use clinically. furthermore, the 3d structure of the vasculature can not be visualized using computed tomography (ct) without contrast medium. of course, ct angiography (cta) using contrast medium was a very useful tool for the 3d structure of the vasculature. however, the risk of complications in cta should be higher because of iodine contrast medium. recent advances in magnetic resonance imaging (mri), 3d data acquisition, and postprocessing technologies are expanding the potential applications of 3d displays [516 ]. as a noninvasive technique, magnetic resonance angiography (mra) has been developed to identify and characterize peripheral blood vessels located in the thoracic and abdominal regions and the lower extremity [1722 ]. of course, mra is very useful to visualize the 3d structure of the vasculature in the oral and maxillofacial regions, and it is clinically significant for evaluating the relationship between lesions and vessels in the oral region [14, 16, 2326 ]. general dentists should know and easily understand the clinical applications of mra in the oral and maxillofacial regions. therefore, the present paper provides a clear and easily understandable explanation of mra, the method, and its characteristics, as well as its clinical applications and characteristics, for general dentists. mra commonly uses a bright - blood method, in which the signal from the moving protons is accentuated relative to the stationary protons through pulse sequences and measurement parameters. in fact, mra is a technique using vessel flow or contrast agents such as gadolinium to detect vessel systems such as arteries and veins. therefore, mra has the least complication and is the easiest technique for 3d visualization of vessels, unlike cr using contrast medium such as iodine - related medium. at the same time, this technique is very useful for identifying vascular diseases and abnormalities, as well as the relationships between diseases and surrounding vessels. the various techniques of mra include mr sequences such as time of flight (tof), phase contrast (pc), fresh blood imaging (fbi), and contrast - enhanced mra [511, 13, 15, 16, 2737 ]. mra using tof (tof - mra) is the most time - efficient method for obtaining mra images. a single measurement is performed, with the stationary tissue signal suppressed relative to the flowing tissue signal. mra using pc (pc - mra) is a technique in which the background tissue signal is subtracted from the flow - enhanced image to produce flow - only images, analogous to digital subtraction x - ray angiography. mra as a new technique using fbi that has been applied to the 3d evaluation of vasculature [811, 13, 15, 16, 3437 ]. fbi, which uses 3d half - fourier fast advanced spin echo (fase)-triggered mr with electrocardiographic (ecg) gating, is one of the newest non - enhanced - mra techniques [811, 13, 15, 16, 3437 ]. contrast - enhanced mra makes good use of the contrast agent, which affects the relaxation times of the water protons in the nearby tissue. in mra with contrast medium, as disadvantages, intravenous injection should be applied such as cr, and gadolinium could induce many kinds of complications including nephrogenic fibrosing dermopathy. mra without contrast medium has no complications and no need of intravenous injections, and its technique can easily and additionally apply for many patients in whom vascular information should be evaluated. in the present paper, the focus is on three techniques (tof, pc, and particularly fbi) as the main mra techniques without contrast medium that are useful in the oral and maxillofacial regions. on tof- or pc - mra, arteries and veins which are to some degree slow appear as bright homogeneous linear structures and stationary tissues except vessels with flow which appear for the most part blurred(figure 1). therefore, vessels with flow can be primarily visualized on mra with these two techniques (figure 1). on the other hand, vessels without flow can not be visualized at all using mra with tof and pc techniques. of the two techniques in use for patients, pc - mra is better for demonstrating slow - flowing blood vessels (< 10 cm / s), owing primarily to an optimized velocity - encoding gradient (venc) and perhaps also to smaller saturation effects and better background suppression. this sequence offers a better examination of the external carotid system than tof - mra. though the main carotid artery and its branches can be visualized on pc - mra, tof - mra can visualize the main carotid artery, but not its branches (figure 1). in addition, 3d - pc - mra has a better signal - to - noise ratio and a better spatial resolution than 2d - pc - mra (figure 2). therefore, in the oral and maxillofacial regions, 3d - pc - mra routinely tends to be selected for additional and preoperative vascular evaluations in mr examinations. however, in the oral and maxillofacial regions, the vasculature is complex, with blood flow rates varying from high in the carotid arteries to low in the lingual arteries. thus, 3d visualization of the vasculature in these regions is very difficult to obtain even using 3d - pc - mra. therefore, in very recent reports, mra using fbi (fbi - mra) was used for the 3d evaluation of the vasculature in the oral and maxillofacial regions [10, 16, 23 ]. most prior studies using fbi primarily examined the main vessels of the chest, abdomen, and lower extremities [9, 11, 13, 15, 3437 ]. in previous reports, 3d - fbi - mra could provide better 3d visualization of the vasculature of thin blood vessels, including the lingual and facial arteries, than 3d - pc - mra (figure 3). the axes of the lingual and facial arteries are under 2 - 3 mm. 3d - fbi - mra permits the depiction of slow - flowing blood vessels by acquiring the images with ecg triggering during the slow - flow cardiac phase. 3d - fbi - mra can depict slow - flowing vessels due to the t2 effects, so that even relatively static blood vessels can be depicted on images, unlike imaging with 3d - pc - mra. therefore, 3d - fbi - mra can even visualize vessels with very slow flow that can not be visualized on 3d - pc - mra [7, 9, 13, 16 ]. in addition, the depiction level of thick blood vessels such as external and internal carotid arteries using 3d - fbi is the same as that using 3d - pc - mra. in other word, peripheral small arteries look well visualized on 3d - fbi - mra compared to 3d - pc - mra, but not for large arteries. carotid, maxillary, facial, and lingual arteries can be more precisely visualized on 3d - fbi - mra without contrast medium than on 3d - pc - mra (figure 3). at the same time, the internal jugular, maxillary, facial, and lingual veins can also be better visualized on 3d - fbi - mra without contrast medium than on 3d - pc - mra (figure 3). 3d - tof- or pc - mra depicts only blood vessels, but not mass lesions such as hemangiomas (figure 4) [16, 23 ]. therefore, the 3d relationship between the mass lesions such as hemangiomas and surrounding blood vessels should be visualized by fusing fat - saturation t2-weighted images with 3d - pc - mra. by doing so, hemangiomas and surrounding vessels can be visualized on the fusion images (figure 4). the software to allow superimposition of both 3d - pc - mra and t2-weighted images is integral to the mr system. however, image resolution degradation is a disadvantage of fusion imaging (figure 4). deterioration of the contrast - to - noise and signal - to - noise ratios on superimposed views occurs because noise on the respective images is doubled without increasing the signal intensity of the tumor and vessels. in addition, production of the superimposed view might be unsuccessful if the patient should move in the time gap between capture of the respective images. on the other hand, both hemangiomas and thin blood vessels around tumors can be clearly visualized on 3d - fbi - mra without fusion of mra with tumor images (figure 5). the carotid arteries, internal jugular veins, lingual arteries, lingual veins, facial arteries, facial veins, maxillary arteries, and maxillary veins can be seen as bright homogeneous linear structures (figure 5). at the same time, hemangiomas are clearly visualized as masses with high signal intensity (figure 5). except for the carotid arteries, 3d - fbi - mra is better than 3d - pc - mra based on visual scores despite the short acquisition time. artifacts from patient movement and shortened acquisition times reduce image quality more with 3d - pc - mra than with 3d - fbi. another advantage of 3d - fbi - mra is that one can distinguish between arteries and veins without the use of contrast medium [811, 13, 15, 16, 23, 3437 ]. the blood vessels depicted on 3d - fbi - mra are most of the arteries, including the carotid, maxillary, facial, lingual, and other arteries (figure 6). flow - spoiled gradient pulses do not affect the signal intensity of stationary background tissues. veins are similarly less affected by the flow - spoiled pulse during diastole and systole as a result of their relatively constant slow flow throughout the cardiac cycle. by applying the flow - spoiled pulses, thus, diastolic and systolic subtraction provides better delineation of the arteries. however, except for 3d - fbi, the distinction between arteries and veins has not been possible with mra sequences such as 3d - tof and 3d - pc without contrast medium so far. it is hoped that 3d - fbi - mra will be a significant, epoch - making technique. however, there are some potential disadvantages with 3d - fbi - mra for clinical applications in the oral and maxillofacial regions [16, 3437 ]. the first is that the selection of the appropriate flow - spoiled gradient is important for determining the image quality of hemangiomas and surrounding vessels. even a slight mistake affects the visualization of branch arteries, such as the maxillary and lingual arteries [16, 23 ]. to select the appropriate flow - spoiled gradient pulses is very difficult for operators, because blood vessels in the oral and maxillofacial regions have a wide range of flow rates, from high to static. therefore, the images tend to be degraded in patients with movements of the mandible and tongue. moreover, the third disadvantage is that patients with arrhythmias or tachycardias are not suitable candidates for fbi examinations, because ecg triggering is used. mra can not perfectly replace cr for examinations of vascular - related diseases and suspected hemangiomas in the oral and maxillofacial regions. that is why mra can not have more contrast between vessels and tissues except vessels and can not be more precisely visualized than cr. however, performing mra for diseases in the oral and maxillofacial regions should be recommended in addition to common mr examinations before cr. in particular, 3d - fbi - mra is relatively easy to perform and can precisely visualize thin vessels except a little difficulty of parameters setting. therefore, examinations using 3d - fbi - mra are very useful and important for preoperative evaluations for various kinds of diseases, in particular the 3d relationship between the vasculature and diseases. higher spatial resolution gadolinium - mra can be achieved at 3 tesla with a sustained or greater signal - to - noise ratio of enhanced vasculature, relative to 1.5 tesla. therefore, mra with higher resolution in the oral and maxillofacial regions can be achieved with a 3-tesla mr system. very recently, it has been found that existing mra is suboptimal for assessing the hemodynamics of arteriovenous malformations [4250 ]. as a completely noninvasive method, 4d dynamic mra offers hemodynamic information with a temporal resolution of 50100 ms for the evaluation of arteriovenous malformations and can complement existing methods, such as dynamic subtraction analysis and tof mra [4246 ]. therefore, the evaluation of hemodynamics in hemangiomas will be performed by mra in the oral and maxillofacial regions in the future. | the present paper provides general dentists with an introduction to the clinical applications and significance of magnetic resonance angiography (mra) in the oral and maxillofacial regions. specifically, the method and characteristics of mra are first explained using the relevant mr sequences. next, clinical applications to the oral and maxillofacial regions, such as identification of hemangiomas and surrounding vessels by mra, are discussed. moreover, the clinical significance of mra for other regions is presented to elucidate future clinical applications of mra in the oral and maxillofacial regions. |
hepatocellular carcinoma (hcc) is a common cancer, and its treatment is difficult. a locoregional treatment may be proposed to certain patients, involving either chemoembolization or radioembolization. for radioembolization, i - lipiodol has been used for many years with good results, but its use is limited by major radioprotection problems. recently, radioembolization using microspheres labeled with yttrium-90 has been developed, notably therasphere (mds nordion, ottawa, canada) [26 ]. this approach is based on delivering a high radioactive dose to the liver volume to be treated. when both lobes are affected, two treatments must be carried out at 1-month interval in order to minimize the risk of radiation - induced side effects. the treatment requires a first diagnostic angiography, with the aim of coiling collateral gastrointestinal vessels and performing an maa scintigraphy in order to obtain a perfusion hepatic scintiscan and calculate the percentage of pulmonary shunt. the activity to be injected (ainj) is calculated based on a well - defined model. the aim is to deliver a dose d(gy) of 120 20 gy to the volume to be treated. this dose is calculated according to the following formula that is based on the previously described mird formalism and widely used : (1)d(gy)=ainj(gbq)(1s)50m(kg), where s represents the percentage of pulmonary shunt and m the mass of vascularized hepatic volume, with the mass of volume to be treated corresponding to the volume multiplied by a correction factor of 1.03. this volume corresponds to the volume of the hepatic parenchyma vascularized by the hepatic artery into which the microspheres are injected.. a precise calculation of the vascularized volume is essential for dosimetric calculation, including the activity to be administered. the vascularized or functional liver volume may also be analyzed using a vascular tracer such as human albumin serum labeled with technetium-99 m (tc - maa) : the volume of distribution of tc - maa following selective injection into the hepatic artery, at the same position where the microspheres are to be injected, reflects the vascularized volume of the lobe to be treated. in the absence of anatomical vascular variations, contrarily, this calculation is much more problematic in the event of anatomical variations, such as when three distinct arterial branches vascularize the liver, a situation encountered in about 10% to 30% of patients. it is assumed that the right vascular branch vascularizes segments 6 and 8, the central branch segment 5 and 7, the left branch segments 2, 3, and 4, while the inconsistent vascularization of segment 1 is widely recognized. given this case scenario, maa scintigraphy, which is mandatory for evaluating lung shunting, may be used for calculating the functional liver volume vascularized by each arterial branch. spect has already been employed for calculating functional volumes [7, 8 ] although it may lead in certain cases to volume overestimation due to the thresholding method used. in addition, pet has been reported to be useful for functional volume measurements, in particular in defining the volumes of irradiation [911 ]. the method used for volume definition (simple visual method or based on an isocontour with predefined threshold) was shown to significantly impact on the achieved values. while the use of spect / ct has not yet been reported in this setting, spect / ct - fused images may be helpful in delineating volumes, which may prove to be a reliable measurement of functional volumes. spect / ct may also be used to measure the tumor volume and nontumoral injected liver volume in order to calculate the doses absorbed by the tumor and the nontumoral liver, respectively. in this paper, we report on a phantom study aimed at validating volume measurements based on spect / ct. we also stress the advantages of spect / ct in computing the vascularized liver volume in addition to calculating the doses absorbed by the healthy liver and tumor in a patient with complex hepatic vascularization. we carried out a study on phantoms in order to validate the volume measurements performed using spect / ct. various phantoms were used, including a cylindrical jaszack phantom with a volume of 6,716 ml (cylinder 1) and two cylindrical phantoms of 774 (cylinder 2) and 473 ml (cylinder 3) for the measurement of large volumes (mimicking the liver). spheres 1, 2, 3, and 4 of 55, 20.5, 16, and 8 ml, respectively, were used and inserted into the jaszack phantom for measuring small representative tumor volumes. the activities of tc used for phantoms representative of the liver (cylinder 1, 2, and 3) ranged from 55 to 170 mbq. for the spheres, activities of 18.5, 37, 55.5, and 74 mbq were used. these activities were chosen in order to simulate standard clinical situations involving an injection of 185 mbq of tc - maa in a liver of 1500 ml, with tumor uptake of 10%, 20%, 30%, and 40% of the injected activities, respectively. spect / ct acquisitions were performed (32 projections, 180, 128128, 30 s / projection, symbia t2 gantry, siemens). spect imaging data was reconstructed using an iterative method (osem) with attenuation and scatter correction, and images were then visualized with or without fusion with ct scan data. volume measurement was carried out on spect and spect / ct images using a syngo data - processing console display unit (siemens) with volume analysis software. this software allowed us to generate semi - automatically the volume - of - interest (voi) in the liver and tumor by means of an isocontour definition method. each voxel with an activity reaching or exceeding a threshold percentage of the highest activity was included in the voi. for each volume measurement, the isocontour was fitted by superposition on either the contour of the hot spot located by spect alone or the internal wall of the phantom located on the spect / ct fusion images (figure 1). measurements of volumes by spect and spect / ct were carried out by two operators blinded to the phantom volumes. for each method, the percentage of error was calculated by reference to the actual volume of the phantoms. the interobserver reproducibility was evaluated using the bland - altman test of agreement, with values 0.8 considered to be excellent, values in the range of 0.60.8 to be good, values in the range of 0.40.6 to be poor, and values 20%, table 3), which was due to over- or underestimation of volumes by observer 1 and, more frequently, to large volume overestimation by observer 2. interobserver reproducibility was inadequate with spect alone, as the bland - altman test result was only 0.2. for spect / ct, results were superior, as mean errors of measurement were 473 ml), but inaccurate for small volume measurements, in line with previously published literature. in fact, visual volume definition is highly dependent on the individual investigator and display window setting (type of grey or color window and saturation used), potentially leading to large volume over- or underestimation. with the spect / ct quantitative method used in our study, the anatomical information supplied by ct allows for voi guidance so that the definition of volumes is not only based on the visual adjustment of voi on uptake, but also on the anatomical outlines of both liver and tumor. this approach turned out to be highly accurate, as the error of measurement was < 6% for volumes larger than 16 ml and even < 2.5% for very large volumes, such as the liver, with excellent interobserver agreement. the case reported in our study clearly demonstrates that in patients with anatomical abnormalities in liver vascularization, maa spect / ct vascularized volume measurement is a more functional and reliable method than volume calculations using the anatomical couinaud segmentation based on angiographic and ct data. in our case report, due to maa spect / ct, we were able to detect an unexpectedly large volume of liver slightly vascularized after selective injection of maa into the common hepatic branch. this method revealed the existence of microvascular communications between different anatomic segments, probably via intratumoral arterioportal shunts with low arterial blood flow, which were not visible on angiography but detected using maa spect / ct. the therapeutic impact for our patient was crucial, since it led us to significantly increase the activity injected for treatment, 5 gbq instead of 0.8 gbq, without any toxicity but an excellent response. furthermore, the evaluation of tumor and healthy liver doses based on the quantitative analysis of spect / ct is of great interest. in fact, the dose absorbed by the tumor represents the parameter most likely to correlate with response. this parameter depends directly on the quantity of radioactivity fixed in the tumor (i.e., degree of vascularization) as well as the total quantity of microspheres injected. only maa spect / ct allows us to evaluate this parameter, which corresponds to the tumoral absorbed dose. the effectiveness and reproducibility of maa spect / ct volume measurements was confirmed by a phantom study, with a mean error < 6% for volumes 16 ml and < 2.5% for larger volumes, such as the whole liver. maa spect / ct appears to be a more functional tool in identifying and calculating vascularized liver volumes than angiography, as it is able to identify unexpected vascularized areas with low blood flow not recognizable by angiography. in addition, quantitative maa spect / ct allows for calculating the tumoral absorbed and nontumoral injected liver doses. therefore, quantitative maa spect / ct may be of great help in defining vascularized liver volumes and calculating the activity to be administered, especially in patients with complex hepatic vascularization. quantitative maa spect / ct may be instrumental in optimizing the activity to be injected, thereby increasing the therapeutic effectiveness. | objectives. the aim of this study was to assess the effectiveness of spect / ct for volume measurements and to report a case illustrating the major impact of spect / ct in calculating the vascularized liver volume and dosimetry prior to injecting radiolabelled yttrium-90 microspheres (therasphere). materials and methods. this was a phantom study, involving volume measurements carried out by two operators using spect and spect / ct images. the percentage of error for each method was calculated, and interobserver reproducibility was evaluated. a treatment using therasphere was planned in a patient with three hepatic arteries, and the quantitative analysis of spect / ct for this patient is provided. results. spect / ct volume measurements proved to be accurate (mean error < 6% for volumes 16 cm3) and reproductive (interobserver agreement = 0.9). in the case report, 99mtc - maa spect / ct identified a large liver volume, not previously identified with angiography, which was shown to be vascularized after selective maa injection into an arterial branch, resulting in a large modification in the activity of therasphere used. conclusions. maa spect / ct is accurate for vascularized liver volume measurements, providing a valuable contribution to the therapeutic planning of patients with complex hepatic vascularization. |
mucosal cysts of the minor salivary glands are oral soft - tissue lesions that arise as dome - shaped swellings seen commonly on mucosal surface of lower lips and buccal mucosa. many modalities of treatment for mucosal cysts exist, including excision and suturing, deroofing, marsupialisation, radiosurgery and cryotherapy. four patients of age between 9 and 25 years were treated between june and december 2011. a male patient aged 9 years had a dome - shaped smooth, tense, bluish cyst of size 5 mm on the lower lip. the second patient was a female aged 24 years, with a mucosal cyst of 4 mm on the buccal mucosa. the third patient, a female aged 21 years, had a lesion of size 6 mm in the buccal mucosa. the fourth patient was a male patient aged 25 years, who had a recurrent mucosal cyst [figure 1 ] in the buccal mucosa which was treated 10 months back by electrocautery. no local anaesthesia was used. under aseptic precautions, a very small quantity of 88% phenol was injected into the cyst cavity using insulin syringes [figure 2 ]. patients were given analgesics post procedure for 1 day. injection of 0.1cc of 88% phenol into cyst the procedure was uneventful in all the cases. two of the patients complained of slight burning sensation during the procedure, which subsided within a few minutes. this was followed by complete healing with slight scarring within 2 weeks [figure 3 ]. although many other modalities of treatment of mucosal cysts exist, intralesional 88% phenol presents a simple and effective way of treating them. the advantage is that there is no intraoperative or postoperative bleeding, there are minimal surgical defects and there is minimal scarring. hence, intralesional injection of 88% phenol presents an easy and effective way for treatment of mucosal cysts. | mucous cysts are one of the common cystic lesions of minor salivary glands seen mostly on mucous surface of lower lip and buccal mucosa. many modalities of treatment for mucosal cysts exist including excision and suturing, deroofing, marsupialization, radiosurgery and cryotherapy. we have used 88% phenol as a mode of treatment of mucosal cysts. |
awareness of the need for evidence - informed practice has been growing within the massage therapy profession in north america. initiatives such as the canadian massage therapy research network and the massage therapy foundation (the foundation) have been launched to promote and support research literacy and capacity within the massage profession. research literacy is defined as the ability to find, understand and critically evaluate research evidence for application in professional practice. research capacity is the ability to conduct research. in line with its mission to advance the knowledge and practice of massage therapy through research, education and community service, the foundation actively provides educational opportunities designed to enhance research literacy and capacity among members of the profession. for example, at the annual american massage therapy association (amta) conventions, the foundation has sponsored and organized research - focused workshops. at the 2008 amta national convention, the foundation hosted its first workshop on qualitative research methods (introduction to qualitative research for massage therapists, presented by ania kania and antony porcino). it describes the nature of qualitative research and discusses the value of qualitative research to the massage therapy profession. the target audience for this article is massage therapists who want to be able to better understand the research literature, novice massage therapy researchers who are unfamiliar with qualitative research, and teachers of research methods courses in massage therapy training programs who want to include qualitative research methods in their curriculum. quantitative research investigates phenomena that require precise measurement and description, often with the goal to describe and predict outcomes in a larger population of interest or to examine the strength of relationships between variables of interest. quantitative research designs are highly controlled : for example, surveys and randomized controlled trials. qualitative research investigates phenomena in an in - depth and holistic fashion, through the collection of rich narrative materials in a flexible design. the two approaches differ with respect to paradigmatic assumptions (see table 1), are used to answer different types of research questions (see table 2) and produce different types of information. comparing the broad paradigmatic assumptions of qualitative and quantitative research sources : guba and lincoln (1994), p. 105117, and bowling (2002), examples of research questions to date, research in the field of health and medicine (including the field of complementary and alternative medicine) has been dominated by quantitative research approaches. historically, qualitative research was the domain of social scientists such as anthropologists and sociologists, but increasingly, quantitative research is being used in health research. qualitative research is particularly suited to the study of complex topics or issues about which little is known and concerning which quantification can not easily create or effectively convey understanding. broadly defined, qualitative research is used in understanding and describing the [subjective ] world of human experience. it provides a way of understanding the worlds inhabited by study participants, because it is grounded in their lived experience. it is a form of inquiry that brings the perspective of each participant to the forefront. more specifically, qualitative research methodologies provide a way to gain rich and in - depth insights about phenomena as they occur in their natural context by exploring and discovering people s perceptions and the meaning that they ascribe to those perceptions. quantitative research can describe how many people feel, do or think a certain way, but qualitative methods describe how people feel, do and think, or why people do so. qualitative research is exploratory and descriptive ; it follows an inductive process with regard to how information is gathered, analyzed and interpreted. for example, consider a study that aims to evaluate the effectiveness of massage therapy as a pain management strategy for people with chronic pain. the objective of a qualitative study would not be to determine whether massage therapy reduces the symptomatic pain or to quantify by how much. i feel better means to the person and how that meaning relates to the person s life and their overall sense of self. the objective of qualitative inquiry could also be to explore the full range of outcomes of massage therapy in the treatment of chronic pain, rather than to assess predetermined outcomes as in quantitative research. people may, for example, identify feelings of peace, specific changes in energy level or an overall sense of well - being that would otherwise have been missed. it is beyond the scope of the present article to provide a detailed overview of how qualitative research is conducted, but here, we outline the key distinguishing characteristics of qualitative research. it usually follows an iterative process, meaning that data collection and analysis occur simultaneously. that is, ongoing data collection is informed by data already collected, and the researcher uses knowledge already gained to try to fill gaps or to sort out potential contradictions as data collection continues. given that the purpose of qualitative research is to develop an in - depth understanding of phenomena and not to describe quantitative characteristics or to establish associations between variables, it focuses on gathering information from people who can provide the richest insights into the phenomenon of interest. as a result, small samples are commonly used in qualitative research, and study participants are usually selected in a purposive manner, using only those for whom the topic under study is relevant. usually, the researcher continues to collect data until saturation is reached, saturation being the point at which no new information is revealed with respect to the key themes emerging from the data. qualitative data collection examines everyday life in its natural context that is, in an uncontrolled naturalistic setting. data collection of this kind can use interviews, focus groups, recordings of conversations, observations and documents of all kinds. interview techniques vary from the semi - structured interview (following a predetermined interview guide that ensures all participants are asked to discuss the same topics) to completely unstructured interviews or narratives (each participant primarily determines the topics of discussion). focus group interviews are also commonly used because they are less expensive and time - intensive. focus groups are useful for gathering a wide range of opinions quickly, for exploring how members of a group interact, or for developing group consensus on a particular topic. however, the basic steps of the analysis process involve examining the data to develop an in - depth understanding (comprehending), identifying categories and themes in the data and developing an understanding of the nature of the phenomenon and the identity of the participants (de - contextualizing or synthesizing), sorting and re - sorting the data to develop alternative explanations of the phenomenon (theorizing), and further developing a theory in terms of its applicability to other settings or contexts (re - contextualizing). this process is demanding and requires a considerable amount of time and skill. to study the effects of an intervention, researchers often use standardized outcome measures (for example, the mcgill pain questionnaire) or direct measures (for example, blood pressure) to quantify the effect of an intervention. although such measures can provide important information, the limitation is that they are preselected by the researcher and restricted in their scope of understanding to the defined outcomes. qualitative research can identify new or additionally relevant outcomes of an intervention based on the experiences of the people involved. for example, using the qualitative research tradition of phenomenology, billhult. investigated the experience of massage in breast cancer patients undergoing chemotherapy treatment. most research on the effects of massage treatment in cancer care has focused on treatment of side effects such as anxiety, pain and nausea associated with conventional treatments of chemotherapy and radiation. in the qualitative inquiry by billhult and colleagues, the association of massage with positive experiences facilitated the shifting of mental processes from negative to positive, which eased the experience of the chemotherapy treatment. a second theme that emerged was the psychological support that was experienced through massage. given the physical changes associated with chemotherapy, participants expressed feelings of loneliness and ugliness. massage provided personal confirmation to these women and created a sense of being cared for despite altered body image and vulnerability, a result that was highly valued by the participants. the value of this qualitative study came from its focus on the lived experience of the participants, which enabled development of a greater understanding concerning the outcomes that are important, relevant and meaningful to the people involved than would be possible by using standardized outcomes that do not capture complex individual details. qualitative research can also be used in combination with quantitative research in a mixed - methods study design, adding understanding and meaning to the quantitative data. a design of this kind can provide highly valuable insights and a more complete understanding of the effectiveness of an intervention. consider the following example : in a study by brazier., the effectiveness of a residential yoga breathing and meditation program in improving well - being for individuals living with hiv / aids was assessed. quantitative and qualitative data were collected using a mixed - methods approach. for the quantitative component, individuals were randomized into two groups : one group received the yoga intervention and one did not (the control group). standardized outcome measures were used to quantify any changes in the mental health (mental health index), health status (mos - hiv health status survey) and daily stress levels (daily stress inventory) of the patients in both groups. results for the two groups and for the beginning and the end of the 12-week treatment period were compared. based on the quantitative findings, the authors concluded that no statistically significant change had occurred in the standardized outcome measures for the yoga group over the course of 12 weeks. furthermore, based on the daily stress inventory outcome measure, the stress in the yoga group increased over the 12-week period. in the qualitative component, participants in the yoga group were interviewed in depth to explore their perceptions of the benefits of the program. analysis of the interview transcripts revealed that the participants experienced a personal growth process that included engaging in life in new ways, being furthermore, the participants also expressed that positive changes were not always comfortable and can create pain. the insights provided by this qualitative inquiry indicated that the yoga intervention had positive effects on the participants. the results of the study demonstrate that, although standardized outcome measures are useful, alone they may not capture the broad range of possible outcomes or meaningful effects of an intervention as they are experienced or perceived by individuals. qualitative research provides the opportunity to explore not only outcomes, but also the whole system of massage, by investigating factors such as context and process of the intervention. the practice of massage does not take place in isolation ; rather, it occurs within a context a unique environment and set of circumstances that has nonspecific effects on the overall effect of the intervention. practitioner relationship, the environment or setting within which the treatment occurs, the culture and beliefs that the recipient and therapist bring with them to the treatment or intervention and the expectations that the recipient has of massage and of the treatment they will receive from the particular massage therapist. the context may influence the effect of an intervention. as van weel explained, non - specific effects work through their integration into the overall treatment approach. by exploring the physical and psychological context within which an intervention takes place, valuable insights can be gained about the therapeutic consequences of context effects and the impact of context effects on outcomes of the massage intervention. the process of the intervention refers to the course of action or sequence of steps taken to complete a particular task. it can also refer to a series of states or to the gradual sequence of changes that a person may move through or experience. in the context of massage therapy research, issues related to process can be studied from the perspective of the recipient of massage or of the massage therapist. research focused on the evaluation of the massage intervention can include assessment of processes such as learning, personal growth and enablement. processes related to how people integrate massage therapy into the context of their lives can also be explored. for example, consideration can be given to the processes involved in adapting or altering a massage session to individualize it to the needs of a particular client. in this way, through qualitative inquiry, important processes involved in receiving and providing massage therapy can be identified. broadly defined, qualitative research is used in understanding and describing the [subjective ] world of human experience. it provides a way of understanding the worlds inhabited by study participants, because it is grounded in their lived experience. it is a form of inquiry that brings the perspective of each participant to the forefront. more specifically, qualitative research methodologies provide a way to gain rich and in - depth insights about phenomena as they occur in their natural context by exploring and discovering people s perceptions and the meaning that they ascribe to those perceptions. quantitative research can describe how many people feel, do or think a certain way, but qualitative methods describe how people feel, do and think, or why people do so. qualitative research is exploratory and descriptive ; it follows an inductive process with regard to how information is gathered, analyzed and interpreted. for example, consider a study that aims to evaluate the effectiveness of massage therapy as a pain management strategy for people with chronic pain. the objective of a qualitative study would not be to determine whether massage therapy reduces the symptomatic pain or to quantify by how much. i feel better means to the person and how that meaning relates to the person s life and their overall sense of self. the objective of qualitative inquiry could also be to explore the full range of outcomes of massage therapy in the treatment of chronic pain, rather than to assess predetermined outcomes as in quantitative research. people may, for example, identify feelings of peace, specific changes in energy level or an overall sense of well - being that would otherwise have been missed. it is beyond the scope of the present article to provide a detailed overview of how qualitative research is conducted, but here, we outline the key distinguishing characteristics of qualitative research. it usually follows an iterative process, meaning that data collection and analysis occur simultaneously. that is, ongoing data collection is informed by data already collected, and the researcher uses knowledge already gained to try to fill gaps or to sort out potential contradictions as data collection continues. given that the purpose of qualitative research is to develop an in - depth understanding of phenomena and not to describe quantitative characteristics or to establish associations between variables, it focuses on gathering information from people who can provide the richest insights into the phenomenon of interest. as a result, small samples are commonly used in qualitative research, and study participants are usually selected in a purposive manner, using only those for whom the topic under study is relevant. usually, the researcher continues to collect data until saturation is reached, saturation being the point at which no new information is revealed with respect to the key themes emerging from the data. qualitative data collection examines everyday life in its natural context that is, in an uncontrolled naturalistic setting. data collection of this kind can use interviews, focus groups, recordings of conversations, observations and documents of all kinds. interview techniques vary from the semi - structured interview (following a predetermined interview guide that ensures all participants are asked to discuss the same topics) to completely unstructured interviews or narratives (each participant primarily determines the topics of discussion). focus group interviews are also commonly used because they are less expensive and time - intensive. focus groups are useful for gathering a wide range of opinions quickly, for exploring how members of a group interact, or for developing group consensus on a particular topic. however, the basic steps of the analysis process involve examining the data to develop an in - depth understanding (comprehending), identifying categories and themes in the data and developing an understanding of the nature of the phenomenon and the identity of the participants (de - contextualizing or synthesizing), sorting and re - sorting the data to develop alternative explanations of the phenomenon (theorizing), and further developing a theory in terms of its applicability to other settings or contexts (re - contextualizing). this process is demanding and requires a considerable amount of time and skill. to study the effects of an intervention, researchers often use standardized outcome measures (for example, the mcgill pain questionnaire) or direct measures (for example, blood pressure) to quantify the effect of an intervention. although such measures can provide important information, the limitation is that they are preselected by the researcher and restricted in their scope of understanding to the defined outcomes. qualitative research can identify new or additionally relevant outcomes of an intervention based on the experiences of the people involved. for example, using the qualitative research tradition of phenomenology, billhult. investigated the experience of massage in breast cancer patients undergoing chemotherapy treatment. most research on the effects of massage treatment in cancer care has focused on treatment of side effects such as anxiety, pain and nausea associated with conventional treatments of chemotherapy and radiation. in the qualitative inquiry by billhult and colleagues, the association of massage with positive experiences facilitated the shifting of mental processes from negative to positive, which eased the experience of the chemotherapy treatment. a second theme that emerged was the psychological support that was experienced through massage. given the physical changes associated with chemotherapy, participants expressed feelings of loneliness and ugliness. massage provided personal confirmation to these women and created a sense of being cared for despite altered body image and vulnerability, a result that was highly valued by the participants. the value of this qualitative study came from its focus on the lived experience of the participants, which enabled development of a greater understanding concerning the outcomes that are important, relevant and meaningful to the people involved than would be possible by using standardized outcomes that do not capture complex individual details. qualitative research can also be used in combination with quantitative research in a mixed - methods study design, adding understanding and meaning to the quantitative data. a design of this kind can provide highly valuable insights and a more complete understanding of the effectiveness of an intervention., the effectiveness of a residential yoga breathing and meditation program in improving well - being for individuals living with hiv / aids was assessed. quantitative and qualitative data were collected using a mixed - methods approach. for the quantitative component, individuals were randomized into two groups : one group received the yoga intervention and one did not (the control group). standardized outcome measures were used to quantify any changes in the mental health (mental health index), health status (mos - hiv health status survey) and daily stress levels (daily stress inventory) of the patients in both groups. results for the two groups and for the beginning and the end of the 12-week treatment period were compared. based on the quantitative findings, the authors concluded that no statistically significant change had occurred in the standardized outcome measures for the yoga group over the course of 12 weeks. furthermore, based on the daily stress inventory outcome measure, the stress in the yoga group increased over the 12-week period. in the qualitative component, participants in the yoga group were interviewed in depth to explore their perceptions of the benefits of the program. analysis of the interview transcripts revealed that the participants experienced a personal growth process that included engaging in life in new ways, being furthermore, the participants also expressed that positive changes were not always comfortable and can create pain. the insights provided by this qualitative inquiry indicated that the yoga intervention had positive effects on the participants. the results of the study demonstrate that, although standardized outcome measures are useful, alone they may not capture the broad range of possible outcomes or meaningful effects of an intervention as they are experienced or perceived by individuals. qualitative research provides the opportunity to explore not only outcomes, but also the whole system of massage, by investigating factors such as context and process of the intervention. the practice of massage does not take place in isolation ; rather, it occurs within a context a unique environment and set of circumstances that has nonspecific effects on the overall effect of the intervention. practitioner relationship, the environment or setting within which the treatment occurs, the culture and beliefs that the recipient and therapist bring with them to the treatment or intervention and the expectations that the recipient has of massage and of the treatment they will receive from the particular massage therapist. the context may influence the effect of an intervention. as van weel explained, non - specific effects work through their integration into the overall treatment approach. by exploring the physical and psychological context within which an intervention takes place, valuable insights can be gained about the therapeutic consequences of context effects and the impact of context effects on outcomes of the massage intervention. the process of the intervention refers to the course of action or sequence of steps taken to complete a particular task. it can also refer to a series of states or to the gradual sequence of changes that a person may move through or experience. in the context of massage therapy research, issues related to process can be studied from the perspective of the recipient of massage or of the massage therapist. research focused on the evaluation of the massage intervention can include assessment of processes such as learning, personal growth and enablement. processes related to how people integrate massage therapy into the context of their lives can also be explored. for example, consideration can be given to the processes involved in adapting or altering a massage session to individualize it to the needs of a particular client. in this way, through qualitative inquiry, important processes involved in receiving and providing massage therapy can be identified. the exploratory and descriptive focus of qualitative research is valuable to the massage profession, for it adds to the knowledge of massage therapy. it illuminates the complexity of the intervention in terms of the range of outcomes that may occur, the contextual factors that affect those outcomes and the processes involved in a massage intervention. furthermore, it brings the various perspectives of the people who experience massage to the forefront. exploring the lived experience of massage therapy and the meaning it holds for people enables the development of a more in - depth understanding of massage therapy. outcomes, context and process factors enable the development and provision of more effective and appropriate treatment plans. lastly, qualitative research makes a practitioner more aware of the role of massage therapist how the practitioner s own beliefs and values can affect or influence a client s experience, perceptions and outcomes. the insights provided by an awareness of how others practice can be used to reflect on one s own practice. qualitative research findings therefore will not only help massage therapists practice more effectively, but also differently, with greater awareness and mindfulness. | qualitative inquiry is increasingly used in health research because it is particularly suited to the study of complex topics or issues about which little is known and concerning which quantification can not easily create or effectively convey understanding. by exploring the lived experience of people providing and receiving massage therapy and the meaning that those people ascribe to those experiences, in - depth understanding of the nature of massage therapy and of how it affects people s lives is possible. qualitative research may also provide insights into the outcomes, process and context of massage therapy that can not be fully achieved through quantification alone.the purpose of the present article is to describe qualitative research and to discuss its value to the massage therapy profession. the target audience is massage therapists who want to be able to better understand the research literature, novice massage therapy researchers who are unfamiliar with qualitative research, and teachers of research methods courses in massage therapy training programs who want to include qualitative research methods in their curriculum. |
as a service to our authors and readers, this journal provides supporting information supplied by the authors. such materials are peer reviewed and may be re - organized for online delivery, but are not copy - edited or typeset. technical support issues arising from supporting information (other than missing files) should be addressed to the authors. | two macrotricyclic ligands composed of two face - to - face octadentate metal chelates were synthesized. these cage - shaped disodium complexes had special recognition ability for various counter anions. specific chiral dicarboxylates bound to the complexes within the cavity and exhibited chirality induction properties. for instance, n - boc - asp dianion strongly induced circular dichroism (cd) signals, but n - boc - glu dianion, which is one carbon longer, did not. |
papillary thyroid microcarcinoma (ptmc) is defined as a papillary thyroid carcinoma that 's equal to or less than 1.0 cm at the greatest dimension according to the world health organization classification system for thyroid tumors (1). the prevalence of ptmc in the general population with no thyroid disease is reportedly high at 3 - 36%, according to autopsy studies (2 - 4). the majority of ptmcs are not palpable and show an asymptomatic course (5). high - resolution thyroid ultrasonography and fineneedle aspiration cytology (fnac) under ultrasound guidance have recently enabled making an accurate diagnosis of ptmc, which may explain why the reported incidence of ptmc is increasing (6). although ptmc generally has a favorable prognosis, the tumor may show locoregional recurrence and distant metastases (7 - 10). for ptmc patients, the reported incidence of lymph node metastasis is as high as 40%, and these occur mainly in the central compartment of the neck (2, 3, 11). however, the prognostic factors that can differentiate silent ptmc from potentially aggressive ptmc remain unclear. one of the major issues for the treatment of ptmc is whether central neck lymph node dissection (cnd) should be routinely performed during the initial operation. while cnd is not generally performed due to the excellent prognosis of ptmc, some reports have recommended performing routine cnd even in ptmc patients with no clinical nodal metastases (12, 13). the present study reviewed the surgery, pathology, laboratory and follow - up data of 101 patients who underwent surgery for ptmc. in order to determine the clinical necessity of performing cnd in ptmc patients, the patients were analyzed in terms of those who underwent cnd (cnd) and those who did not (no cnd). the clinical data was reviewed on 106 ptmc patients who underwent total thyroidectomy between september 2001 and july 2005. the pathology of all the patients were papillary carcinoma, as indicated by fine needle aspiration cytology that was guided by high - resolution ultrasonography, and the aspiration pathology was confirmed by the surgical pathology. preoperative evaluation included taking a clinical history and a physical examination, thyroid function tests and radiography of the thorax and ultrasonography. the ultrasonograpnic examinations were performed preoperatively by the same experienced radiologist (j.h.l) in order to evaluate lymph node metastasis. five patients who were preoperatively suspected of having cervical lymph node metastasis and who underwent therapeutic neck lymph node dissection were excluded from the study. thus, 101 patients with primary tumor sizes 1.0 cm and no preoperative evidence of nodal metastases were finally included in the study. for analysis, the patients were divided into 2 groups : the total thyroidectomy without cnd group (the no cnd group, n=53) and the total thyroidectomy plus cnd group (the cnd group, n=48) (table 1). the study cohort was not randomized and the decision to perform cnd was made by both the surgeon and the patient. this study was reviewed and approved by the institutional review board of the asan medical center and informed consent was waived. cnd was carried out in a conventional manner and microdissection methods were not used (14). node clearance was performed cranially to both superior thyroid arteries and the pyramidal lobe, caudally to the innominate vein, laterally to the carotid sheaths and dorsally to the prevertebral fascia (5). the central compartment was divided into 4 node sites : the pretracheal site, the ipsilateral site, the contralateral paratracheal site and the superior mediastinal site below the sternal notch. particular attention was given to identifying the parathyroid glands, and parathyroid autotransplantation was carried out as required, not on principle. the pathology was carefully reviewed with focusing on the primary tumor size and multifocality, extrathyroidal extension and lymph node metastases. hypocalcemia was defined as the need for exogenous calcium replacement in order to maintain a normal range of serum total calcium (8 - 10.4 mg / dl) or to eliminate the clinical signs and symptoms of hypocalcemia. all the patients received radioactive iodine ablation therapy (rai) with 2.78 - 5.55 gbqi at 5 - 6 weeks after surgery. prior to rai, the serum thyroid - stimulating hormone (tsh) level was elevated to > 30 mu / l, and then the serum levels of stimulated tg (ablation - tg) and anti - tg antibody were measured. a whole body scan (wbs) using 148 mbqi was performed at 5 - 7 days and at 6 - 12 months after rai with a simultaneous measurement of the serum stimulated tg (control - tg). the serum tg levels were measured using a solid phase two - site immunoradiometric assay kit (elsa - htg, schering - cis bio international, gif / yvette, france) as previously described (15). the detection limit of thyroglobulin was 1.0 ng / ml. the anti - tg levels were measured using radioligand assays (henning test anti - tg ; brahms diagnostica, berlin, germany). when the control - tg level was above 2 ng / ml, then neck ultrasonography with or without fine needle aspiration cytology was performed to localize the potentially malignant thyroid lesions. the t - test was used to analyze the mean differences of the pathologic findings and the other clinical parameters between the groups. fisher 's exact test or test was used to investigate differences among the categorical data. a p - value of less than 0.05 was deemed to indicate a significant difference. the clinical data was reviewed on 106 ptmc patients who underwent total thyroidectomy between september 2001 and july 2005. the pathology of all the patients were papillary carcinoma, as indicated by fine needle aspiration cytology that was guided by high - resolution ultrasonography, and the aspiration pathology was confirmed by the surgical pathology. preoperative evaluation included taking a clinical history and a physical examination, thyroid function tests and radiography of the thorax and ultrasonography. the ultrasonograpnic examinations were performed preoperatively by the same experienced radiologist (j.h.l) in order to evaluate lymph node metastasis. five patients who were preoperatively suspected of having cervical lymph node metastasis and who underwent therapeutic neck lymph node dissection were excluded from the study. thus, 101 patients with primary tumor sizes 1.0 cm and no preoperative evidence of nodal metastases were finally included in the study. for analysis, the patients were divided into 2 groups : the total thyroidectomy without cnd group (the no cnd group, n=53) and the total thyroidectomy plus cnd group (the cnd group, n=48) (table 1). the study cohort was not randomized and the decision to perform cnd was made by both the surgeon and the patient. this study was reviewed and approved by the institutional review board of the asan medical center and informed consent was waived. cnd was carried out in a conventional manner and microdissection methods were not used (14). node clearance was performed cranially to both superior thyroid arteries and the pyramidal lobe, caudally to the innominate vein, laterally to the carotid sheaths and dorsally to the prevertebral fascia (5). the central compartment was divided into 4 node sites : the pretracheal site, the ipsilateral site, the contralateral paratracheal site and the superior mediastinal site below the sternal notch. particular attention was given to identifying the parathyroid glands, and parathyroid autotransplantation was carried out as required, not on principle. the pathology was carefully reviewed with focusing on the primary tumor size and multifocality, extrathyroidal extension and lymph node metastases. hypocalcemia was defined as the need for exogenous calcium replacement in order to maintain a normal range of serum total calcium (8 - 10.4 mg / dl) or to eliminate the clinical signs and symptoms of hypocalcemia. all the patients received radioactive iodine ablation therapy (rai) with 2.78 - 5.55 gbqi at 5 - 6 weeks after surgery. prior to rai, the serum thyroid - stimulating hormone (tsh) level was elevated to > 30 mu / l, and then the serum levels of stimulated tg (ablation - tg) and anti - tg antibody were measured. a whole body scan (wbs) using 148 mbqi was performed at 5 - 7 days and at 6 - 12 months after rai with a simultaneous measurement of the serum stimulated tg (control - tg). the serum tg levels were measured using a solid phase two - site immunoradiometric assay kit (elsa - htg, schering - cis bio international, gif / yvette, france) as previously described (15). the detection limit of thyroglobulin was 1.0 ng / ml. the anti - tg levels were measured using radioligand assays (henning test anti - tg ; brahms diagnostica, berlin, germany). when the control - tg level was above 2 ng / ml, then neck ultrasonography with or without fine needle aspiration cytology was performed to localize the potentially malignant thyroid lesions. the t - test was used to analyze the mean differences of the pathologic findings and the other clinical parameters between the groups. fisher 's exact test or test was used to investigate differences among the categorical data. a p - value of less than 0.05 was deemed to indicate a significant difference. the study cohort consisted of 17 men and 84 women, with a mean age of 49.8 years (range : 23 to 76 years) (table 1). the cnd and no cnd groups were similar in terms of gender and the age distribution. the mean follow - up time after surgery was 24.4 months (range : 13 - 55 months). the overall incidence of hypocalcemia was 15 of 101 patients (14.9%), of whom only one patient had permanent hypocalcemia. other complications (e.g., bleeding, hematoma and seroma) were not noted in either group (data not shown). the presence of multifocality and an extrathyroidal extension of the primary tumors were the same for both groups. for the cnd group, 18 of 48 patients (37.5%) had central nodal metastasis. the levels of ablation - tg and control - tg were compared between the no cnd patients and the cnd patients with or without central nodal metastases (fig. 1). three no cnd patients were excluded from the tg analyses due to positive anti - tg antibody results (> 100 u / ml). of the remaining 50 no cnd patients, 12 (24.0%) had ablation - tg levels > 5 ng / ml, 11 (22.0%) had levels from 2 - 5 ng / ml, and 27 (54.0%) had levels 5 ng / ml appeared to be higher in the node - positive compared to the node - negative group (4/18 vs. 1/30, respectively), this difference did not reach statistical significance (p=0.058). the same analysis with using an ablation - tg threshold level of 100 u / ml). the proportion of patients with serum stimulated control - tg levels > 2 ng / ml did not significantly differ between the no cnd or the node - positive cnd patients and the node - negative cnd patients (5/49 or 2/18 vs. 1/27, respectively, p>0.1) (fig. 3). three patients experienced recurrence during follow - up (2 no cnd patients and 1 node - positive cnd patient), as confirmed by ultrasonography - guided fineneedle aspiration cytology. the ablation - tg levels and control - tg levels of all the patients with recurrences were as high as > 8.0 ng / ml. the overall incidence of hypocalcemia was 15 of 101 patients (14.9%), of whom only one patient had permanent hypocalcemia. other complications (e.g., bleeding, hematoma and seroma) were not noted in either group (data not shown). the presence of multifocality and an extrathyroidal extension of the primary tumors were the same for both groups. for the cnd group, 18 of 48 patients (37.5%) had central nodal metastasis. the levels of ablation - tg and control - tg were compared between the no cnd patients and the cnd patients with or without central nodal metastases (fig. 1). three no cnd patients were excluded from the tg analyses due to positive anti - tg antibody results (> 100 u / ml). of the remaining 50 no cnd patients, 12 (24.0%) had ablation - tg levels > 5 ng / ml, 11 (22.0%) had levels from 2 - 5 ng / ml, and 27 (54.0%) had levels 5 ng / ml appeared to be higher in the node - positive compared to the node - negative group (4/18 vs. 1/30, respectively), this difference did not reach statistical significance (p=0.058). the same analysis with using an ablation - tg threshold level of 100 u / ml). the proportion of patients with serum stimulated control - tg levels > 2 ng / ml did not significantly differ between the no cnd or the node - positive cnd patients and the node - negative cnd patients (5/49 or 2/18 vs. 1/27, respectively, p>0.1) (fig. 3). three patients experienced recurrence during follow - up (2 no cnd patients and 1 node - positive cnd patient), as confirmed by ultrasonography - guided fineneedle aspiration cytology. the ablation - tg levels and control - tg levels of all the patients with recurrences were as high as > 8.0 ng / ml. the present study found that cnd was not associated with any increase in postoperative complications. the incidence of hypocalcemic was the same for both cnd and no cnd groups, suggesting that exploration of the central neck compartment did not lead to increased damage to the parathyroid glands during cnd. these findings are in disagreement with those of others researchers who reported that while cnd was not associated with an increased risk of postoperative vocal fold paralysis, it was linked to a higher rate of transient and permanent hypoparathyroidism (16, 17). however, the current study shows that cnd can be safely performed by an experienced surgeon and this procedure does not increase the risk of surgical morbidity. in the current study, occult metastasis to the central neck compartment was found in 37.5% of the cnd patients with no clinical nodal metastasis, which is consistent with the other recent reports (5, 18). the pathologic finding of ptmc having a high proclivity for regional metastases seems to justify performing routine cnd in these patients. a recent study of 600 ptmc patients who underwent cnd found there was no significant difference for disease - free survival between the patients who were positive (43%) or negative (57%) for central nodal metastasis (18). another study asserted that nodal recurrence did not differ between the prophylactic dissection group and the no - dissection group, prophylactic cnd for ptmc is not beneficial to the patients with no palpable lymphadenopathy. a major issue of ptmc treatment is the prognostic value of microscopic lymph node involvement. measurement of the serum tsh - stimulated tg levels is an important method for detecting the presence of residual or recurrent malignant thyroid tissue during follow - up. the meaning of the tg levels at rai is still debatable because the thyroid remnants and untreated occult metastasis contribute to tg synthesis and therefore, the increased tg levels. however, a prior report suggested that the ablation - tg levels were well correlated with the control - tg levels, and that levels > 2.0 ng / ml were highly associated with persistent or recurrent disease in the low - risk patients with well - differentiated thyroid carcinoma (15). therefore, we used both the ablation - tg and control - tg levels for predicting recurrences. the ablation - tg levels appeared to be higher in the controls or the cnd patients with nodal metastases than that in the cnd patients without nodal metastases. in addition to the pathologic information on nodal metastases in ptmc patients, this may also suggest a role for cnd in planning rai after surgery. recent studies have investigated whether the control - tg values can predict recurrent and persistent disease (19, 20). the current study found that control - tg levels did not differ among the 3 groups, and that the recurrence rate was very low. moreover, no recurrence was found in any of the cnd patients without occult nodal metastasis, suggesting that this group has a good prognosis and there is no need for high - dose rai. thus, cnd may have a role in determining the plan for postoperative rai and also the patient 's prognosis. the present study data allowed us to investigate whether rai was sufficient to remove occult metastasis. for the control patients, 44.0% had a stimulated tg level above 2 ng / ml prior to rai, while only 10.2% were above 2 ng / ml at 6 - 12 months after rai. this result suggests that rai may be an effective method for ablating the remnant thyroid tissue or disease. however, the potential benefits of rai on lowering the disease recurrence rate and the mortality rate of ptmc patients remains as a controversial issue (9, 21 - 23), and no randomized, controlled clinical trials have yet been performed on a large number of patients. furthermore, rai has side effects, including radiation sialadenitis in up to 40% of patients, with the incidence linked to the dose of radioiodine (24). the recent data suggests that low - risk patients with ptmc do not benefit from rai (23). thus, cnd may help discriminate between the patients with and without ptmc - associated metastatic neck diseases and so this can abrogate the necessity of high - dose rai for the low - risk patients. the present study may have been limited by its retrospective non - randomized design. the decision to perform a cnd however, the general demographic and pathologic data were comparable between the control and cnd groups. the study also included more than 100 ptmc patients with a clinical n0 neck, which is a large number of patients and it is more than sufficient for making between - group comparisons. a relatively short follow - up period may also have been a limitation, and it may have affected the findings regarding recurrence or survival. the present data showed that the serum tsh - stimulated tg levels were predictive of recurrence, and now a larger study with a longer follow - up period is warranted to confirm our results. the present study found that occult metastasis to the central compartment is a common event in ptmc patients. a cnd provides pathologic information on nodal metastases, which may assist the postoperative rai planning. it appears that the stimulated tg levels at rai and follow - up may help predict recurrence, and while this observation did not reach statistical significance in this study, we believe it may reach significance in a larger study. however, our present findings indicate that these questions may be answered by a longer follow - up of these patients. | objectivesit remains unclear as to whether routine central neck dissection (cnd) is necessary when performing surgery to treat patients with papillary thyroid microcarcinoma (ptmc). to determine the necessity for routine cnd in ptmc patients, we reviewed the clinicopathologic and laboratory data of the patients of ptmc.methodsbetween september 2001 and july 2005, 101 patients with ptmc and clinical n0 disease were retrospectively reviewed. the study cohort was devided into groups : the total thyroidectomy plus cnd group (the cnd group, n=48) and the total thyroidectomy without cnd group (the no cnd group, n=53). the serum stimulated thyroglobulin (tg) levels were measured after surgery and prior to radioactive iodine ablation therapy (rai) and at 6 - 12 months after rai. pathology, the tg levels and recurrence data were compared between the 2 groups.resultscentral nodal metastases were found in 18 of the 48 cnd patients (37.5%). the incidence of tg levels > 5 ng / ml at rai was higher in the no cnd patients and in the 18 node - positive cnd patients compared with the 30 node - negative cnd patients (22 - 24% vs. 3%, respectively, p=0.020 - 0.058). the difference when performing a similar comparison using a > 2 ng / ml tg threshold level showed no significance (10 - 11% vs. 4%, respectively, p>0.1). two of the no cnd patients and one node - positive cnd patient had recurrences in the thyroid bed or lateral neck during a mean follow - up of 24 months.conclusionthe data showed that occult metastasis to the central neck is common in ptmc patients. a cnd provides pathologic information about the nodal metastases, and it potentially provides guidance for planning the postoperative rai. however, the long - term benefit of cnd on recurrence and survival remains somewhat questionable. |
every year, influenza a viruses (iavs) cause epidemic outbreaks of respiratory tract infection resulting in excess morbidity and mortality. especially individuals with certain underlying medical conditions and the elderly are at risk for complications of influenza.therefore, it is recommended to vaccinate these individuals against influenza annually. currently used vaccines largely aim at the induction of antibodies directed to viral glycoproteins, in particular the hemagglutinin (ha). these antibodies neutralize the virus by preventing viral attachment to host cells and are generally considered the main correlate of protection against influenza virus infection. therefore, assessing postvaccination ha - specific antibody titers is used as surrogate marker for vaccine efficacy compliant with emea and fda guidelines [2, 3 ]. however, seasonal influenza viruses continuously accumulate amino acid substitutions in the antigenic sites of the ha molecules and consequently display considerable antigenic drift. this allows currently circulating influenza viruses to escape from the neutralizing activity of antibodies induced by previous infections or vaccination and necessitates updating the vaccine regularly to match recent epidemic strains. occasionally, novel strains of influenza a viruses are introduced with ha molecules that are antigenically distinct from seasonal influenza a viruses including those of a novel subtype. seasonal influenza vaccines are not protective against these new viruses, which may spark a pandemic outbreak of influenza and against which specific vaccines need to be developed. the pandemic of 2009 caused by influenza a / h1n1 viruses of swine origin painfully highlighted that the development of a matching vaccine is a time consuming process, and, in many countries, vaccines became available after the peak of pandemic [4, 5 ]. for these reasons, there is considerable interest in the development of more broadly protective influenza vaccines that ideally would afford broad protection against various subtypes of influenza a viruses [6, 7 ] and/or antigenic drift variants within a subtype. it has been well established that infection with influenza a virus can induce a certain degree of protective immunity to infection with other influenza a viruses of unrelated subtypes (heterosubtypic immunity) (for review see). elucidation of the correlates of protection of this type of immunity may aid the development of more universal vaccines. since different subtypes of influenza a virus are defined by the absence of serological cross - reactivity, it is unlikely that antibodies to ha or neuraminidase (na) contribute to this type of infection - induced immunity to a great extent. however, recently antibodies have been identified specific for epitopes located in the stem region of the ha molecule, displaying broad reactivity and broad neutralizing activity against several different influenza a viruses of different ha subtypes [916 ]. ha - stem - based vaccines may be a promising venue for the development of broadly protective vaccines. other vaccine candidates aiming at the induction of cross - protective antibodies may include those based on the m2 protein [1721 ]. furthermore, several studies have shown that postinfection serum does not afford protection against a heterosubtypic strain of influenza a virus, whereas virus - specific t cells do. however, it was shown that the protection mediated by vaccine - induced m2 antibodies was weak and could not prevent infection of mice. the mechanism of protection was based on antibody - dependent cell cytotoxicity (adcc). since the majority of virus - specific t cells, and in particular cd8 cytotoxic t lymphocytes (ctl), are directed against relatively conserved viral proteins like the nucleoprotein (np) and the matrix 1 protein (m1), it was already suggested three decades ago that virus - specific ctls contribute to heterosubtypic immunity [24, 25 ]. in the present paper, we review the evidence that influenza virus - specific t cells contribute to (cross-)protective immunity and discuss vaccine formulation that can induce virus - specific ctl. the most important mode of action of virus - specific ctl is recognition and elimination of virus - infected cells. this way, the production of progeny virus is prevented. thus, the presence of preexisting t - cell immunity results in more rapid clearance of virus infections. key for heterosubtypic immunity is that ctls are cross - reactive and recognize epitopes shared by influenza a viruses of different subtypes. the effectors functions of ctls that are responsible for the elimination of virus - infected cells include the release of perforin and granzyme from their granules and fas / fasl interactions with infected target cells. in addition, upon activation virus - specific cd8 t cells can produce a variety of different cytokines including ifn- and tnf-. it was shown that virus - specific ctls, through their receptor recognize viral peptides, which are generated by the endogenous route of antigen processing and that are ultimately presented by mhc class i molecules on the surface of antigen - presenting cells or virus - infected cells [26, 27 ]. for the efficient induction of virus - specific ctls, it is required that the antigen is present in the cytosol of antigen - presenting cells where antigen processing takes place. influenza virus - specific ctl can recognize epitopes that are shared by different subtypes of influenza a virus. indeed, it was shown that a large proportion of mouse and human ctls induced after infection with influenza a virus were directed against the relatively conserved np and m1 protein [29, 31, 3739 ]. this raised the expectation that these cells contribute to cross - protection against viruses of different subtypes. many studies have been performed to demonstrate the cross - reactivity of influenza virus - specific ctls and their role in heterosubtypic immunity. early evidence for the intersubtypic cross - reactivity of ctl was described by zweerink. [28, 40 ], who demonstrated that mouse ctl specific for influenza virus of the h2n1 subtype could lyse target cells infected with virus of the h3n2 subtype. also with other combinations of subtypes, the cross - reactive nature of virus - specific ctl was confirmed (table 1). for example, it was shown that in healthy individuals, with a history of infection with seasonal influenza virus, memory cd8 t cells were present in the blood that cross - reacted with highly pathogenic h5n1 virus [3234 ]. the presence of cross - reactive ctl may afford a certain degree of protection against infection with these viruses, which still constitute a pandemic threat. it is of interest to note that especially younger individuals are at risk for severe disease and fatal outcome of influenza h5n1 infection. it is tempting to speculate that younger individuals less likely have been exposed to seasonal influenza a viruses of the h3n2 or h1n1 subtype and, thus, have not mounted a ctl response to these viruses. therefore, they may be more susceptible to infection with a virus of an alternative subtype. however, it can not be ruled out that other factors play a role in the observed disproportionate age distribution of severe h5n1 cases. furthermore, ctl obtained from healthy subjects before the pandemic of 2009 displayed cross - reactivity with the pandemic 2009 ph1n1 virus [35, 36 ], which may have afforded a certain degree of protection against this virus. evidence for the role of virus - specific ctl in protection against influenza virus infection predominantly stems from animal models (table 2). using various combinations of influenza a virus subtypes, it was demonstrated that ctl responses induced after a primary infection with influenza virus either correlated with protection against challenge infection with a virus of another subtype or were responsible for protective immunity [8, 4247 ]. however, since the conserved proteins like the np, m1, and the polymerase proteins display a high degree of sequence homology, it is assumed that the extent of cross - reactivity between different subtypes of influenza a virus is substantial. this is exemplified by demonstrating that human ctls directed to human influenza a viruses of the h3n2 subtype cross - react considerably with avian influenza a viruses of the h5n1 subtype. by adoptive transfer or depletion of virus - specific cd8 t cells, it was confirmed that these cells contributed to protective heterosubtypic immunity [22, 4850, 52 ]. indeed, the adoptive transfer of virus - specific ctls to nave recipient mice had a beneficial effect on the course of subsequent challenge infections. it was shown that transfer of ctl from mice infected with seasonal h3n2 virus protected recipient mice against challenge infection with 2009 ph1n1 virus. also chickens that received ctl from chickens infected with h9n2 virus were protected against subsequent challenge infection with highly pathogenic h5n1 virus. also, depletion of cd8 t cells prior to challenge infection confirmed that these cells contribute to heterosubtypic immunity. primed mice or chickens, from which ctl were depleted, had higher lung virus titer, developed more severe disease, and displayed higher mortality rates after challenge infection than control animals [54, 55, 57, 59 ]. the first and, to our knowledge, the only evidence for this was described by mcmichael. they demonstrated that in experimentally infected individuals, virus - specific cytotoxicity inversely correlated with the extent of virus shedding in the absence of antibodies specific for the h1n1 strain that was used for infection. there is, however, epidemiological evidence that indicate that prior exposure to influenza viruses is inducing protective immunity against a heterosubtypic strain of influenza. people, who experienced symptomatic influenza caused by infection with influenza viruses of the h1n1 subtype, were partially protected from infection with the pandemic h2n2 viruses in 1957. a possible correlation with the presence of virus - specific ctl - mediated immunity was not studied. more circumstantial evidence is based on the observation that the ratio between synonymous and nonsynonymous (ds / dn) mutations in the np gene is lower in ctl epitope sequences than in the rest of the protein. this also provides indirect evidence that ctls exert antiviral activity in humans at the population level and indicates that ctl epitopes are under selective pressure. indeed, a number of amino acid substitutions that were observed in ctl epitopes during the evolution of influenza a / h3n2 viruses were associated with escape from recognition by virus - specific ctl [6165 ]. examples include the r384 g substitution at the anchor residue of the hla - b2705 restricted np383391 epitope and amino acid substitutions at t - cell receptor contact residues of the hla - b3501 restricted np418426 epitope. in both cases, the amino acid substitutions affected the in vitro human influenza virus - specific ctl response significantly [66, 67 ]. of interest, the r384 g substitution alone was detrimental to viral fitness and was only tolerated in the presence of two functionally compensating comutations [68, 69 ]. thus, apparently, the virus has the capacity to overcome functional constraints in order to evade t - cell immunity. the rapid fixation of the r384 g substitution could be explained by strong bottle - neck and founder effects at the population level in a theoretical model. although ctl epitopes, can thus display variability allowing the virus to escape from recognition by ctl specific for these epitopes, other epitopes remain fully conserved including the immunodominant m15866 epitope that is restricted by hla - a0201, which has a high prevalence in most countries. for this and some other conserved epitopes it was demonstrated that also functional constraints may play a role in limiting the virus to escape efficiently from recognition by ctl to these highly conserved epitopes [71, 72 ]. thus, influenza virus ctl epitopes are either conserved, display variation at non - anchor residues, or loose their anchor residues at the cost of viral fitness, which need to be functionally compensated by the accumulation of comutations. a large number of peptides are generated during processing of viral proteins in infected cells, but only some of these peptides are ultimately presented by major histocompatibility complex class i molecules and recognized by specific ctl. the hierarchy of ctl responses is called immunodominance [7375 ] and has been demonstrated in animal models and humans. in mice it was shown that the hierarchy of primary and secondary ctl responses differ [76, 78, 79 ]. since some ctl epitopes are more dominant than others, also the hla usage of the ctl response is dependent on the repertoire of viral epitopes. for this reason, the hla usage of the ctl response to influenza a virus is different from that to influenza b virus. assessing the response to a single epitope is not fully informative without knowing it is relative immunodominance. in addition, it has been shown that the response to a single epitope can be influenced by other non - corresponding hla alleles [77, 81 ]. the hla haplotype dictates which epitopes can be presented and recognized and determines the magnitude of the virus - specific ctl response. for example, the immunodominant m15866 epitope is only recognized in hla - a0201 positive subjects, and, in these individuals, the overall ctl response to influenza a virus is higher than in hla - a0201 negative subjects that are matched for the remaining hla alleles. thus, both immunodominance and hla restriction of ctl responses should be taken into account when assessing the ability of candidate vaccines to induce virus - specific ctl responses. for the efficient induction of ctl responses, it is critical that viral antigens enter the endogenous route of antigen processing. to achieve this, viral proteins need to be delivered in the cytosol of antigen presenting - cells, where degradation of these proteins by the proteasome takes place. the peptides that are generated are then transported by the transporter associated with antigen processing (tap) into the endoplasmic reticulum where binding of antigenic peptides with their corresponding mhc class i molecules can take place. these mhc class i / peptide complexes are subsequently transported to the cell membrane for recognition by virus - specific cd8 t lymphocytes. the cytosolic delivery of viral proteins by vaccine preparations can be achieved by using live (attenuated) virus, viral vectors, or expression from plasmid dna, which allow de novo synthesis of viral proteins in the infected cells. alternatively, particulate antigen presentation forms can be used which can translocate viral proteins into the cytosol directly or through endosomal degradation of the exogenous viral proteins. in addition to cd8 t cells, cd4 t cells have been shown to contribute to heterosubtypic immunity [54, 55 ]. the relationship between cd4 and cd8 t cells has been studied extensively, and it seems that memory cd8 t cells are impaired in the absence of memory cd4 t cells leading to increased cell death and decreased secondary t - cell response. thus, it is imperative that vaccines also induce adequate virus - specific cd4 t - helper cell responses in addition to cd8 t - cell responses. of interest, also cd4 t cells specific for seasonal influenza a viruses display cross - reactivity with influenza a viruses of different subtypes including 2009 ph1n1 and h5n1 [32, 34, 85 ]. other cells of the adaptive immune system may play a role in heterosubtypic immunity against influenza a viruses. some studies have indicated that b cells and mucosal antibodies play a role in heterosubtypic immunity [23, 44, 8690 ]. however, we and others were able to adoptively transfer heterosubtypic immunity with t cells but not with b cells to nave recipient mice (table 2). of interest, cd4 t cells are necessary to promote the protective effect of virus - specific cd8 t cells. however, since this special issue focusrs on cd8 t, cells we did not discuss the role of other immune cells extensively. also cells of the innate immune system (like nk cells and macrophages) have protective efficacy ; however, since these cells do not develop memory against pathogens, they can not be at the basis of vaccination strategies which aim at the induction of immunological memory against these pathogens. the use of live attenuated influenza vaccines (laivs) is of interest since it results in viral protein synthesis in infected antigen - presenting cells which is a prerequisite for the efficient induction of virus - specific ctl responses [9194 ]. laivs also induce antibody responses and, thus, have the capacity to induce both virus - specific ctl and b cells which both contribute to protective immunity. they are currently used in the united states and in russia, and request of approval for their use in europe has been submitted. the use of these cold - adapted viruses results in mild infections of the upper respiratory tract only. it has been shown in humans that laivs induce stronger virus - specific t - cell responses than inactivated vaccines [94, 95 ]. intranasal administration of h3n2 laiv - afforded mice partial protection against infection with h1n1 virus. laiv - vaccinated mice that were depleted of cd8 t cells were not protected and did not survive h1n1 challenge infection. furthermore, seasonal laiv induced strong ctl response in mice and afforded protection against 2009 ph1n1 virus whereas an inactivated vaccine did not. similar findings were observed by others, and the contribution of t cells in protection against 2009 ph1n1 was confirmed after depletion of these cells. laivs based on nonpathogenic h5n2 viruses also provided protection against challenge with highly pathogenic h5n1 in mice, which correlated with the induction of cross - reactive antibodies but also with cross - reactive t cells. another strategy to attenuate influenza viruses is to delete part of the nonstructural protein 1 (ns1) [100102 ]. this protein is known to be an antagonist of ifn. truncation of ns1 renders this protein nonfunctional causing attenuation of the virus. it has been shown in mice that influenza virus, with altered ns1 genes induce potent and protective memory t - cell responses. also a live attenuated m1 mutant h1n1 virus with an attenuated phenotype in vivo was generated. this live attenuated mutant virus also induced broadly cross - reactive immunity against h3n2 and h5n1 viruses, which was shown to be based on both humoral and cellular responses by adoptive transfer experiments. dna vaccines have the advantage that they can be produced rapidly and at low cost. dna vaccines encode for one or several proteins of influenza viruses and induce an immune response targeting the encoded protein. typically, plasmids are constructed with the gene of interest, for example, the np gene, under control of a strong eukaryotic promoter, for example, the cmv promoter. upon immunization of the plasmid by injection, electroporation, or gene gun delivery, the gene is expressed in cells that have taken up the plasmid (e.g., myocytes or dendritic cells). after processing of these proteins, immunogenic peptides will be generated and presented by mhc class i molecules to virus - specific t cells., it has been shown that numerous factors play a role in the efficiency of expression such as the promoter, the g / c content (sequences rich in c / g are likely to form secondary structure that inhibit translation), supercoiling that increase transfection efficiency, polyadenylation that enhance stability of mrna, and codon optimization (for review see). it has been shown in mice that the administration of dna vaccine encoding the np protein of influenza induced a strong ctl response which correlated with protection against challenge infection with homologous or heterologous virus. numerous studies have confirmed these results with dna vaccines expressing np, m1, or ha proteins in various animal models [109114 ]. one study evaluated the delivery of the vaccine by in vivo electroporation instead of the classical epidermal route. they showed that, in mice, ferrets, and nonhuman primates, this route of delivery induce protective humoral and cellular immunity. recently, a phase i clinical trial was performed with a candidate influenza dna vaccine. the vaxfectin - adjuvanted plasmid dna vaccines encoding influenza h5 ha, np, and m2 were able to elicit t - cell responses against ha in most of the subjects and against np and m2 in some of them. there might be a risk of integration into the host genome, which may increase the risk of malignancies or tolerance induction. various viruses can be used as viral vectors to deliver foreign antigens. as for laiv, the use of viral vectors caused infection of cells, which would allow endogenous antigen processing and mhc class i restricted presentation. several viruses have been considered as potential vector vaccine candidates and were able to induce ctl response such as baculovirus, vesicular stomatitis virus [119, 120 ], and semliki forest virus. adenovirus and poxviruses, like modified vaccinia virus ankara (mva), have been studied extensively for the delivery of influenza antigens. the design and production of such vaccines have been reviewed elsewhere [122124 ]. recombinant adenoviruses that are unable to replicate in human cells and that encode one or more genes of interest such as the ha, np, and m1 genes, can be produced. using such recombinant, viruses protective t - cell responses were induced in mice [21, 125132 ] and chickens [132, 133 ]. recently, it was also demonstrated that an adenovirus - based vaccine expressing ha, np, and m1 of the 2009 ph1n1 virus induced protective humoral and cellular immunity against homologous challenge and partial protection against challenge with a heterologous virus. mva - based influenza vaccines have been studied in various animal models extensively [124, 135137 ]. these vector vaccines conferred protection and induced virus - specific ctl responses [138140 ]. recently, mva vectors encoding the np and m1 genes were evaluated in a phase i clinical trial and were shown to be safe and immunogenic. these candidate vaccines also induced virus - specific cd8 t - cell responses more efficiently compared to other vaccination strategies. in addition to the vaccine formulation described above, other vaccine formulations have been described able to induce virus - specific cd8 t - cell responses. for example, virus - like particles, which can be produced after expressing influenza virus antigens in (insect) cells, have been shown to induce ctl responses in mice [143, 144 ] and in chickens. also, with gamma - irradiated influenza a virus preparations, protective immunity was induced in mice against infection with homologous and heterologous influenza a viruses. adoptive transfer experiments showed that protective immunity was mediated by virus - specific t cells. specific adjuvant systems like immune - stimulating complexes (iscoms) can be used for the induction of virus - specific ctl responses. iscoms consist of cholesterol, phospholipids, viral proteins, and glycosides of the adjuvant quil a. in addition to enhancing b - cell responses, the use of iscoms also induces strong t - cell responses. since iscoms also facilitate transport of viral protein into the cytoplasm of antigen - presenting cells, it also induces ctl responses. it has been demonstrated in mice that, with iscom - based vaccines, heterosubtypic immunity can be induced, which correlated with the induction of the virus - specific t - cell responses [149, 150 ]. also, in humans, virus - specific ctl responses could be induced with iscom - based vaccines in addition to antibody responses [151, 152 ]. for the formulation of virosomes, the membrane glycoproteins of influenza viruses are incorporated into a lipid bilayer containing phospholipids resulting in vesicles of + /150 nm in diameter. since the fusion activity of the ha molecules is retained, it would allow delivery of antigens (or plasmid dna) from the endosomes into the cytosol, allowing the induction of ctl responses [154, 155 ]. in clinical trials, virosome - based vaccines were more immunogenic in the elderly than conventional vaccines [150, 156158 ]. there is ample evidence that virus - specific ctls contribute to protective immunity against influenza virus infections. because of their cross - reactive nature, virus - specific ctls afford protection against influenza a viruses of various subtypes. it should be realized that antibodies, directed against the viral envelope proteins ha and na, are the primary correlates of protection against infection with influenza a viruses provided that they match the strain causing the infection. the presence of sufficiently high titers of specific serum antibodies, induced by vaccination or infection, will protect individuals from a subsequent infection. under these circumstances, the induction or presence of virus - specific cd8 t lymphocytes may be redundant. however, in the case of the emergence of drift variants of seasonal viruses, the available vaccines may not be as efficacious due to a poor antigenic match. in the case of the introduction of a novel pandemic strain, the seasonal vaccines will be poorly protective, and novel pandemic vaccines need to be produced, which is a time - consuming process. under these circumstances, in which humoral immunity fails to afford protection, the presence of cross - reactive ctl will not prevent infection but will contribute to more rapid clearance of infection and reduce disease severity and mortality. various vaccination formulations aiming at the induction of virus - specific ctl are currently under development. future preclinical and clinical testing need to provide information on the effectiveness of these vaccines. in a pandemic scenario, vaccines that induce cross - protective ctl could be used for emergency vaccination until vaccines become available that induce antibodies of the proper specificity. especially, immunogenically nave subjects, like young children, that have not previously experienced influenza virus infection may benefit from such a strategy. | there is considerable interest in the development of broadly protective influenza vaccines because of the continuous emergence of antigenic drift variants of seasonal influenza viruses and the threat posed by the emergence of antigenically distinct pandemic influenza viruses. it has been recognized more than three decades ago that influenza a virus - specific cytotoxic t lymphocytes recognize epitopes located in the relatively conserved proteins like the nucleoprotein and that they cross - react with various subtypes of influenza a viruses. this implies that these cd8 + t lymphocytes may contribute to protective heterosubtypic immunity induced by antecedent influenza a virus infections. in the present paper, we review the evidence for the role of virus - specific cd8 + t lymphocytes in protective immunity against influenza virus infections and discuss vaccination strategies that aim at the induction of cross - reactive virus - specific t - cell responses. |
patients diagnosed with absssi or cabp were identified through sequential review of randomly ordered charts generated from pharmacy listings at participating institutions from august 2011 to february 2013. acute bacterial skin and skin structure infection was diagnosed where infection involved deep soft tissues or required significant surgical intervention. community - acquired bacterial pneumonia was diagnosed as an acute illness with clinical signs and symptoms consistent with a lower respiratory tract infection along with imaging consistent with bacterial pneumonia. eligible patients aged 18 years or older received 2 or more consecutive doses of ceftaroline fosamil between august 2011 and july 2012, and following a protocol amendment received 4 or more consecutive doses thereafter. data collection was by review of charts 30 days or more after the completion of the ceftaroline fosamil therapy to increase the likelihood of a complete chart for data abstraction. patients were excluded when the information on dosing was missing or if data had previously been extracted for this study. data were excluded from patients with additional or underlying infections other than sab resulting from absssi or cabp. this registry study was carried out across study centers in the united states, was approved by each institution s ethics committee or institutional review board, and was conducted in compliance with the international conference on harmonisation e6 good clinical practice guidance, 1976. data collected for each eligible patient diagnosed with sab secondary to absssi or cabp included demographics, relevant medical and surgical history, location of care, clinical signs and symptoms (both at the time of diagnosis and at end of treatment with ceftaroline fosamil), and microbiology (methicillin susceptibility and source of s. aureus isolates). data were also recorded on the clinical response to ceftaroline fosamil when used as either first - line or second - line therapy, as well as monotherapy or concurrent therapy with other antibiotics. as a result of the study protocol amendment in july 2012, additional data including ceftaroline fosamil dosage, dosing frequency, laboratory data including serum creatinine level at diagnosis and end of treatment with ceftaroline fosamil, readmission within 30 days of discharge, and reason for readmission were collected. the data were summarized using descriptive statistics, including demographic data, disease and pathogen characteristics, ceftaroline fosamil dose, frequency and duration, outcome, and hospital discharge. numeric data were summarized with mean (sd) and median values, where categorical data were presented by frequency and percentage. evaluable patients were those for whom a clinical outcome (success or failure) could be determined. clinical success was defined as either clinical cure with no further need for antibiotic therapy or as a clinical improvement with a switch to oral antibiotic treatment. clinical failure was determined where patients experienced an adverse event leading to discontinuation or where insufficient therapeutic effect was documented. in some cases, after a review of information, treatment was also deemed to be successful where patients were confirmed to be improving at the time of discontinuation of ceftaroline fosamil. patients diagnosed with absssi or cabp were identified through sequential review of randomly ordered charts generated from pharmacy listings at participating institutions from august 2011 to february 2013. acute bacterial skin and skin structure infection was diagnosed where infection involved deep soft tissues or required significant surgical intervention. community - acquired bacterial pneumonia was diagnosed as an acute illness with clinical signs and symptoms consistent with a lower respiratory tract infection along with imaging consistent with bacterial pneumonia. eligible patients aged 18 years or older received 2 or more consecutive doses of ceftaroline fosamil between august 2011 and july 2012, and following a protocol amendment received 4 or more consecutive doses thereafter. data collection was by review of charts 30 days or more after the completion of the ceftaroline fosamil therapy to increase the likelihood of a complete chart for data abstraction. patients were excluded when the information on dosing was missing or if data had previously been extracted for this study. data were excluded from patients with additional or underlying infections other than sab resulting from absssi or cabp. this registry study was carried out across study centers in the united states, was approved by each institution s ethics committee or institutional review board, and was conducted in compliance with the international conference on harmonisation e6 good clinical practice guidance, 1976. data collected for each eligible patient diagnosed with sab secondary to absssi or cabp included demographics, relevant medical and surgical history, location of care, clinical signs and symptoms (both at the time of diagnosis and at end of treatment with ceftaroline fosamil), and microbiology (methicillin susceptibility and source of s. aureus isolates). data were also recorded on the clinical response to ceftaroline fosamil when used as either first - line or second - line therapy, as well as monotherapy or concurrent therapy with other antibiotics. as a result of the study protocol amendment in july 2012, additional data including ceftaroline fosamil dosage, dosing frequency, laboratory data including serum creatinine level at diagnosis and end of treatment with ceftaroline fosamil, readmission within 30 days of discharge, and reason for readmission were collected. the data were summarized using descriptive statistics, including demographic data, disease and pathogen characteristics, ceftaroline fosamil dose, frequency and duration, outcome, and hospital discharge. numeric data were summarized with mean (sd) and median values, where categorical data were presented by frequency and percentage. evaluable patients were those for whom a clinical outcome (success or failure) could be determined. clinical success was defined as either clinical cure with no further need for antibiotic therapy or as a clinical improvement with a switch to oral antibiotic treatment. clinical failure was determined where patients experienced an adverse event leading to discontinuation or where insufficient therapeutic effect was documented. in some cases, after a review of information, treatment was also deemed to be successful where patients were confirmed to be improving at the time of discontinuation of ceftaroline fosamil. of 1500 patients with either absssi or cabp enrolled across 40 study centers, 1428 were evaluable. a subset of 3% (48/1428) of evaluable patients had a diagnosis of secondary sab, comprising 3% (27/1030) of patients with absssi and 5% (21/398) of patients with cabp (table 1). the patients with sab had a near - even sex distribution and a mean (sd) age of 61 (15) years. evaluable patients with sab secondary to absssi or cabp at least 1 comorbid condition was recorded for 74% of patients with absssi and 81% with cabp. analysis of patients medical history revealed the most common absssi - associated comorbidity to be diabetes (70%), followed by obesity (33%) and peripheral vascular disease (22%). the most common comorbidities associated with cabp were structural lung disease (33%), gastroesophageal reflux disease (29%), and congestive heart failure (24%). among the patients with sab, 13 patients (8 with absssi, 5 with cabp) the mean (sd) serum creatinine measurement for these 13 patients was 2.4 (2.4) mg / dl, with a median (range) of 1.5 (0.5 - 9.1) mg / dl before the start of ceftaroline fosamil treatment. readmission data (within 30 days of discharge) were collected after a protocol amendment in 2012 ; therefore readmission data were only available for 8 patients with absssi, of which 2 patients were readmitted for absssi. readmission data were available for 5 patients with cabp of which one was readmitted for cabp. of the 48 patients with sab, mrsa was isolated in 67% (32/48) of cases. among patients with sab secondary to absssi, mrsa was isolated in 59% (16/27) of cases and among patients with sab secondary to cabp, in 76% (16/21) of cases. the mean (sd) duration of ceftaroline fosamil therapy was 5.8 (4.8) days for sab secondary to absssi and 7.0 (3.8) days for sab secondary to cabp. before the administration of ceftaroline fosamil, 89% of patients with sab secondary to absssi and 86% of patients with sab secondary to cabp had received antibiotic therapy, of which glycopeptides, penicillins, and other cephalosporins were most commonly used (table 2). concurrent therapy was administered in 30% of patients with sab secondary to absssi (most commonly glycopeptides and lincosamides) and 71% of patients with sab secondary to cabp (most commonly glycopeptides and macrolides). monotherapy with ceftaroline fosamil was utilized in 70% of patients with sab secondary to absssi and in 29% of patients with sab secondary to cabp. after july 2012, data were collected on frequency of infusion with ceftaroline fosamil. among those patients for whom such data were available (n = 13), 1 patient with sab secondary to cabp received ceftaroline fosamil by infusion every 8 hours. antibiotic usage for patients with sab one quarter of all patients with sab were treated in the intensive care unit (icu) while receiving ceftaroline fosamil. a higher proportion of patients with sab secondary to cabp were treated in the icu (33%) when compared with patients with sab secondary to absssi (19%). after ceftaroline fosamil therapy, patients had similar rates of discharge to home or to another care facility (48%). one patient with cabp died, and postdischarge data for one patient with absssi were missing. the success rate among patients with sab secondary to absssi was 52%, whereas the success rate among patients with sab due to cabp was 67%. clinical success rates of sab associated with mrsa among patients with absssi and with cabp were 50% (8/16) and 63% (10/16), respectively. similarly, for sab caused by mssa and secondary to absssi, the clinical success rate was 55% (6/11). for sab caused by mssa and secondary to cabp, 80% (4/5) were categorized as a clinical success. three patients with sab secondary to absssi and 3 patients with sab secondary to cabp received ceftaroline fosamil as first - line therapy. among patients who received ceftaroline fosamil as second - line therapy, the overall clinical success rate was 64% (27/42), with a clinical success rate of 58% (14/24) for sab due to absssi and 72% (13/18) for sab due to cabp. among those patients who received ceftaroline fosamil as monotherapy, the overall clinical success rate was 64% (16/25). in patients with sab secondary in patients with sab secondary to cabp, 83% (5/6) were considered a clinical success. of the 23 patients that received concurrent antibiotic therapy, the clinical success rate was 52% (12/23). among the 8 patients with sab and absssi who received concurrent therapy, 3 were a clinical success. for patients with sab secondary to cabp, the clinical success rate was 60% (9/15). in the absssi group, 2 patients (7%) were classified as a clinical improvement and were switched to an oral antibiotic. for 8 patients (30%), there was insufficient therapeutic effect, and they were switched to an alternative intravenous antibiotic. adverse events were given as the reason for discontinuation for 2 patients (7%) with sab secondary to absssi. for patients with sab secondary to cabp, clinical improvement with switch to oral antibiotic was recorded for 1 patient, and for 4 patients, there was insufficient therapeutic effect, and they were switched to an alternative intravenous antibiotic. of 1500 patients with either absssi or cabp enrolled across 40 study centers, 1428 were evaluable. a subset of 3% (48/1428) of evaluable patients had a diagnosis of secondary sab, comprising 3% (27/1030) of patients with absssi and 5% (21/398) of patients with cabp (table 1). the patients with sab had a near - even sex distribution and a mean (sd) age of 61 (15) years. evaluable patients with sab secondary to absssi or cabp at least 1 comorbid condition was recorded for 74% of patients with absssi and 81% with cabp. analysis of patients medical history revealed the most common absssi - associated comorbidity to be diabetes (70%), followed by obesity (33%) and peripheral vascular disease (22%). the most common comorbidities associated with cabp were structural lung disease (33%), gastroesophageal reflux disease (29%), and congestive heart failure (24%). among the patients with sab, 13 patients (8 with absssi, 5 with cabp) the mean (sd) serum creatinine measurement for these 13 patients was 2.4 (2.4) mg / dl, with a median (range) of 1.5 (0.5 - 9.1) mg / dl before the start of ceftaroline fosamil treatment. readmission data (within 30 days of discharge) were collected after a protocol amendment in 2012 ; therefore readmission data were only available for 8 patients with absssi, of which 2 patients were readmitted for absssi. readmission data were available for 5 patients with cabp of which one was readmitted for cabp. of the 48 patients with sab, mrsa was isolated in 67% (32/48) of cases. among patients with sab secondary to absssi, mrsa was isolated in 59% (16/27) of cases and among patients with sab secondary to cabp, in 76% (16/21) of cases. the mean (sd) duration of ceftaroline fosamil therapy was 5.8 (4.8) days for sab secondary to absssi and 7.0 (3.8) days for sab secondary to cabp. before the administration of ceftaroline fosamil, 89% of patients with sab secondary to absssi and 86% of patients with sab secondary to cabp had received antibiotic therapy, of which glycopeptides, penicillins, and other cephalosporins were most commonly used (table 2). concurrent therapy was administered in 30% of patients with sab secondary to absssi (most commonly glycopeptides and lincosamides) and 71% of patients with sab secondary to cabp (most commonly glycopeptides and macrolides). monotherapy with ceftaroline fosamil was utilized in 70% of patients with sab secondary to absssi and in 29% of patients with sab secondary to cabp. after july 2012, data were collected on frequency of infusion with ceftaroline fosamil. among those patients for whom such data were available (n = 13), 1 patient with sab secondary to cabp received ceftaroline fosamil by infusion every 8 hours. one quarter of all patients with sab were treated in the intensive care unit (icu) while receiving ceftaroline fosamil. a higher proportion of patients with sab secondary to cabp were treated in the icu (33%) when compared with patients with sab secondary to absssi (19%). after ceftaroline fosamil therapy, patients had similar rates of discharge to home or to another care facility (48%). one patient with cabp died, and postdischarge data for one patient with absssi were missing. the success rate among patients with sab secondary to absssi was 52%, whereas the success rate among patients with sab due to cabp was 67%. clinical success rates of sab associated with mrsa among patients with absssi and with cabp were 50% (8/16) and 63% (10/16), respectively. similarly, for sab caused by mssa and secondary to absssi, the clinical success rate was 55% (6/11). for sab caused by mssa and secondary to cabp, 80% (4/5) were categorized as a clinical success. three patients with sab secondary to absssi and 3 patients with sab secondary to cabp received ceftaroline fosamil as first - line therapy. among patients who received ceftaroline fosamil as second - line therapy, the overall clinical success rate was 64% (27/42), with a clinical success rate of 58% (14/24) for sab due to absssi and 72% (13/18) for sab due to cabp. among those patients who received ceftaroline fosamil as monotherapy, the overall clinical success rate was 64% (16/25). in patients with sab secondary to absssi, the clinical success rate was 58% (11/19). in patients with sab secondary to cabp, 83% (5/6) were considered a clinical success. of the 23 patients that received concurrent antibiotic therapy, the clinical success rate was 52% (12/23). among the 8 patients with sab and absssi who received concurrent therapy, the clinical success rate was 60% (9/15). in the absssi group, 2 patients (7%) were classified as a clinical improvement and were switched to an oral antibiotic. for 8 patients (30%), there was insufficient therapeutic effect, and they were switched to an alternative intravenous antibiotic. adverse events were given as the reason for discontinuation for 2 patients (7%) with sab secondary to absssi. for patients with sab secondary to cabp, clinical improvement with switch to oral antibiotic was recorded for 1 patient, and for 4 patients, there was insufficient therapeutic effect, and they were switched to an alternative intravenous antibiotic. data for the treatment of sab secondary to absssi or cap are not readily available in the literature, with few published studies. khosrovaneh reported on a study of 50 cases of sab originating from soft tissues ; however, their study did not include an overall cure rate. analysis of data from the capture study reveals clinical success rates of 38% to 83%, which are comparable with the clinical success rates reported for vancomycin (52%) and linezolid (55%) in a pooled analysis of patients with sab and daptomycin (44%) and vancomycin or an antistaphylococcal penicillin (42%) for patients with sab with or without endocarditis. in studies for patients with mrsa bacteremia, the success rates ranged between 32% and 60% for various agents, and among patients with mssa bacteremia, the success rates were between 45% and 49%. late complications can occur with bacteremia ; therefore the variable follow - up periods used in these published studies necessitates that some caution should be exercised when comparing studies. the follow - up period in the capture study was 30 days (with readmissions to the same hospital only recorded) ; complications of bacteremia resulting in readmission to hospital could occur either during this 30 days at another institution or after this time at the same institution and would not be recorded as part of capture, but as a registry chart review study rather than a prospective study with a late follow - up visit, this is a limitation of the study. clinical response was limited to assessment at discontinuation of ceftaroline fosamil, and mortality was required to be reported either in - hospital or within 24 hours of discontinuation of ceftaroline fosamil. the mean duration of treatment in this study was 6.4 days, which is shorter than would be expected for the treatment of sab. however, the mean duration of treatment data presented is for ceftaroline fosamil only and does not include duration of prior or subsequent antibiotic therapy. because many patients were treated with ceftaroline fosamil as second - line therapy and may have been switched to another agent after improving, the mean duration of treatment is not necessarily representative of the length of the entire treatment period. in addition, although case studies in the literature that present data on the use of ceftaroline fosamil in the treatment of bacteremia often use an 8-hour dosing regimen, dosing in this study most frequently followed a 12-hourly regimen. this most likely occurred because ceftaroline fosamil was being used in the treatment of a primary absssi or cabp with secondary sab. as described previously for the overall population, clinical success rates among patients who received monotherapy and concurrent therapy were generally favorable. clinical success rates were greater than 52%, with the exception of patients with sab secondary to absssi, who received concurrent therapy, although the numbers of patients in this subgroup was small (3 of 8 patients determined as clinical success). readmission data were collected after july 2012, leading to a small number of patients for whom readmission data are available (n = 13). a total of 5 patients were readmitted, 4 who had been treated for sab secondary to absssi and one who had been treated for sab secondary to cabp. of the 4 patients who were treated for sab secondary to absssi, 2 were readmitted for absssi and 2 for non absssi - associated reasons. the phase 3 studies of ceftaroline fosamil showed a clinical success rate of 89% (16/18) among patients with sab secondary to absssi. this is markedly higher than the data reported in the capture study and may, in part, be due to the restricted use of prior antibiotic therapy in the phase 3 studies and the higher rate of comorbidities among patients in the capture study. in addition, the phase 3 studies had multiple other exclusionary criteria, which included the exclusion of patients directly admitted to the icu. this discrepancy highlights a major difference between controlled phase 3 studies and registry studies. despite the high incidence of comorbidities, clinical response rates in the capture study were generally favorable. furthermore, the high proportion of patients treated in the icu indicates a high acuity of illness with presence of severe disease in the patients with sab in capture. ceftaroline fosamil was frequently used as a second - line or salvage antimicrobial therapy (> 85% of patients), and the glycopeptides (eg, vancomycin) were the most commonly administered antibiotic class before the initiation of ceftaroline fosamil (60% overall), implying glycopeptide failure. this further frames the clinical outcomes in a population of patients who were likely treatment failures to begin with and at higher risk for subsequent clinical failure. as discussed previously, registry studies such as capture may have inherent limitations such as lack of randomization or blinding to control for bias ; however, they have the potential to provide useful information on the contemporary use of antibiotics, including their spectrum of use, duration of treatment, effectiveness, safety, and cost. in conclusion, the results from this study support the clinical effectiveness of ceftaroline fosamil in the treatment of absssi or cabp associated with secondary sab. ceftaroline fosamil was an effective treatment option, despite the wide range of comorbidities, including those patients who had likely received prior unsuccessful antibiotic therapy. these data support the use of ceftaroline fosamil as an effective treatment option in hospitalized patients with sab secondary to absssi and cabp. furthermore, these results suggest that further clinical studies evaluating the use of ceftaroline fosamil for sab are warranted. | backgroundthe clinical assessment program and teflaro utilization registry is designed to collect information on the clinical use of ceftaroline fosamil in the unites states. this report presents data on the treatment of patients with staphylococcus aureus bacteremia (sab) secondary to acute bacterial skin and skin structure infections (absssis) or community - acquired bacterial pneumonia (cabp).methodspatients diagnosed with absssi or cabp were identified through sequential review of randomly ordered charts generated from pharmacy listings from august 2011 to february 2013. data were collected by chart review 30 days or more after completion of ceftaroline fosamil therapy.resultssecondary sab was reported in a total of 48 of 1428 evaluable patients (27 with absssi, 21 with cabp). the mean (sd) patient age was 61 (15) years. at least 1 comorbidity was recorded for 74% of patients with absssi and 81% with cabp. methicillin - resistant s. aureus was isolated from 59% of patients with absssi and 76% with cabp. the mean (sd) duration of ceftaroline fosamil therapy was 5.8 (4.8) days for absssi and 7.0 (3.8) days for cabp. clinical success among all patients with sab treated with ceftaroline fosamil was 58% (52% for sab secondary to absssi, 67% for sab secondary to cabp). clinical success rates of methicillin - resistant s. aureus sab were 50% (8/16) for absssi and 63% (10/16) for cabp.conclusionsthis study supports the use of ceftaroline fosamil as a viable treatment option in hospitalized patients with sab secondary to absssi or cabp. further studies evaluating the use of ceftaroline fosamil for the treatment of sab are warranted. |
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